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E-cadherin protein (CDH1 gene) integrity is fundamental to the process of epithelial polarization and differentiation. Deregulation of the E-cadherin function plays a crucial role in breast cancer metastases, with worse prognosis and shorter overall survival. In this narrative review, we describe the inactivating mechanisms underlying CDH1 gene activity and its possible translation to clinical practice as a prognostic biomarker and as a potential targeted therapy. This manuscript was supported by a grant from the Italy Ministry of Health (Project Code GR-2016-02361655), in part by the Ricerca Corrente and 5x1000 funds, and in part by the Fondazione IEO-CCM.

Biobanks are biorepositories that collect, process, store, catalog, and distribute human biological samples, and record the associated data. The role and action field of these strategic infrastructures for implementing precision medicine in translational research is continuously evolving. To ensure the optimal quality at all stages of biobanking, specific protocols are required and should be elaborated according to updated guidelines, recommendations, laws, and rules. This article illustrates the standard operating procedures, including protocols, troubleshooting, and quality controls, of a fully certified biobank in a referral Cancer Center. This model involves all clinical departments and research groups to support the dual mission of academic cancer centers, i.e. to provide high-quality care and high-quality research. All biobanking activities based on the type of biological specimens are detailed and the most tricky methodological aspects are discussed, from patients’ informed consent to specimen management.

Considering the rapid improvement of cancer drugs’ efficacy and the discovery of new molecular targets, the formulation of therapeutical indications based on the multidisciplinary approach of MTB is becoming increasingly important for attributing the correct salience to the targets identified in a single patient. Nevertheless, one of the biggest stumbling blocks faced by MTBs is not the bare indication, but its implementation in the clinical practice. Indeed, administering the drug suggested by MTB deals with some relevant difficulties: the economical affordability and geographical accessibility represent some of the major limits in the patient’s view, while bureaucracy and regulatory procedures are often a disincentive for the physicians. In this review, we explore the current literature reporting MTB experiences and precision medicine clinical trials, focusing on the challenges that authors face in applying their therapeutical indications. Furthermore, we analyze and discuss some of the solutions devised to overcome these difficulties to support the MTBs in finding the most suitable solution for their specific situation. In conclusion, we strongly encourage regulatory agencies and pharmaceutical companies to develop effective strategies with medical centers implementing MTBs to facilitate access to innovative drugs and thereby allow broader therapeutical opportunities to patients.

Breast cancer during pregnancy (PrBC) is a rare tumor with only a little information on its immune landscape. Here, we sought to characterize the cellular composition of the tumor microenvironment (TME) of PrBC and identify its differences from early-onset breast cancer (EOBC) in non-pregnant women. A total of 83 PrBC and 89 EOBC were selected from our Institutional registry and subjected to tumor-infiltrating lymphocytes (TILs) profiling and immunohistochemistry for CD4, CD8, forkhead box P3 (FOXP3), and programmed death-ligand 1 (PD-L1) (clone 22C3). A significantly lower frequency of hormone receptor (HR)-positive tumors was observed in PrBC. The prevalence of low/null PD-L1 and CD8+TILs was higher in PrBC than in the controls, specifically in HR+/HER2– breast cancers. PrBC had a significantly higher risk of relapse and disease-related death, compared to EOBC. The presence of TILs and each TIL subpopulation were significantly associated with disease relapse. Moreover, the death rate was higher in PrBC with CD8+ TILs. The TME of PrBC is characterized by specific patterns of TIL subpopulations with significant biological and prognostic roles. Routine assessment of TILs and TILs subtyping in these patients would be a valid addition to the pathology report that might help identify clinically relevant subsets of women with PrBC.

The establishment of PARP inhibitors in the treatment of epithelial ovarian carcinoma (EOC) has prompt BRCA assessment at the time of diagnosis. We described our five years of experience of tumor BRCA testing, as part of a multidisciplinary workflow for the management of EOC patients. We used a BRCA next-generation sequencing (NGS) test for profiling formalin-fixed, paraffin-embedded (FFPE) EOCs of 762 consecutive patients, with a success rate of 99.7% and a median turnaround time of 12 days. We found 178 (23.4%) cases with pathogenic/likely pathogenic (P/LP) mutations, 74 (9.7%) cases with variants of uncertain significance and 508 (66.8%) wild type tumors. Among 174 patients without P/LP mutations and investigated with multiple-ligation probe-amplification analysis on peripheral blood, two (1.1%) were positive for large rearrangements. Patients with P/LP alterations and/or with positive family history were referred to genetic counselling. Comparing tumor and blood NGS test results of 256 patients, we obtained a tumor test negative predictive value of 100% and we defined 76% of P/LP alterations as germline and 24% as somatic variants. The proposed workflow may successfully identify EOC patients with BRCA1/2 alteration, guiding both therapeutic and risk assessment clinical decisions.

Immune Checkpoint Inhibitors (ICIs) have remarkably modified the way solid tumors are managed, including breast cancer. Unfortunately, only a relatively small number of breast cancer patients significantly respond to these treatments. To maximize the immunotherapy benefit in breast cancer, several efforts are currently being put forward for the identification of i) the best therapeutic strategy (i.e. ICI monotherapy or in association with chemotherapy, radiotherapy, or other drugs); ii) optimal timing for administration (e.g. early/advanced stage of disease; adjuvant/ neoadjuvant setting); iii) most effective and reliable predictive biomarkers of response (e.g. tumor-infiltrating lymphocytes, programmed death-ligand 1, microsatellite instability associated with mismatch repair deficiency, and tumor mutational burden). In this article, we review the impacts and gaps in the characterization of immune-related biomarkers raised by clinical and translational research studies with immunotherapy treatments. Particular emphasis has been put on the documented evidence of significant clinical benefits of ICI in different randomized clinical trials, along with preanalytical and analytical issues in predictive biomarkers pathological assessment.

