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North America Cancer Biopsy Market By Type (Liquid Biopsy, Tissue Biopsy), By Product Type (Instruments, Kits & Consumables, Others), By Application (Breast Cancer, Lung Cancer, Others), By Country, Competition, Forecast and Opportunities, 2019-2029F

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Description

Table of contents.

North America Cancer biopsy Market was valued at USD 10.71 Billion in 2023 and is anticipated to project impressive growth in the forecast period with a CAGR of 7.85% through 2029. The North America cancer biopsy market plays a vital role in the region's healthcare landscape, crucial for cancer diagnosis and treatment. Biopsies, involving tissue sample extraction for examination, are pivotal in identifying cancer presence, type, and extent, guiding personalized treatment plans for physicians. Recent years have seen significant market growth driven by several factors. Rising cancer incidence remains a major public health concern, with various types like breast, lung, colorectal, and prostate cancers steadily increasing. Early detection's importance fuels demand for precise diagnostic tools such as biopsies. Technological advancements, including liquid biopsy, offer less invasive alternatives, especially for challenging cancers. Advanced imaging and robotic-assisted biopsy techniques enhance accuracy and efficiency. Personalized medicine emphasis spurs molecular diagnostics adoption, tailoring treatment to tumor genetics, enhancing therapeutic precision and efficacy. Thus, biopsy samples serve not only as diagnostics but also critical genetic information sources, shaping targeted therapies and cancer care evolution.

Government initiatives and healthcare policies in North America have significantly influenced the cancer biopsy landscape. Regulatory frameworks and reimbursement policies facilitate the integration of advanced biopsy technologies into routine clinical practice. National cancer control programs prioritize preventive and early diagnostic measures, highlighting the importance of biopsy procedures in cancer care. The market is competitive, with key players driving innovation and expansion. Companies specializing in biopsy tools, imaging technologies, and laboratory services collaborate to enhance diagnostic capabilities. Strategic partnerships and mergers foster research and development, enabling the introduction of novel biopsy techniques.

Key Market Drivers

Rising cancer incidence drives demand for precise diagnostics

The North America cancer biopsy market is experiencing a significant surge in demand driven primarily by the escalating incidence of cancer across the region. With cancer rates on the rise, there is an urgent need for precise diagnostics to facilitate timely and accurate detection, thus enabling appropriate treatment strategies to be implemented. Biopsy procedures play a pivotal role in this scenario, serving as indispensable tools in confirming the presence of cancer, determining its specific type, and assessing the extent of its spread within the body. In the context of comprehensive cancer care, reliable diagnostic information is paramount. Biopsy techniques provide clinicians with crucial insights into the nature of the disease, guiding treatment decisions and ensuring that patients receive the most effective interventions tailored to their individual circumstances. From guiding surgical interventions to informing the selection of targeted therapies, biopsy results serve as the foundation upon which personalized treatment plans are built. The increasing demand for precise diagnostics underscores the critical importance of biopsy procedures within the cancer care continuum. As healthcare providers strive to meet the growing needs of patients, there is a corresponding emphasis on enhancing the accessibility, accuracy, and efficiency of biopsy techniques. This has led to ongoing advancements in biopsy technology, including the development of minimally invasive approaches such as liquid biopsies, which offer less discomfort and faster turnaround times compared to traditional tissue biopsies. The integration of advanced imaging modalities and molecular diagnostics has further expanded the diagnostic capabilities of biopsy procedures, enabling clinicians to obtain more detailed and comprehensive information about tumor characteristics and molecular profiles. As a result, the North America cancer biopsy market is poised for continued growth, driven by the imperative to meet the escalating demand for precise diagnostics in the fight against cancer.

Continuous technological advancements enhance biopsy accuracy

The North America cancer biopsy market is experiencing significant growth, largely driven by continuous technological advancements that have revolutionized cancer diagnostics. These innovations have ushered in a new era of precision and reliability in biopsy procedures, empowering healthcare professionals with more accurate and actionable insights into the nature of cancerous tumors. State-of-the-art imaging technologies, such as MRI-guided biopsies and robotic-assisted procedures, have emerged as cornerstones of modern biopsy techniques. These advanced imaging modalities allow for precise targeting of suspicious lesions, ensuring optimal tissue sample extraction with minimal invasiveness. As a result, clinicians can obtain high-quality biopsy specimens, thereby enhancing diagnostic accuracy and reducing the likelihood of sampling errors. The introduction of molecular diagnostics and liquid biopsy techniques has significantly expanded the diagnostic capabilities of cancer biopsies. Molecular profiling of tumor specimens enables oncologists to glean valuable information about specific genetic mutations and biomarkers associated with different cancer types. This deeper understanding of tumor biology not only facilitates more accurate cancer classification but also informs the selection of targeted therapies tailored to individual patients' molecular profiles. The integration of these advanced biopsy technologies into routine clinical practice has been transformative for cancer care. Early and accurate cancer diagnoses are now achievable, allowing for timely initiation of appropriate treatment strategies. The ability to tailor treatment plans based on molecular insights obtained from biopsy specimens has led to more personalized therapeutic interventions, resulting in improved patient outcomes and enhanced overall survival rates. The dynamic nature of the North America cancer biopsy market is sustained by a commitment to research and development. Industry leaders continue to invest in cutting-edge technologies aimed at further refining biopsy procedures and expanding diagnostic capabilities. As a result, the market remains at the forefront of delivering sophisticated and precise diagnostic solutions that are instrumental in the ongoing fight against cancer.

Key Market Challenges

Accuracy and Sensitivity

The Cancer Biopsy Market is confronted with the formidable challenge of upholding high levels of accuracy and sensitivity in diagnostic procedures. The precision and sensitivity of biopsy results are paramount in guiding oncologists toward making informed and effective treatment decisions for patients battling cancer. However, several factors pose hurdles to achieving consistent accuracy in biopsy outcomes. One significant challenge is the variability in sample quality obtained during biopsy procedures. Factors such as tissue preservation techniques, specimen handling, and processing methods can impact the integrity and quality of the tissue sample, potentially compromising the accuracy of diagnostic assessments. The inherent heterogeneity of tumors presents a formidable obstacle to achieving uniformity and reliability in biopsy results. Tumors may exhibit diverse cellular compositions and genetic mutations within different regions, leading to sampling errors and inaccuracies in diagnosis. Innovations in technology, including advanced imaging modalities and molecular diagnostics, hold promise for enhancing the sensitivity and accuracy of biopsy procedures. Advanced imaging techniques, such as MRI-guided biopsies and PET-CT scans, enable precise targeting of suspicious lesions, improving the likelihood of obtaining representative tissue samples. Likewise, molecular diagnostics offer insights into the genetic makeup of tumors, allowing for more refined and personalized treatment strategies based on individual tumor profiles. Despite these advancements, challenges persist in ensuring consistent accuracy and sensitivity in biopsy results. Addressing these challenges requires ongoing research and development efforts aimed at refining biopsy techniques, optimizing sample collection and processing protocols, and leveraging emerging technologies to overcome tumor heterogeneity and sampling errors. By surmounting these obstacles, the Cancer Biopsy Market can continue to provide reliable and actionable diagnostic information, ultimately contributing to improved patient outcomes in the dynamic landscape of cancer diagnostics.

Invasive Nature

The Cancer Biopsy Market faces a significant hurdle posed by the invasive nature of traditional biopsy procedures. These invasive techniques, while essential for obtaining tissue samples for diagnostic evaluation, present several drawbacks and challenges. One major concern is the discomfort and pain experienced by patients undergoing invasive biopsies, which can lead to increased anxiety and reluctance to undergo the procedure. The invasive biopsies carry the risk of complications such as bleeding, infection, and damage to surrounding tissues, particularly in sensitive or difficult-to-access anatomical locations. Another challenge associated with invasive biopsies is the potential for sampling errors, particularly in cases where obtaining adequate tissue specimens is challenging or where tumor heterogeneity complicates accurate diagnosis. These sampling errors can result in misdiagnosis or incomplete characterization of the tumor, leading to suboptimal treatment decisions and outcomes. In response to these challenges, there is growing interest in exploring non-invasive or minimally invasive biopsy alternatives in the Cancer Biopsy Market. These alternative approaches aim to reduce patient discomfort, minimize procedural risks, and improve the overall accessibility and acceptance of biopsy procedures in cancer diagnostics. Techniques such as liquid biopsies, which involve the analysis of circulating tumor cells or cell-free DNA in blood samples, offer a less invasive option for obtaining diagnostic information. Similarly, minimally invasive imaging-guided biopsy techniques, such as ultrasound-guided core needle biopsies or endoscopic biopsies, allow for targeted tissue sampling with reduced procedural invasiveness. Overcoming the invasiveness of traditional biopsy procedures remains a priority for advancing biopsy methodologies in the Cancer Biopsy Market. By developing and refining non-invasive and minimally invasive biopsy techniques, healthcare providers can improve patient experience, enhance diagnostic accuracy, and ultimately optimize treatment outcomes for individuals undergoing cancer diagnostics.

Key Market Trends

Liquid Biopsy Advancements

Bioprocessing automation is emerging as a transformative trend in the Cancer Biopsy Market, revolutionizing the way cell culture workflows are conducted. With automation, production processes are streamlined, leading to improved efficiency, reduced errors, and enhanced product quality. Advanced robotic systems, coupled with integrated sensors and data analytics, optimize every stage of cell culture workflows, from media preparation to cell harvesting. By minimizing human intervention and standardizing procedures, automation helps lower labor costs and ensures consistent results across batches. One of the significant advantages of bioprocessing automation is its ability to increase throughput and scalability. With automated systems, manufacturers can handle larger volumes of samples and meet the growing demand for cell-based products and therapies. This scalability is particularly crucial in the Cancer Biopsy Market, where the need for precise and timely diagnostic solutions continues to rise. The automation plays a pivotal role in improving reproducibility. By eliminating manual errors and variability, automated processes generate standardized results, enhancing the reliability of diagnostic tests and research outcomes. This consistency is essential for ensuring accurate diagnoses and treatment decisions in cancer care. As the Cancer Biopsy Market embraces Industry 4.0 principles, the integration of automation is poised to drive further growth and innovation. By leveraging data-driven insights and advanced technologies, automated systems accelerate research efforts, facilitate rapid prototyping of new diagnostic tools, and enable the development of cutting-edge therapies. Ultimately, bioprocessing automation is reshaping the future of cell culture technologies, paving the way for more efficient, reliable, and scalable solutions in cancer diagnostics and treatment.

Precision Oncology Integration

Precision oncology integration represents a significant advancement in the Cancer Biopsy Market, offering personalized treatment approaches tailored to individual patients' genetic makeup. This trend emphasizes the analysis of tumor tissue or liquid biopsy samples to identify specific genetic alterations, mutations, or biomarkers that drive cancer progression. By obtaining insights into the molecular characteristics of each patient's cancer, oncologists can make informed decisions regarding targeted therapies that precisely target the underlying drivers of tumor growth. The implementation of precision oncology aims to optimize treatment efficacy while minimizing the risk of adverse effects commonly associated with traditional chemotherapy and radiation therapy. By selecting therapies that specifically target the genetic vulnerabilities of the cancer cells, precision oncology holds the potential to improve patient outcomes and survival rates. Advancements in cancer biology and genomic technologies have been instrumental in facilitating the seamless integration of precision oncology into routine cancer biopsy practices. With increasingly sophisticated diagnostic tools and bioinformatics platforms, healthcare providers can efficiently analyze complex genomic data and identify actionable mutations or biomarkers that inform treatment decisions. As precision oncology continues to evolve, driven by ongoing research and technological innovation, it is expected to play an increasingly prominent role in guiding personalized cancer care. By harnessing the power of genomic information, precision oncology has the potential to revolutionize cancer treatment paradigms and improve outcomes for patients worldwide.

Segmental Insights

Type Insights

Based on the type, tissue biopsy remains a dominant method in the Cancer Biopsy Market, serving as a gold standard for diagnosing and characterizing various cancers. Tissue biopsy stands as a fundamental procedure in cancer diagnostics, involving the extraction of a minute sample of abnormal tissue for microscopic analysis. This approach offers invaluable insights into various aspects of cancer, including its type, stage, and molecular features. While innovative techniques like liquid biopsy have gained attention, tissue biopsy remains indispensable, particularly for solid tumours where obtaining tissue samples is essential for accurate diagnosis and characterization. The comprehensive data derived from tissue specimens play a pivotal role in guiding clinicians in devising personalized treatment strategies tailored to individual patients' unique cancer profiles. From selecting targeted therapies to assessing prognosis, tissue biopsy serves as a cornerstone in the holistic management of cancer patients, ensuring that they receive the most effective and appropriate care based on their specific disease characteristics and needs.

Product Type Insights

In the Cancer Biopsy Market, kits & consumables play a dominant role by providing essential tools for sample collection, preservation, and analysis. Biopsy kits play a pivotal role in modern cancer diagnostics, encompassing a range of essential components such as biopsy needles, containers, fixatives, and other consumables necessary for efficient tissue or liquid biopsy procedures. As biopsy techniques continue to advance, there is a corresponding increase in demand for specialized kits tailored to these evolving methodologies. These kits offer several advantages, including convenience, reliability, and compatibility, which are instrumental in ensuring the success of cancer biopsy procedures. Their dominance in the market underscores the growing emphasis on streamlined workflows and the critical importance of obtaining high-quality samples for precise molecular diagnostics and the development of personalized cancer treatment strategies. By providing clinicians with standardized tools and materials, biopsy kits facilitate consistent and accurate sample collection, thereby enhancing the reliability and effectiveness of biopsy-based diagnostic approaches in the realm of oncology.

Regional Insights

The United States holds a dominant position in the Cancer Biopsy Market, primarily due to its robust healthcare infrastructure, advanced diagnostic technologies, and high incidence of cancer cases. With a significant emphasis on healthcare research and development, coupled with substantial investments in oncology research, the U.S. remains at the forefront of cancer diagnostics and treatment globally. The country benefits from a proactive regulatory environment and strong industry collaborations, which foster continuous innovation in cancer biopsy techniques and technologies. The presence of leading biotech and pharmaceutical companies further contributes to the market's prominence, facilitating the development and commercialization of cutting-edge biopsy products and services. The well-established healthcare system in the U.S. ensures widespread adoption of biopsy techniques across various healthcare settings, including hospitals, clinics, and research institutions. As a result, the United States serves as a central hub for advancements in cancer biopsy practices, driving market growth and shaping the trajectory of cancer diagnostics on a global scale.

Key Market Players

Baxter International Inc

B. Braun Medical Inc.

Fresenius Medical Care North America

Medtronic, Inc

Asahi Kasei Homes North America, Inc.

Nipro Medical Corporation - North America Division

Becton Dickinson & Co

Nikkiso America, Inc.

Report Scope:

In this report, the North America Cancer Biopsy Market has been segmented into the following categories, in addition to the industry trends which have also been detailed below:

North America Cancer Biopsy Market, By Type:

North america cancer biopsy market, by product type:, north america cancer biopsy market, by application:, north america cancer biopsy market, by country:, competitive landscape.

Company Profiles: Detailed analysis of the major companies presents in the North America Cancer Biopsy Market.

Available Customizations:

North America Cancer Biopsy market report with the given market data, Tech Sci Research offers customizations according to a company's specific needs. The following customization options are available for the report:

Company Information

Detailed analysis and profiling of additional market players (up to five).

1. Product Overview

2. research methodology, 3. executive summary, 4. north america cancer biopsy market outlook, 5. united states cancer biopsy market outlook, 6. canada cancer biopsy market outlook, 7. mexico cancer biopsy market outlook, 8. market dynamics, 9. market trends & developments, 10. north america cancer biopsy market: swot analysis, 11. porter's five forces analysis, 12. competitive landscape, 12.10 angle plc, 13. strategic recommendations, 14. about us & disclaimer.

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Evaluating the Breast Cancer Therapeutics Market

An analyst at citeline provided pharmanewsintelligence with insights into current trends, gaps, and predictions in the breast cancer therapeutics market..

• Veronica Salib, Assistant Editor

As breast cancer awareness month continues, gathering a comprehensive understanding of the breast cancer therapeutics market is critical, including current trends and market players, gaps, and predictions for the coming year. Understanding the therapeutics market and market dynamics can help companies position themselves for prompt regulatory approval or successful market entry.

According to the World Health Organization (WHO), in 2020, there were approximately 2.3 million breast cancer diagnoses worldwide. Additionally, the global prevalence of breast cancer mortality was nearly 700,000.

The organization explained, "Breast cancer cells begin inside the milk ducts and/or the milk-producing lobules of the breast. The earliest form (in situ) is not life-threatening. Cancer cells can spread into nearby breast tissue (invasion). This creates tumors that cause lumps or thickening.”

Therapeutic interventions for breast cancer may include surgical tumor removal, radiation therapy, or medications, including hormonal therapies, chemotherapy, and targeted biologic therapies.

PharmaNewsIntelligence sat down with Millie Gray, Healthcare Analyst at Citeline, a Norstella company, to discuss the market for breast cancer therapies and treatments.

Breast Cancer Therapeutics Market

A recent market report published by Precedence Research, which analyzed from the base year 2022 to the forecast period of 2023–2032, revealed that in 2022 the breast cancer market size was estimated to be $28.8 billion (USD). Research projections anticipate the market will be valued at $73.68 billion (USD) by 2032 with a compound annual growth rate (CAGR) of 9.9%.

The report revealed that the largest breast cancer market is in North America (US, Canada, and Mexico), with Asia Pacific (Japan, China, Australia, India, South Korea, etc.) in second place. However, researchers also explored other regions, including Europe (Germany, France, United Kingdom, Italy, Spain, etc.), Latin America, the Middle East, and Africa (Brazil, South Africa, Saudi Arabia, etc.).

Analysts segmented the breast cancer market by distribution channel, cancer type, and therapy, evaluating trends in each segment.

According to the report, key players in the breast cancer market include Merck & Co., Bristol Myers Squibb, Kyowa Kirin, Eisai Co. Ltd, Sanofi, Pfizer Inc, AstraZeneca PLC, Novartis AG, Eli Lilly and Company, Genentech, Mylan Laboratories, Celltrion, Fresenius Kabi, Baxter Healthcare Corporation, Halozyme Inc., and GlaxoSmithKline.

In a 2021 Nature market research article , analysts divided breast cancer treatments depending on cancer subtypes, which included the following:

• HER2-positive
• HR-positive/HER2-negative
• Triple-negative breast cancer

Key Market Focuses

PharmaNewsIntelligence asked Gray to provide insights into the key market focuses in breast cancer.

“Last year and the year before, the focus was more on the metastatic setting,” she revealed.

According to Gray, there have been significant advancements in metastatic breast cancer, especially triple-negative breast cancer (TNBC), over the past year.

“TNBC is a subtype of breast cancer, which, before these approvals, essentially was a hundred percent treated by chemotherapy. There was very little use of targeted therapy for TNBC. So that's a huge area of focus and development there,” explained Gray.

Considering the limited options, companies saw the potential for market growth and pursued treatments focused on this aggressive breast cancer subtype.

Early-Stage Focus

Beyond targeting TNBC, Gray noted increasing therapeutic interventions intended to treat breast cancer in its early stages.

“Pharma companies are now focusing on getting their drugs approved in the earlier stages of breast cancer,” she added. “Now, quite a few therapies are approved across all the subtypes of breast cancer. They're focusing on the early stage setting because it is the largest among all breast cancers. Approximately 90% of breast cancers get diagnosed at the early stages.”

Gray explains that targeting early-stage breast cancer allows pharmaceutical companies to reach the greatest portion of patients — meaning that they can make significant revenues even if they comprise a small market share .

Beyond the financial incentives to focus on early-stage breast cancers , Gray adds that early stages are the only stage where physicians could potentially cure the condition.

“In the metastatic setting, patients aren't treated with curative intent; they're treated with palliative attempts to manage the symptoms and potentially keep the patient alive longer,” she explained. “The development of better therapies, in the early-stage setting, is looking at being able to cure these patients or, at the very least, extend their life longer than the chemotherapies and endocrine therapies approved there before.”

Regulatory Approval and Competitive Landscape

In addition to describing therapeutic goals and target changes, Gray explored the competitive landscape and how increased competition and market saturation may impact regulatory approval. As more breast cancer therapies are researched, approved, and launched , market growth may disadvantage new researchers seeking regulatory approval. Companies and scientists are entering a saturated market.

“There can be issues with the approval of drugs. If there are a lot of therapies approved in one setting, there isn't a need for more,” noted Gray. “First of all, physicians probably aren't going to use it even if it does get improved.”

It is increasingly essential for companies to have an intelligent trial methodology to get approval. She explained that trials need a comparator arm showing clinically meaningful data that the experimental treatment is beneficial over the current standard of care. Researchers would submit trial data to the US Food and Drug Administration (FDA) in the US.

