Standardized molecular pathology workflow for ctDNA-based ESR1 testing in HR+/HER2- metastatic breast cancer.

Mutations in the estrogen receptor alpha gene (ESR1) can lead to resistance to endocrine therapy (ET) in hormone receptor-positive (HR+)/ HER2- metastatic breast cancer (MBC). ESR1 mutations can be detected in up to 40 % of patients pretreated with ET in circulating tumor DNA (ctDNA). Data from prospective randomized trials highlight those patients with HR+/HER2- MBC with detectable ESR1 mutations experience better outcomes when receiving novel selective estrogen receptor degraders (SERDs). There is a high need for optimizing ESR1 testing strategies on liquid biopsy samples in HR+/HER2- MBC, including a hugh quality workflow implementation and molecular pathology reporting standardization. Our manuscript aims to elucidate the clinical and biological rationale for ESR1 testing in MBC, while critically examining the currently available guidelines and recommendations for this specific type of molecular testing on ctDNA. The objective will extend to the critical aspects of harmonization and standardization, specifically focusing on the pathology laboratory workflow. Finally, we propose a clear and comprehensive model for reporting ESR1 testing results on ctDNA in HR+/HER2- MBC.

The Evolving Role of Genomic Testing in Early Breast Cancer: Implications for Diagnosis, Prognosis, and Therapy.

Multigene prognostic genomic assays have become indispensable in managing early breast cancer (EBC), offering crucial information for risk stratification and guiding adjuvant treatment strategies in conjunction with traditional clinicopathological parameters. The American Society of Clinical Oncology (ASCO) guidelines endorse these assays, though some clinical contexts still lack definitive recommendations. The dynamic landscape of EBC management demands further refinement and optimization of genomic assays to streamline their incorporation into clinical practice. The breast cancer community is poised at the brink of transformative advances in enhancing the clinical utility of genomic assays, aiming to significantly improve the precision and effectiveness of both diagnosis and treatment for women with EBC. This article methodically examines the testing methodologies, clinical validity and utility, costs, diagnostic frameworks, and methodologies of the established genomic tests, including the Oncotype Dx Breast Recurrence Score®, MammaPrint, Prosigna®, EndoPredict®, and Breast Cancer Index (BCI). Among these tests, Prosigna and EndoPredict® have at present been validated only on a prognostic level, while Oncotype Dx, MammaPrint, and BCI hold both a prognostic and predictive role. Oncologists and pathologists engaged in the management of EBC will find in this review a thorough comparison of available genomic assays, as well as strategies to optimize the utilization of the information derived from them.

Early Breast Cancer Risk Assessment: Integrating Histopathology with Artificial Intelligence.

Effective risk assessment in early breast cancer is essential for informed clinical decision-making, yet consensus on defining risk categories remains challenging. This paper explores evolving approaches in risk stratification, encompassing histopathological, immunohistochemical, and molecular biomarkers alongside cutting-edge artificial intelligence (AI) techniques. Leveraging machine learning, deep learning, and convolutional neural networks, AI is reshaping predictive algorithms for recurrence risk, thereby revolutionizing diagnostic accuracy and treatment planning. Beyond detection, AI applications extend to histological subtyping, grading, lymph node assessment, and molecular feature identification, fostering personalized therapy decisions. With rising cancer rates, it is crucial to implement AI to accelerate breakthroughs in clinical practice, benefiting both patients and healthcare providers. However, it is important to recognize that while AI offers powerful automation and analysis tools, it lacks the nuanced understanding, clinical context, and ethical considerations inherent to human pathologists in patient care. Hence, the successful integration of AI into clinical practice demands collaborative efforts between medical experts and computational pathologists to optimize patient outcomes.

PIK3CA testing in hormone receptor-positive/HER2-negative metastatic breast cancer: real-world data from Italian molecular pathology laboratories.