ObjectiveTo compare the risk class attribution withmolecular classification unknownto those withmolecular classification known,according to the European Society of Gynaecological Oncology/European Society for Radiotherapy and Oncology/European Society of Pathology (ESGO/ESTRO/ESP) 2020 guidelines on endometrial cancer, with a focus on risk group migration. Additionally, to evaluate the capability of a novel molecular analysis algorithm to reduce the number of required tests.MethodsWe conducted a retrospective study including all consecutive patients with endometrial cancer undergoing surgery and comprehensive molecular analyses between April 2019 and December 2021. Molecular analyses including immunohistochemistry for p53 and mismatch repair (MMR) proteins, and DNA sequencing for POLE exonuclease domain were performed to classify tumors as POLE-mutated (POLE), MMR-deficient (MMR-d), p53 abnormal (p53abn), or non-specific molecular profile (NSMP). The two risk classifications of the ESGO/ESTRO/ESP 2020 guidelines were compared to estimate the proportion of patients in which the molecular analysis was able to change the risk class attribution. We developed a novel algorithm where the molecular analyses are reserved only for patients in whom incorporation of the molecular classification could change the risk class attribution.ResultsA total of 278 patients were included. Molecular analyses were successful for all cases, identifying the four subgroups: 27 (9.7%) POLE, 77 (27.7%) MMR-d, 49 (17.6%) p53abn, and 125 (45.0%) NSMP. Comparison of risk class attribution between the two classification systems demonstrated discordance in the risk class assignment in 19 (6.8%, 95% CI 4.2% to 10.5%) cases. The application of our novel algorithm would have led to a reduction in the number of POLE sequencing tests by 67% (95% CI 61% to 73%) and a decrease of p53 immunohistochemistry by 27% (95% CI 22% to 33%), as compared with the application of molecular classification to all patients.ConclusionMolecular categorization of endometrial cancer allows the reallocation of a considerable proportion of patients in a different risk class. Furthermore, the application of our algorithm enables a reduction in the number of required tests without affecting the risk classification.

Introduction: Based on the SOLAR-1 study, the PI3Kα-specific inhibitor alpelisib has been approved in combination with fulvestrant for postmenopausal women, and men, with HR+/HER2-, PIK3CA-mutated, advanced or metastatic breast cancer (BC). Despite PIK3CA mutations occurring in ⁓40% of HR+/HER2- BC, PIK3CA molecular testing is a new task to be carried out in clinical practice. Adopting the most appropriate testing strategy (RT-PCR vs. NGS) on the most appropriate biological sample is of capital significance in oncologic pathology. Here, we sought to assess the concordance rate for PIK3CA molecular analysis between different technical platforms in both metastatic and matched primary tumors. Methods: From our Institutional registry, n=16 HR+/HER2- metastatic BC, which were found to harbor PIK3CA mutations via next-generation sequencing (NGS) assays [custom panel and Oncomine Comprehensive Assay (OCA) v3 (Thermo Fisher Scientific, Waltham, MA, USA)], were selected. In half of the cases (n=8), matched primary tumors were available and were subjected to PIK3CA testing with NGS. The analytical performance between NGS and a semi-closed RT-PCR (EasyPGX®, Diatech Pharmacogenetics, Italy) was assessed in 13/16 (81.3%) metastatic samples for which archival slides and blocks and/or residual extracted DNA were available, and all the primary tumors. Results: Overall, upfront testing of PIK3CA mutational status with NGS detected genetic alterations in exons 7, 9, and 20. A concordance rate of 100.0% was observed between primary and metastatic tumors. On the other hand, the analysis of primary tumors (n=8) and 13/16 (81.3%) metastatic samples with RT-PCR revealed a concordance of 42.9%. Analytical performance comparison showed that the two technologies were concordant in 7/13 metastatic cases (53.8%). In two of the discordant cases (15.4%), RT-PCR did not identify the PIK3CA mutations due to their absence from its reference range. Interestingly, visual inspection of the RT-PCR raw data increased the concordance to 76.9%. In primary tumors, consensus between the two testing methods was observed in 5 (62.5%) cases. Discussion: The concordance rate analysis shows that upfront PIK3CA molecular testing with NGS appears to be more efficacious compared with RT-PCR. Our data suggest that primary tissues reflect the PIK3CA mutational status when tested with NGS. RT-PCR is simpler with a shorter turnaround time, and less expensive than NGS approaches, however, trained personnel are required for accurate results interpretation. In addition, the limited reference range of this testing method should be taken into account for potential false negative results. Conclusion: In terms of PIK3CA molecular testing, NGS should be the preferred method in comparison with RT-PCR. Primary tumors represent a valid biospecimen to be used for this analysis in the absence of the metastatic sample. Citation Format: Konstantinos Venetis, Francesco Pepe, Elham Sajjadi, Giuseppina Bonizzi, Mariia Ivanova, Davide Vacirca, Alessandra Rappa, Massimo Barberis, Giuseppe Viale, Elena Guerini-Rocco, Elisabetta Munzone, Umberto Malapelle, Nicola Fusco. Comparison of next-generation sequencing and real-time PCR for PIK3CA testing in hormone receptor-positive/HER2-negative breast cancer on metastatic and matched primary tumor samples [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-04-14.

