CDH1 Genotype Exploration in Women With Hereditary Lobular Breast Cancer Phenotype.

Importance: Pathogenic or likely pathogenic (P/LP) germline CDH1 variants are associated with risk for diffuse gastric cancer and lobular breast cancer (LBC) in the so-called hereditary diffuse gastric cancer (HDGC) syndrome. However, in some circumstances, LBC can be the first manifestation of this syndrome in the absence of diffuse gastric cancer manifestation. Objectives: To evaluate the frequency of germline CDH1 variants in women with the hereditary LBC (HLBC) phenotype, somatic CDH1 gene inactivation in germline CDH1 variant carriers' tumor samples, and the association of genetic profiles with clinical-pathological data and survival. Design, Setting, and Participants: This single-center, longitudinal, prospective cohort study was conducted from January 1, 1997, to December 31, 2021, with follow-up until January 31, 2023. Women with LBC seen at the European Institute of Oncology were included. Testing for germline CDH1, BRCA1, and BRCA2 genes was performed. Somatic profiling was assessed for germline CDH1 carriers. Main Outcomes and Measures: Accurate estimates of prevalence of germline CDH1 variants among patients with HLBC and the association of somatic sequence alteration with HLBC syndrome. The Kaplan-Meier method and a multivariable Cox proportional hazards regression model were applied for overall and disease-free survival analysis. Results: Of 5429 cases of primary LBC, familial LBC phenotype accounted for 1867 (34.4%). A total of 394 women with LBC were tested, among whom 15 germline CDH1 variants in 15 unrelated families were identified. Among these variants, 6 (40.0%) were P/LP, with an overall frequency of 1.5% (6 of 394). Of the 6 probands with P/LP CDH1 LBC, 5 (83.3%) had a positive family history of BC and only 1 (16.7%) had sporadic juvenile early-onset LBC. No germline BRCA1 and BRCA2 variants were identified in CDH1 carriers. An inactivating CDH1 mechanism (second hit) was identified in 4 of 6 explored matched tumor samples (66.7%) in P/LP germline carriers. The P/LP CDH1 LBC variant carriers had a significantly lower age at diagnosis compared with the group carrying CDH1 variants of unknown significance or likely benign (42.5 [IQR, 38.3-43.0] vs 51.0 [IQR, 45.0-53.0] years; P = .03). Conclusions and Relevance: In this cohort study, P/LP germline CDH1 variants were identified in individuals not fulfilling the classic clinical criteria for HDGC screening, suggesting that identification of these variants may provide a novel method to test women with LBC with early age at diagnosis and/or positive family history of BC.

PD-L1 testing in metastatic triple-negative breast cancer: Interobserver and interplatform reproducibility of CE-IVD assays for CPS and IC scores.

PD-L1 test is recommended in different types of tumors to select patients eligible for immune checkpoint inhibitors (ICI) therapy. Several factors make this test challenging in metastatic triple-negative breast cancer (mTNBC). Different assays and platforms are available, each associated with distinct scoring systems and threshold values specific to the ICI compound used, i.e. CPS≥10 for pembrolizumab and IC ≥ 1 % for atezolizumab. Our objective was to assess the consistency of PD-L1 testing in mTNBC by examining interobserver and interassay reproducibility. We assessed n = 60 mTNBC samples for PD-L1 testing using 22C3 pharmDx assay on a Dako Autostainer Link 48 and VENTANA PD-L1 (SP263) on a Ventana BenchMark Ultra. Additionally, a subset of n = 19 samples was tested using the SP142 assay, also on the Ventana BenchMark Ultra. CPS with both 22C3 and SP263 was independently evaluated by five pathologists, all certified PD-L1 trainers. The IC with SP142 was assessed by three of these pathologists, who have particular expertise in breast pathology. Following the computation of the intraclass correlation coefficient (ICC) for each assay and their respective thresholds, we assessed the agreement between different raters and assays using Fleiss's κ, with a 95 % confidence interval (CI). Overall, we observed a significant (p < 0.001) ICC with both CPS assays [22C3 = 0.939 (CI:0.913-0.96); SP263 = 0.972 (CI:0.96-0.982); combined 22C3-SP263 = 0.909 (CI:0.874-0.938)]. Fleiss's κ confirmed an almost perfect agreement among pathologists and assays: 22C3 = 0.938 (CI:0.857-1.018); SP263 = 0.972 (CI:0.890-1.052); combined 22C3-SP263 = 0.907 (CI:0.869-0.945). Perfect inter-rater agreement was reached considering IC. This study establishes the reliability of assessing CPS in mTNBC using either the 22C3 pharmDx, as employed in the KEYNOTE studies, or the VENTANA SP263 assay. Each assay must be used on its designated platform, namely the Dako for 22C3 pharmDx and the Ventana for VENTANA SP263. It is important to remark that CPS and IC identify different patient cohorts and, therefore, are not interchangeable.