Introduction: PIK3CA gene mutations occur in approximately 40% of hormone receptor-positive/HER2-negative (HR+/HER2-) metastatic breast cancers (MBCs), electing them to targeted therapy. Testing PIK3CA status is complex due to selection of biological specimen and testing method. Materials & methods: This work investigates real-life experience on PIK3CA testing in HR+/HER2- MBC. Clinical, technical and molecular data on PIK3CA testing were collected from two referral laboratories. Additionally, the results of a nationwide PIK3CA survey involving 116 institutions were assessed. Results: Overall, n = 35 MBCs were PIK3CA-mutated, with mutations mostly occurring in exons 9 (n = 19; 51.4%) and 20 (n = 15; 40.5%). The nationwide survey revealed significant variability across laboratories in terms of sampling methodology, technical assessment and clinical report signing healthcare figures for PIK3CA molecular testing in diagnostic routine practice. Conclusion: This study provides insights into the real-world routine of PIK3CA testing in HR+/HER2- MBC and highlights the need for standardization and networking in predictive pathology.

Standardized pathology report for HER2 testing in compliance with 2023 ASCO/CAP updates and 2023 ESMO consensus statements on HER2-low breast cancer.

Since the release of the DESTINY-Breast04 (DB-04) trial findings in June 2022, the field of pathology has seen a renaissance of HER2 as a predictive biomarker in breast cancer. The trial focused on patients with metastatic breast cancer who were classified as "HER2-low," i.e., those with immunohistochemistry (IHC) HER2 1 + or 2 + and negative in situ hybridization (ISH) results. The study revealed that treating these patients with trastuzumab deruxtecan (T-DXd) instead of the oncologist's chosen chemotherapy led to outstanding improvements in survival. This has challenged the existing binary HER2 pathological classification system, which categorized tumors as either positive (overexpression/amplification) or negative, as per the ASCO/CAP 2018 guideline reaffirmed by ASCO/CAP 2023 guideline update. Given that DB-04 excluded patients with HER2 IHC score 0 status, the results of the ongoing DB-06 trial may shed further light on the potential benefits of T-DXd therapy for these patients. Roughly half of all breast cancers are estimated to belong to the HER2-low category, which does not represent a distinct or specific subtype of cancer. Instead, it encompasses a diverse group of tumors that exhibit clinical, morphological, immunohistochemical, and molecular variations. However, HER2-low offers a distinctive biomarker status that identifies a specific therapeutic regimen (i.e., T-DXd) linked to a favorable prognosis in breast cancer. This unique association emphasizes the importance of accurately identifying these tumors. Differentiating between a HER2 IHC score 0 and score 1 + has not been clinically significant until now. To ensure accurate classification and avoid misdiagnosis, it is necessary to adopt standardized procedures, guidelines, and specialized training for pathologists in interpreting HER2 expression in the lower spectrum. Additionally, the utilization of artificial intelligence holds promise in supporting this endeavor. Here, we address the current state of the art and unresolved issues in assessing HER2-low status, with a particular emphasis on the score 0. We explore the dilemma surrounding the exclusion of HER2-zero patients from potentially beneficial therapy based on traditional HER2 testing. Additionally, we examine the clinical context, considering that DB-04 primarily involved heavily pretreated late-stage metastatic breast cancers. We also delve into emerging evidence suggesting that extrapolating HER2-low status from the original diagnosis may lead to misleading results. Finally, we provide recommendations for conducting high-quality testing and propose a standardized pathology report in compliance with 2023 ASCO/CAP updates and 2023 ESMO consensus statements on HER2-low breast cancer.

ESR1 mutations in HR+/HER2-metastatic breast cancer: Enhancing the accuracy of ctDNA testing.