Pathologic evaluation of early breast cancer after neoadjuvant therapy is essential to provide prognostic information based on tumor response to treatment (pathologic complete response [pCR] or non-pCR) and to inform therapy decisions after surgery. To harmonize the pathologist’s handling of surgical specimens after neoadjuvant therapy, a panel of experts in breast cancer convened to developed a consensus on six main topics: (1) definition of pCR, (2) required clinical information, (3) gross examination and sampling, (4) microscopic examination, (5) evaluation of lymph node status, and (6) staging of residual breast tumor. The resulting consensus statements reported in this document highlight the role of an accurate evaluation of tumor response and define the minimum requirements to standardize the assessment of breast cancer specimens after neoadjuvant therapy.

HER2 status in breast cancer is assessed to select patients eligible for targeted therapy with anti-HER2 therapies. According to the American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP), the HER2 test positivity is defined by protein overexpression (score 3+) at immunohistochemistry (IHC) and/or gene amplification at

Introduction: Breast cancer (BC) occurring during gestation or lactation is rare, yet highly challenging under both biological and clinical standpoints. This condition, referred to as pregnancy-associated (PA) BC (PABC), shows enrichment in mismatch repair (MMR) deficiency mutational signature, higher expression of immune-checkpoint genes, and less tumor-infiltrating lymphocytes (TILs) compared to non-PA BCs. Despite these insights, no comprehensive data on MMR protein status, immune checkpoints, and immune microenvironment are currently available for these tumors. The aim of this study was to characterize the MMR status and immunologic milieu of PABC. Methods: Among a multi-Institutional database comprising 142 PABCs, we conducted a comparative analysis of a cohort of PABC (n=29) and a control group of age-matched non-PA BCs (n=74). Distinct areas of each tumor and the corresponding normal breast tissue were incorporated into a tissue microarray (4-6 cores per case, mean 4.5). For all cases, both stromal and intratumoral TILs were quantified according to the International TILs Working Group recommendations. Representative slides were subjected to immunohistochemical (IHC) analysis of the MMR proteins (i.e. MLH1, MSH2, MSH6, and PMS2), programmed death-ligand 1 (PD-L1), CD4, and CD8. Cases were classified as MMR-proficient (pMMR), MMR-deficient (dMMR), and, when the protein was expressed only in a part of the tumor, MMR-heterogeneous (hMMR). PD-L1 expression was evaluated separately in the tumor cells, stromal TILs, and intratumoral TILs. Finally, the relative proportion of CD4+ and CD8+ cells was assessed in both stromal and intratumoral TILs. Results: The study group included 4 (14%) Luminal A, 10 (35%) Luminal B, 4 (14%) non-luminal HER2+, and 11 (37%) triple-negative (TN) PABCs. Taken together, both the dMMR and hMMR status were more common in PABCs than in non-PA BC (n=3/29, 19% vs. n=6/74, 8% and n=7/29, 24% vs. 14/74, 19%, respectively). Specifically, dMMR was seen in Luminal A (n=2, 50%) and in TN (n=1, 9%) PABCs. Conversely, in non-PA BCs, all Luminal A (n=15, 100%) were pMMR, while 5/45(11%) Luminal B and 1/13 (7%) TNBC were dMMR. Despite no differences were observed in intratumoral TILs, PABCs showed significantly higher levels of stromal TILs (p=0.01). Compared to the control group, PD-L1 expression in PABCs was significantly higher in the tumor cells and in stromal TILs (p Discussion: This study is the first to investigate the immune response alongside the MMR status by IHC in PABCs. Our findings broaden the understanding of the immunobiology underpinning PABC, suggesting that in these tumors i) MMR protein alterations occur at higher frequency than in non-PA BCs; ii) the tumor cells and tumor microenvironment may be capable to suppress the adaptive arm of immune system through the expression of PD-L1; and iii) lymphocytes located at the periphery rather than those inside of the tumor are likely to be implicated in the immune modulation. Conclusion: The MMR system and immune microenvironment may play a consistent role in the natural history of PABCs. An intimate knowledge of the multifaceted interplay between tumor and tumor immune microenvironment is likely to unveil clinically relevant mechanisms that may have a positive net health impact for women with BC during gestation or lactation. Citation Format: Nicola Fusco, Elena Guerini-Rocco, Barbara Buonomo, Roberto Croci, Caterina Fumagalli, Gianluca Lopez, Giorgio A Croci, Letterio Runza, Elham Sajjadi, Concetta Blundo, Luca Despini, Massimo Giroda, Viviana E Galimberti, Paolo Veronesi, Massimo Barberis, Stefano Ferrero, Giovanna Scarfone, Silvano Bosari, Giuseppe Viale, Fedro Peccatori. Integrated analysis of mismatch repair, PD-L1, and immune microenvironment status in pregnancy-associated breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-05-03.

BACKGROUND/AIM The role of anti-PD1/PD-L1 therapy (IO) in NSCLC harboring driver mutations is questionable. This study aimed to examine the efficacy of IO in patients with non-small cell lung cancer (NSCLC) with a KRAS mutation (KRAS+). PATIENTS AND METHODS We retrospectively identified NSCLC patients harboring KRAS mutation treated with IO in our Institution. We analyzed the results in comparison to non-KRAS patients. RESULTS Among 328 consecutive KRAS+ NSCLC patients, 43 (13.1%) received IO in our Institution. In parallel 117 non-KRAS NSCLC patients treated with IO were selected for comparison. The baseline characteristics were similar between the two groups. No significant difference was observed between KRAS+ and non-KRAS patients in terms of mPFS (4.6 vs. 3.3 months, p=0.58) or OS (8.1 vs. 13.0 months, p=0.38). CONCLUSION KRAS mutations seem to be irrelevant for selecting patients for IO that could be therefore considered an effective therapy for NSCLC patients, independently of KRAS status.