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.

Clinicopathological features and survival outcomes of luminal-like breast tumors with estrogen receptor loss at metastatic recurrence: A case-control study.

INTRODUCTION: Estrogen receptor (ER) loss at metastatic relapse occurs in up to 20% of luminal-like primary breast tumors. Data about clinicopathological features associated with ER loss and its prognostic significance are limited. METHODS: In a nested-case-control study, we compared clinicopathological characteristics and clinical outcomes between a cohort of 51 patients with primary ER+ /HER2- and paired triple-negative metastasis (LUM-TN) and two control cohorts of paired early-metastatic ER+ /HER2- (LUM-LUM, n = 50) and triple-negative (TN-TN, n = 49) breast cancers. Stromal tumor-infiltrating lymphocytes (TILs) were assessed according to the TILs Working Group recommendations as continuous and discrete variables with cutoffs (20%, 40%). RESULTS: LUM-TN tumors had lower ER expression than LUM-LUM tumors, but lower grade and Ki67 than TN-TN cases. Median distant-metastasis free survival was similar for LUM-TN and LUM-LUM cohorts, but significantly longer than in TN-TN cases (log-rank P < 0.001). LUM-TN and TN-TN cohorts had a comparable survival from the time of metastatic recurrence, which was significantly shorter than in patients with LUM-LUM tumors (log-rank P < 0.001). High TILs were associated with worse outcomes in patients with ER loss (P < 0.001). CONCLUSIONS: Breast tumors with ER loss at metastatic relapse have intermediate features and outcomes compared with metastatic luminal-like and ab initio triple-negative tumors. Further investigation on the biological mechanisms underpinning the loss of ER expression is ongoing.

Immune microenvironment dynamics in breast cancer during pregnancy: impact of gestational age on tumor-infiltrating lymphocytes and prognosis.

Background: Breast cancer during pregnancy (PrBC) is a rare condition known for its aggressive clinical behavior. The presence of tumor-infiltrating lymphocytes (TILs) has been shown to have a significant impact on the prognosis of these patients. Despite some biological characteristics of the tumor that may differ depending on the gestational age, little is known about the dynamics of the immune landscape within the tumor microenvironment (TME) in PrBC. Therefore, in this study, our objective was to gain comprehensive insights into the relationship between gestational age at breast cancer diagnosis and the composition of the TME. Methods: n = 108 PrBC were selected from our institutional registry and categorized based on the gestational age by trimester. For all cases, TILs were profiled according to the International TILs Working Group recommendations, and subtyped by CD4, CD8, and forkhead box P3 (FOXP3) immunohistochemistry. PD-L1 was tested according to the combined positive score (CPS) using the IHC 22C3 pharmDx assay, with a cutoff value of ≥10 for positivity. The statistical approach encompassed Fisher's and Chi-squared tests, with appropriate adjustments for multiple comparisons, logistic regression models, and survival analyses based on the Kaplan-Meier method. Results: The proportion of patients with poorly differentiated (G3) neoplasms increased as the gestational age advanced (first trimester, n = 25, 56.8%; second trimester, n = 27, 69.2%; third trimester, n = 21, 87.5%; p = 0.03). The histologic subtypes as well as the hormone receptor (HR) and HER2 status did not show significant changes across different pregnancy trimesters. In the HR+/HER2- subtype, there was a higher proportion of tumors with high/moderate TILs in the early phases of pregnancy, similar to FOXP3 expression (TILs: first trimester, n = 10, 35.7%; second trimester, n = 2, 10.5%; third trimester, n = 0; p = 0.02; FOXP3: first trimester, n = 10, 40%; second trimester, n = 3, 15.8%; third trimester, n = 0; p = 0.03). The median follow-up for our cohort was 81 months. Patients who relapsed after a breast cancer diagnosis during the first trimester were more frequently PD-L1-negative, unlike those with no disease recurrence (n = 9, 100% vs. n = 9, 56.3%; p = 0.03; hormone therapy and n = 9, 100% vs. n = 7, 53.9%; p = 0.02; chemotherapy). No statistically significant differences were seen among the three trimesters in terms of survival outcome. Conclusion: The TME dynamics of HR+/HER2- PrBC vary based on gestational age, suggesting that immune tolerance expression during later gestational age could explain the increased aggressiveness of tumors diagnosed at that stage.