“There are already so many options; it's difficult for physicians to differentiate between them. If it's not addressing an unmet need — either by being better than what is currently approved or finding its niche, such as a strong benefit in a certain biomarker population — there's almost no point in getting it approved.”

Although there is approval potential for drugs that can be used down the line, Gray explains that the market for these therapies is slim.

“As patients progress down the lines, they will get sicker and sicker. Fewer patients are going to be treated. So the treatment rates in fourth or fifth lines will be minimal,” she explained.

Companies generally do not intend to be fourth- or fifth-line treatments because of the small market size and limited revenue options. Late-stage breast cancer patients are most commonly treated with generic chemotherapies.

Beyond that, Gray adds, “Payers will have less of an issue treating these patients with these chemotherapies. The issue is that these patients are incredibly sick and would rather be treated with these cheaper, already-established chemotherapies this far down the line. The majority of them will eventually stop being treated.”

Gaps In the Market

Despite a relatively saturated market in some areas, Gray explained that the currently approved drugs do not meet all needs. She explored existing gaps in the market, identifying areas where new companies or research can address unmet needs.

“Triple-negative breast cancer is the area of breast cancer that has the biggest gaps in the market right now,” emphasized Gray.

So far, Keytruda (Merck & Co.) , Lynparza (AstraZeneca and Merck), and Talzenna (Pfizer Oncology) have been approved for triple-negative breast cancer.

“They are all approved for specific biomarker populations. So, Keytruda is only approved for PD-1-positive patients. Talzenna and Lynparza are approved for BRCA-mutated patients. There isn't a targeted therapy in triple-negative breast cancer that has applicability across the population that isn't biomarker-limited, except famously toxic chemotherapies,” Gray added.

According to the American Society of Clinical Oncology (ASCO), , including fatigue, nausea, diarrhea, mouth sores, hair loss, and anemia. The Centers for Disease Control and Prevention (CDC) also describes emotional and neurological impacts, including depression, anxiety, stress, sleep disturbances, and cognitive issues.

Gray identifies this as a critical unmet need in the breast cancer drug market: finding breast cancer treatments beyond chemotherapy that can address a broad range of triple-negative breast cancer cases.

“In the same vein, efficacy in TNBC is an issue,” she continued.

Although she notes that Keytruda and Lynparaza have generally strong efficacy data, Gray also emphasizes that TNBC is a highly aggressive form of breast cancer.

“Patients still don't live in the metastatic setting of TNBC longer than a year, which isn't necessarily the case in the other two breast cancer subtypes.”

HR-Positive/HER2-Negative

Additionally, Gray told PharmaNewsIntelligence that another unmet need is pre-treated metastatic hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer, another highly aggressive form. Beyond developing new and efficacious therapeutics , researchers should work toward developing less painful treatments.

Currently, researchers are exploring oral selective estrogen receptor degraders (SERDs), a hormonal therapy similar to Fulvestrant, as an alternative to the standard of care.

However, Gray explains, “Fulvestrant is off-patent; it's cheap, and it's been a standard of care for over 10 years. So, physicians’ familiarity is solid there. Oral SERDs must have impressive trial results to replace that.”

HER2-Positive

One of the most significant unmet needs in HER2-positive breast cancer is a therapeutic intervention to address brain metastasis, a substantial issue in this subtype. An article in Nature revealed that nearly 20% of all breast cancers are HER2-positive breast cancer.

Gray revealed that Herceptin (trastuzumab), manufactured by Roche, has been the standard of care across nearly every HER2-positive breast cancer setting. The drug was the first targeted treatment for this subtype, with the approval in 1998. However, the US patent expired in 2019 , introducing pricing pressures on the market.

“There are now far cheaper biosimilars . Herceptin's market share has dropped rapidly, meaning that it is going to be far harder to get approval for a novel therapy in this market, given that patients and insurers are going to prefer using this cheaper biosimilar that's available,” noted Gray, when asked to provide an example of how patent expiration may impact market entry.

Market Predictions

PharmaNewsIntelligence asked Gray to provide insights into the three biggest trends coming to the breast cancer market. She touched on multiple areas of focus, including a focus on early-stage therapeutics, increased segmentation of patients, and slowed development.

She explained that five years ago, few breast cancer therapies targeted early treatment stages, especially in HR-positive and triple-negative breast cancer.

“It’s a very lucrative area with so many patients. So, pharma companies are increasingly targeting this area to maximize profit.”

Another trend she predicted is increased market segmentation as research advances. For example, Keytruda targets PD-1-positive patients, while capivasertib and Piqray target patients with PI3K mutations.

“There are increasing numbers of trials looking at smaller and smaller subsets of patients with certain biomarkers. For example, in non-small cell lung cancer, [treatment] is heavily divided by the types of mutations a patient will have. That is going to happen in breast cancer,” predicted Gray. “All these different biomarkers are going to emerge, and it's going to be a far more segmented indication than it is now.”

Companies may do additional market research to understand segments with unmet needs and how new products could fit into the global market.

Finally, Gray emphasized that the saturated market will prompt slower development as companies navigate regulatory approval and product differentiation challenges.

“Particularly for HER2-positive and HR-positive, development will slow down over the next 10 years. They are relatively crowded at the moment. It will become increasingly difficult to show a strong efficacy benefit over what's currently approved. And, as patents start to expire, it'll be more difficult to justify the pricing of a novel therapeutic, which generally will be high,” she concluded.

Editor's Note: This article has been edited to correctly reference capivasertib.

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JSmol Viewer

The effects of radiotherapy on the sequence and eligibility of breast reconstruction: current evidence and controversy.

Simple Summary

1. introduction, 2. breast reconstruction techniques, 2.1. overview of immediate reconstruction options, 2.2. overview of delayed reconstruction options, 3. influence of post-mastectomy radiotherapy on choice of reconstruction, 4. complications.

5. Influence of Post-Mastectomy Radiotherapy on Survival Outcomes

Impact of nodal and molecular subtypes on locoregional recurrence (lrr) and survival, 6. influence of post-mastectomy radiotherapy on cosmetic outcomes and patient satisfaction, 7. future directions and premrt, 8. conclusions, author contributions, conflicts of interest.

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Campbell, A.R.; Didier, A.J.; Sheikh, T.M.; Ansari, S.; Watkins, D.E.; Fahoury, A.M.; Nandwani, S.V.; Rashid, M. The Effects of Radiotherapy on the Sequence and Eligibility of Breast Reconstruction: Current Evidence and Controversy. Cancers 2024 , 16 , 2939. https://doi.org/10.3390/cancers16172939

Campbell AR, Didier AJ, Sheikh TM, Ansari S, Watkins DE, Fahoury AM, Nandwani SV, Rashid M. The Effects of Radiotherapy on the Sequence and Eligibility of Breast Reconstruction: Current Evidence and Controversy. Cancers . 2024; 16(17):2939. https://doi.org/10.3390/cancers16172939

Campbell, Andrew R., Alexander J. Didier, Taha M. Sheikh, Sami Ansari, Dean E. Watkins, Alan M. Fahoury, Swamroop V. Nandwani, and Mohammad Rashid. 2024. "The Effects of Radiotherapy on the Sequence and Eligibility of Breast Reconstruction: Current Evidence and Controversy" Cancers 16, no. 17: 2939. https://doi.org/10.3390/cancers16172939

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Breast cancer survival improved in Gaza between 2017–2020, finds study

by King's College London

A recent study shines light on the treatment and survival outcomes for women with breast cancer in Gaza before the current conflict. While over half of reported diagnoses were made at advanced stages, a high short-term survival rate was discovered, highlighting the positive impact of international investment and collaboration in recent years before the conflict.

The paper, published in JCO Oncology and authored by Dr. Shaymaa Alwaheidi and Professors Richard Sullivan and Elizabeth Davies, looks at women in Gaza who were diagnosed with breast cancer between 2017 and 2018. It describes the careful follow up of women using paper medical records and interview data and the calculation of their survival until the end of 2020. This detailed analysis was needed due to the significant gaps in the available research on breast cancer outcomes in Gaza.

Results showed that women in Gaza with breast cancer had a 95.4% chance of survival after the first year, and 86.6% chance of survival after two years. The short-term survival rates (as of 2020) had improved from previous reports, which were lower than some of their neighboring countries in the Middle East including Lebanon and Jordan.

However, 52% of diagnoses were at the advanced stages III or IV. The authors suggest that this high proportion of advanced stage diagnoses could be due to a combination of women who delay seeking a diagnosis, and delays from the health care system , both of which need to be resolved by creating clearer referral guidelines.

The increase in short-term survival could be attributed to increased awareness of breast cancer symptoms among women during this period, leading to their earlier presentation, quicker diagnoses and better treatment. The authors also note that it could be due to changes to the treatment offered and the reduction of modified radical mastectomy.

These changes coincided with investment into the cancer care system, most notably the development of a multidisciplinary breast cancer team at Al-Shifa Hospital and collaborative efforts from the international community as shown by the support of visiting surgeons from the UK.

These positive results highlight how such investments and collaborations had significant benefits for women with breast cancer in Gaza, at least prior to the current conflict. The authors hope that it can encourage more investments and collaborations in breast cancer in Gaza, and more broadly encourage efforts in places throughout the world where women need greater medical support with breast cancer.

"Our results show how much had been achieved for Gazan women with breast cancer before the current conflict. Sadly, they now emphasize how the international community must find more robust ways of stepping up to protect vital cancer services during conflict ," says Davies.

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Volume 201, Issue 1

No more disparities among regions in italy: recent approval of genomic test reimbursability for early breast cancer patients in the country.

• Sara Bravaccini
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Identifying miRNA biomarkers for breast cancer and ovarian cancer: a text mining perspective

Advances of neuroimaging in chemotherapy related cognitive impairment (CRCI) of patients with breast cancer

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Heterogeneous circulating tumor cells correlate with responses to neoadjuvant chemotherapy and prognosis in patients with locally advanced breast cancer

• Jingyi Wang

Modeling the novel SERD elacestrant in cultured fulvestrant-refractory HR-positive breast circulating tumor cells

• Taronish D. Dubash
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Conventional specimen radiography in breast-conserving therapy: a useful tool for intraoperative margin assessment after neoadjuvant therapy?

• Benedikt Schäfgen
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Radiofrequency localization of nonpalpable breast cancer in a multicentre prospective cohort study: feasibility, clinical acceptability, and safety

• Anke Christenhusz
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Impact of non-adherence to endocrine therapy on recurrence risk in older women with stage I breast cancer after breast-conserving surgery

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Survival benefits associated with being adherent and having longer persistence to adjuvant hormone therapy across up to five years among U.S. Medicare population with breast cancer

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Treatment patterns and outcomes associated with sequential and non-sequential use of CDK4 & 6 inhibitors in patients with HR+, HER2− MBC in the real world

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Development of cardiometabolic risk factors following endocrine therapy in women with breast cancer

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Surviving the COVID-19 pandemic: navigating the recovery of breast imaging services in a safety-net hospital

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Validating the PROMIS cognitive function short form in cancer survivors

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Is there any association between declined creatinine clearence and higher endoxifen levels in older patients with non-metastatic breast cancer?

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It is the impact of racism, not race, that causes breast cancer inequities

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• Introduction
• Conclusions
• Article Information

Tornado diagram shows results of one-way sensitivity analysis for key model inputs. The gray portion of each bar represents the incremental cost-effectiveness ratio (ICER) range when the specified model input is lower than its base-case value. The black portion of each bar represents the ICER range when the specified model input is higher than its base-case value. Low and high values for all parameters are presented in Table 1. The first vertical line reflects the base-case value of each input, where the ICER is $110 962 per quality-adjusted life-year (QALY) gained comparing adjuvant olaparib with no adjuvant olaparib. The second vertical line reflects the value of each input that would yield an ICER of $150 000 per QALY, holding all other inputs constant. Threshold values above or below which the ICER exceeded $150 000 per QALY were 0.694 for the hazard ratio for recurrence with olaparib; 0.048 for the discount rate; and 0.743 for the utility of no recurrence/no olaparib, holding all other parameters constant at their base-case values. EV indicates expected value; WTP, willingness to pay.

Incremental cost-effectiveness ratios (ICERs) from 10 000 simulations in probabilistic sensitivity analysis. Quality-adjusted life-year (QALY) threshold indicated by dashed line. In all simulations, adjuvant olaparib was both more effective and more costly than no adjuvant olaparib. The oval depicts a 95% confidence ellipse. WTP indicates willingness to pay.

Curves indicate the proportion of simulations (n = 10 000) in probabilistic sensitivity analysis for which each strategy is preferred, as a function of the threshold willingness to pay for an additional quality-adjusted life-year.

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Zettler CM , De Silva DL , Blinder VS , Robson ME , Elkin EB. Cost-Effectiveness of Adjuvant Olaparib for Patients With Breast Cancer and Germline BRCA1/2 Mutations. JAMA Netw Open. 2024;7(1):e2350067. doi:10.1001/jamanetworkopen.2023.50067

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Cost-Effectiveness of Adjuvant Olaparib for Patients With Breast Cancer and Germline BRCA1/2 Mutations

• 1 COTA Healthcare, New York, New York
• 2 Peter MacCallum Cancer Centre, Parkville Familial Cancer Centre, Melbourne, Victoria, Australia
• 3 Thoracic Oncology Service, Memorial Sloan Kettering Cancer Center, New York, New York
• 4 Breast Medicine Service and Immigrant Health and Cancer Disparities Service, Memorial Sloan Kettering Cancer Center, New York, New York
• 5 Breast Medicine Service, Memorial Sloan Kettering Cancer Center, New York, New York
• 6 Department of Health Policy and Management, Columbia University Mailman School of Public Health, New York, New York

Question   Is adjuvant olaparib cost-effective in patients with early-stage breast cancer with high-risk disease and germline BRCA1/2 mutations?

Findings   In an economic evaluation using a Markov model, adjuvant olaparib therapy was associated with a 1.25-year increase in life expectancy and a 1.20-quality-adjusted life-year (QALY) increase at an incremental cost of $133 133 compared with no olaparib, yielding a discounted incremental cost-effectiveness ratio of approximately $111 000 per QALY gained. Results were sensitive to assumptions about the effectiveness of olaparib and quality of life for patients with no disease recurrence.

Meaning   The findings of this study suggest that, at a willingness-to-pay threshold of $150 000 per QALY, adjuvant olaparib may be cost-effective at its 2021 price.

Importance   The OlympiA trial found that 1 year of adjuvant olaparib therapy can improve distant disease-free survival and overall survival from early-stage breast cancer in patients with a germline BRCA1/2 mutation. However, olaparib, an oral poly-adenosine diphosphate ribose polymerase inhibitor, is estimated to cost approximately $14 000 per month in the US.

Objective   To estimate the incremental cost-effectiveness of adjuvant olaparib compared with no olaparib in eligible patients.

Design, Setting, and Participants   In an economic evaluation from a health care system perspective, the cost-effectiveness of adjuvant olaparib was analyzed using a Markov state-transition model. The model simulated costs and lifetime health outcomes of 42-year-old women with high-risk early-stage breast cancer and a known BRCA1/2 mutation who completed definitive primary therapy and neoadjuvant or adjuvant systemic therapy. The study was conducted from August 2021 to July 2023. The effectiveness of olaparib was based on the findings of the OlympiA randomized clinical trial, and other model parameters were identified from the literature. The model was calibrated to the 1-, 2-, 3-, and 4-year distant disease-free and overall survival observed in the OlympiA trial, and olaparib was assumed to reduce the risk of distant recurrence only in the first 4 years.

Exposure   One year of adjuvant olaparib or no adjuvant olaparib.

Main Outcome and Measure   Incremental cost-effectiveness ratio (ICER) in 2021 US dollars per quality-adjusted life-year (QALY) gained. All outcomes were discounted by 3% annually.

Results   In the base case, adjuvant olaparib was associated with a 1.25-year increase in life expectancy and a 1.20-QALY increase at an incremental cost of $133 133 compared with no olaparib. The resulting ICER was approximately $111 000 per QALY gained. At a willingness-to-pay threshold of $150 000 per QALY, olaparib was cost-effective at its 2021 price and in more than 92% of simulations in probabilistic sensitivity analysis. The results were sensitive to assumptions about the effectiveness of olaparib and quality of life for patients with no disease recurrence.

Conclusions and Relevance   In this study, from a US health care system perspective, adjuvant olaparib was a cost-effective option for patients with high-risk, early-stage breast cancer and a germline BRCA1/2 mutation.

Olaparib, a poly-adenosine diphosphate ribose polymerase (PARP) inhibitor, was first approved by the US Food and Drug Administration in 2014 for the treatment of advanced ovarian cancer in patients with a deleterious or suspected deleterious germline BRCA1/2 mutation (gBRCAm). In 2018, it was approved in the US for ERBB2 (previously HER-2 )-negative metastatic breast cancer associated with a gBRCAm in patients who previously received chemotherapy. 1

In June 2021, interim results of the randomized, double-blind OlympiA clinical trial were published, demonstrating the efficacy of olaparib for treating breast cancer in the adjuvant setting. 2 Patients with pathogenic or likely pathogenic gBRCAm and nonmetastatic, ERBB2 -negative primary breast cancer were randomized to receive twice-daily oral olaparib or placebo for 1 year following completion of definitive local treatment and neoadjuvant or adjuvant chemotherapy. In March 2022, an updated interim analysis 3 with 1836 patients, 330 invasive-disease-free survival events, and a median of 3.5 years of follow-up reported that patients receiving olaparib had superior 4-year distant disease-free survival (DDFS) (86.5% vs 79.1% for placebo) and overall survival (OS) (89.8% vs 86.4% for placebo). The hazard ratio (HR) for distant disease or death at 4 years was 0.61 (95% CI, 0.48-0.77). Olaparib was subsequently approved for use in the adjuvant setting among patients similar to those in the OlympiA trial.

While approximately 3% of all breast cancers in the US are associated with a gBRCAm, pathogenic variants of these genes are prevalent in more than 10% of patients with triple-negative breast cancer (TNBC)—tumors that lack estrogen and progesterone receptors and do not overexpress ERBB2 protein. 4 - 6 Triple-negative breast cancer generally has more limited therapeutic options and poorer prognosis than other types of breast cancer. 7 , 8 In the OlympiA trial, more than 80% of patients in both arms had TNBC.

The results of the OlympiA trial indicate that adjuvant olaparib provides a clinically and statistically significant health benefit for patients with early-stage breast cancer and a known gBRCAm. Our goal was to assess the value of these improvements relative to olaparib’s cost.

In an economic evaluation conducted from the health care system perspective, we created a Markov model to estimate the lifetime costs, life-years, and quality-adjusted life-years (QALYs) associated with adjuvant olaparib or no olaparib for early-stage breast cancer with known gBRCAm. The model included 4 health states: no recurrence, metastatic recurrence, death due to breast cancer, and death from other causes. All patients started in the no recurrence health state after completion of definitive primary therapy and neoadjuvant or adjuvant chemotherapy. From no recurrence, patients could transition to metastatic recurrence or die from other causes. Patients could only die from breast cancer in the metastatic recurrence health state. Local recurrences and other primary cancers were not modeled. The model used 1-month cycles and simulated the DDFS and OS observed in the OlympiA trial for the first 48 months. We extrapolated beyond this period by applying monthly estimates of metastatic recurrence from studies of patients with breast cancer with similar tumor features. 9 , 10 The model was created in TreeAge Pro 2020 software (TreeAge Software LLC). The study was conducted from August 2021 to July 2023. The study was conducted using information from published reports in the peer-reviewed literature and did not involve new collection or analysis of data from humans; therefore, the study did not require institutional review board review or exemption according to the Common Rule (45 CFR §46). We followed the Consolidated Health Economic Evaluation Reporting Standards ( CHEERS ) reporting guideline.

Consistent with the OlympiA trial, 2 we assumed that patients received either oral olaparib, 300 mg, twice daily or no olaparib. The starting age was 42 years, which was the median age of patients in the olaparib arm of the trial. We also examined starting ages of 50 and 60 years.

The probability of metastatic recurrence without olaparib was derived from the DDFS results observed in the placebo arm of the OlympiA study. We estimated the annual rate of metastatic recurrence in each of the first 4 years and translated these year-specific rates into constant monthly probabilities. 11 We modeled the association between olaparib and risk of metastatic recurrence in the first 4 years using the stratified HR for distant disease or death observed in the trial (HR, 0.61; 95% CI, 0.48-0.77). We assumed that olaparib did not reduce the risk of metastatic recurrence after 4 years. Consistent with the literature 9 , 10 on recurrence risks for patients with TNBC, we assumed that the risk of metastatic recurrence decreased after the first 4 years and remained constant after 10 years.