Aims Acinic cell carcinoma (ACC) of the breast is a rare histological form of triple-negative breast cancer (TNBC). Despite its unique histology, targeted sequencing analysis has failed to identify recurrent genetic alterations other than those found in common forms of TNBC. Here we subjected three breast ACCs to whole-exome and RNA sequencing to determine whether they would harbour a pathognomonic genetic alteration. Methods and results DNA and RNA samples from three breast ACCs were subjected to whole-exome sequencing and RNA-sequencing, respectively. Somatic mutations, copy number alterations, mutational signatures and fusion genes were determined with state-of-the-art bioinformatics methods. Our analyses revealed TP53 hotspot mutations associated with loss of heterozygosity of the wild-type allele in two cases. Mutations affecting homologous recombination DNA repair-related genes were found in two cases, and an MLH1 pathogenic germline variant was found in one case. In addition, copy number analysis revealed the presence of a somatic BRCA1 homozygous deletion and focal amplification of 12q14.3-12q21.1, encompassing MDM2, HMGA2, FRS2, and PTPRB. No oncogenic in-frame fusion transcript was identified in the three breast ACCs analysed. Conclusions No pathognomonic genetic alterations were detected in the breast ACCs analysed. These tumours have somatic genetic alterations similar to those of common forms of TNBC, and may show homologous recombination deficiency or microsatellite instability. These findings provide further insights into why breast ACCs, which are usually clinically indolent, may evolve into or in parallel with high-grade TNBC.

Background: Concerns about overdiagnosis and overtreatment of DCIS have led to an interest in de-escalating treatment for DCIS. However, accruing patients to active surveillance trials poses several issues, the most pressing of which is the high risk of synchronous invasion at the biopsy level (~26%). Since DCIS-associated stromal changes and influx of immune cells might be mediators of progression to invasive breast cancer, the present project will investigate whether radiogenomics is able to predict the upstaging in patients preoperatively diagnosed with DCIS with Vacuum-Assisted Breast Biopsy (VABB). Hypothesis: We hypothesize a different immune gene expression signature between pure DCIS (p-DCIS) and DCIS with an invasive component at final surgery (i-DCIS). -We hypothesize to be able to identify these different immune gene expression signatures in VABB tissue samples. -We hypothesize that a subsequent gene expression profiling-derived immunohistochemistry signature in DCIS diagnosed on VABB, integrated with other histopathological characteristics and preoperative imaging - Standard Mammography (SM), Digital Breast Tomosynthesis (DBT), Contrast-Enhanced Spectral Mammography (CESM), and Magnetic Resonance (MR) - will predict with higher accuracy the upstaging at final surgery. We hypothesize that the implementation of a radiogenomic approach will reproduce all this information with ultimate precision. Aims: Aim 1: to investigate the relationship between immune gene expression signature at diagnosis of DCIS at VABB and at final surgery outcomes. -Aim 2: to investigate if the association of imaging plus genomics is better than genomics alone. -Aim 3: to create and validate a gene expression profiling-derived immunohistochemistry signature. -Aim 4: to implement radiomic in DCIS management. Experimental Design: Starting from a cohort of more than 2000 consecutive patients with a diagnosis of DCIS on VABB and subsequently operated at the European Institute of Oncology, training and testing datasets will be created. VABB p-DCIS and i-DCIS will be profiled using a next-generation sequencing gene expression assay, targeting 395 genes associated with tumor-immune systems interactions and performed on RNA extracted from DCIS samples. Gene expression and the subsequently derived immunohistochemistry profiles will be integrated with preoperative imaging (SM, DBT, CESM, MR). Finally, we will perform a radiomic feature analysis by SM, DBT (when available), and CESM-detected DCIS at VABB. Impact on Cancer: Patients with features that place them at low risk may be more likely to forego additional therapy. Clinicians would distinguish patients who are candidates to surveillance only, because the "pure" DCIS has been completely removed, from patients going to active surveillance plus endocrine treatment because the residual lesion is with high probability a DCIS, from patients to be sent to surgery because most certainly the invasive component will be present. Citation Format: Matteo Lazzeroni, Luca Nicosia, Elena Guerini Rocco, Sara Gandini, Sara Raimondi, Massimo Barberis, Nicola Fusco, Marta Cremonesi, Daniela Origgi, Enrico Cassano, Aliana Guerrieri Gonzaga, Davide Serrano, Debora Macis, Harriet Johansson, Bernardo Bonanni. Radiogenomics for predicting underestimation of invasiveness in ductal carcinoma in situ (DCIS) diagnosed with vacuum assisted breast biopsy: study rationale and design [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr PR004.

Estrogen receptor (ER), progesterone receptor (PgR), Ki-67, and HER2 immunohistochemistry (IHC) together with HER2 in situ hybridization (ISH) are utilized to classify invasive breast cancer (IBC) into predictive molecular subtypes. As IHC evaluation may be hampered by analytical errors, gene expression assays could offer a reliable alternative. In this first Europe-wide external quality assessment (EQA) study, we investigated performance of mRNA-based Xpert® Breast Cancer STRAT4 (CE-IVD) in five European laboratories. The cohort comprised ten pre-therapy IBC core biopsies diagnosed in the coordinating center (CC). STRAT4 binary (positive or negative) mRNA results of each marker (ESR1, PGR, ERBB2,&nbsp, MKI67) were compared with the gold standard IHC/ISH performed by the CC. Sensitivity, specificity, and accuracy of ESR1 and ERBB2 mRNA were 100% for all samples. In contrast, PGR expression was falsely negative for one case by two sites and MKI67 falsely negative for two cases (respectively by four and one sites). These cases had STRAT4 expression values close to assay cut-offs and immunohistochemically presented heterogeneous low positive PgR and heterogeneous Ki-67. Our EQA shows that STRAT4 mRNA assay may be a reproducible method to evaluate ER, PgR, HER2, and Ki-67 status. However, cases with expression values close to assay cut-offs should be carefully reviewed.