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.

Characterization of the immune environment in pregnancy-associated breast cancer.

Pregnancy-associated breast cancer (PrBC) is a rare and clinically challenging condition. Specific immune mechanisms and pathways are involved in maternal-fetal tolerance and tumor-host immunoediting. The comprehension of the molecular processes underpinning this immune synergy in PrBC is needed to improve patients' clinical management. Only a few studies focused on the immune biology of PrBC and attempted to identify bona fide biomarkers. Therefore, clinically actionable information remains extremely puzzling for these patients. In this review article, we discuss the current knowledge on the immune environment of PrBC, in comparison with pregnancy-unrelated breast cancer and in the context of maternal immune changes during pregnancy. A particular emphasis is given to the actual role of potential immune-related biomarkers for PrBC clinical management.

Pregnancy-associated breast cancer (PrBC) affects about 4% of women with breast cancer who are of childbearing age. Managing these tumors is difficult due to interactions between the mother, fetus and tumor. These interactions cause changes in the immune system of patients with PrBC. Understanding how the immune system responds to PrBC may lead to better ways of managing the disease. This review focuses on the current knowledge of the immune system in PrBC, including which components can be used as biomarkers to improve clinical management.

Immune plasticity in pregnancy-associated breast cancer tumorigenesis.

Pregnancy-associated breast cancer (PrBC) is a rare tumor that requires complex management. The coexistence of cancer and pregnancy involves several proliferative, invasive, and immune tolerance mechanisms that are shared between the two conditions. In normal pregnancy, successful fetal development is achieved through suppression of the maternal immune response toward the fetus. Similar immunosuppressive patterns during the malignant transformation supporting tumor growth, progression, and metastasis are also exhibited by tumors. An improved understanding of the immunosuppressive mechanisms and pathways underlying the immunological synergy in PrBC could lead to the identification of novel biomarkers that potentially improve patients' clinical management. In this review article, we outline some of the paramount features of immune plasticity during pregnancy, discussing the similarities shared between normal pregnancy and breast cancer in terms of immune suppression mechanisms. Emphasis is also placed on how the current knowledge of the immune milieu of these conditions may be translated into consequent therapeutic opportunities.

Computational pathology to improve biomarker testing in breast cancer: how close are we?

The recent advancements in breast cancer precision medicine have highlighted the urgency for the precise and reproducible characterization of clinically actionable biomarkers. Despite numerous standardization efforts, biomarker testing by conventional methodologies is challenged by several issues such as high inter-observer variabilities, the spatial heterogeneity of biomarkers expression, and technological heterogeneity. In this respect, artificial intelligence-based digital pathology approaches are being increasingly recognized as promising methods for biomarker testing and subsequently improved clinical management. Here, we provide an overview on the most recent advances for artificial intelligence-assisted biomarkers testing in breast cancer, with a particular focus on tumor-infiltrating lymphocytes, programmed death-ligand 1, phosphatidylinositol-3 kinase catalytic alpha, and estrogen receptor 1. Challenges and solutions for this integrative analysis in pathology laboratories are also provided.

Pathological identification of HER2-low breast cancer: Tips, tricks, and troubleshooting for the optimal test.

The introduction of novel anti-HER2 antibody-drug conjugates (ADC) for the treatment of HER2-low breast cancers has transformed the traditional dichotomy of HER2 status to an expanded spectrum. However, the identification of HER2-low (i.e., immunohistochemistry (IHC) score 1 + or IHC score 2+, without gene amplification) tumors is challenged by methodological and analytical variables that might influence the sensitivity and reproducibility of HER2 testing. To open all possible therapeutic opportunities for HER2-low breast cancer patients the implementation of more accurate and reproducible testing strategies is mandatory. Here, we provide an overview of the existing barriers that may trouble HER2-low identification in breast cancer and discuss practical solutions that could enhance HER-low assessment.

The molecular landscape of breast mucoepidermoid carcinoma.