Monthly rates of metastatic recurrence in the model were iteratively adjusted to recreate the 12-, 24-, 36-, and 48-month DDFS and OS observed in the OlympiA trial. We converted the reported annual recurrence probabilities to constant monthly rates within each year, then increased or decreased each monthly rate by increments of 0.0001 until the rates converted to monthly probabilities yielded the arm- and year-specific DDFS and OS estimates from the trial. The DDFS rates for the olaparib arm in the OlympiA trial were 94.4% at 12 months, 90.6% at 24 months, 88.0% at 36 months, and 86.5% at 48 months. 3 For the no olaparib arm, the DDFS rates were 90.3% at 12 months, 84.0% at 24 months, 81.0% at 36 months, and 79.1% at 48 months. The OS based on this interim analysis for the olaparib arm was 98.0% at 12 months, 95.0% at 24 months, 92.8% at 36 months, and 89.8% at 48 months. For the no olaparib arm, the OS was 96.9% at 12 months, 92.8% at 24 months, 89.1% at 36 months, and 86.4% at 48 months.

Age-specific non–breast cancer mortality was estimated from 2018 US life tables. 12 The monthly probability of death after metastatic recurrence was based on a published estimate of 13.3 months median survival in patients with TNBC. 13

Using a health care system perspective, we estimated direct medical costs related to breast cancer, including olaparib, routine oncology visits, annual surveillance imaging, and the cost of care for metastatic recurrence. Olaparib was discontinued after the first of metastatic recurrence or 1 year of treatment. We assumed that patients receiving olaparib would see their oncologist monthly during the year of treatment, then every 6 months for 5 years after completing systemic therapy, and annually thereafter. Patients not receiving olaparib were assumed to have an oncologist visit every 6 months through year 5 and annually thereafter. We assumed that patients who had at least 1 breast (53.5% of patients in the OlympiA trial) would receive a diagnostic mammogram and breast magnetic resonance imaging with contrast annually. The costs of managing serious adverse events were not considered, given the tolerability profile of olaparib found in the OlympiA trial. Treatment costs for metastatic recurrence did not differ by receipt of adjuvant olaparib.

The costs of olaparib, routine oncology visits, breast imaging, and care for metastatic recurrence were identified from previously published studies, inflated to 2021 US dollars, and from the 2021 Medicare Fee Schedule ( Table 1 ). 15 - 18 Because most patients in the OlympiA trial had TNBC in both the olaparib (81.5%) and placebo (82.8%) arms, estimates for this specific patient population were used when available. All costs and health outcomes were discounted by 3% annually. 19

The utility weight, which reflects health-related quality of life on a scale of 0 (worst imaginable health) to 1 (best imaginable health), for patients not receiving olaparib was assumed to be equivalent to the utility associated with no recurrence after completion of adjuvant treatment. This utility was 0.98, based on published estimates. 14 The utility weight for patients receiving olaparib was estimated as a multiplicative function of the utility for patients not receiving olaparib and a factor less than 1. The utility for metastatic recurrence was estimated from published studies. 14

We performed 1-way sensitivity analysis to evaluate the uncertainty around model inputs. Ranges of values were obtained from published studies or were varied ±25% from their base-case value ( Table 1 ). We also performed probabilistic sensitivity analysis, assigning probability distributions to model inputs and conducting 10 000-second-order Monte Carlo simulations. Costs were characterized by γ distributions and utilities by β distributions. The HR for the association between olaparib and metastatic recurrence was modeled using a log-normal distribution, and the monthly rate of death following metastatic recurrence was modeled with a β distribution. Probabilistic sensitivity analysis results were plotted in a scatterplot of ICERs and a cost-effectiveness acceptability curve, varying the willingness-to-pay threshold from 0 to $300 000 per QALY.

In the base-case analysis, treatment with olaparib yielded 17.94 life-years and 17.40 QALYs at an average cost of $314 789 compared with no olaparib, which yielded 16.69 life-years and 16.20 QALYs at a cost of $181 655 ( Table 2 ). Adjuvant olaparib was associated with a 1.25-year increase in life expectancy and a 1.20-QALY increase at an incremental cost of $133 133. The resulting ICERs were $106 506 per life-year and $110 962 per QALY gained. At a willingness-to-pay threshold of $150 000 per QALY gained, olaparib would be considered cost-effective at its 2021 price. Varying the age of the cohort resulted in a modest change in the results. Holding constant assumptions about disease recurrence and drug efficacy, adjuvant olaparib was associated with ICERs of $124 897 per QALY in patients aged 50 years and $154 921 per QALY in those aged 60 years.

The ICER was most sensitive to assumptions about the effectiveness of olaparib, the discount rate, and the utility of the no recurrence health state ( Figure 1 ). At the upper bound of the 95% CI for the HR for distant disease or death estimated in the OlympiA trial (0.77), adjuvant olaparib was associated with a cost of $212 303 per QALY gained. At the lower bound of the 95% CI (0.48), the ICER was $75 999 per QALY gained. When olaparib cost 25% more than the base case value ($18 154), the ICER was $145 868 per QALY gained. At the lower bound of the cost of olaparib ($10 892), the ICER was $76 056 per QALY gained. Results were least sensitive to the utility weight for metastatic recurrence and costs of routine oncology visits and surveillance mammograms.

In probabilistic sensitivity analysis, we found that, in all 10 000 simulations, adjuvant olaparib was both more costly and more effective than no olaparib ( Figure 2 ). In more than 92% of the simulations, olaparib was preferred to no olaparib at a willingness-to-pay threshold of $150 000 per QALY gained. The probability that adjuvant olaparib was cost-effective, compared with no olaparib, exceeded 50% at a willingness-to-pay threshold of approximately $111 000 per QALY and exceeded 80% at a threshold of approximately $130 000 per QALY ( Figure 3 ). At a threshold of $100 000 per QALY, the probability of adjuvant olaparib being cost-effective was approximately 30%.

Since the approval of trastuzumab for ERBB2 -positive tumors in 1998, targeted systemic therapy remains a paramount goal of drug discovery and clinical care for breast cancer treatment. 20 PARP inhibitors represent one of the newer classes of targeted therapies for breast and other solid tumors. The OlympiA trial indicates that adjuvant olaparib improves both DDFS and OS in patients with high-risk, early-stage breast cancer with gBRCAm. Our economic evaluation suggests that olaparib is also cost-effective in this setting.

In our base-case analysis from a health care system perspective, adjuvant olaparib was associated with an ICER of approximately $111 000 per QALY gained. This estimate compares favorably with currently accepted thresholds of willingness to pay for health gains in the US and with estimates of the cost-effectiveness of olaparib in other tumor types and settings. 21 In its original indication for recurrent ovarian cancer in women with gBRCAm, estimates of the cost-effectiveness of olaparib in the US have varied from approximately $193 000 to $287 000 per progression-free year of life saved. 22 - 24 For newly diagnosed advanced ovarian cancer, the ICER could be as low as $51 000 per QALY. 25 In metastatic prostate cancer, the estimated cost-effectiveness of olaparib has varied from approximately $117 000 to just under $250 000 per QALY. 26 , 27

It is notable that more than 80% of patients in each arm of the OlympiA trial had TNBC. Given the relatively poor prognosis for patients with this tumor profile, adjuvant therapies that can reduce recurrence risk in TNBC are especially meaningful. Not only did olaparib have clinical benefit in this patient population, our results suggest that, despite its relatively high cost, it also represents a good value.

Our results were sensitive to assumptions about the effectiveness of olaparib. Within the 95% CI of the HR for DDFS found in the OlympiA trial, the ICER for adjuvant olaparib varied nearly 3-fold, from as low as $76 000 per QALY gained to as high as $212 000 per QALY gained. Now that olaparib is approved for adjuvant breast cancer treatment in patients with gBRCAm, it will be important to monitor its effectiveness and cost-effectiveness in the clinical practice setting. 1

Our model was agnostic to specific therapies received in the metastatic setting. We used an average monthly cost estimate for treating metastatic TNBC based on a mix of agents and regimens, which may have included a PARP inhibitor, and our estimate of breast cancer mortality following distant recurrence was based on evidence from cohorts with TNBC who were treated before the advent of PARP inhibitors. It is not yet clear whether oncologists will routinely administer PARP inhibitors in the metastatic setting following disease recurrence in patients who received adjuvant olaparib. As physicians begin incorporating adjuvant olaparib into their treatment protocols, we will learn more about the subsequent use and effectiveness of PARP inhibitors for treating metastatic disease. It is also not clear how the effectiveness and cost-effectiveness of adjuvant olaparib might be affected by the use of other agents, including pembrolizumab, capecitabine, and abemaciclib. Some of these medications have become standard of care in the neoadjuvant or adjuvant settings for patients with TNBC or other tumor features. 28 As newer data on costs and outcomes of different treatment combinations and sequences become available, estimates of olaparib effectiveness and cost-effectiveness can be updated to consider the impact of these other systemic therapies.

This study has several limitations. We did not explicitly model local and regional recurrences or subsequent primary cancers. Although these events were uncommon during the available follow-up, and the OlympiA trial was not powered to test differences in these end points, observed rates favored the adjuvant olaparib arm. Similarly, we did not explicitly model adverse events or treatment discontinuation. The rate of serious adverse events—events causing death, hospitalization, or major disability—was similar in the 2 study arms. While the frequency of adverse events leading to treatment discontinuation was greater among patients receiving olaparib (10.8% vs 4.6% of those receiving placebo), the absolute difference was relatively small. Given the substantial impact of olaparib on DDFS and OS, explicitly incorporating treatment discontinuation would not likely alter our conclusions. Moreover, our base-case analysis assumed that adjuvant olaparib was, on average, associated with slightly diminished quality of life compared with no olaparib. Although there was no significant difference between treatment arms in quality of life during the first 24 months following randomization in the OlympiA trial, more patients in the olaparib arm experienced a grade 3 or 4 adverse event, particularly anemia. Even when the utility weight for olaparib was assumed to be as low as 80% of the utility of no olaparib (ie, olaparib utility, 0.784; no olaparib utility, 0.98), the ICER for adjuvant olaparib was less than $150 000 per QALY gained.

In some instances, we used Medicare reimbursement rates as a proxy for health care costs, although most patients in the model would not be eligible for Medicare, since we assumed a starting age of 42 years, consistent with the OlympiA trial. 2 However, sensitivity analysis on these model inputs—costs of routine oncology visits and surveillance mammograms and breast magnetic resonance imaging—suggested that they had almost no impact on the estimated ICER of adjuvant olaparib.

In extrapolating results beyond the OlympiA trial, we conservatively assumed that olaparib had no association with DDFS and OS after 4 years. If olaparib has longer-term benefit, then our analysis would yield an overestimate of the ICER, and therefore an underestimate of the cost-effectiveness of the drug in this setting.

In addition, our base-case analysis was conducted from the health care system perspective, and thus did not include costs associated with informal caregiving or lost productivity. Patients receiving olaparib might incur greater costs associated with lost workdays due to a greater number of physician visits during the year of treatment. However, these costs would be insubstantial compared with the costs of informal caregiving in patients at the end of life and productivity losses associated with morbidity and premature mortality due to metastatic cancer. Such productivity losses could be considerable given the age distribution of these patients with breast cancer. 29 However, since adjuvant olaparib should decrease these costs on average by reducing the risk of metastatic recurrence, an analysis conducted from a societal perspective would likely find a more favorable ICER.

We assumed that gBRCAm status is known for every patient entering the model. Despite calls for universal germline genetic testing in all patients with newly diagnosed breast cancer, National Comprehensive Cancer Network guidelines still recommend testing based on risk factors associated with clinical presentation, personal health history, and family history. 30 - 32 The availability and effectiveness of adjuvant olaparib for patients with gBRCAm is likely to prompt increased testing among patients with newly diagnosed early-stage breast cancer. A recent study of strategies to identify patients with breast cancer eligible for olaparib treatment reported that universal testing in patients with TNBC was cost-effective (ICER<$60 000 per QALY) compared with a selective testing strategy, and expanding testing to all patients with ERBB2-negative disease had a favorable ICER as well. 33 Universal germline testing might have even greater value when cascade testing in family members is considered. However, ensuring equitable access to adjuvant olaparib may require dedicated efforts to reduce racial disparities in the use of germline genetic testing. 5 , 34 , 35

Expensive new cancer drugs such as olaparib can pose a challenge for both payers and patients. 36 , 37 Ensuring access to this effective and cost-effective agent is critical if we are to attenuate, rather than exacerbate, disparities in breast cancer outcomes. This is especially important in the context of TNBC, which is disproportionately common and lethal in Black women. 38 , 39 The findings of this economic evaluation suggest that with an ICER less than $150 000 per QALY gained, adjuvant olaparib in eligible patients represents a good value at its 2021 price.

Accepted for Publication: November 6, 2023.

Published: January 3, 2024. doi:10.1001/jamanetworkopen.2023.50067

Open Access: This is an open access article distributed under the terms of the CC-BY License . © 2024 Zettler CM et al. JAMA Network Open .

Corresponding Author: Elena B. Elkin, PhD, MPA, Department of Health Policy and Management, Mailman School of Public Health, 722 W 168th St, New York, NY 10032 ( [email protected] ).

Author Contributions: Ms Zettler and Dr Elkin had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Concept and design: All authors.

Acquisition, analysis, or interpretation of data: Zettler, De Silva, Elkin.

Drafting of the manuscript: Zettler, De Silva, Robson, Elkin.

Critical review of the manuscript for important intellectual content: De Silva, Blinder, Robson, Elkin.

Statistical analysis: Zettler, De Silva, Elkin.

Obtained funding: Elkin.

Administrative, technical, or material support: De Silva, Elkin.

Supervision: Elkin.

Conflict of Interest Disclosures: Ms Zettler reported receiving personal fees from Columbia University Mailman School of Public Health during the conduct of the study and current employment by and equity ownership in COTA Healthcare Inc. Dr Robson reported receiving grants and nonfinancial support from AstraZeneca and Pfizer, and grants from Merck outside the submitted work. Dr Elkin reported receiving institutional research funding from Pfizer. No other disclosures were reported.

Funding/Support: Ms Zettler’s work on this study was supported by research funds from the Department of Health Policy and Management at the Mailman School of Public Health, Columbia University.

Role of the Funder/Sponsor: Columbia University had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Meeting Presentation: A preliminary version of this study was presented at the Annual Meeting of the American Society of Clinical Oncology; June 6, 2022; Chicago, Illinois.

Data Sharing Statement: See the Supplement .

Additional Contributions: Ling Chen provided statistical assistance to parameterize distributions for probabilistic sensitivity analysis.

Additional Information: Ms Zettler was a full-time student at the Mailman School of Public Health at the time this research was conducted; COTA Healthcare was not involved in this study.

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• Volume 14, Issue 8
• Qualitative exploration of patients’ experiences with Intrabeam TARGeted Intraoperative radioTherapy (TARGIT-IORT) and External-Beam RadioTherapy Treatment (EBRT) for breast cancer
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• http://orcid.org/0000-0002-7573-6712 Sandeep Kumar Bagga 1 ,
• Natalie Swiderska 2 ,
• Charlotte Hooker 1 ,
• Jennifer Royle 3 ,
• Marie Ennis-O'Connor 4 ,
• Siobhan Freeney 5 ,
• Dympna Watson 4 ,
• Robin Woolcock 6 ,
• George Lodge 7 ,
• Siobhan Laws 7 ,
• http://orcid.org/0000-0003-1760-1278 Jayant S Vaidya 8
• 1 Research , MediPaCe , London , UK
• 2 Patient Engagement , MediPaCe , London , UK
• 3 Strategy , MediPaCe , London , UK
• 4 Independent Patient Advocate , Dublin , Ireland
• 5 Lobular (Breast Cancer) Ireland , Dublin , Ireland
• 6 Triple Negative Breast Cancer Foundation Inc , London , UK
• 7 Royal Hampshire County Hospital , Winchester , UK
• 8 Division of Surgery and Interventional Science , University College London , London , UK
• Correspondence to Dr Sandeep Kumar Bagga; sandeep{at}medipace.com

Objective To gather a deep qualitative understanding of the perceived benefits and impacts of External-Beam RadioTherapy (EBRT) and TARGeted Intraoperative radioTherapy (TARGIT-IORT) using Intrabeam to assess how the treatments affected patient/care partner experiences during their cancer treatment and beyond.

Design and participants A patient-led working group was established to guide study design and to help validate findings. Patients with experience of receiving EBRT or TARGIT-IORT were purposively sampled by Hampshire Hospitals NHS Foundation Trust. These patients had been offered both regimens as per their clinical features and eligibility. Semistructured interviews were conducted with 29 patients and care partners with lived experience of either EBRT (n=12, 5-day FAST-Forward regimen and n=3, 3-week regimen) or TARGIT-IORT (n=14). Thematic analysis was then carried out by two coders generating 11 themes related to EBRT or TARGIT-IORT.

Setting Semistructured interviews were conducted virtually via Zoom during February and March 2023.

Results A number of procedural grievances were noted among EBRT patients. EBRT was perceived as being disruptive to normal routines (work, home and travel) and caused discomfort from side effects. TARGIT-IORT was perceived by patients and care partners as the safer option and efficient with minimal if any disruptions to quality of life. The need for timely accessible information to reduce anxieties was noted in both cohorts.

Conclusions This qualitative study found that patients perceived EBRT as being greatly disruptive to their lives. In contrast, the one-off feature of TARGIT-IORT given while they are asleep during surgery gives them the feeling of stamping out the cancer without conscious awareness. These insights can help healthcare staff and policy-makers further justify the incorporation of the treatment favoured by these patient perceptions (TARGIT-IORT) more widely in routine practice. Further research is planned to explore TARGIT-IORT in more diverse populations and in the 35 countries where it is an established treatment option.

• breast cancer
• radiotherapy
• qualitative research
• breast surgery
• quality of life

Data availability statement

Data are available upon reasonable request. Raw data such as interview transcripts are not publicly available due to participant confidentiality and risk of compromising privacy but can be made available to researchers if appropriate confidentiality, ethics, regulatory and consent processes can be put in place.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See:  http://creativecommons.org/licenses/by-nc/4.0/ .

https://doi.org/10.1136/bmjopen-2023-081222

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STRENGTHS AND LIMITATIONS OF THIS STUDY

This qualitative study included the two routinely offered radiotherapy treatment options (External-Beam RadioTherapy and TARgeted Intraoperative radioTherapy using Intrabeam) allowing for assessment of patients’ perceptions and experiences in each.

Methodological strengths include measures to prevent researcher bias, such as producing reflexive accounts, independent coding, and exploring both patient and care partner perspectives .

Extensive involvement of a patient-led working group ensures the study design and delivery is robust yet sensitive and respectful.

A limitation is the lack of diversity in the study population, being predominantly white and of higher socioeconomic status from a single English location because of which we are planning to explore these concepts in more diverse populations and in the 35 other countries where TARGIT-IORT is already a well-established treatment option.

The COVID-19 pandemic during which the patients were treated may have introduced some confounding factors, but they did provide useful insights into patient isolation issues.

Introduction

Conventionally, radiotherapy treatment for breast cancer has involved patients undergoing External-Beam Radiotherapy (EBRT) several weeks or months after their surgical removal (lumpectomy). EBRT is usually delivered postoperatively to the whole breast. For external beam radiotherapy, patients are required to attend 15 treatment sessions, each lasting about 15 min, 5 days a week over 3–6 weeks. 1 , 2 In 2020, the FAST-Forward protocol, administering radiotherapy over five sessions, was adopted in some parts of UK, partly as a response to the COVID-19 pandemic and even before the results of the FAST-Forward trial were published. 3 An additional 5–8 days of tumour bed boost is given in about a quarter of cases who are found to have higher-risk disease. 3

Targeted Intraoperative Radiotherapy (TARGIT-IORT) using Intrabeam offers an alternative to women with early breast cancer that is currently being used in a small number of hospitals across England. This approach, first used in 1998, delivers a single dose of radiotherapy directly to the breast tissue surrounding the tumour immediately after the tumour has been removed and the patient is still under the same anaesthetic in the operating theatre. The long-term results of the international randomised TARGIT-A trial (n=2298) in which TARGIT-IORT was compared with EBRT found TARGIT-IORT to be effective as whole breast radiotherapy, reduced non-breast cancer deaths and improved overall survival in those with grade 1 and grade 2 cancers. 4–12

To date, several studies have investigated patients’ experiences with TARGIT-IORT quantitatively. 13–18 These studies gathered information about patients’ quality of life (QoL) during and after treatment via questionnaires and have concluded that patients receiving TARGIT-IORT report high QoL scores 13 and better emotional well-being, less pain, fewer breast and arm symptoms compared with patients receiving EBRT. 14 19 The social impact of reducing the repeated journeys to the radiotherapy centre for both the patient and their care partner has been established. 20 Patient preferences have also been explored in studies based in the USA and Australia. 21–24 However, qualitative insights can give researchers and practitioners an in-depth understanding of patient perceptions that can help explain, with confidence, the reasoning behind the difference in QoL experienced by patients having these treatments.