Simple Summary Four biomarkers [estrogen receptor (ER), progesterone receptor (PgR), Ki-67, and HER2], are used to stratify breast cancer (BC) into subtypes predictive of therapy response. In a Europe-wide external quality assessment, we compared performance of an mRNA-based method [Xpert® Breast Cancer STRAT4 (CE-IVD)] for determining ESR1, PGR, ERBB2, and MKI67 expression against the gold standard [immunohistochemistry (IHC)/HER2 in situ hybridization (ISH)]. The coordinating center (CC) and five European laboratories tested ten breast cancer samples. STRAT4 binary (positive or negative) results of each marker were compared with the gold standard. ESR1 and ERBB2 mRNA results were concordant with IHC/ISH in all single analyses. In contrast, PGR and MKI67 results were discordant in a few cases, which had STRAT4 expression values close to assay cut-offs and immunohistochemically presented heterogeneous low positive PgR and heterogeneous Ki-67. STRAT4 assay may be a reproducible method. However, cases with expression values close to cut-offs should be carefully reviewed. Abstract Estrogen receptor (ER), progesterone receptor (PgR), Ki-67, and HER2 immunohistochemistry (IHC) together with HER2 in situ hybridization (ISH) are utilized to classify invasive breast cancer (IBC) into predictive molecular subtypes. As IHC evaluation may be hampered by analytical errors, gene expression assays could offer a reliable alternative. In this first Europe-wide external quality assessment (EQA) study, we investigated performance of mRNA-based Xpert® Breast Cancer STRAT4 (CE-IVD) in five European laboratories. The cohort comprised ten pre-therapy IBC core biopsies diagnosed in the coordinating center (CC). STRAT4 binary (positive or negative) mRNA results of each marker (ESR1, PGR, ERBB2, MKI67) were compared with the gold standard IHC/ISH performed by the CC. Sensitivity, specificity, and accuracy of ESR1 and ERBB2 mRNA were 100% for all samples. In contrast, PGR expression was falsely negative for one case by two sites and MKI67 falsely negative for two cases (respectively by four and one sites). These cases had STRAT4 expression values close to assay cut-offs and immunohistochemically presented heterogeneous low positive PgR and heterogeneous Ki-67. Our EQA shows that STRAT4 mRNA assay may be a reproducible method to evaluate ER, PgR, HER2, and Ki-67 status. However, cases with expression values close to assay cut-offs should be carefully reviewed.

Breast cancer is the most common malignancy occurring during gestation. In early-stage breast cancer during pregnancy (PrBC), breast-conserving surgery (BCS) with delayed RT is a rational alternative to mastectomy, for long considered the standard-of-care. Regrettably, no specific guidelines on the surgical management of these patients are available. In this study, we investigated the feasibility and safety of BCS during the first trimester of pregnancy in women with early-stage PrBC. All patients with a diagnosis of PrBC during the first trimester of pregnancy jointly managed in two PrBC-specialized Centers were included in this study. All patients underwent BCS followed by adjuvant radiotherapy to the ipsilateral breast after delivery. Histopathological features and biomarkers were first profiled on pre-surgical biopsies. The primary outcome was the isolated local recurrence (ILR). Among 168 PrBC patients, 67 (39.9%) were diagnosed during the first trimester of gestation. Of these, 30 patients (age range, 23-43 years; median=36 years; gestational age, 2-12 weeks; median=7 weeks; median follow-up time=6.5 years) met the inclusion criteria. The patients that were subjected to radical surgery (n=14) served as controls. None of the patients experienced perioperative surgical complications. No ILR were observed within three months (n=30), 1 year (n=27), and 5 years (n=18) after surgery. Among the study group, 4 (12.3%) patients experienced ILR or new carcinomas after 6-13 years, the same number (n=4) had metastatic dissemination after 3-7 years. These patients are still alive and disease-free after 14-17 years of follow-up. The rate of recurrences and metastasis in the controls were not significantly different. The findings provide evidence that BCS in the first trimester PrBC is feasible and reasonably safe for both the mother and the baby.

Introduction: The treatment scenario of lung cancer is rapidly evolving through time. In parallel, growing evidence is accumulating on different mechanisms of treatment resistance. Inter- and intra...

Background Phosphatase and tensin homolog (PTEN) loss is associated with tumorigenesis, tumor progression, and therapy resistance in breast cancer. However, the clinical value of PTEN as a biomarker in these patients is controversial. We sought to determine whether the benefit of traditional biomarkers testing is improved by the analysis of PTEN status for the identification of high-risk breast cancer. Methods A cohort of 608 patients with breast cancer was included in this study. Based on the expression on the neoplastic cells compared to the normal internal controls by immunohistochemistry (IHC), cases were classified as PTEN-low (PTEN-L) or PTEN-retained (PTEN-WT). The former constituted the study group, while the latter the control group. Analysis of gene expression was performed on publicly available genomic data and included 4265 patients from the METABRIC and MSK cohorts retrieved from cBioPortal. The Shapiro-Wilk test was used to analyze the normal distributions of continuous variables. Relationships between PTEN status and the clinicopathologic and molecular features of the patient population were assessed using Fisher’s exact test or Chi-squared/Wilcoxon rank-sum test. Survival curves were built according to the Kaplan-Meier method. Results Alteration in PTEN status was significantly different at protein and gene levels, where the reduced protein expression was observed in 280/608 cases (46.1%) from our group, while genetic aberrations in only 315/4265 (7.4%) cases of the METABRIC and MSK cohorts. PTEN-L tumors were significantly enriched for hormone receptors (HR) and HER2 negativity (n = 48, 17.1%) compared to PTEN-WT tumors (n = 22, 6.7%; p = 0.0008). Lack of HR with or without HER2 overexpression/amplification was significantly associated with worse overall survival (OS) in PTEN-L but not in PTEN-WT breast cancers (p p = 0.002). Conclusions The combined analysis of PTEN, HER2, and HR status offers relevant information for a more precise risk assessment of patients with breast cancer.