Mucoepidermoid carcinoma (MEC) of the breast is an extremely rare salivary gland-type tumor characterized by epidermoid, basaloid, intermediate, and/or mucinous cells arranged in solid and cystic patterns. Despite their triple-negative phenotype, breast MECs are generally considered low-risk malignancies but their biology is largely unexplored; therefore, guidelines for clinical management are lacking. Here, we sought to characterize the molecular landscape of breast MECs. Thirteen cases were histologically reviewed, characterized for tumor-infiltrating lymphocytes (TILs), and were subjected to immunohistochemistry for programmed death-ligand 1 (PD-L1, clone 22C3), EGFR, and amphiregulin (AREG). Rearrangements in MAML2 and EWSR1 were investigated by fluorescent in situ hybridization. Targeted next-generation sequencing of 161 genes was performed on eight cases. Most MECs had low histological grade (n = 10, 77%), with the presence of TILs (n = 9/12; 75%) and PD-L1 combined positive score ranging from 10 to 20 (n = 4/6; 67%). All cases showed EGFR and AREG overexpression and were fusion negative. Enrichment of genetic alterations was observed in PI3K/AKT/mTOR and cell cycle regulation pathways, while only one case harbored TP53 mutations. This is the first study providing extensive molecular data on breast MECs and the largest collection of cases available to date in the literature. Breast MECs lack TP53 mutations found in high-grade forms of triple-negative breast cancers and MAML2 or EWSR1 rearrangements pathognomonic of salivary MECs. Triple-negativity and PD-L1 positivity suggest a window of opportunity for immunotherapy in these patients. The EGFR/AREG axis activation, coupled with the mutational patterns in PI3K/AKT/mTOR and cell cycle pathways warrants caution in considering MECs as low-risk neoplasms.

An Italian Multicenter Perspective Harmonization Trial for the Assessment of MET Exon 14 Skipping Mutations in Standard Reference Samples.

Lung cancer remains the leading cause of cancer deaths worldwide. International societies have promoted the molecular analysis of MET proto-oncogene, receptor tyrosine kinase (MET) exon 14 skipping for the clinical stratification of non-small cell lung cancer (NSCLC) patients. Different technical approaches are available to detect MET exon 14 skipping in routine practice. Here, the technical performance and reproducibility of testing strategies for MET exon 14 skipping carried out in various centers were evaluated. In this retrospective study, each institution received a set (n = 10) of a customized artificial formalin-fixed paraffin-embedded (FFPE) cell line (Custom METex14 skipping FFPE block) that harbored the MET exon 14 skipping mutation (Seracare Life Sciences, Milford, MA, USA), which was previously validated by the Predictive Molecular Pathology Laboratory at the University of Naples Federico II. Each participating institution managed the reference slides according to their internal routine workflow. MET exon 14 skipping was successfully detected by all participating institutions. Molecular analysis highlighted a median Cq cut off of 29.3 (ranging from 27.1 to 30.7) and 2514 (ranging from 160 to 7526) read counts for real-time polymerase chain reaction (RT-PCR) and NGS-based analyses, respectively. Artificial reference slides were a valid tool to harmonize technical workflows in the evaluation of MET exon 14 skipping molecular alterations in routine practice.

Malignancies in Patients with Celiac Disease: Diagnostic Challenges and Molecular Advances.

Celiac disease (CD) is a multiorgan autoimmune disorder of the chronic intestinal disease group characterized by duodenal inflammation in genetically predisposed individuals, precipitated by gluten ingestion. The pathogenesis of celiac disease is now widely studied, overcoming the limits of the purely autoimmune concept and explaining its hereditability. The genomic profiling of this condition has led to the discovery of numerous genes involved in interleukin signaling and immune-related pathways. The spectrum of disease manifestations is not limited to the gastrointestinal tract, and a significant number of studies have considered the possible association between CD and neoplasms. Patients with CD are found to be at increased risk of developing malignancies, with a particular predisposition of certain types of intestinal cancer, lymphomas, and oropharyngeal cancers. This can be partially explained by common cancer hallmarks present in these patients. The study of gut microbiota, microRNAs, and DNA methylation is evolving to find the any possible missing links between CD and cancer incidence in these patients. However, the literature is extremely mixed and, therefore, our understanding of the biological interplay between CD and cancer remains limited, with significant implications in terms of clinical management and screening protocols. In this review article, we seek to provide a comprehensive overview of the genomics, epigenomics, and transcriptomics data on CD and its relation to the most frequent types of neoplasms that may occur in these patients.

Biobank for Translational Medicine: Standard Operating Procedures for Optimal Sample Management.

Biobanks are key research infrastructures aimed at the collection, storage, processing, and sharing of high-quality human biological samples and associated data for research, diagnosis, and personalized medicine. The Biobank for Translational and Digital Medicine Unit at the European Institute of Oncology (IEO) is a landmark in this field. Biobanks collaborate with clinical divisions, internal and external research groups, and industry, supporting patients' treatment and scientific progress, including innovative diagnostics, biomarker discovery, and clinical trial design. Given the central role of biobanks in modern research, biobanking standard operating procedures (SOPs) should be extremely precise. SOPs and controls by certified specialists ensure the highest quality of samples for the implementation of science-based, diagnostic, prognostic, and therapeutic personalized strategies. However, despite numerous efforts to standardize and harmonize biobanks, these protocols, which follow a strict set of rules, quality controls, and guidelines based on ethical and legal principles, are not easily accessible. This paper presents the biobank standard operating procedures of a large cancer center.