Activating mutations of the estrogen receptor alpha gene (ESR1) are common mechanisms of endocrine therapy (ET) resistance in hormone receptor-positive (HR + )/Human Epidermal Growth Factor Receptor 2 (HER2)-negative metastatic breast cancer (MBC). Recent clinical findings emphasize that both old and new generations of selective ER degraders (SERDs) demonstrate enhanced clinical effectiveness in patients with MBC who have detectable ESR1 mutations via liquid biopsy. This stands in contrast to individuals with MBC carrying these mutations and undergoing conventional endocrine monotherapies like aromatase inhibitors (AIs). Liquid biopsy, particularly the analysis of circulating tumor DNA (ctDNA), has emerged as a promising, minimally invasive alternative to conventional tissue-based testing for identifying ESR1 mutations. Within the context of the PADA-1 and EMERALD trials, distinct molecular methodologies and assays, specifically digital droplet PCR (ddPCR) and next-generation sequencing (NGS), have been employed to evaluate the mutational status of ESR1 within ctDNA. This manuscript critically examines the advantages and indications of various ctDNA testing methods on liquid biopsy for HR+/HER2-negative MBC. Specifically, we delve into the capabilities of ddPCR and NGS in identifying ESR1 mutations. Each methodology boasts unique strengths and limitations: ddPCR excels in its analytical sensitivity for pinpointing hotspot mutations, while NGS offers comprehensive coverage of the spectrum of ESR1 mutations. The significance of meticulous sample handling and timely analysis is emphasized, acknowledging the transient nature of cfDNA. Furthermore, we underscore the importance of detecting sub-clonal ESR1 mutations, as these variants can exert a pivotal influence on predicting both endocrine therapy resistance and responsiveness to SERDs. In essence, this work discusses the role of ctDNA analysis for detecting ESR1 mutations and their implications in tailoring effective therapeutic strategies for HR+/HER2- MBC.

Circulating tumour DNA testing in metastatic breast cancer: Integration with tissue testing.

Breast cancer biomarker profiling predominantly relies on tissue testing (surgical and/or biopsy samples). However, the field of liquid biopsy, particularly the analysis of circulating tumour DNA (ctDNA), has witnessed remarkable progress and continues to evolve rapidly. The incorporation of ctDNA-based testing into clinical practice is creating new opportunities for patients with metastatic breast cancer (MBC). ctDNA offers advantages over conventional tissue analyses, as it reflects tumour heterogeneity and enables multiple serial biopsies in a minimally invasive manner. Thus, it serves as a valuable complement to standard tumour tissues and, in certain instances, may even present a potential alternative approach. In the context of MBC, ctDNA testing proves highly informative in the detection of disease progression, monitoring treatment response, assessing actionable biomarkers, and identifying mechanisms of resistance. Nevertheless, ctDNA does exhibit inherent limitations, including its generally low abundance, necessitating timely blood samplings and rigorous management of the pre-analytical phase. The development of highly sensitive assays and robust bioinformatic tools has paved the way for reliable ctDNA analyses. The time has now come to establish how ctDNA and tissue analyses can be effectively integrated into the diagnostic workflow of MBC to provide patients with the most comprehensive and accurate profiling. In this manuscript, we comprehensively analyse the current methodologies employed in ctDNA analysis and explore the potential benefits arising from the integration of tissue and ctDNA testing for patients diagnosed with MBC.

Advancing the PD-L1 CPS test in metastatic TNBC: Insights from pathologists and findings from a nationwide survey.

Pembrolizumab has received approval as a first-line treatment for unresectable/metastatic triple-negative breast cancer (mTNBC) with a PD-L1 combined positive score (CPS) of ≥ 10. However, assessing CPS in mTNBC poses challenges. Firstly, it represents a novel analysis for breast pathologists. Secondly, the heterogeneity of PD-L1 expression in mTNBC further complicates the assessment. Lastly, the lack of standardized assays and staining platforms adds to the complexity. In KEYNOTE trials, PD-L1 expression was evaluated using the IHC 22C3 pharmDx kit as a companion diagnostic test. However, both the 22C3 pharmDx and VENTANA PD-L1 (SP263) assays are validated for CPS assessment. Consequently, assay-platform choice, staining conditions, and scoring methods can significantly impact the testing outcomes. This consensus paper aims to discuss the intricacies of PD-L1 CPS testing in mTNBC and provide practical recommendations for pathologists. Additionally, we present findings from a nationwide Italian survey elucidating the state-of-the-art in PD-L1 CPS testing in mTNBC.