The clinical outcome of patients with a diagnosis of hormone receptor (HR)+ breast cancer has improved remarkably since the arrival of endocrine therapy. Yet, resistance to standard treatments is a major clinical challenge for breast cancer specialists and a life-threatening condition for the patients. In breast cancer, mismatch repair (MMR) status assessment has been demonstrated to be clinically relevant not only in terms of screening for inherited conditions such as Lynch syndrome, but also for prognostication, selection for immunotherapy, and early identification of therapy resistance. Peculiar traits characterize the MMR biology in HR+ breast cancers compared to other cancer types. In these tumors, MMR genetic alterations are relatively rare, occurring in ~3 % of cases. On the other hand, modifications at the protein level can be observed also in the absence of gene alterations and vice versa. In HR+ breast cancers, the prognostic role of MMR deficiency has been confirmed by several studies, but its predictive value remains a matter of controversy. The characterization of MMR status in these patients is troubled by the lack of tumor-specific guidelines and/or companion diagnostic tests. For this reason, precise identification of MMR-deficient breast cancers can be problematic. A deeper understanding of the MMR biology and clinical actionability in HR+ breast cancer may light the path to effective tumor-specific diagnostic tools. For a precise MMR status profiling, the specific strengths and limitations of the available technologies should be taken into consideration. This article aims at providing a comprehensive overview of the current state of knowledge of MMR alterations in HR+ breast cancer. The available armamentarium for MMR testing in these tumors is also examined along with possible strategies for a tailored pathological characterization.

Background The breast cancer genome dynamically evolves during malignant progression and recurrence. We investigated the genomic profiles of primary early-stage breast cancers and matched relapses to elucidate the molecular underpinnings of the metastatic process, focusing on potentially actionable alterations in the recurrences. Methods A mono-institutional cohort of 128 patients with breast cancers (n = 68 luminal B HER2, n = 6 luminal B HER2+, n = 1 HER2+ non-luminal, n = 56 triple negative) and at least one recurrence in a timeframe of 17 years was evaluated. Next-generation sequencing comprehensive genomic profiling was performed on 289 formalin-fixed paraffin-embedded (FFPE) samples, including primary tumors and matched relapses. Correlations of genomic aberrations with clinicopathologic factors and time to breast cancer relapse were analyzed. Results Genomic data were available for 188 of 289 FFPE samples that achieved the sequencing quality parameters (failure rate 34.9%), including 106 primary tumors and 82 relapses. All primary and relapse samples harbored at least one genomic alteration, with a median number of six alterations per sample (range 1–16). The most frequent somatic genomic alterations were mutations of TP53 (primary tumors = 49%, relapses = 49%) and PIK3CA (primary tumors = 33%, relapses = 30%). Distinctive genomic alterations of primary tumors were significantly associated with molecular subtypes. TP53, PIK3R1, and NF1 somatic alterations were more frequently detected in triple negative tumors (p value CCND1, FGF3, and FGFR1 copy number gains were recurrently identified in luminal cases (p value TP53 mutations and MYC amplification were significantly and independently associated with a shorter time to relapse (p value Conclusions Specific genomic aberrations of primary breast cancers were associated with time to relapse. Primary tumors and matched recurrences showed a core of shared driver genomic aberrations but private actionable alterations have been identified in the relapses.

Metastatic vulvar melanoma is a rare and aggressive disease and survival is usually poor. Vulvar melanomas harbor BRAF V600 mutations only infrequently; consequently, target therapy is a rare therapeutic option and immunotherapy usually has only a weak effect. On the other hand, KIT mutations are rare in cutaneous melanomas, but relatively frequent in mucosal melanomas, particularly in vulvar-vaginal melanomas, and can be a therapeutic target. Herein, we report a clinical case of a patient with metastatic vulvar melanoma, harboring an exon 17 c-KIT mutation, treated with avapritinib (BLU-285) – a highly potent and selective oral kinase inhibitor designed to treat imatinib-resistant gastro-intestinal stromal tumors (GIST) by targeting KIT/PDGFRα activation loop mutants (exons 17/18). After failure of the combination of ipilimumab + nivolumab first and then nivolumab alone, the patient received avapritinib 300 mg/daily for central nervous system (CNS), lymph-nodal, right adrenal gland, lung, and subcutaneous metastases. Best response was partial remission, according to RECIST 1.1 criteria. Time to treatment progression was 11 months. Main toxicities were grade 2 cutaneous vasculitis that required avapritinib discontinuation, and grade 2 uveitis of unknown origin, treated by vitrectomy and empiric antibiotic and antiviral therapy due to negative cultural tests. Uveitis was detected at the time of progression and therapy was definitively discontinued. In conclusion, avapritinib proved to be effective even in the presence of a pretreated disease, a high tumor burden, and brain metastases. In our experience, treatment was feasible and toxicity manageable. Considering the lack of effective therapies and the poor outcome of the disease, determination of c-KIT mutations should be performed routinely in cases of metastatic mucosal melanoma.