Much of the literature on patients’ experiences of receiving radiotherapy has focused on EBRT alone where qualitative studies have used various methods such as workshops, interviews and diary entry analysis. Recurring themes include the need for adequate information provision, healthcare professionals’ knowledge of breast or arm lymphoedema (sluggish drainage of lymph fluid), perceived lack of choice, experiences of being naked and feelings of disempowerment, 25 psychological burdens of impact (and the resources required to support patients), 26 impact of side effects such as skin toxicity on patients’ QoL, life and health after radiotherapy and feeling mystified by radiotherapy and how it works. 27 28 While there are other studies investigating breast cancer patients’ lived experiences of receiving the diagnosis, treatment perceptions, experiences of survivorship and symptoms from radiotherapy, 29–34 they do not focus on lived experiences of receiving EBRT specifically.

In addition, the literature review has highlighted that no qualitative comparison of patients’ experiences of TARGIT-IORT and EBRT has been conducted although one qualitative study, exploring overdiagnosis of breast cancer, did briefly describe the experiences of patients having TARGIT-IORT and EBRT. 35 Rich descriptions of authentic experience can help to place the treatment pathway in the context of patients’ everyday world and to truly understand the perceived barriers, benefits and personal consequences of treatment. Therefore, this study is designed to gather a deep understanding of how patients define the benefits and impacts of each therapeutic regimen and how this qualitatively affects patients’ and/or care partners’ experiences. As a secondary aim, the study will also identify where there have been unnecessary treatment-related impacts on QoL and areas of potential improvement.

Methodology

Study design.

This study used a qualitative research design with semistructured interviews as the primary research instrument. Researchers adopted a phenomenological approach which encourages a bracketing off of researchers’ own preconceptions and opinions to help mitigate bias and promotes a special importance to individual human experience where multiple realities exist (based on participants’ own subjective experiences).

At the inception of the study, a patient-led working group was established to ensure the research was designed and conducted in a respectful and sensitive manner. Figure 1 outlines the research process. The authors have used the Consolidated criteria for Reporting Qualitative research to report the study 36 (see online supplemental file 1 ).

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Overview of research process. EBRT, External-Beam RadioTherapy; TARGIT-IORT, TARGeted intraoperative radiotherapy; REC, Research Ethics Committee.

Working group

Four patient advocates (three patients who had been treated for breast cancer and one care partner) with lived experiences of radiotherapy were invited to participate in a working group with the researchers. An initial meeting was held on 19 August 2022. In this meeting, the research design was discussed which included reviewing the study aims, the need for a comparison group, data collection method and participant recruitment channels. The second meeting, on 30 May 2023, focused on validating the emerging themes from the analysis. Between these meetings, the researchers shared early drafts of the core research material (eg, participant information sheet and consent form) to obtain members’ feedback and suggestions for amendments. The working group has also coauthored this paper.

Recruitment of participants and consent

A key outcome of the first working group meeting was to ensure the study design had a comparison group. This meant recruiting patients or care partners with lived experience of receiving EBRT to enable a comparison to those who had received TARGIT-IORT. The eligibility criteria are based on criteria used previously in TARGIT-IORT clinical trials ( table 1 ). All patients were, at the time of their cancer diagnosis, eligible for both TARGIT-IORT and EBRT.

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Eligibility criteria for interview participants

Participants were first identified by SL and GL at Hampshire Hospitals NHS Foundation Trust in accordance with the eligibility criteria, using purposive sampling (NHS stands for National Health Service, which is provided free for cost to patients in the United Kingdom). This Trust recruited patients to the randomised TARGIT-A trial between 2000 and 2012. Since the National Institute of Health and Care Excellence (NICE) recommendation to offer TARGIT-IORT to suitable patients, they have been offering the procedure to their patients. For this study, GL compiled a list of all patients who received either TARGIT-IORT or EBRT. Prospective participants were stratified first into rural and urban subgroups and then by age (50–60 and 60–70). A randomiser was then applied to these subgroups to ensure the final selection process was free from bias from clinicians who had treated patients. Cover letters and recruitment advertisements (approved by the ethics committee) were posted by GL to 58 eligible patients. Those interested in participating in the study contacted researchers voluntarily. Subsequently, the researchers shared a participant information sheet, consent form and provided further information during a short introductory call where participants also had the opportunity to ask further questions about the purpose and conduct of the research. In total 29 participants responded, and all were successfully recruited to the study. Participant characteristics can be found in table 2 .

Sample characteristics of interview participants

Semistructured interviews

Working group members agreed semistructured interviews should be used to gain rich descriptive accounts of experiences with EBRT and TARGIT-IORT. Members felt discussing sensitive and privileged information, namely people’s experiences of receiving the cancer diagnosis and treatment, would be more uncomfortable in, for example, a focus group environment. Therefore, two discussion guides (one for each type of radiotherapy) were developed and refined with the help of working group members (see online supplemental files 2 and 3 ). Interviews were conducted between 9 February 2023 and 2 March 2023 by researchers SKB, NS and JR who are experienced in using qualitative research methods as part of their professional roles. Each interview lasted approximately 60 min and was conducted virtually through Zoom and either digitally video recorded or audio recorded depending on participants’ wishes. The identity of the interviewer can positively or negatively affect the interviewer–interviewee relationship. Participants were, therefore, asked whether they would prefer an interviewer of the same sex, with the default position being that a female interviewer (NS or JR) will conduct the interview with patients and similarly, a male interviewer (SKB) for male care partners. During the interviews, informal member checking took place by interviewers routinely summarising what participants had said to check for accuracy and understanding.

Recordings were transcribed verbatim, with potentially identifying details anonymised and assigned a unique identifier.

In keeping with a phenomenological approach to analysis, researchers began by writing reflexive accounts. This involved reflecting on their own experiences, preconceptions and assumptions that have the potential to influence interpretations of participants’ accounts. This process helps to create the self-awareness required when attempting to consciously bracket out thoughts and opinions that could lead to bias.

Reflexive thematic analysis based on Braun and Clarke’s 37–39 six-step approach was used to analyse the qualitative data and to identify recurring themes related to patient and care partner experience ( figure 2 ). The process of data familiarisation took place during data collection, postinterview reflections, transcribing and re-reading the transcriptions and interview field notes. Initial transcripts were individually coded (identifying units of meaning) by two researchers (SKB and NS) who then reviewed the other’s codes. Through subsequent discussion and reflection and agreement that theoretical saturation was sufficiently achieved, codes were finalised and applied throughout the remaining transcripts. Through an iterative process, descriptive and interpretative codes were categorised to form 28 subthemes and 11 major themes. Microsoft Word and Excel were used to facilitate coding, grouping and text retrieval to identify illustrative quotes.

Reflexive thematic analysis (adapted from Braun and Clarke, 2020). 37

Patient and public involvement

Patients were involved in refining the research question, study design and outcome measures. Their contributions during these discussions were informed by their lived experiences, priorities and preferences during the first working group meeting (described above in the ‘Methodology, Working group’ section). Participant identification and recruitment channels were also discussed with the patient-led working group though they were not directly involved in recruitment into the study nor in the conduct of the study. The results of the study will be disseminated to the study participants once the peer-reviewed paper is published. The burden of the intervention was assessed by the patients themselves for this qualitative study.

The following section presents findings from the thematic analysis which looked at EBRT and TARGIT-IORT separately and the outputs from discussions of the second working group meeting ( table 3 ). Participants’ quotes have been labelled with identifiers (eg, P1, P2) not known to anyone other than the researchers (ie, not the hospital staff, participants themselves or anyone else).

Themes and subthemes arising from the interviews

Themes coming out of interviews with patients who had EBRT

Dissatisfaction with unalterable elements of ebrt procedure.

The majority of EBRT participants expressed discontent with many of the standard elements of the EBRT procedure. Some participants felt intimidated by the size of the room being ‘disturbing’ (P22) and the radiotherapy machine being ‘scary’ (P19):

…the room that you go into where the machine is, is cold…it could be a bit warmer. Now, some of that could be psychological because you're in a big white room with a big, huge machine… (P3) …the 2 nurses go into another side room, so, you feel so alone, and you know, and this machine sort of moving around you. It’s, it is quite scary to deal with. (P19)

Four participants also described the challenge associated with needing to hold one’s breath during sessions. This is done with the hope that the heart may receive less radiation by pushing the chest wall and the breast away from it. Participants described it as saying, ‘ that was the worst bit’ (P12), ‘ it’s going to be difficult’ (P15), ‘ I don’t want to be zapped on my heart’ (P21) and another felt it was ‘ really claustrophobic’ (P19) or causing ‘ panic’ (P19). The planning appointment required for EBRT was met with similar dissatisfaction. While there is a clear appreciation for healthcare staff and their workload, participants were unhappy with the dehumanising nature of these appointments:

You become another face… you do feel like a slab of meat while they're trying to get you in the right position and it’s not a pleasant experience. (P19)

These experiences resonated with working group members’ recollections: ‘silent’ and ‘cold, dark room’ and finding it difficult, a ‘physical challenge’ to maintain position after surgery. Another member felt that while healthcare staff were pleasant, the experience of receiving radiotherapy itself is ‘quite traumatic’ and emotional, ‘I remember lying there and tears came from nowhere…’.

As with study participants, working group members acknowledged that while healthcare staff themselves are not at fault, the ‘system’ causes the dehumanising elements described by participants referring to poor staffing levels and a high-pressure work environment within healthcare.

In contrast, one participant positively describes the EBRT sessions, ‘ there’s music on, and I didn’t find any cause to worry at all’ (P15). The relaxing effect of music was echoed by a working group member who also recalled how music helped in which she felt was ‘ brilliant’ and stated ‘ it helped my head’ .

Finally, one-third of participants expressed a strong objection to being tattooed which was required to ensure radiation is delivered to the right location.

What really did wind me up actually, I had to have the 3 dots tattooed on me and I didn’t want tattoos. (P3)

Participants were frustrated by the fact that it is permanent, the colour used and two participants felt it affected their confidence in wearing certain clothes, ‘ I can still see that now, if I wear a swimsuit or something’ (19 ). One working group member felt that patients are often uninformed about radiotherapy and that patients’ preferences are not listened to. She concluded that this was a good example of an area that required adequate information sharing at the right time.

Unanticipated disruptions cause helplessness

One-third of EBRT participants experienced either delays on the day of the EBRT session or extensions to their course owing to either machine failure or staff absences. The impacts on patients and care partners include stress, aggravation and disappointment with knock-on consequences proving to be burdensome:

…the machine broke down…but I couldn't find [the new hospital], and I got really tired and upset. I was trying to find where I was supposed to go, and nobody seemed to know, and I just managed to grab the team before they went home. I was like, ‘Give me my last radiotherapy now!’ It was down in some basement I mean. Location S is a maze. So, that was a bit stressy. (P1)

Patients who received their EBRT during the COVID-19 pandemic were unable to have their care partners (in most cases husbands) with them at key treatment stages. This isolation caused additional anxieties in the EBRT cohort as one care partner stated, ‘ it’s disappointing and it would have been nicer for me to be able to support her more…’ (P23). Similarly, a patient participant states:

…it’s anxiety level of just having that, that security blanket of having somebody there with you. (P19)

The working group discussed isolation and the emotional impact during EBRT sessions. Since EBRT continues for days and indeed weeks in some cases (5–6 weeks for all members), patients truly feel alone during this phase. They recalled overwhelming feelings of sadness during the sessions with thoughts such as, ‘ how did I get here’ . One member expressed empathy with study participants who would have had to endure further isolation during the COVID-19 pandemic period.

Straightforward sessions were met with surprise but travelling for EBRT was still a concern

Four out of the 15 participants interviewed from the EBRT cohort, stated that no side effects or other complications were experienced with one participant saying she felt like ‘ one of the lucky ones’ (P20). In the absence of side effects such as pain, burning or tiredness, patients experience a sense of surprise and relief after having received prior warnings from either clinical staff or hearing stories from friends and family.

…they said to put a couple of tubes of aloe vera, and keep in the fridge, and put it on religiously. But I was fine. I was fine. I literally had no burning. No rash, no nothing. I’d heard about friends having burns, but I just didn’t, I didn’t. (P12)

Similarly, three participants were appreciative of the fact that individual sessions of EBRT were in fact ‘ very straightforward’ (P1) and quick:

I did find the time, actually, went quite quick. It wasn’t very long; it might have been an hour. Yeah, it wasn’t too long. (P1)

Although these four participants did not experience radiation-related side effects, it should be noted that two of the four participants did, express frustrations about the burden of travelling during EBRT sessions and that the overall radiotherapy course was ‘ time-consuming’ (P2).

Disruption to normal life routines

A few participants were either employed or self-employed and described how EBRT impacted their own work performance (eg, tiredness, weakness in arm) with one person concluding, ‘ I’m an office worker but if I’d be doing a manual job, I think it might have impacted more.’ (P3). There is also the realisation that patients would likely need to adjust work sessions to accommodate for the EBRT sessions and the related side effects: ‘ I’ve worked out a part time basis to get back into work.’ (P19).

Participants shared concerns about the impact on work colleagues. One participant states: ‘ I’m the only person that does my job. So, I was acutely aware that when I’m not at work other people are picking up my job’ (P19). Similarly:

…we were a short-staffed team, I was aware that when I wasn’t there, it was putting work onto other people, and I felt I should have been there…. (P3)

The EBRT cohort also revealed the emotional toll of work-related worries and impacts, and the work guilt associated with the impact on colleagues. Similarly, the potential impact on the care partner’s work is also, clearly, a significant consideration for patients:

The first night after my first session I was in so much pain, I mean I didn’t sleep a wink the first night. It was absolute agony…it was my self-confidence, and everything was destroyed…and I didn’t dare, I didn’t want to wake my husband up. He had to go to work early, so then he could take me to radiotherapy in the afternoon. (P19)

These impacts on employment were corroborated during working group discussions. One member, commenting in particular on self-employed people, described the impact on financial standing and home life as ‘catastrophic’.

In contrast, those who had flexible hours of working were less affected, as one patient participant describes her care partner: ‘ luckily he was doing a job where could sort of pick and choose what he did and his hours, so it was all right.’ (P3).

The repetitive nature of the EBRT sessions, the travel involved and the side effects experienced all also impacted on participants’ normal daily routines. For instance, home activities such as shopping, gardening and caring responsibilities were impacted:

I think we might have cut down [caring for elderly parents] to once a week instead of two or three times… (P3)

One participant mentioned a close family member taking a week off from work to support looking after her, her husband, family pets and ‘ doing a few household chores like pushing the Hoover around’ (P22).

Severe pain brought on by EBRT was described as ‘ agony’ (P19) both during sessions and after sessions (P7). Ongoing pain months and years after the radiotherapy means patients need to settle into a new norm, now constantly having to be aware of their own ‘ limitations’ (P6):

I’m still aching like mad from [radiotherapy], that’s two years later and I’m still achy and in pain. (P6)

For working group members, these experiences were familiar. One member clarified that although there were no specific side effects from the radiotherapy the disruption to life was ‘ hugely problematic’ .

Many of the patients who received the FAST-Forward protocol included sessions on either side of a weekend resulting in a total of 7 days to complete the course. No participants complained specifically about the weekend being involved in this way however many described dedicated activities for the weekend, for example, food shopping, family commitments and short trips away which would undoubtedly be affected while in active treatment.

Conversations with participants revealed personalities play a role in patients’ cancer journey. One participant felt the emotional impact of radiotherapy treatment (and the subsequent withdrawal of routine contact with healthcare staff) more than all the others. The anxieties associated with this perceived void and the mental health impact itself are disruptive to re-engaging with normal life and routines:

…you’ve got people checking on you as in consultants or the breast care specialist nurses, your GP, the radiographer, people ringing you, checking on you, you’re seeing them all the time. After the radiotherapy, I’m suddenly though on my own now. I didn’t realise it was a real security blanket…it was so reassuring to seeing the consultant and seeing this nurses every day. That was the bit where I took a bit of a dive…I had two or three days where I couldn’t stop crying, I thought, ‘Oh I’m on my own now’… (P12)

There were also those who demonstrated a pragmatic mindset viewing any discomfort and impactful delays associated with EBRT sessions as realities to, in effect, take in their stride, ‘ any inconvenience, you just get on with it.’ (P20 )

Travel is clearly an uncomfortable reality and a demanding aspect of receiving EBRT. Many participants complained about the repeated journeys required for EBRT sessions. The burden has been described as ‘ dreadful’ (P7) with people feeling ‘ exhausted’ particularly where the effects of radiotherapy (tiredness and pain) are felt. The burden of travel also manifests as experiencing a longer day overall as well as the sheer cost of public transport used to make the trips independently:

It’s a pain having to go to Location S, there’s a hospital there where the bus doesn’t even go. So, from Location O, I have to get a bus from here to Location H, then wait half an hour, then from Location H to Location W, bus station, then a taxi to the hospital. That all costs 30 quid and is time-consuming, and when you’re sore, it’s not ideal. (P2)

In the majority of cases, there is a reliance on others (the husband but on occasion friends or children) to drive participants to the EBRT sessions. While there are no direct statements indicating a burden to care partners, it is important to note that in these cases both the patient and the care partner endure the repeated journeys:

My husband drove…I’m not a good driver…I certainly can’t park so it was good that he went with me because in case, if something went wrong or something because he’s like my rock he is. (P3)

The location of the EBRT sessions is critical to the quality of patient experience and when participants were presented with two hospitals to choose from it was clearly valued:

…it’s the same surgeons, all the same team, who were in Hospital W or B. I opted for Hospital B, it’s nearer to me and I could get to Hospital B easily, whereas Hospital W was an ordeal for me. (P21)

Two participants who were retired and for who the location of the hospital was particularly close indicated that travel was not a burden and considered themselves ‘ very lucky’ (P22), though of note, one of these participants experienced no EBRT side effects which may have contributed to relatively positive experience.

In contrast to many of the experiences described above, one working group member shared that she was relieved to have regained her independence since she was able to travel to her EBRT sessions herself. However, the working group notes that all their experiences included a difficult period of receiving chemotherapy first where radiotherapy was viewed as the ‘lesser of evils’.

Experience characterised by discomfort from side effects

A wide range of side effects, clearly attributed to EBRT, were reported by the vast majority of participants and this characterises an important part of the EBRT experience. These included varying intensities of tiredness; burning (from warm sensation to blistered and sore); skin-related conditions (dryness, itchiness, rash); pain; and breast size and density changes. The most common complaint was tiredness:

…there were 3 or 4 days the following week when I just had to go to bed, or just have to, you know, lie on the sofa in the afternoon, or you know I was just really bombed out, and I’m not someone who goes to bed and afternoon normally, I’m always busy, and but I had to. I was knackered. (P12)

EBRT sessions can be very uncomfortable. Severe pain, described as ‘ agony’ (P7, P19) was experienced by two participants. One individual, unable to withstand the pain and the tiredness experienced, made an independent decision to stop attending the sessions for a few days:

I was very tired…I think I might have missed a few days, because I couldn’t make it in between…I thought I’ve done so much now, I’m not going to go anymore because it was really, really hurting…I just wanted it to end and go away? And not think about it anymore basically. (P7)

Patients experienced some side effects such pain in the breast or weakness in the arm for a prolonged period—months and years after the EBRT sessions. There were also reports of new symptoms requiring follow-up that were attributed to EBRT. One participant (P8), who felt uninformed about the ‘long-term, lasting and late effects of radiotherapy’ had developed a new pain under her ribs—she reflected:

I would not have had radiotherapy, and I would not be beating myself up about having had it now had I been given the full information about the long-term effects. You know, it’s life-shortening, radiotherapy is life shortening in itself like chemotherapy. (P8)

During discussions at the second working group meeting, members were not surprised by the insights captured from study participants and felt strongly that there were ‘no positives’ from EBRT (particularly when compared with TARGIT-IORT). One member reflecting on their experience with EBRT stated they came away thinking ‘almost anything is better than this.’ (WG member)

Specific anxieties about receiving EBRT

Discussions with the EBRT cohort revealed three main anxieties associated with receiving radiotherapy. First, while there is evidence that more information early on (particularly from consultants) helps to reduce worry, stories of radiotherapy experiences from friends and family members can raise concerns and anxiety levels:

I only knew what I knew as a lay person, you know, various friends have had radiotherapy, unfortunately, people know a lot of people who have all these sort of things… I’d heard about friends having burns… (P12) …and actually, talking to another friend, she said she would do chemotherapy any day over radiotherapy because of how the radiotherapy, the pushing around and making you feel like a piece of meat, how it how it made her feel. (P19)

The second concern was the potential for radiation to cause harm: (a) so soon after surgery and (b) to healthy tissue and organs: ‘ You’ve got to heal a bit; you can’t go straight into radiotherapy because obviously you’re as raw as hell’ (P6). Two participants in particular experienced discomfort in their arms during their EBRT sessions—one participant had a number of lymph nodes removed from the armpit and one participant developed a seroma which is an abnormal accumulation of fluid following surgery. A working group member shared a similar experience where the development of seroma delayed the start of her radiotherapy course.