Analytical Performance of Next-Generation Sequencing and RT-PCR on Formalin-Fixed Paraffin-Embedded Tumor Tissues for PIK3CA Testing in HR+/HER2- Breast Cancer.

Somatic mutations in PIK3CA are present in ~40% breast cancers (BC); their detection in hormone receptor (HR)+/HER2- tumors allows for selecting patients with advanced disease eligible for PIK3CA targeting with alpelisib. The choice of what type of PIK3CA testing approach to adopt and which tissue sample to analyze is a new task in breast pathology. In this methodological study, we sought to assess the performance of next-generation sequencing (NGS) and RT-PCR for PIK3CA testing on archival formalin-fixed paraffin-embedded (FFPE) primary tumors and corresponding metastases. Sixteen HR+/HER2- BC with known PIK3CA-mutated status (ex. 7, 9, and 20) on metastatic samples by means of amplicon-based targeted NGS were selected, and n = 13 of these samples were re-tested with a commercially available CE-IVD RT-PCR assay. All available primary tumors (n = 8) were tested with both methods. NGS detected mutations in all samples, while RT-PCR in n = 2 sample-pairs and overall, in n = 5/8 (62.5%) primary tumors and 7/13 (53.8%) metastases (κ = 0.09; 95% CI, -0.69-0.87). Slight agreement (κ = 0; 95% CI, -0.59-0.59) was observed between NGS and RT-PCR, with the former being generally more sensitive in cases with low DNA quality and quantity. Post hoc visual inspection of the RT-PCR data increased the concordance to 76.9%. Targeted NGS offers reliable and robust PIK3CA testing on both tumor and metastasis FFPE samples; the accuracy of RT-PCR depends on the DNA quantity and quality. In HR+/HER2- BC, both the selection of the PIK3CA testing strategy of FFPE tissues and which sample to analyze should consider several technical parameters and should be tailored for each case.

Osteoclast-like stromal giant cells in breast cancer likely belong to the spectrum of immunosuppressive tumor-associated macrophages.

Background: Breast cancer with osteoclast-like stromal giant cells (OSGC) is an exceedingly rare morphological pattern of invasive breast carcinoma. The tumor immune microenvironment (TIME) of these tumors is populated by OSGC, which resemble osteoclasts and show a histiocytic-like immunophenotype. Their role in breast cancer is unknown. The osteoclast maturation in the bone is regulated by the expression of cytokines that are also present in the TIME of tumors and in breast cancer tumor-associated macrophages (TAMs). TAMs-mediated anti-tumor immune pathways are regulated by miRNAs akin to osteoclast homeostasis. Here, we sought to characterize the different cellular compartments of breast cancers with OSGC and investigate the similarities of OSGC with tumor and TIME in terms of morphology, protein, and miRNA expression, specifically emphasizing on monocytic signatures. Methods and Results: Six breast cancers with OSGC were included. Tumor-infiltrating lymphocytes (TILs) and TAMs were separately quantified. The different cellular populations (i.e., normal epithelium, cancer cells, and OSGC) were isolated from tissue sections by laser-assisted microdissection. After RNA purification, 752 miRNAs were analyzed using a TaqMan Advanced miRNA Low-Density Array for all samples. Differentially expressed miRNAs were identified by computing the fold change (log2Ratio) using the Kolmogorov-Smirnov test and p values were corrected for multiple comparisons using the false discovery rate (FDR) approach. As a similarity analysis among samples, we used the Pearson test. The association between pairs of variables was investigated using Fisher exact test. Classical and non-classical monocyte miRNA signatures were finally applied. All OSGC displayed CD68 expression, TILs (range, 45-85%) and high TAMs (range, 35-75%). Regarding the global miRNAs profile, OSGC was more similar to cancer cells than to non-neoplastic ones. Shared deregulation of miR-143-3p, miR-195-5p, miR-181a-5p, and miR-181b-5p was observed between OSGC and cancer cells. The monocyte-associated miR-29a-3p and miR-21-3p were dysregulated in OSGCs compared with non-neoplastic or breast cancer tissues. Conclusion: Breast cancers with OSGC have an activated TIME. Shared epigenetic events occur during the ontogenesis of breast cancer cells and OSGC but the innumophenotype and miRNA profiles of the different cellular compartmens suggest that OSGC likely belong to the spectrum of M2 TAMs.