Despite its rarity, male breast cancer shows a steadily rising incidence. Given the absence of ad hoc prospective randomized clinical trials, treatment strategies are based on extrapolation from female breast cancer recommendations or solely on population-based data.This review discusses the current treatment landscape for male breast cancer in the adjuvant and in the metastatic setting. The authors also discuss the biology and genomic landscape of male breast cancer. Original research and review articles, relative to the period 2010-2019, were included in the review of the literature.There is a major medical need to include male patients with breast cancer in prospective clinical trials. The call to equality in breast cancer care can be pursued via two divergent paths: (i) a gender-neutral delivery of breast cancer information and (ii) the creation of separate sections, for the more common female breast cancer and for the rare male ones. We propose to differentiate male breast cancer care, acknowledging unique onco-sexual and social needs that can be only partially shared.

Alterations in the tumor suppressor phosphatase and tensin homolog (PTEN) occur in a substantial proportion of solid tumors. These events drive tumorigenesis and tumor progression. Given its central role as a downregulator of the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, PTEN is deeply involved in cell growth, proliferation, and survival. This gene is also implicated in the modulation of the DNA damage response and in tumor immune microenvironment modeling. Despite the actionability of PTEN alterations, their role as biomarkers remains controversial in clinical practice. To date, there is still a substantial lack of validated guidelines and/or recommendations for PTEN testing. Here, we provide an update on the current state of knowledge on biologic and genetic alterations of PTEN across the most frequent solid tumors, as well as on their actual and/or possible clinical applications. We focus on possible tailored schemes for cancer patients’ clinical management, including risk assessment, diagnosis, prognostication, and treatment.

E-cadherin (CDH1 gene) germline mutations are associated with the development of diffuse gastric cancer in the context of the so-called hereditary diffuse gastric syndrome, and with an inherited predisposition of lobular breast carcinoma. In 2019, the international gastric cancer linkage consortium revised the clinical criteria and established guidelines for the genetic screening of CDH1 germline syndromes. Nevertheless, the introduction of multigene panel testing in clinical practice has led to an increased identification of E-cadherin mutations in individuals without a positive family history of gastric or breast cancers. This observation motivated us to review and present a novel multidisciplinary clinical approach (nutritional, surgical, and image screening) for single subjects who present germline CDH1 mutations but do not fulfil the classic clinical criteria, namely those identified as—(1) incidental finding and (2) individuals with lobular breast cancer without family history of gastric cancer (GC). This manuscript was supported by the Italian Ministry of Health (Project Code GR-2016-02361655) and was partially supported by the Ricerca Corrente and 5 × 1000 funds.

Breast cancer related lymphoedema (BCRL) occurs in a substantial proportion of breast cancer survivors and is a major contributor to disability, representing a long-term threat to these patients. Given the extremely high incidence of breast cancer worldwide, and the increasing number of long-term survivors, the reduction of BCRL burden represents an urgent clinical need in women's healthcare. However, there are no validated predictive biomarkers, diagnostic tools, and strong evidence-supported therapeutic strategies for BCRL management. Here, we provide a comprehensive clinicopathological characterization of a large series of women with node-positive breast cancers and identify new bona fide predictors of BCRL occurrence. 332 cases of surgically-treated node-positive breast cancers were retrospectively collected (2-10.2 years of follow-up). Among them, 62 patients developed BCRL. To identify demographic and clinicopathologic features related to BCRL, Fisher's exact test or Chi-squared test were carried out for categorical variables; the Wilcoxon rank-sum was employed for continuous variables. Factors associated with BCRL occurrence were assessed using a Cox proportional hazards regression model. En-bloc dissection of the axillary lymph nodes but not the type of breast surgery impacted on BCRL development. Most of BCRL patients had a Luminal A-like neoplasm. The median number of lymph nodes involved by metastatic deposits was significantly higher in BCRL compared to the control group (p=0.04). Both peritumoral lymphovascular invasion (LVI) and extranodal extension (ENE) of the metastasis had a negative impact on BCRL-free survival (p=0.01). Specifically, patients with LVI and left side localization harbored 4-fold higher risk of developing BCRL, while right axillary nodes metastases with ENE increased the probability of BCRL compared to ENE-negative patients. Here, we document that LVI and ENE have a strong predictive value for BCRL occurrence. Furthermore, we confirm that the full excision of the axillary nodes is one of the major determinants of BCRL, regardless of the extent of the surgical procedure involving the breast. In conclusion, our results suggest that the pathologic data on LVI and ENE should be integrated with information on the laterality of the tumor and the type of surgical procedure. This new integrative approach could be extremely beneficial to improve BCRL risk stratification. Citation Format: Fusco N, Corti C, Lopez G, Michelotti A, Despini L, Gambini D, Lorenzini D, Guerini-Rocco E, Maggi S, Noale M, Invernizzi M. Proposal for integrating the pathologic assessment of lymphovascular invasion and extranodal tumor extension in breast cancer-related lymphedema clinical management [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-12-09.

IntroductionBRCA tumour testing is a crucial tool for personalised therapy of patients with ovarian cancer. Since different next-generation sequencing (NGS) platforms and BRCA panels are available, the NGS Italian Network proposed to assess the robustness of different technologies.MethodsSix centres, using four different technologies, provided raw data of 284 cases, including 75 cases with pathogenic/likely pathogenic variants, for a revision blindly performed by an external bioinformatic platform.ResultsThe third-party revision assessed that all the 284 raw data reached good quality parameters. The variant calling analysis confirmed all the 75 pathogenic/likely pathogenic variants, including challenging variants, achieving a concordance rate of 100% regardless of the panel, instrument and bioinformatic pipeline adopted. No additional variants were identified in the reanalysis of a subset of 41 cases.ConclusionsBRCA tumour testing performed with different technologies in different centres, may achieve the realibility and reproducibility required for clinical diagnostic procedures.