Another participant who was particularly concerned about unnecessary radiation exposure requested that only half the breast be irradiated because she wanted ‘ the absolute minimum’ (P8). Similarly, another care partner described his wife’s concerns:

One thing is that my wife was worried about was the radiotherapy because obviously there is this thing with radiotherapy, particularly on the breast, of potential damage to the lungs and she was very concerned about that. (P23)

Third, since many of the participants received their EBRT during the COVID-19 pandemic, a few expressed worries about potential delays either due to staff shortages, protocol-driven cancellations (ie, limiting patient numbers) or themselves contracting COVID-19 (since multiple visits are required with EBRT) and thereby being unable to attend hospital:

But COVID was going on and I remember being so scared that my appointments would be cancelled. (P12)

Targeted Intraoperative radiotherapy using Intrabeam

Perception that targit-iort is efficient and aggravation-free.

Of the 14 TARGIT-IORT participants interviewed, 11 indicated the one-off feature of the procedure was appealing. There are many references to how quickly the procedure was completed ‘ it’s lovely to get it all done and finished with on the day’ (P26). Similarly:

…having it done and dusted, and then then waving goodbye at the hospital gates, it was like why, why would I say, ‘No thank you’? (P16)

As a consequence of this efficiency, there is relief that the procedure permitted radiotherapy to be administered without any COVID-19-related delays, or exposure to the COVID-19 virus during travel or hospital during the multiple visits for EBRT, or complications for which three participants had expressed initial concerns:

I was just delighted that it was dealt with really, really quickly, because back at that time the news was full of things where, you know, because of COVID-19, you know, everything has been delayed, and people not getting cancer treatment and that was one of my, I remember having that conversation with the consultant and said, ‘Look are we going to be delayed’. (P25)

There is a similar relief detected in participants discussing the potential positive impact TARGIT-IORT can have on patient’s mental health as one care partner states:

…the alternative would have been [EBRT]… her symptoms of depression are she gets very, very tired… so intuitively our reaction to [TARGIT-IORT] was… actually quite a good idea. (P24)

Going through a cancer diagnosis and receiving treatment was clearly an emotional time. One participant was impressed with TARGIT-IORT precisely because the efficient delivery of radiotherapy facilitates her moving on quickly:

…the beauty of intraoperative radiotherapy is that I could say ‘OK, been there, done that, move on. (P9)

Convenience of performing TARGIT-IORT during surgery is valued

Most participants from the TARGIT-IORT cohort shared why they preferred to receive radiotherapy at the same time as the surgery. There is a recognition of the convenience that TARGIT-IORT brings as a result of not having to attend hospital on multiple occasions, for example, less travel and car parking and supporting independence (particularly for retired individuals):

It’s my choice to have [TARGIT-IORT] because I thought that it was a better option for me particularly because I live on my own and it would allow me to be more independent. (P18)

While the majority of participants were retired, those who did have young children felt TARGIT-IORT supports their caring responsibilities: ‘ I’ve got a [child] and I’ve got to look after him… This is a better way to go…’ (P14). Additionally, one retired participant who valued the independence TARGIT-IORT facilitated concluded it would also suit younger, busy, women well:

…particularly for younger women this would be an extremely good thing, if they're working, it allows them to get back to work without that constant interruption and if they've got a young family. (P18)

Many participants were able to draw from stories and experiences they had heard from friends and families. The apparent inconvenience and impact of daily radiotherapy doses discouraged patients from EBRT when TARGIT-IORT was presented as an option. One participant whose father received daily doses for prostate cancer felt she would ‘ rather get it all over in one go’ (P10). Similarly:

[TARGIT-IORT] was perfect, because it just meant I didn't have to queue up in the car park with the other poor people having radiotherapy, and I did have friends who had serious cancers who were having radiotherapy at the time, and it was just miserable. (P9)

There is also a perception that with TARGIT-IORT recovery times are likely to be faster since it would signify the end of their cancer treatment: ‘ I’m going to get [TARGIT-IORT] and it’s done’ (P16) and ‘ I can just then get on and recover’ (P4). Another participant summarises her main reasons for opting to receive TARGIT-IORT:

So, there were probably 3 reasons I went for [TARGIT-IORT]. You know, COVID, convenience, and the fact that I thought, you know, ultimately, I’d probably recover quicker. (P9)

Only one participant from the TARGIT-IORT cohort, a care partner, described a significant logistical impact due to his wife’s cancer treatment in general:

…created quite a challenge really for me, I mean, I was never going to moan about it, I wasn’t the one who just had cancer surgery! But you know, it meant the days suddenly got very challenging… (P25)

Perception TARGIT-IORT is a safer alternative to standard practice

Five participants felt that they did not experience any complications as a result of TARGIT-IORT and were able to resume their normal activities quickly. While there are a few cases of soreness and itchiness that participants specifically attributed to TARGIT-IORT, most participants did not report the range of side effects seen in the EBRT cohort. As a result, participants gave their endorsements for TARGIT-IORT, respectively:

I moved around, I got up, got changed, got dressed. It was surprising actually, this is why I’ve decided to do this, if this is what it gives you then everyone should have it. You know you don't need to feel debilitated, and you can carry on with your life. I've got a [child], and I've got to look after him. So, if you can, why not. This is a better way to go if the prognosis allows it. (P14) There were no, no after-effects, no problems. It all healed up very well, because it was quite a small incision anyway and very, very successful. (P28)

The majority of participants felt the procedure prevented healthy tissue and organs from being unnecessarily exposed to radiation because ‘ the radiotherapy is directed immediately where the lump [is]’ (P17).

I confess I heard that and thought ‘God, that’s a bloody good idea, why don’t they do that more often?’. Because obviously if you don’t have to beam through loads of flesh and muscle to get at what you're aiming for then that’s got to be better to be honest. (P24)

A few participants described side effects (soreness, tiredness), precautions (new bra needed, seatbelt cushion) and restrictions (no pressure, sport, lifting), however, they were unable to clearly attribute whether these were related to the surgery or the TARGIT-IORT procedure since both occur at the same time.

…yeah, my arm was a little bit sore…I’m sure it must have been the radiotherapy or the operation, I don’t know. (P29) …a special seat belt cushion that protects your breast from the seat belt and I had one another cushion under my breast supporting it… (P11)

Novel nature of TARGIT-IORT impresses while prompting early caution

Although it has been in use for the last 25 years since the first case was done in 1998 TARGIT-IORT is seen as novel and innovative with advantages acknowledged over EBRT. The decision to proceed with TARGIT-IORT is widely considered ‘ easy’ (P28) or ‘ intuitive’ (P24) or a ‘ no brainer’ (P4):

…well, you’re in there, so you might as well get on and do it and that would surely save the need for me having to come back, I can then just get on and recover basically…it was a no brainer for me, an absolute no brainer. (P4)

However, a few participants described their initial concerns since TARGIT-IORT was introduced by the consultants as a clinical trial and was largely unheard or ‘ unknown’ (P9). Care partners, often husbands and sometimes participants’ children wanted to carry out their own research to help making an informed decision about TARGIT-IORT. One participant had already felt she was convinced by the consultant’s explanation and the advantages over EBRT, however, her daughter, who worked in healthcare, stated ‘ …‘hold on a minute, we need to look at the statistics and the recovery times, side effects’…’ (P10). Similarly, another participant’s husband wanted an opportunity to ask the consultant more questions to help feel more reassured:

…but [care partner] just wanted to have the conversation around the intraoperative radiotherapy because it was an unknown really. (P9)

It should be noted that many of the participants were either themselves or their close family (eg, husband) highly educated, often with a science-based background and were able to explore clinical study papers and statistics: ‘ I’ve got a little statistical training…so I looked at the stats and what the mean variation was…what the levels of certitude at either end of the scale were…’ (P24).

TARGIT-IORT patients have high information needs

As mentioned above, due to the relative novelty of TARGIT-IORT and in the absence of experiences of TARGIT-IORT among participants’ friends and family, reliable information from trustworthy sources is critical. The majority of participants (in both EBRT and TARGIT-IORT cohorts) displayed high levels of trust in their consultant. Receiving adequate information from them about TARGIT-IORT, particularly due to its initial availability via a clinical trial, was appreciated:

I think what was good was the way that it was explained in the first place and what the pros and cons were, or if in fact, there weren't any cons really at all…So, you know, we were told that the treatment, doing it during the operation, is just as effective but it would mean that you would have no subsequent radiotherapy and, you know, of course I’m young and foolish, I assume that to be true, we trust the doctor… (P25) [The consultant] said’ ‘This is this, that is that…pluses and minus’…gone through pros and cons and I had made up my mind that that was a good way to go. (P14)

Working group members could relate closely to this subtheme of trust. They explained that the retrospective perception of TARGIT-IORT was always likely to be a ‘no brainer’, however, for a patient going through the highly emotionally charged process of receiving their diagnosis and treatment, at a time when they are already overwhelmed with new information, the relationship with the doctor is important:

…if it’s being offered to you, it’s important how it’s being offered to you. We put out trust in, so much, our doctors. (WG member)

Two participants described receiving explanations from radiation oncologists during their presurgery appointment, however, these discussions were not influential in helping to decide which type of radiotherapy they would receive. A few participants were wary of using the Internet to search for information related to their treatment options: ‘ I’m very cautious of what information I take in from Google’ (P4). However, the majority did conduct their own Internet searches to bolster their understanding of TARGIT-IORT:

I then went away and looked the bugger up, and then you could learn for yourself a little bit, reading between the technical stuff, what it’s all about, the success rate is there or there about the same, it’s not wonderful but for me, it was a no brainer. (P16)

The provision of information was discussed on a number of occasions by working group members. Simple and clear language is particularly important at a time when patients are already in a vulnerable, stressed and emotional state:

…you are so blindsided…the normal way you operate doesn’t necessarily apply. (WG member)

Working group members pointed to the need for information sources to be created adequately in the first place, for example, being written by patients/care partners who possess the lived experiences and so are able to elaborate on the areas that matter.

…there should never be a need for a patient to go home and want to Google, you should go home with the information in hand or go home with reputable evidence-based sources of information. (WG member)

The primary finding of this study is that the subjective experiences of patients and care partners receiving EBRT or TARGIT-IORT differ significantly. Strong recurring themes of appreciation and recognition of innovation, convenience, absence of side effects and lack of disruption to life have emerged from the TARGIT-IORT cohort while in the EBRT cohort, we have largely heard about discomfort and disruption to life. These themes—centring around (a) treatment procedure itself; (b) impact on QoL and (c) information needs—were presented to and were validated by a patient-led working group.

Patients and care partners involved in this study described numerous challenges, concerns and dissatisfaction with elements of the EBRT procedure while processing a difficult and emotional diagnosis. These findings are consistent with the existing literature on EBRT experience. 25 Probst et al 25 also identified procedural grievances, for instance, patients described the radiotherapy sessions as ‘dehumanising’, ‘emotionally draining’ and complained about the tattoos being a permanent reminder of the cancer. Previous studies exploring patient-perceived barriers to radiotherapy include patients’ fear surrounding radiation toxicity which can result in non-compliance and insufficient treatment. 13 40 In fact, research has identified that fears and anxiety regarding the EBRT experience can influence a patient’s decision to opt for a mastectomy over EBRT, despite the latter having equivalent if not non-inferior survival rates. 30 Several studies have demonstrated that as the distance from radiotherapy centre increases, the rate of mastectomy also increases. 41–45 Indeed, this was the primary patient-centric reason that the TARGIT-IORT procedure was originally conceived. 10–12 46

Our study demonstrates the need for improvements in the way EBRT is delivered and has implications for practice that extend to cases where patients are not eligible for TARGIT-IORT. In stark contrast, those receiving TARGIT-IORT have no awareness or recollection of the procedure since radiation is administered during surgery. Patients and care partners found this feature particularly appealing which contributed to their decision to opt for TARGIT-IORT. Indeed, TARGIT-IORT has been widely adopted elsewhere and treated 45 000 patients across 38 countries. 6

A high proportion of our EBRT cohort (12/15) received the FAST-Forward regimen. This regimen of highly compressed higher-dose-per-fraction radiotherapy was adopted in the UK even before the results of the FAST-Forward trial were published with the aim of reducing waiting time pressures during the early part of the COVID-19 pandemic. We recognise this 5-day regimen is indeed not adopted elsewhere in the world—it has much higher toxicity—19 times higher fibrosis and a quarter of women reporting hardened breasts 3 and this toxicity is seen even with the short follow-up of the FAST-Forward trial. 47 48 It is noteworthy that a large part of the patient’s perceived benefit came from the immediacy of TARGIT-IORT due to its administration during the same anaesthetic as their lumpectomy, the resulting convenience and the absence of additional hospital visits for radiotherapy that would be otherwise required for EBRT. In our study, this benefit of TARGIT-IORT was perceived by patients even though the majority of the comparator group received EBRT over just 5 days rather than the international standard of 3 weeks. It is, therefore, likely that the contrasting experience of patients may have even higher significance and the perceived benefit may be greater when TARGIT-IORT is compared with 3 weeks of EBRT.

Patients in the study who received TARGIT-IORT had been given the option to have it because they fulfilled the eligibility criteria ( table 1 ). Since patients made a conscious choice, it is plausible that the results of this study could be biased favouring TARGIT-IORT. However, the authors of this study submit that patients should be given a choice. Our study shows that those who choose TARGIT-IORT have a positive perception of treatment and the overall experience is better than those who opted for EBRT. Others have shown that if given a choice between no radiotherapy, mastectomy, EBRT and TARGIT-IORT, 75% of patients preferentially choose TARGIT-IORT. 24

It is evident that QoL-related benefits and impacts are a central component of radiotherapy lived experiences. Compared with TARGIT-IORT, EBRT has a prolonged impact on patients and perhaps a compounded impact on QoL where patients live alone (lack emotional or practical support), do not drive (reliance on others or public transport with additional costs and travel time) or have caring responsibilities (partners, parents, children and pets). Travel and mobility issues have been recognised as barriers already 49 as has the inconvenience of a prolonged treatment plan which can affect those living in remote areas even more. 13 Our findings demonstrate the advantages TARGIT-IORT offers to those who are eligible. All participants in our study acknowledged the efficiency of the procedure with many drawn to the option (over EBRT) because it was considered ‘straightforward’ and ‘over-and-done’ during surgery. The benefits of TARGIT-IORT to patients in terms of cost, travel time and distance have been demonstrated, in principle, elsewhere. 49 50 Furthermore, the environmental and social impact of the substantially more travel required for EBRT, and a huge reduction in carbon footprint from cancer treatment by use of TARGIT-IORT has also been well documented. 20

In our study, inconveniences and logistical complications were exacerbated by EBRT side effects which were recognised as a key characteristic of the EBRT patient experience and have implications on QoL. Stanton et al 51 investigated factors affecting QoL during and after radiotherapy and found that functional impacts of treatment, particularly breast-specific pain (eg, mobility) are important correlates of QoL. In addition, Schnur et al 27 showed that key patient concerns include the timing of side effects and the impact of side effects on self-esteem affecting patients’ perception of being attractive, good workers, patients and parents. Another study supports these findings and also shares one case of such extreme physical discomfort (pain, burning, etc) that the patient admitted she had considered ending treatment and another saying she would never choose to have radiotherapy again due to the burning sensation. 28 Our study has captured similar cases. This study also underscores the emotional toll, anxieties and stresses that disruptions to life (eg, work-related) cause and have been heard at a NICE Committee meeting. 52 There were fewer reports of side effects directly attributed to TARGIT-IORT in our findings. This is consistent with a study comparing TARGIT-IORT with EBRT (quantitatively) in which patients receiving TARGIT-IORT also reported less pain, fewer breast, and arm symptoms, and better everyday functioning when compared with patients receiving EBRT. 14 We recognise that our study has found stark differences in patient experience and perception between TARGIT-IORT and EBRT. This can seem obvious because patients with TARGIT-IORT have almost no poor experience in relation to actually receiving the treatment (mainly because they are under general anaesthetic when it is given). Our findings resonate with others who also report the negative patient experiences with EBRT and have suggested interventions to improve them. 25 The important qualitative patient benefits identified in this study are of course in addition to the quantitatively proven significant reduction in non-breast cancer deaths, and an improved overall survival in patients with grade 1 and grade 2 cancers within the randomised TARGIT-A trial. 7 However, our study does detect apparent patient obscurity between surgery-linked or TARGIT-IORT-linked side effects—this clearly needs to be addressed through appropriate education and adequate information provision.

Results from our study cohorts point to the need for improvements in communication and information provision. The role of high-quality communication by healthcare staff and access to emotional support services, particularly when radiotherapy treatment ends has been highlighted already. 26 Previous research has also identified that patients can often feel mystified by radiotherapy (EBRT), how it works and will have anxieties about life and health after radiotherapy 27 or feel disempowered and lacking the ability to make an informed choice. 25 In our study, working group members emphasised the importance of trust in connection with information provision, particularly during an emotional cancer diagnosis. Members felt a number of study findings could be addressed adequately by effectively communicating the right information at the right time. Examples include letting participants know clearly that tattoos will be permanent; what the immediate and long-term side effects of both radiotherapy types are; understanding the side effects of surgery thereby avoiding confusion with TARGIT-IORT; ensuring TARGIT-IORT explanations are always supplemented with lay language overviews of the efficacy and safety profile compared with EBRT. One study showed that more than 90% of patients felt that if they were more informed about radiotherapy, they would be less scared about it. 30 The working group advocated for any shared information, such as leaflets, to be written by patients, that is, those who have experiences of receiving radiotherapy, and therefore, have an awareness of where there are likely to be challenges in understanding treatments and their impacts clearly. Similarly, considerations also ought to be given to ensuring people with learning disabilities and communication difficulties are able to make an informed choice by developing accessible information. To satisfy ‘valid consent’, doctors in the UK are now obliged to follow the new GMC guidelines underlining the essential nature of adequate patient information, 53 about all proven treatment options, even if they are not available at their own centre. In the UK, this powerful principle is now fully enshrined in law (Montgomery vs Lancashire Health Board, 2015). 54 55 The substantially better patient perception and experience documented in this study need to be included during consultations with patients when discussing treatment options before they have their surgery for breast cancer.

This qualitative study, co-led by patients, uncovered detailed lived experiences of receiving either EBRT or TARGIT-IORT from patients treated for early breast cancer, as well as those of their care partners. The research demonstrated a patient-perceived superiority of TARGIT-IORT over EBRT—it is considered more efficient with less disruption to life routines. The paper also illustrates the importance of provision of accessible information about all radiotherapy treatment options from trusted sources, at the right time (before breast cancer surgery), to reduce initial anxieties and help patients make informed choices. These new insights need to be taken together with the established quantitative survival and QoL benefits of TARGIT-IORT over EBRT. We believe that these deep insights into the patient’s perspective will substantially improve our understanding of the lived experiences of patients with breast cancer and will help clinicians, patients and policy-makers to comprehensively consider how access to better treatments can improve patients’ lives.

Ethics statements

Patient consent for publication.

Not applicable.

Ethics approval

This study involves human participants and was approved by Health and Social Care Research Ethics Committee B (HSC REC B), Office for Research Ethics Committees Northern Ireland (ORECNI). IRAS Project ID number: 320976. Participants gave informed consent to participate in the study before taking part.

Acknowledgments

The authors thank the patients and care partners who volunteered to help in development of the research question, helping design the study and outcome measures. The authors thank patients and care partners (the study participants) who volunteered to participate in this study. We are grateful to them for giving up their time and sharing their treatment experiences and valuable insights during a difficult period in their lives for the benefit of science and clinical research.

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X @jsvaidya

Contributors SKB, NS, CH, ME-O'C, SF, DW and RW were responsible for the study concept and design. SL and GL identified patients who met the eligibility criteria. SL, GL and JSV contributed to the study design. GL posted cover letters and recruitment adverts to all identified patients. SKB, NS and JR collected the data. SKB and NS analysed and conducted the thematic analysis from the data. ME-O'C, SF, DW and RW approved the initial report. SKB and CH wrote the first draft of this manuscript. JSV and all other authors were involved in interpreting the data and made substantial contributions to the intellectual content of the manuscript and approved the final version. The authors took full responsibility for the manuscript. SKB is responsible for the overall content (as guarantor).

Funding The study was sponsored by MediPaCe. Unrestricted funding was provided to MediPaCe by Carl Zeiss Medtech AG. The manufacturers of the Intrabeam device (Carl Zeiss Medtech AG) did not have any part in concept, design, or management of the study, or in data analysis, data interpretation, or writing of the report. A grant/award number was not issued for the funder.