Immune Biomarkers in Triple-Negative Breast Cancer: Improving the Predictivity of Current Testing Methods.

Triple-negative breast cancer (TNBC) poses a significant challenge in terms of prognosis and disease recurrence. The limited treatment options and the development of resistance to chemotherapy make it particularly difficult to manage these patients. However, recent research has been shifting its focus towards biomarker-based approaches for TNBC, with a particular emphasis on the tumor immune landscape. Immune biomarkers in TNBC are now a subject of great interest due to the presence of tumor-infiltrating lymphocytes (TILs) in these tumors. This characteristic often coincides with the presence of PD-L1 expression on both neoplastic cells and immune cells within the tumor microenvironment. Furthermore, a subset of TNBC harbor mismatch repair deficient (dMMR) TNBC, which is frequently accompanied by microsatellite instability (MSI). All of these immune biomarkers hold actionable potential for guiding patient selection in immunotherapy. To fully capitalize on these opportunities, the identification of additional or complementary biomarkers and the implementation of highly customized testing strategies are of paramount importance in TNBC. In this regard, this article aims to provide an overview of the current state of the art in immune-related biomarkers for TNBC. Specifically, it focuses on the various testing methodologies available and sheds light on the immediate future perspectives for patient selection. By delving into the advancements made in understanding the immune landscape of TNBC, this study aims to contribute to the growing body of knowledge in the field. The ultimate goal is to pave the way for the development of more personalized testing strategies, ultimately improving outcomes for TNBC patients.

Breast Cancer with Brain Metastasis: Molecular Insights and Clinical Management.

Breast cancer is the most frequently diagnosed malignancy worldwide and the leading cause of cancer-related death among women. Brain metastases are a primary contributor to mortality, as they often go undetected until late stages due to their dormant nature. Moreover, the clinical management of brain metastases is complicated by the relevant issue of blood-brain barrier penetration. The molecular pathways involved in the formation, progression, and colonization of primary breast tumors and subsequent brain metastases are diverse, posing significant hurdles due to the heterogeneous nature of breast cancer subtypes. Despite advancements in primary breast cancer treatments, the prognosis for patients with brain metastases remains poor. In this review, we aim to highlight the biological mechanisms of breast cancer brain metastases by evaluating multi-step genetic pathways and to discuss currently available and emerging treatment strategies to propose a prospective overview of the management of this complex disease.

Think "HER2" different: integrative diagnostic approaches for HER2-low breast cancer.

This work explores the complex field of HER2 testing in the HER2-low breast cancer era, with a focus on methodological aspects. We aim to propose clear positions to scientific societies, institutions, pathologists, and oncologists to guide and shape the appropriate diagnostic strategies for HER2-low breast cancer. The fundamental question at hand is whether the necessary tools to effectively translate our knowledge about HER2 into practical diagnostic schemes for the lower spectrum of expression are available. Our investigation is centered on the significance of distinguishing between an immunohistochemistry (IHC) score 0 and score 1+ in light of the clinical implications now apparent, as patients with HER2-low breast cancer become eligible for trastuzumab-deruxtecan treatment. Furthermore, we discuss the definition of HER2-low beyond its conventional boundaries and assess the reliability of established diagnostic procedures designed at a time when therapeutic perspectives were non-existent for these cases. In this regard, we examine potential complementary technologies, such as gene expression analysis and liquid biopsy. Ultimately, we consider the potential role of artificial intelligence (AI) in the field of digital pathology and its integration into HER2 testing, with a particular emphasis on its application in the context of HER2-low breast cancer.