Competing interests This qualitative study was initiated by MediPaCe, a patient engagement and patient research company. The manufacturers of the TARGIT-IORT device (Carl Zeiss Medtech AG) did not have any part in concept, design, or management of the study, or in data analysis, data interpretation, or writing of the report. Authors SKB, NS, CH and JR are employed at MediPaCe. MediPaCe received payment to independently plan, coordinate and conduct this study. JSV declares Support from University College London Hospitals (UCLH)/ UCL Comprehensive Biomedical Research Centre, UCLH Charities, HTA, NIHR, National Institute for Health Research (NIHR) Health Technology Assessment (HTA) programme, Department of Health and Social Care, UK Ninewells Cancer Campaign and Cancer Research Campaign (now Cancer Research UK); Research grant from Photoelectron Corp (1996–1999) and for supporting data management at the University of Dundee (Dundee, UK, 2004–2008) and travel reimbursements and honorariums from Carl Zeiss. SL and GL declare no conflicts of interest.

Patient and public involvement Patients and/or the public were involved in the design, or conduct, or reporting, or dissemination plans of this research. Refer to the Methods section for further details.

Provenance and peer review Not commissioned; externally peer reviewed.

Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

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• Published: 21 August 2024

Proteomic insights into breast cancer response to brain cell-secreted factors

• Shreya Ahuja 1 &
• Iulia M. Lazar 1 , 2 , 3 , 4  

Scientific Reports volume  14 , Article number:  19351 ( 2024 ) Cite this article

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• Breast cancer

The most devastating feature of cancer cells is their ability to metastasize to distant sites in the body. HER2 + and TN breast cancers frequently metastasize to the brain and stay potentially dormant for years until favorable conditions support their proliferation. The sheltered and delicate nature of the brain prevents, however, early disease detection and effective delivery of therapeutic drugs. Moreover, the challenges associated with the acquisition of brain biopsies add compounding difficulties to exploring the mechanistic aspects of tumor development. To provide insights into the determinants of cancer cell behavior at the brain metastatic site, this study was aimed at exploring the early response of HER2 + breast cancer cells (SKBR3) to factors present in the brain perivascular niche. The neural microenvironment was simulated by using the secretome of a set of brain cells that come first in contact with the cancer cells upon crossing the blood brain barrier, i.e., endothelial cells, astrocytes, and microglia. Cytokine microarrays were used to investigate the secretome mediators of intercellular communication, and proteomic technologies for assessing the changes in the behavior of cancer cells upon exposure to the brain cell-secreted factors. The cytokines detected in the brain secretomes were supportive of inflammatory conditions, while the SKBR3 cells secreted numerous cancer-promoting growth factors that were either absent or present in lower abundance in the brain cell cultures, indicating that upon exposure the SKBR3 cells may have been deprived of favorable conditions for optimal growth. Altogether, the results suggest that the exposure of SKBR3 cells to the brain cell-secreted factors altered their growth potential and drove them toward a state of quiescence, with broader overall outcomes that affected cellular metabolism, adhesion and immune response processes. The findings of this study underscore the key role played by the neural niche in shaping the behavior of metastasized cancer cells, provide insights into the cellular cross-talk that may lead cancer cells into dormancy, and highlight novel opportunities for the development of metastatic breast cancer therapeutic strategies.

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Introduction.

Brain metastasis accounts for more than half of the intracranial tumors in adults, making it one of the primary causes of brain cancer in patients 1 . Tumors of the lung, breast, and kidney, in addition to melanoma and colorectal cancer, have the highest propensity to spread to the brain, with approximately 80% of cancers metastasizing to the cerebral hemispheres due to favorable blood flow patterns 1 . Moreover, recent statistics indicate that among the breast cancer patient cohort, the triple negative and HER2 + breast cancer subtypes are the most common to metastasize to the brain with a median incidence rate of 31% and 32%, respectively 2 . In order to colonize the brain, the breast cancer cells must breach the blood brain barrier (BBB), invade the neural matrix, and engage in a cross-communication of signaling molecules with the brain cells to evade the eliminatory signals from the host matrix. Crossing the BBB is a challenge in itself, as the endothelial cells of the BBB are linked together by tight junctions that restrict the paracellular movement of most large molecules and cells. Once the BBB has been breached, the tumor cells attach themselves to the brain microvasculature, a necessary process for extracting nourishment and proliferation cues 3 . In this stage, the cancer cells can stay dormant for years 4 . To create favorable growth conditions and further enable successful colonization, the cancer cells engage with the brain cells through direct physical interactions or via paracrine signaling. The neurons and astrocytes produce large amounts of extracellular matrix which accounts for ~ 20% of the CNS volume, alongside a broad spectrum of neural stimulants like trophic factors, neurotransmitters, cytokines, etc., that render a fertile ground for tumor outgrowth 5 . For example, the gap junction connexin Cx43 that interconnects the network of astrocytes has been found to also instigate cell survival and proliferative signaling in cancer cells 6 . Nevertheless, the brain also contains its own population of macrophages, the microglia, that survey the CNS for pathogenic insults and initiate immune responses. In this unfamiliar environment, the metastatic cancer cells face unique challenges, and, in response, express anti-inflammatory mediators that can suppress the immune activation of microglia and reprogram them into a tumor-supportive type. Several studies have underscored the complex interactions that occur between tumor cells and their microenvironment, and that lead to the activation of processes and pathways that favor the development of metastases 7 , 8 . Very little is known, however, about the early molecular responses which are activated in cancer cells when they encounter the foreign neural niche. To gain insights into this process, the objective of this study was to explore the response of SKBR3/HER2 + breast cancer cells to factors expressed by three main cell types present in the neurovascular unit, i.e., microglia, astrocytes, and brain endothelial cells, with which the cancer cells come in contact shortly after entering the brain parenchyma. The selection of cells was driven by their most relevant functions in the brain, i.e.: (a) endothelial cells, due to their role in angiogenic processes that provide nutrients to cancer cells; (b) microglia, due to their role as “immune barrier” cells essential to triggering immune responses; and (c) astrocytes, due to their role in maintaining homeostasis and providing biochemical support to brain cells such as neurons and endothelial BBB cells.

The “neural niche” conditions were replicated by treating the cancer cells with conditioned media (CM) collected from in vitro cultured cells that contain secreted factors such as proteins, cytokines, growth factors, as well as extracellular vesicles (EVs) released by the cultured cells 9 . These components provide the ground for establishing the intercellular communication and promote processes that can accelerate the malignant growth of cells. In one such study, for example, the authors reported the presence of angiogenic factors FGF2 and VEGF in EVs shed by astrocytes in culture 10 . Astrocytes have been also found to interact with the invading tumor cells in early stages of brain metastases by secreting factors such as MMP9 and SDF1 that in turn support cancer progression 11 . Another group demonstrated that the astrocyte-conditioned medium contained exosomes with high levels of microRNAs which inhibit the expression of tumor suppressor PTEN in metastatic breast cancer cells, leading to aggressive tumor formation in the brain 12 . Together with astrocytes, microglia too secrete a variety of cytokines and factors that contribute to immunosuppression, increased invasiveness, angiogenesis, and cancer cell proliferation 13 , 14 . Furthermore, microglial EVs have been found to induce survival and provide metabolic support to other cells 15 . Owing to their role in promoting neurogenesis, maintenance of neuronal circuitry and blood brain barrier functions, microglia and astrocytes secrete neurotrophic factors to carry out neuroprotective and developmental functions in the brain 16 , 17 , 18 . Previous studies have highlighted the brain-derived neurotrophic factor (BDNF) released by astrocytes, which activates the tropomyosin-related kinase B (TrkB) receptor on metastasized breast cancer cells to promote cell proliferation and colonization of the brain 19 . Therefore, we hypothesized that the factors that are present in the CM collected from brain cells would alter the proliferative capacity of breast cancer cells and induce changes in the proteome that are suggestive of increased metastatic potential.

Despite the fact that the SKBR3 cells were not harvested from a brain-metastatic site, there are several features that made this cell line attractive for this study. It is a HER2 + cell line, and HER2 + breast cancers metastasize frequently to the brain. Previous studies in mice that used SKBR3/HER2 + cells as a brain metastatic model were able to demonstrate that metabolic diversity and plasticity can determine metastatic fitness 20 (doi.org/ https://doi.org/10.1016/j.cmet.2021.12.001 ). SKBR3 is also a useful preclinical model system to screen for the effectiveness of therapeutic agents that target HER2 cancers and to investigate resistance mechanisms that evolve in response to HER2-targeted therapies 21 . Last, in contrast to other brain metastasized breast cancers, the SKBR3 cells were not exposed prior to brain factors, and we speculated that the observable impact would be highest in this case.

To test our aforementioned hypothesis, we first explored the cytokine and growth factor profile of the conditioned medium collected from the three brain cell cultures by using a panel of capture antibodies spotted on a membrane-based cytokine array. Next, we used high-resolution mass spectrometry (MS)-based proteomics to measure the change in protein abundances between the CM-treated and non-treated SKBR3 cells. Finally, we mapped these changes to biological processes that are reflective of cancer growth and progression. This study provides preliminary, yet novel, insights into the early response of breast cancer cells exposed to in vitro conditions that mimic the neural niche. Our results suggest that the exposure of SKBR3 cells to neural niche factors may usher the cells into a more advanced state of quiescence, which is a possible result of inflammatory mediators and reduced levels of certain growth factors in the CM from brain cells.

Experimental methods

Human HER2 + breast cancer cells (SKBR3), microglia (HMC3), brain endothelial cells (HBEC5i), and penicillin–streptomycin (PenStrep) solution were purchased from ATCC (Manassas, VA, USA). Normal human astrocytes (NHA), Clonetics AGM™ Astrocyte Growth Medium BulletKit™ (comprised of ABM-astrocyte basal medium and SingleQuots™ growth factors, cytokines, and supplements pack), and astrocyte ReagentPack™ subculture reagents were procured from Lonza (Morristown, NJ). Minimum essential medium (MEM), McCoy’s 5A (Modified) medium, DMEM/F-12, DMEM high glucose (HG), Dulbecco’s phosphate buffered saline (DPBS) and trypsin–EDTA were obtained from Gibco (Gaithersburg, MD). BenchMark Fetal Bovine Serum (FBS) was purchased from Gemini Bio Products (West Sacramento, CA). Endothelial cell growth supplements (ECGS) and all commonly used reagents (Na 2 VO 3 , NaF, DTT, NH 4 HCO 3 , TFA, urea) were from Sigma Aldrich (St. Louis, MO). Acetonitrile, HPLC grade, was from Fischer Scientific (Fair Lawn, NJ), and high purity water was prepared from DI water, in-house, by distillation.

Cell culture

The cells were initially propagated in the manufacturer’s suggested growth media, i.e., SKBR3 in McCoy 5A, HMC3 in EMEM, HBEC5i in DMEM/F-12/ECGS, and NHA in ABM with SingleQuots growth supplements (FBS, EGF, insulin, L-glutamine, ascorbic acid, and gentamycin). After the third passage, 50% of the culture media content was replaced with DMEM-HG, and after yet another 48 h the cell cultures were continued in 100% DMEM-HG ( Fig.  1 A ) . All culture media were supplemented with FBS (10%) and PenStrep (0.5%) at all times, the astrocytes with the additional growth supplements, and the endothelial cells with ECGS. At ~ 80–100% confluence, the cell cultures were serum-starved for 24 h by growing them in DMEM-HG/PenStrep. Serum-free conditioned media (CM) collected from HMC3, HBEC5i and NHA were mixed and spun down by centrifugation at 500 × g for 5 min to remove the floating cells. Clarified CM was added to 24 h serum-starved SKBR3 cells which were grown for another 24 h in the presence of the secreted factors. The SKBR3 cells that were used as control were left under serum-starved conditions for a total of 48 h. Morphological differences between treated and non-treated SKBR3 cells were visualized by using phase-contrast and immunostaining microscopy and a Nikon Eclipse TE2000 U epi-fluorescence inverted microscope (Nikon Instruments Inc., Melville, NY) equipped with the NIS-Elements Advanced Research imaging platform. Mouse monoclonal anti-HER2 primary antibody (Santa Cruz sc-08) and mouse IgG-kappa binding proteins conjugated to CruzFluor488, as well as all Western blot antibodies, were purchased from Santa Cruz Biotechnology (Dallas, TX). The cells were harvested by trypsinization and spun down to pellets which were flash frozen at − 80 °C until further use. DNA content analysis for cell cycle stage assessment was performed by FACS using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Three independent biological replicates of control and treated cells were performed [control: 69–72% in G1 (CV = 9%), 8–13% in S, 2–7% in G2; treatment: 70–90% in G1 (CV = 13%), 11–14% in S and 0.3–4% in G2].

Overview of the experimental setup. ( A ) Schematic outline of the cell culture. Three biological replicates were established for HMC3, HBEC5i and NHA. All cells were grown in DMEM-HG medium under serum-deprived conditions for 24 h after which the media was collected and pooled from the three brain cell types to treat the SKBR3 cells for another 24 h. SKBR3-control cells were maintained in serum-free media for 48 h. ( B ) Phase contrast microscopy image of non-treated SKBR3 cells under 20X magnification. ( C ) Immunofluorescence microscopy of non-treated SKBR3 cells labeled with anti-HER2 antibody and detected using an anti-mouse IgG secondary antibody conjugated to CruzFluor488. ( D ) Phase contrast microscopy image of CM-treated SKBR3 cells under 20X magnification shows development of filopodia/lamellipodia type protrusions from the surface of the cells. ( E ) Immunofluorescence microscopy of treated SKBR3 cells labeled with anti-HER2 antibody and detected using an anti-mouse IgG secondary antibody conjugated to CruzFluor488.

Cytokine array analysis

Protein profiling of the secreted factors from each cell culture was performed using the Proteome Profiler Human XL Cytokine array kit (R&D systems, Minneapolis, MN) according to the manufacturer’s recommended protocol. CM from HMC3, NHA and HBEC5i, at 90% confluence, was collected after 24 h of serum starvation. CM from SKBR3 cells was collected after 24 h arrest + 24 h of stimulation with CM from brain cells (treatment), and after 48 h of serum starvation alone (control). After collection, the conditioned media were centrifuged at 500 × g for removal of floating cells, and 2 mL of the supernatant solution was added to the blocked membrane arrays and left to incubate overnight at 4 °C. After rinsing three times with wash buffer, the membranes were incubated with the antibody cocktail for 1 h at room temperature followed by a 30 min incubation with Streptavidin-HRP solution. The membranes were once again rinsed with the wash buffer and the signal was developed using the ChemiReagent mix provided in the kit. The chemiluminescent signal was captured using a ChemiDoc™ Imaging System (BioRad, Hercules, CA) and the raw images were analyzed for pixel intensities using the EMPIRIA Studio software ( https://www.licor.com/bio/empiria-studio/ ). Background subtraction was performed by the software’s adaptive background subtraction method to minimize variability and improve the consistency of data from each array. The signal intensities from the duplicate cytokine spots on each array were averaged and normalized to the average of reference spots from all arrays that were exposed to the secretome of serum-starved cells. Two independent microarray measurements were acquired from two biological replicates of each cell line and further averaged for measuring differences in protein abundance. Only spots with averaged normalized pixel intensities > 2000 were used for comparative purposes.

Cellular fractionation into nuclear and cytoplasmic proteins

The cells were separated into nuclear and cytoplasmic fractions prior to MS analysis 22 , 23 . The isolation of nuclear proteins was achieved with the CelLytic™ NuCLEAR™ extraction kit (Sigma Aldrich, St. Louis, MO) using the manufacturer’s recommended protocol. In brief, the cells were disrupted under ice-cold conditions with a hypotonic buffer in the presence of protease inhibitors (Sigma product P8340), Na 2 VO 3 /NaF phosphatase inhibitors (1 mM), DTT (1 mM), and Igepal. The lysed cells were centrifuged at 4 °C for 1 min at 15,000 × g. The clarified supernatant containing the cytoplasmic components was set aside until further use. The nuclear pellet was further disrupted with a high-salt containing buffer, supplemented with the same inhibitors, by applying constant agitation using a vortex mixer for 30 min at 4 °C. The lysate was sonicated and centrifuged at 4 °C for 10 min at 15,000 × g to generate the nuclear protein fraction. The total protein concentration in the cell extracts was measured using the Bradford assay.

Protein digestion and sample preparation for MS analysis

For MS analysis, the protein extracts were denatured for 1 h at 57 °C, pH ~ 8, in the presence of urea (8 M) and DTT (5 mM). The samples were diluted tenfold with 50 mM NH 4 HCO 3 prior to tryptic digestion. In-solution digestion was performed overnight on a shaker set at 37 °C using sequencing grade trypsin (Promega, Madison, WI) at ~ 1:25 w/w enzyme/protein ratio. The reaction was quenched with TFA and the peptides were isolated and desalted using reverse phase C18 and SCX cartridges (Agilent technologies, Santa Clara, CA). Tryptic peptide mixtures were evaporated with speed vacuum and dissolved in 2% acetonitrile solution acidified with 1% TFA at a final concentration of 2 μg/uL.

LC–MS analysis

The peptide samples (1–1.5 μL) were analyzed with a Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with a nano-electrospray source operating at 2 kV at a capillary temperature of 320 °C and coupled to an EASY-nLC 1200 UHPLC system (Thermo Fisher Scientific) 23 . The nano-LC separation was performed on a 75 μm i.d. reverse-phase EASY-Spray column ES802A (250 mm long) packed with 2 μm C18/silica particles (Thermo Fisher Scientific). Column equilibration was performed with buffer A (H 2 O 96%, CH 3 CN 4%, TFA 0.01%) and the peptides were eluted in a 2 h long gradient of buffer B (H 2 O 10%, CH 3 CN 90%, TFA 0.01%) at a 250 nL/min flow rate. The MS was operated in a data-dependent acquisition mode with a full-scan range of 400–1600 m/z, resolution 70,000, AGC target 3E6, and maximum IT 100 ms. Tandem MS/HCD spectral acquisition was performed with a quadrupole isolation width of 2.4 m/z, 30% normalized collision energy, resolution of 17,500, AGC target 1E5, maximum IT 50 ms, loop count of 20, and dynamic exclusion 10 s. To improve the consistency and reproducibility of MS measurements, each sample was injected three times. Relevant quantitative results were validated via parallel reaction monitoring (PRM) accomplished with quadrupole isolation width of 2 m/z, resolution of 35,000, AGC target (1–2)E5, and maximum IT of 110 ms.

Database searching and protein identifications

Tandem mass spectra raw files were processed with the Proteome Discoverer 2.5 software package (Thermo Fisher Scientific, Waltham, MA), using the Sequest HT search engine, a minimally redundant/reviewed Homo sapiens database (UniProtKB, 2019 download), and two processing workflows that utilized either (a) the Percolator or (b) the Target/Decoy peptide spectrum match (PSM) validator nodes to discriminate between correct and incorrect PSMs and calculate the associated scores and statistical features (e.g., q-values, FDRs). The Sequest search parameters were set for fully tryptic precursor peptides with a maximum of 2 missed cleavages, min of 6 and max of 144 amino acids, 400–5000 m/z, dynamic modifications allowed on Met (+ 15.995 Da/oxidation) and on the protein N-terminal (+ 42.011 Da/acetylation), and precursor mass tolerance of 15 ppm and fragment ion tolerance of 0.02 Da. The Percolator node was run by using a concatenated Target/Decoy strategy and default parameters, i.e., q-value based validation, max delta Cn of 0.05, and strict/relaxed peptide FDRs of 0.01/0.05. Chromatographic peak identification across the different samples was performed by adding the Minora Feature Detector node in the processing workflow. Parameters for this node were set to min trace length of 5, S/N threshold of 1, max deltaRT 0.2, and PSM confidence set to at least high (FDR < 0.01) for feature to ID mapping. The Target/Decoy PSM Validator node also used a concatenated Target/Decoy selection strategy, but the PSM and peptide strict/relaxed FDRs were set to 0.01/0.03. In the consensus workflows, the PSMs with Xcorr < 1 were filtered out, the peptide group modification site probability threshold was 75, only rank 1 peptides were matched to parent proteins, and protein grouping was performed by enabling the strict parsimony principle. The Protein Validator nodes had the confidence thresholds set to FDRs of 0.01/0.05 when using the Percolator, and to 0.01/0.03 when using the Target/Decoy Validator nodes, respectively, in the two processing workflows.

Label-free quantification and statistical analysis

Six datasets, i.e., three biological replicates of the treated and three of the non-treated cells, were considered for quantitation. Nuclear and cytoplasmic datasets were treated separately at the processing stage but combined later to enable a better understanding of the biological context. Protein abundances were calculated based on the summed peptide peak areas assigned to a particular protein, when using the Percolator node, and based on the count of correct PSMs, when using the Target/Decoy node. For peak area measurements, the chromatographic peaks were aligned with the Feature Mapper node, allowing for a max RT shift of 15 min and a peptide mass tolerance of 15 ppm. Global drifts in peptide abundance measurements were adjusted by normalizing the data based on total peptide abundance in a given sample (all peptides were used for normalization). Missing values were handled by enabling the imputation mode in the Precursor Ions Quantifier node with the low abundance resampling parameter (lower 5% of detected values). Log 2 fold changes (FC) of protein abundances in SKBR3 treated vs. non-treated datasets were calculated by using a pairwise approach, i.e. , by calculating the protein ratios as the median of all possible pairwise peptide ratios (unique and razor, excluding modified peptides). Statistically significant changes were derived by applying a t-test (background based), with multiple testing being performed based on the Benjamini–Hochberg correction method. For PSM count-based abundance measurements, the datasets were normalized based on the average of total PSMs identified in each of the six datasets, one PSM was added to each protein to account for the missing values, and a t-test was performed for assessing significance. For both scenarios, only proteins that were matched by at least two unique peptide sequences, and for which the Log 2 (FC) was either ≥ 1 or ≤ (− 1) with p -value < 0.05, were included in downstream analysis for biological interpretation. The PRM data were analyzed with Skyline ( https://skyline.ms ) 24 , using mass spectral libraries created from the SKBR3 MS raw files. Peptides having a minimum of 5 transitions with a dot product ( dotp ) score > 0.9 were considered for validation.

Biological data interpretation

The biological relevance and visualization of proteins that displayed statistically significant differences in peak areas or PSM counts was performed by using publicly available bioinformatics tools provided by STRING, GeneCards, and Cytoscape. GO biological process enrichment was explored with the STRING web interface. Biological schematics were created with tools provided by Biorender.com.

To explore the early response of HER2 + breast cancer cells to the biological effects of the brain microenvironment, the SKBR3 cells were cultured using serum-deprived conditions in the presence (T-treated) and absence (C-control) of conditioned medium combined from serum-deprived astrocytes (NHA), microglia (HMC3) and brain endothelial (HBEC5i) cells ( Fig.  1 A ) . It was anticipated that the combined CM from all three brain cells would mimic the microenvironment of the metastasized tumor cells in the perivascular niche. All brain cells were cultured to high confluence to ensure the secretion of factors in sufficient concentration for the stimulation experiments. The study was conducted in the absence of fetal bovine serum for multiple reasons. First, we intended to explore the behavior of cancer cells under conditions that induce cell cycle arrest—a state which is believed to be a major reason for metastatic latency of brain metastasized breast cancer cells, relapse being enabled by re-entering of the cancer cells in the cell cycle 25 , 26 . Under such conditions, it has been shown that adherence to endothelial cells is critical for the survival of single cancer cells that extravasate in the perivascular niche and encounter a foreign environment deprived of typical nutrients present in the blood stream. It has been also shown that after therapy, metastatic growth evolves from preexisting dormant disseminated cancer cells. Second, we intended to avoid the interference of the broad range of components of undefined concentration present in the serum (e.g., growth factors, hormones, enzymes, proteins, carbohydrates, etc.), as well as the variable batch-to-batch composition that can affect the reproducibility of findings 27 . The SKBR3 cells were depleted of serum for 24 h prior to, not just during, treatment to ensure that the impact of the treatment on SKBR3 cells stems only from the neural cell CM and not prior FBS supplementation. The control (C) and treated (T) nuclear (N) and cytoplasmic (C) cellular subfractions were annotated as follows: CN1–CN3 nuclear fractions of control cells, CC1–CC3 cytoplasmic fractions of control cells, TN1–TN3 nuclear fractions of treated cells, and TC1–TC3 cytoplasmic fractions of treated cells.

Morphological characteristics of CM-treated SKBR3 cells

Under basal conditions, the SKBR3 cells displayed a grape-like morphology with a lower intercellular adherence (Fig.  1 B), as it has been confirmed by previous studies 28 . The cells stained strongly for the ERBB2 receptor which is known to be present in high abundance on the surface of the cells (Fig.  1 C). Upon treatment with the CM, we observed the development of protruding structures resembling lamellipodia and filopodia emanating from the surface of cells, which could be also visualized by ERBB2 staining (Fig.  1 D,E). DNA content analysis by flow cytometry revealed small but consistent differences, the treated SKBR3 cells displaying a larger proportion of cells in the G1 stage of the cell cycle in comparison to the controls [83% (CV 13%) treatment vs. 74% (CV 9%) control], suggesting an inhibition of growth upon treatment with the CM. Nonetheless, the proteomic data (see further) revealed a more complex response to the CM treatment that branched out into multiple biological processes beyond cell cycle arrest such as metabolism, adhesion, and immune response.

Analysis of conditioned media using cytokine arrays

Membrane-based cytokine arrays were used to measure the relative concentration of 105 different components in the CM of serum-deprived brain and cancer cells and assess the factors relevant to intercellular communication that may affect the behavior of cells (Fig.  2 ). Detailed results of the replicate cytokine measurements including images and raw and normalized pixel intensities are provided in Supplemental file 1 . The functional impact of components that displayed noticeable pixel intensities (> ~ 2000) was evaluated. A set of ~ 50 proteins of interest emerged from the analysis: chemokines and interleukins, growth factors, and various other factors with roles in growth/development, angiogenesis, adhesion, migration, and immune response. The group of proteins with increased abundance in at least one of the brain cell secretomes included pro-inflammatory chemokines (CCL2, CCL5, CCL7/MCP3, CXCL1, CXCL5, CXCL8/IL8, CXCL10, CXCL11), homeostatic chemokines (CXCL12/SDF1), pro- and inflammatory cytokines and factors (IL6, IL15, TNFSF13B, FLT3LG, RBP4, CD14, CSF1/2), pro-inflammatory inhibitors and homeostatic cytokines (IL18BP, PTX3), growth and cell activator factors (BDNF, IGFBP2/3, PDGFA, GDF15, TNFSF13B, ANGPT1/ANGPT2, CSF1/2, CHI3L1, SPP1, DKK1), and cell adhesion and extracellular matrix (ECM) remodeling molecules (ICAM1, VCAM1, MMP9, THBS1, SERPINE1, PLAUR) (Fig.  2 A–C,F).

Results from the cytokine microarray profiling of conditioned medium collected from serum-deprived cell cultures of ( A ) HBEC5i, ( B ) HMC3, ( C ) NHA, ( D ) SKBR3-control, and ( E ) SKBR3-stimulated cells. ( F ) Bar chart showing the average pixel intensities of the set of cytokines identified in the CM collected from the serum-starved brain and SKBR3-control cells (pixel intensities > 2000). Color code: blue-HBEC5i, green-HMC3, yellow-NHA, orange-SKBR3.

The group of proteins with increased abundance in the SKBR3 secretome (not necessarily unique to SKBR3) included cytokines (pro-inflammatory MIF, CCL20, IL17A, and anti-inflammatory TFF3) and several groups of factors and receptors with relevance to growth and development (PDGFB, FGF19, TFRC), ECM remodeling, and invasiveness (LCN2, BSG) (Fig.  2 D, F). Angiogenesis factors such as VEGFA, ANG and ENG were secreted by all cells, with ENG being more abundant in the HBEC5i and SKBR3 secretomes. A schematic representation of a subgroup of cytokines that provided visually discernable spots (pixel intensity > 7000), and of which some displayed discrepancies in abundance in the cell secretomes, is provided in a node/edge networked configuration in Fig.  3 A with edges indicating detectability in either of the four cell lines. A cutoff of ~ threefold change (FC) in normalized pixel intensities in the group of HBEC5i/HMC3/NHA brain cells and SKBR3 was used to indicate unique association with a particular cell line.

Inflammatory cytokines

With few exceptions, all pro-inflammatory cytokines with elevated abundance in the brain cell secretomes were secreted at the basal level by the HBEC5i cells, with only a few being secreted in similar abundance by the HMC3 (CCL2, CCL5, CXCL8/IL-8, PTX3, RBP4, FLT3LG, CD14) and NHA (CCL7, CXCL8) cells. Owing to their prime location in the bloodstream, the endothelial cells are the first to come in direct contact with any infectious entity in the body. Therefore, these cells advanced mechanisms for the recognition of damage-associated molecular patterns (DAMPs) for inciting an immune response and for recruiting immune cells to the site of pathogenesis 29 . Many of these functions are facilitated by the inflammatory cytokines and chemokines that are released by the endothelial cells in their microenvironment. Elevated levels of TNFSF13B, a cytokine involved in the regulation of immune responses and stimulation of B-/T-cells, were observed only in the HBEC5i cell secretome. IL6 was also preponderantly expressed in the HBEC5i secretome. Along with its well-known inflammatory functions, this cytokine has been implicated in regulating the proliferation, angiogenesis, invasion, and metabolism of cancer cells. IL6 mediates these outcomes primarily via the STAT3 or NFKB signaling axis, and induces the expression of factors which promote enhanced malignancy (GM-CSF, CCL2, MMP, VEGF) 30 . Previous studies have confirmed a basal level expression of CCL2 and CXCL8/IL8 by the human brain endothelial cells, however, CXCL10 and CCL5 were shown to be expressed only upon pro-inflammatory cytokine stimulation 31 . The levels of CCL2 were relatively constant in all cell secretomes, but there is accumulating evidence that CCL2 induces angiogenesis via increased VEGFA expression, while CXCL5, CXCL8/IL8 and CXCL1 directly affect tumor growth and metastasis by enhancing blood vessel supply through neovascularization 32 . On the other hand, CXCL10 and CXCL11 exert angiostatic effects and initiate T-cell or NK-cell mediated immune responses 32 . Previous studies suggested that CCL7 could be involved in promoting tumor invasion and metastasis, but tumor suppressor effects of this cytokine have been also identified 33 . The detection of CCL7 in this work was, however, just above the intensity threshold setting in HBEC5i and NHA cells. IL15 was also observable in low abundance, and only in the secretome of HBEC5i cells. By stimulating the proliferation and activation of immune cells (NK-, B- and T-cells), IL15 has many protective and anti-tumor roles. It is explored as a potential therapeutic agent by itself or as a target for the development of IL15 agonists and a number of immunotherapies 34 . IL18BPa (IL18 binding protein) which is an inhibitor of the proinflammatory IL18 cytokine was upregulated in HBEC5i, while the homeostatic/tissue remodeling PTX3 (Pentraxin-3) in the HMC3 and HBEC5i secretomes. IL18BPa is believed to play a buffering role for IL18 35 , and PTX3 to exert multiple tumor suppressive and supportive roles via complex involvement in mediating immune responses, ECM remodeling and angiogenic programs 36 , 37 . CXCL12 (SDF1α) was elevated in the HBEC5i secretome relative to SKBR3, and while this is a homeostatic chemokine, it is known to exert various roles in pathogenic conditions 38 , 39 . Different splice variants have specific activities, which are also controlled by PTMs 38 , and hypoxia and growth arrest in different cell types can elevate the expression level of this cytokine 38 . Increased CXCL12 levels produced under hypoxia by ovarian cancers have been shown to promote the expansion of endothelial cells and angiogenesis 40 . Altogether, aberrant expression of CXCL12 in the tumor microenvironment was correlated with tumor growth, proliferation, inhibition of apoptosis, and driving cancer cell migration to distant sites 39 . The cytokine signals along the MAPK, PI3K/AKT, Wnt and NFKB pathways to induce the expression of VEGF, FGF, cyclooxygenase-2 and IL6, all of which are critical mediators of angiogenesis 39 . CXCL12 was also observed in the NHA medium. Additional pro-inflammatory proteins such as the Fms-Related Tyrosine Kinase 3 Ligand and growth factor (FLT3LG), retinol binding protein 4 (RBP4), and receptor CD14, albeit in lower abundance than the other cytokines, complemented the ability of HBEC5i cells to stimulate the proliferation or activity of immune cells 41 . In addition, the cell adhesion glycoproteins VCAM1 and ICAM1 with roles in mediating cell adhesion, motility and maintaining tissue architecture, were also predominantly expressed in the HBEC5i secretomes, especially VCAM1 (ICAM1 was observable in low abundance). VCAM1 is overexpressed on endothelial cells under inflammatory conditions to allow for the adhesion and rolling of immune cells on the endothelium surface 42 . It has particular relevance during cancer metastasis, as it is one of the molecules that tumor cells bind for transmigrating across the endothelial barrier 42 .

In microglia, the presence of IL6, CXCL8/IL8, CCL5, PTX3 was consistent with the role of these cytokines in the regulation of leukocyte activation and chemotaxis that is invoked in the brain in the case of neuropathological and inflammatory conditions, and the detection of PTX3 and FLT3LG with the involvement of microglia in mediating inflammation and phagocytosis, respectively 43 . Likewise, the secretion of CCL7 by NHA cells was associated with promoting microglia-mediated inflammation after brain injury 44 .

In the SKBR3 secretome, pro-inflammatory cytokines were present, but generally in low abundance (excepting IL17A and MIF which were also present in the brain cell secretomes) or had mostly tumor-promoting functions (CCL20). Cytokines such as IL11, IL22, RETN and TNF which are associated with inflammatory responses were observable at very low intensities, but their emerging role in promoting the progression of cancer has been recognized 45 , 46 , 47 , 48 . The other cytokines have been shown to promote cell renewal, tissue regeneration, recruitment of immune cells and activation of pro-survival and mitogenic signaling (IL17A 49 ); to induce cell invasiveness, angiogenesis and cell survival pathways (macrophage migration inhibitory factor (MIF 50 ); or, to stimulate invasiveness via MMP secretion and impart chemotherapeutic resistance (chemokine CCL20 51 ).

Growth factors

We further assessed the differences in the concentration of growth and stimulating factors between the SKBR3-control and the brain cells (Fig.  2 A–D) 41 , 52 , 53 , 54 , 55 . The analysis revealed that the level of many pro-tumor factors, especially of those that are implicated in growth/proliferation, angiogenesis, migration, and metastatic progression was either similar or higher in the SKBR3 than in the brain cell secretomes (PDGFB, FGF19, GDF15, MIF, LCN2, TFF3, VEGFA, ANG, ENG, ANGPT2, TFRC, SERPINE1, PLAUR, BSG) (Figs.  2 F, 3 A). Specific biological processes sustained by these proteins included regulation of ERK1/ERK2 and MAPK signaling cascades, cell adhesion, cell migration and invasion, blood vessel development and angiogenesis, and response to stress/defense and inflammation. Selected GO biological processes represented by minimum 5 proteins from either of the cell secretomes, with a fold-enrichment of > 4, are provided in Fig.  3 B. The multifunctional platelet derived growth factor subunit B (PDGFB) with roles in cell proliferation/survival and migration, in particular, was highly abundant in SKBR3 and essentially missing from all brain cells. GDF15 was expressed by all cells, and increased levels have been shown to be associated with advanced cancer and poor patient outcomes, as this factor promotes mechanisms of immune evasion in cancer cells and enhances cell viability and metastasis 52 . Others, however, have suggested a conflicting response to GDF15, when it was observed to induce apoptosis in some cancer cells 52 . Further evidence for the secretion of cancer progression supportive factors in SKBR3 was presented by the higher expression of lipocalin-2 (LCN2) which has important ramifications in EMT-related processes, invasion, and cell migration 53 and of the anti-inflammatory trefoil factor 3 (TFF3) which supports invasion and metastasis in several carcinomas 54 .

All three brain cell types secreted some if not all of the above cancer-supportive factors. In a broader context, these proteins are involved in developmental processes, and regulate cell proliferation, mitogenesis, survival, tissue repair, and chemotaxis (Fig.  3 B). A few factors that were identifiable in SKBR3 were present in higher abundance in the brain cell secretomes (IGFBP2/3, PDGFA, SPP1, CSF1/2), some being particularly elevated in NHA (IGFBP2, ANGPT1, CHI3L1, SPP1) or HMC3 (PDGFA, BDNF) (Fig.  3 A). NHA exclusively expressed the angiopoietin (ANGPT1) and chitinase 3 like 1 (CHI3L1) proteins. Together with VEGFA, angiopoietins secreted by astrocytes are important for governing the angiogenic remodeling processes for the development of vasculature in the brain 56 . Increased levels of CHI3L1 have been associated with neurodegenerative and inflammatory brain diseases, but aberrant expression has been also observed in brain tumors and metastasis. It has been suggested that CHI3L1 expression by activated astrocytes may play a role in tumor progression, angiogenesis, immune escape, and resistance to therapeutic drugs 57 . It is also worth highlighting osteopontin (SPP1) which plays a crucial role in driving metastasis, invasion, angiogenesis, chemotaxis, and suppression of anti-tumor immune responses 58 . SPP1 is known to be secreted by both cancer cells and the cells of the TME, and to have an overall positive impact on the metastatic progression of cancer 58 . HMC3 revealed the expression of the BDNF involved in the development and survival of CNS cells. Interestingly, BDNF activates TrkB (Tropomyosin-Related Kinase B or Neurotrophic Receptor Tyrosine Kinase 2) and HER2 receptors and enhances the proliferation and survival of brain metastatic HER2 + breast cancer cells 59 . On the other hand, while the exact function of PDGFA in microglia is not fully understood, this growth factor is recognized for its important contributions to supporting cell proliferation, angiogenesis, migration, as well as chemotaxis 41 . The matrix remodeling proteins and enzymes (SERPINE1, PLAUR, PTX3, LCN2, BSG, THBS1, MMP9), together with the CAMs (ICAM, VCAM1) that were overexpressed in the HBEC5i secretome, formed a group that plays key roles in mediating cell–cell/cell–matrix interactions and ECM remodeling processes that ultimately affect, again, the adhesion, migration, and the invasion capabilities of cancer cells.

The culture medium collected from the SKBR3 cells grown in brain cell-conditioned media generated a signal for almost all factors that produced intense spots in the brain and SKBR3-control secretions (Fig.  2 E). It was therefore difficult to conclusively determine which cytokines and biological processes were upregulated in the SKBR3 secretions after treatment with conditioned media from the brain cells.

Proteomic analysis and quantification of the CM-treated versus non-treated SKBR3 cells

The impact of treating the SKBR3 cells with the brain cell-conditioned media was next evaluated quantitatively at the proteome level. The expected outcome was the identification of novel proteins that mediate an early response in SKBR3 cells to the factors that they encounter in a foreign microenvironment (i.e., the brain TME). Three biological replicates of SKBR3-treated (T) cells were compared to three biological replicates of SKBR3-control (C) cells. Two complementary approaches were used for assessing protein differential expression, i.e., based on LC peak area and PSM count measurements. The two approaches produced rather complementary results due to: (a) the actual use of two distinct methods for assessing protein abundance; (b) the use of two distinct approaches to validate the PSMs, with the area-based measurements being enabled by a workflow that included the Percolator node that uses a semi-supervised machine learning approach to distinguish the correct from the incorrect PSMs, in contrast to the PSM count measurements that relied on using the results from the Target and Decoy database searches; and (c) the possible software-related misrepresentation of area measurements because some chromatographic peaks did not have actual PSMs associated with them in all treatment and control samples. The complementarity of such results is not surprising when only few proteins change abundance or the changes in abundance are small, and it was noted in previous studies, as well 60 . Longer exposure of the SKBR3 cells (> 24 h) to the brain CM could have amplified the changes in the proteome profiles, however, the prolonged lack of serum from the culture medium would have biased the results by inducing an excessive stress response.

On the average, ~ 3200/4700 proteins per cell fraction were identified in the SKBR3 cells when using the Percolator and Target/Decoy validator nodes, respectively, with over 70% of proteins being identified with two or more unique peptides (Supplemental file 2 ). The reproducibility of protein identifications in biological replicates for the workflow that used the Percolator node is illustrated in Fig.  4 (# protein IDs, overlaps, PSM correlations), and the results of the quantitative comparisons in Fig.  5 A–E,G [box and whisker plots of raw and normalized Log10(Protein Abundance) and Volcano plots] and Supplemental file 3 . There were no major overlaps between the proteins that displayed differences in counts in the nuclear and cytoplasmic fractions, demonstrating the complementarity of the data (Fig.  5 F and H). Taken altogether, the area and PSM-based upregulated datasets comprised 96/91 proteins, respectively, and the downregulated ones 256/46 proteins, combined from the analysis of both nuclear and cytoplasmic samples. A few representative proteins are illustrated in Fig.  6 A. Selected proteins were validated using PRM-MS analysis and a few others by Western blotting (Supplemental file 4 ).

Evaluation of reproducibility in protein identifications. ( A ) Stacked bar chart showing the percentage of proteins identified by 2 or more unique peptides in a cell state and fraction (~ 70%, bars shaded in gray). The white boxes in the center display the total number of identified proteins in each cell state and fraction. ( B – E ) Venn diagrams displaying the overlap between the proteins identified with high/medium FDR and ≥ 2 unique peptides in three biological replicates of various SKBR3 cell fractions: ( B ) CN, ( C ) CC, ( D ) TN, and ( E ) TC. ( F – I ) Scatterplots displaying the degree of correlation between the PSMs of two biological replicates for ( F ) CN, ( G ) CC, ( H ) TN, and ( I ) TC datasets. Heatmaps on the right show the Pearson correlation coefficients for every comparison.

Overview of the proteomic label-free differential expression analysis approach. ( A – D ) Boxplots of area-based protein abundances for three biological and three technical replicates of SKBR3 cell fractions: ( A ) before and ( B ) after normalization for the quantitative analysis of TC vs CC fractions; ( C ) before and ( D ) after normalization for the quantitative analysis of TN vs CN fractions. ( E , G ) Volcano plots illustrating the results of differential expression analysis for proteins that displayed minimum 2-FC up- (red) or downregulation (blue) with a p-value ≤ 0.05 in TN vs CN and TC vs CC: ( E ) area-based measurements, and ( G ) PSM count-based measurements. ( F, H ) Venn diagrams showing the overlap between the proteins in the N and C fractions that were either up- or down-regulated in the area-based and PSM count-based measurements.

GO Biological processes represented by the proteins that changed abundance in the proteomic profiling of SKBR3 cells treated with CM medium from brain cells. ( A ) Box-whisker plots displaying the relative spectral counts for the selected proteins identified in the control and treated proteomic datasets. ( B ) Enriched biological processes represented by the combined nuclear/cytoplasmic proteins detected by the area and PSM-count measurements with increased or decreased abundance, respectively, in CM-treated vs non-treated SKBR3 cells. ( C ) Protein counts that emerged from the area and PSM measurements in the CM-treated vs non-treated SKBR3 cells mapped to GO biological processes of relevance to cancer (note: the proteins were mapped to biological processes by using GO controlled vocabulary terms).

Quantification was performed separately for the nuclear and cytoplasmic fractions, however, the results were combined for a thorough understanding of the cellular biology. The short lists of differentially expressed proteins yielded only very few enriched biological functional categories. When combined, however, the nuclear and cytoplasmic results, as assessed either via area or PSM measurements, revealed the main processes that were affected by treating the cells with brain cell conditioned medium. The processes that were sustained by proteins with increased counts were dominated by altered gene expression, chromosome organization, trafficking, protein localization, and interferon signaling. The processes that were supported by proteins with decreased counts (represented mostly by the cytoplasmic cluster generated by area measurements) were broadly associated with various metabolic and transport processes, and included an over-representation of cytoplasmic translation and electron transport/mitochondrial respiratory processes (Fig.  6 B and Supplemental file 5 ). Ribosome biogenesis was represented by proteins with both increased and decreased counts. Many of the above processes are mediated via posttranslational activation/deactivation or protein shuttling between various cellular organelles. Therefore, increased or decreased protein abundance measurements may not be reflective of actual protein up- or down-regulation, but rather of changes in PTMs or cellular location. Altogether, however, the results suggest that the treatment of cells with conditioned medium altered the cellular transcriptional/translational machinery with a net outcome of slowed metabolic processes. Ribosome biogenesis, for example, is an energy-intensive process, and a disruption in the availability of nutrients or energy in the cell that can affect any step of the biogenesis process leading to either up- or down-regulated ribosome components, has been shown to result in altered cell cycle progression or even cell death 61 .

It is important to reiterate that upon treatment with the brain cell-conditioned media the SKBR3 cells were exposed to many inflammatory molecules that were either absent or present in low abundance in the secretome of the SKBR3-control cells. In the same time, the SKBR3-control cells secreted larger amounts of several important growth factors that may or may not have been at optimum levels in the brain cell-conditioned media to elicit a signaling response in the SKBR3 cells. Therefore, to derive a better understanding of the processes that were dysregulated in the SKBR3 treated cells, the proteins were further mapped directly to specific GO terms reflective of cancer-supportive processes and metastatic progression (Fig.  6 C). The complex interplay between these proteins in affecting the cell fate is exemplified in a Sankey diagram that presents a selected list of SKBR3 proteins with changed abundance, matched by at least three unique peptides, mapped to such processes (Fig.  7 and Supplemental file 6 ).

Sankey diagram illustrating the mapping of dysregulated nuclear/cytoplasmic proteins with increased abundance to key biological processes of relevance to metastatic progression of cancer (note: only proteins identified by at least three unique peptides are shown).

Based on our previous work on proteomic profiling of SKBR3 cells that has identified a broad range of receptors in the cell membrane (EGFR, FGFR, CSFR, IL, CD44/Cd47, IL, PDGFRA, TNFR, VEGFA receptor FLT1, ephrin, integrin, neuropilin NRP1, activin, chemokine ACKR2, adiponectin ADIPOR1, sortilin SORT1, low density lipoprotein LRP, syndecan SDC4) 62 , a depiction of the possible interactions between the SKBR3 breast cancer and the brain cells is provided in Fig.  8 . Relevant SKBR3 receptors, along with the most abundant cytokines and growth factors secreted by the brain cells, are listed in the figure. The observed changes in the SKBR3 proteome were further interpreted based on the consideration that the SKBR3 cells that were exposed to the CM from brain cells experienced an environment deprived of optimal levels of growth factors needed for proliferation, but more abundant in inflammatory molecules. Proteins with similar trends in abundance changes, as emerged from the two quantification approaches, are discussed below.

Illustration of receptors and secreted factors that can enable interactions between the SKBR3 breast cancer and brain cells. Relevant SKBR3 receptors were detected by proteomic profiling 58 . Abundant cytokines and growth factors secreted by brain cells, but missing or detected in low abundance in the SKBR3 secretome, are depicted near each cell line. Abundant cytokines and growth factors secreted in common by all brain and/or cancer cells are shown inside the sidebars.

Effect of CM-treatment on cell cycle and proliferation

An interesting behavior of metastatic cancer cells is dormancy, a mechanism through which the metastatic cells temporarily exit the cell cycle to enter a state of quiescence and growth arrest, which allows them to survive the adverse conditions of a foreign niche 63 . Dormancy is activated in cancer cells to cope with environmental stress triggered by growth factor deprivation, oxidative stress, lack of ECM attachment and stromal cell interactions 63 , 64 . Inhibited ERK, autocrine Wnt signaling, and activation of various components of MKK4/p38/JNK, MTOR and hypoxic pathways are reflective of this state. The quiescent cells significantly reduce their metabolic activity including biosynthesis of macromolecules, carbohydrate and lipid metabolism, and mitochondrial respiration 65 . A recent study demonstrated an enrichment of stress response and cell cycle related processes in the transcriptomics data derived from the dormant cancer cells of mouse and human origin 64 . In this work, a number of transcription factors and regulators were affected by the SKBR3 stimulation process with both initiator/activator (GTF2E2, USF1, CDK5RAP3, PML, CEBPB) and repressor roles (GATAD2B, PML, ZBTB7A). In particular, ZBTB7A (zinc finger and BTB domain containing 7A protein) which is a transcriptional repressor of genes implicated in cell cycle progression and known to exert multiple roles in cancer cells that affect NFKB and Notch signaling, glycolysis, OXPHOS, transcription, chromatin organization, and DNA damage 66 , warrants further investigation to provide clarity about its contribution to tumorigenesis. In contrast, CEBPB, a transcription factor involved in the regulation of a number of immune and inflammatory responses (see also later in the text), has been recognized for both its proliferative and anti-proliferative activities 41 . Clarifying the mechanisms of its functionality, however, is challenging because the function of CEBPB in cancer cells is context specific with diverse outcomes in different types of cancers, and because its activity is regulated by a number of different PTMs 67 . While on one hand CEBPB inactivates p53 expression to improve rates of survival and reduce apoptosis in cancer cells, it has also been shown to induce growth arrest in several types of cancers 67 . In this work, CEBPB was measured with increased PSM counts, result that was validated by targeted PRM-MS analysis (Supplemental file 4 ). Several proteins from the dataset also pointed to the alteration of signaling pathways that modulate in concert cell cycle, proliferation, stress and inflammation, such as EGFR (ERBIN—Fig.  6 A), p38/MAPK (TAOK3), PI3K (PI3KR2), DNA damage and repair (RPA2; ASCC—Fig.  6 A; 14-3-3 sigma or stratifin/SFN—Supplemental file 4 ), TGFB (ZBTB7A; SDCBP), NFKB-mediated transcription and host-virus interactions (CDK5RAP3/LZAP), and anti-inflammatory processes (GNG5). The Ser/Thr protein kinase TAOK3 is a regulator and activator of the p38/MAPK14 stress-activated MAPK cascade, with implications in many cellular stress-responses including apoptosis, metabolism, cell cycle, proliferation, cytoskeleton rearrangement, and inflammation 41 , 68 . Alterations in its abundance/activity were confirmed by PRM/MS (Supplemental file 4 ). Recently, it was found to induce drug resistance in breast cancer cells via NFKB activation and pathways that can have diverse outcomes, one of which is blockade of cell cycle progression and development of quiescence or dormancy 69 , 70 . Notable were also the upregulated replication protein A2 (RPA2) that responds to cellular stress by repairing damaged DNA at cell cycle checkpoints 41 , and CDK5RAP3 and PML, due to their involvement in numerous cellular processes such as transcription regulation, DNA repair, growth suppression, apoptosis, and viral defense mechanisms. The last two proteins have been additionally recognized for their potential role in tumor suppression.

Proliferation markers such as KI-67 or PCNA did not display differential expression by MS analysis. This, however, was not unexpected as both cell states were deprived of serum components for 48 h. Under proliferative conditions, in the presence of FBS, the PSMs of KI-67 typically increase ~ 5–tenfold. The mass spectrometry results for a negative regulator of cell cycle, 14-3-3 sigma, indicated changes in the protein abundance, but the changes were small and did not pass all data filtering criteria. Nonetheless, Western blot analysis confirmed an increase in 14-3-3 expression, corroborating the more advanced state of arrest of CM-treated SKBR3 cells (Supplemental file 4 ). Altogether, the findings suggest that the cells demonstrated a more complex response, rather than merely exhibiting a more pronounced cell cycle arrest. The cells possibly initiated adaptive mechanisms to survive the nutrient-limited stress conditions and respond to additional insults inflicted by the brain cell-secreted factors. Cancer cell quiescence has been also linked to increased resistance to apoptosis 71 . The results indicate, however, that although the cells activated biological mechanisms in response to nutritional and inflammatory stress, the cells were not apoptotic.

Effect of CM-treatment on cancer cell metabolism and response to stress

Scarcity of nutrients and unfavorable growth conditions drive cancer cells towards quiescence, which results in reduced metabolic activity, biosynthetic rates and respiration 65 . Recent studies have shown that dormant/quiescent cells switch their carbohydrate metabolism from mitochondrial OXPHOS to fatty acid oxidation, in order to secure a minimal supply of energy and prevent oxidative damage caused by the reactive oxygen species released during mitochondrial respiration 72 , 73 . Several metabolism and oxidative stress associated proteins were downregulated according to the area-based measurements, including the ribosomal subunit proteins involved in translation (RPLs, RPSs), the large neutral amino acid transporter LAT1 (SLC7A5), the mitochondrial OXPHOS/electron transport and ATP production subunit proteins, and various antioxidant proteins (PRDX1). PRM/MS (LAT1, RPL8, PRDX1) and Western blot (LAT1) analysis validated the MS results (Supplemental file 4 ). Ribosome biogenesis is an energy consuming process, necessary for rapid cell growth and division, as a result of which ribosomes have a central role in a number of oncogenic signaling networks 61 , 74 . Under conditions of nutritional stress, cells can curtail the biogenesis of ribosomes to conserve energy, and, consequently, limit the G1/S progression of cells 75 . Reduced levels of solute carriers such as of the nucleotide-sugar transmembrane transporter (SLC35A4), that are implicated in cellular response to stress by regulating translation processes, corroborated the results. Lower expression of LAT1 was also suggestive of impaired proliferation in the CM-treated cells. The LAT1 transporter allows for an increased uptake of essential amino acids from the external microenvironment, in exchange for glutamine, which is pumped out of the cells 76 . LAT1 overexpression was found associated with highly proliferating cancer cells growing under low nutrient conditions and hypoxia, and is being evaluated for its therapeutic target potential 76 . The cluster also included proteins that mediate transport across the mitochondrial membrane, such as the glutamate carrier SLC25A22 and amino acid transporter SFXN1, all of which pointed toward restricted mitochondrial metabolism. These results correlated with the upregulation of the transcriptional repressor of genes involved in OXPHOS and glycolysis (ZBTB7A). Overall, the results indicate that the nutritional stress conditions induced by serum-deprivation and CM-stimulation impeded ribosomal protein synthesis pathways and reduced mitochondrial respiration. Furthermore, the inflammatory conditions mediated by the cytokines and chemokines triggered an overexpression of proteins that respond to these stimuli by regulating changes in cancer cell proliferation, growth and survival.

Effect of CM-treatment on cancer cell adhesion/migration and EMT

Epithelial cells are characterized as having an apical-basal polarity, with junction proteins binding the cells together in a monolayer, attached to the basement membrane 77 . During cancer progression, tumor cells undergo EMT (epithelial to mesenchymal transition) which is marked by the loss of certain adhesion and junction proteins and acquisition of mesenchymal features, such as cytoskeletal rearrangements, that induce invasive and migratory capacities in the cancer cells 77 . The CM treatment of SKBR3 resulted in changes in the expression level of several proteins that have known implications in adhesion, migration and EMT-related processes.

Evidence for EMT type behavior and development of cell motility in the CM-treated SKBR3 cells was provided by the overexpression of syntenin-1 and annexin proteins. Syntenin-1 (SDCBP) activates cancer cell invasion and migration through actin remodeling processes during the late stages of cancer metastasis 78 (Fig.  6 A and Supplemental file 4 ). It colocalizes with growth factor receptors, the focal adhesion kinase complex, and other integrin-associated signal transducers to activate p38MAPK/JNK downstream signaling 78 , 79 . This, ultimately triggers cytoskeleton rearrangement processes, which regulate adhesion and migration in cancer cells 79 . A critical outcome of this pathway is the activation of mesenchymal proteins such as SLUG that induce EMT 79 , 80 . The cytoplasmic fractions of the treated cells displayed an upregulation of annexin A1 (ANXA1), which has the ability to bind the actin cytoskeleton, which in turn is crucial for the initiation of EMT, migration, and invasive properties in cancer cells 81 . The multifunctional ZBTB7A, as noted above, has been shown to also mediate EMT via the NFKB signaling pathway in a number of breast cancer cell lines 82 . As noted earlier, treatment with the CM induced the formation of protruding lamellipodia and filopodia on the surface of SKBR3 cells (Figs.  1 D and 1E). In transmigration assays that exposed the SKBR3 cells to HMC3 secreted factors similar changes in morphology were observed, however, cell migration within 24 h of exposure was very slow, and a Western blot of MIEN1 (Migration and Invasion Enhancer 1) that induces the formation of filopodia 41 did not show a change in the abundance of this protein (Supplemental file 4 ).

A few additional proteins of potential interest included the downregulated YBX1 and LAMP1, and upregulated ICAM1 and S100 Ca-binding proteins. PRM/MS conducted for a few of these proteins, confirmed their change in abundance (Supplemental file 4 ). Notably, the DNA/RNA binding protein YBX1 participates in a range of cellular transcription/translation processes and augments metastatic processes such as invasion and cell migration 83 , development of drug resistance 84 , and elevated glycolysis 85 . Its activity, however, depends on phosphorylation, and further investigation is required to provide clarity on its activation status in the CM-treated SKBR3 cells. YBX1 and the previously discussed TAOK3 are both included in the DRMref database, which provides a comprehensive map of drug resistance mechanisms in human cancer 86 . The lysosome associated membrane glycoprotein LAMP1 provides glycan ligands to selectins and promotes cell–cell adhesion in metastatic cancer cells 87 . ICAM1, an intercellular cell adhesion molecule, is involved in cell–cell and cell-ECM adhesion and was found to be expressed by metastasized cancer cells as they interact with the endothelial cells to undergo trans-endothelial migration. S100 calcium-binding proteins bind to a variety of cell surface receptors on other cells to induce metastatic progression 88 . These proteins carry out, however, a broader range of functions that present relevance to metastatic processes, both in the intra- and extracellular environments. The intracellular functions of S100 proteins include involvement in various aspects of cell cycle regulation, promotion of cell survival and invasion via the NFKB signaling pathway, metabolism, and trafficking. The extracellular functions include involvement in pro-inflammatory, anti-microbial and death-inducing activities 41 . Binding to receptors on macrophages/microglia or endothelial cells facilitates their recruitment and angiogenesis at the site of tumorigenesis 88 . In this study, S100A7 and S100A9 were found upregulated in the CM-treated cells (Fig.  6 A and Supplemental file 4 ), while other isoforms (A4, A6, A13, and A16) appeared to be rather downregulated, pinpointing again to the need for careful interpretation of results and further studies.

Effect of CM-treatment on the mechanisms of immune evasion

Upregulated ISG15 (interferon-induced ubiquitin-like protein) and MX1 (interferon-inducible protein p78 or myxoma resistance protein 1) were two proteins that were potentially reflective of the SKBR3 cell response to exposure to the cytokines from the conditioned medium. ISG15 plays multiple roles in innate immune responses that include antiviral activities, cell–cell signaling, and chemotactic activity 41 . It can act either in free form or by binding to target proteins, including MX1, a protein that impedes viral replication. Previous studies have demonstrated that ISG15 enhances EGFR recycling on the surface of breast cancer cells, as a result of which the cells display an increased membrane localization of EGFR and aberrant tumorigenic signaling 89 . A recent study identified MX1 as a potent inducer of migration, EMT, and invasion in cancer cells due to its association with EGFR signaling 90 . According to the cytokine arrays, a small increase in the abundance of interferon γ in the secretome of CM-treated SKBR3 cells was observable, but further studies would be needed to clarify the complex mechanisms that are responsible for the upregulation of these proteins. In the same time, the lysosomal thiol reductase IFI30 which is involved in MHC/HLA class I/II antigen presentation and which is known to be induced by interferon-γ in non-professional antigen presenting cells, was downregulated, suggesting a loss of HLA presentation. HLA proteins were practically undetectable in the SKBR3 cells, so comparisons between CM-treated and non-treated cells could not be made. The inability to detect HLA class I molecules was, however, not surprising, as cancer cells downregulate the HLA class I presentation machinery to evade detection and destruction by immune cells (T/CD8 cells) 91 . Nonetheless, HLA-E and HLA-C expression was detected in both cell states. HLA-E is a ligand for the CD94/NKG2A inhibitory receptor expressed by NK- and T/CD8-cells, and HLA-C for the NK-cell KIR receptors 92 , 93 . Both proteins were detected with low counts, and changes in their expression were not observed. Complementary cell-membrane protein enrichment protocols could provide additional insights into how the cancer cells evade cytotoxic immune surveillance at a metastatic site.

Conclusions

The findings of this study provide preliminary, yet novel, insights into the early response of breast cancer cells to in vitro conditions that mimic the neural niche. The results led to the hypothesis that unfavorable environmental conditions that support cell cycle arrest, further mediated by inflammatory molecules or suboptimal concentrations of growth factors, drive the cancer cells into quiescence, and possibly dormancy, in the foreign perivascular metastatic niche. The pro-inflammatory chemokines and cytokines secreted by the brain cells have pleiotropic roles with anti-tumor activities and simultaneous functions in facilitating the recruitment of immune cells and promoting neovascularization to enhance blood vessel supply in a hypoxic TME. The pro-tumorigenic growth factors, on the other hand, can counteract the detrimental effects of the pro-inflammatory mediators and promote viability of cancer cells. Altogether, however, the SKBR3 cells secreted cancer-promoting growth factors in higher abundance than the brain cells, and displayed signs of impaired growth when subjected to the brain cell-conditioned medium. Such pilot studies can provide crucial insights into the initial response of cancer cells in a foreign niche, especially as the concept of dormancy is difficult to replicate experimentally under both in vivo and in vitro conditions. Furthermore, such exploratory results can pave the way for future research into critical questions such as how cancer cells adapt and/or mutate to nutritionally deprived conditions, and how the combination of factors in the neural niche protects and later supports the revival of dormant cells. Moreover, the detection of early metastatic markers not just from cancer but the TME will be critical to running accurate diagnostic tests, devising adequate treatment regimens with improved monitoring and better survival outcomes, and minimizing the negative impact on brain function. Finally, the extrapolation of this study to primary cells, fluids or tumors collected from brain metastatic patients can provide a stepping stone in the discovery efforts of novel drug targets and therapeutic strategies that target not just the cancer cells but also the tumor microenvironment.

Data availability

The mass spectrometry raw files were deposited to the ProteomeXchange Consortium via the PRIDE 94 partner repository with the following dataset identifier: PXD046330.

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Acknowledgements

This work was supported by an award from the National Institute of General Medical Sciences (Grant No. 1R01GM121920) to IML.

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SA performed the experiments; SA and IML analyzed the data and wrote the manuscript; IML coordinated the work. All authors reviewed and approved the final version of the manuscript.

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Ahuja, S., Lazar, I.M. Proteomic insights into breast cancer response to brain cell-secreted factors. Sci Rep 14 , 19351 (2024). https://doi.org/10.1038/s41598-024-70386-7

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Received : 22 February 2024

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DOI : https://doi.org/10.1038/s41598-024-70386-7

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