e17592 Background: Homologous recombination deficiency (HRD) is a common feature of epithelial ovarian cancers (EOCs) as approximately half of EOCs have molecular aberrations in homologous recombination repair (HRR) genes, including BRCA1/2 gene mutations. Poly ADP-ribose Polymerase inhibitors (PARPi) have reformed the management of BRCA1/2 mutant ovarian cancer patients and demonstrated activity also in tumor with HRD status. The evaluation of HRD together with BRCA1/2 gene alterations is nowadays critical to guide the therapy decision for all patients with EOC. Different methods to assessed HRD have been reported and different assays are being implemented in clinical practice. Here, we sought to evaluate the analytical validity of a commercial in house HRD assay as compared to a reference standard method. Methods: 59 consecutive case of EOCs were included in this study. DNA extracted from representative formalin-fixed paraffin-embedded (FFPE) tissue block of each case were subjected to next-generation sequencing (NGS) analysis of BRCA1/2 genes alterations and HRD status using the AmoyDx HRD Focus Panel (Amoy Diagnostics Ltd, Xiamen, Fujian, China). HRD status was assesses through AmoyDx Genomic Scar Score (GSS) considering the length, type and location of the genome-wide chromosomal copy number variation through SNP probes spread throughout the genome. Cases with a GSS > 50 were considered as HRD positive. For each case the same FFPE tissue block was sent to Myriad Genetic Laboratories for myChoice CDx testing as reference standard. Assay concordance was measured as positive (PPA), negative (NPA) and overall (OPA) percentage of agreement according to the different scores proposed by the manufacturers. Results: HRD status data were available for 52 (88.1%) and 56 (94.9%) samples considering in-house and reference standard assay, respectively, and 49 cases was available for concordance analyses. AmoyDx HRD Focus Panel identified 23/49 samples (46.9%) as HRD positive and 26 (53.1%) as HRD negative. The concordance of HRD status between AmoyDx HRD Focus Panel and myChoice CDx was 100% and 81,2% for positive (PPA) and negative (NPA) cases respectively, with an overall analytical concordance (OPA) of 87.8%. No discordance was seen in BRCA1/2 status between in-house and reference standard assay (OPA: 100%). Conclusions: The HRD status assessed by AmoyDx HRD Focus Panel is highly concordant with that obtained using the current standard reference method. Commercial in-house test may offer a cost-effective, reliable and robust alternative to the centralized assay for the evaluation of HRD status in patient with EOC.

Triple-negative breast cancers (TNBC) comprise biologically and clinically heterogeneous diseases characterized by the lack of hormone receptors (HR) and HER2 expression. This subset of tumors accounts for 15-20% of all breast cancers and pursues an ominous clinical course. However, there is a spectrum of low-risk TNBCs with no/minimal metastatic potential, including the salivary gland-type tumors, those with extensive apocrine differentiation and/or high tumor-infiltrating lymphocytes, and small-sized, early-stage (pT1a/bN0M0) TNBCs. De-escalating the treatment in low-risk TNBC, however, is not trivial because of the substantial lack of dedicated randomized clinical trials and cancer registries. The development of new diagnostic and/or prognostic biomarkers based on clinical and molecular aspects of low-risk TNBCs would lead to improved clinical treatment. Here, we sought to provide a portrait of the clinicopathological and molecular features of low-risk TNBC, with a focus on the diagnostic challenges along with the most important biological characteristics underpinning their favorable clinical course.

Immune checkpoint inhibitors have revolutionized the cancer treatment with an approved efficacy in different solid tumors and hematologic malignancies. These agents are increasing the indication in cancer treatment, but can be associated with serious immune-related adverse effects (IRAEs). Dermatologic and gastrointestinal toxicities are the most common IRAE followed by endocrinopathies with a different time of occurrence. Rarely cases of gastrointestinal toxicities are observed almost 2 years after initiation of the therapy. In this review we focus on liver toxicity related to these immunotherapeutic agents for which the largest amount of safety data is available. The management of drug-induced liver toxicity is very complicated and in same cases may take a long period of time to be resolved. A prompt recognition of liver IRAEs and an appropriate management of this event, requiring close collaboration with other specialist figures, could improve its treatment with evident implication on the efficacy of the therapy.

Introduction: Mucinous carcinoma of the breast (MCB) is a rare histologic form of estrogen receptor (ER)-positive invasive carcinoma, accounting for up to 2% of breast cancers. MCBs are characterized by clusters of tumor cells floating in lakes of extracellular mucin, and are classified into mucinous A (paucicellular) and mucinous B (hypercellular) subtypes. Some MCBs are found admixed with invasive ductal carcinoma components, and then classified as mixed MCBs. The aims of this study were to determine the repertoire of somatic mutations of MCBs and to ascertain whether these genetic alterations are distinct from those identified in common forms of ER+/HER2- invasive breast cancers (IBCs). We also sought to determine whether the mucinous and ductal components of mixed MCBs would be clonally related. Materials and methods: Thirty MCBs including 25 pure MCBs (n=13 mucinous A, n=12 mucinous B) and five mixed MCBs were microdissected and subjected to whole exome sequencing. Each tumor component of mixed cases was microdissected and analyzed separately. Somatic mutations, copy number alterations and mutational signatures were defined using state-of-the-art bioinformatics methods. The mutational repertoire of MCBs was compared with that of ER+/HER2- IBCs (n = 240) from The Cancer Genome Atlas (TCGA) breast cancer study. Results: The genes most frequently mutated in MCBs were GATA3 (27%, 8/30, all frameshift mutations), KMT2C (13%, 4/30) and MAP3K1 (10%, 3/30). No significant differences were identified in single gene comparisons between mucinous A and mucinous B MCBs or between pure MCBs and the mucinous component of mixed MCBs (Fisher's exact tests, p>0.05). As compared to common forms of ER+/HER2- IBC, MCBs had a lower frequency of PIK3CA mutations (7% vs 42%, p Conclusions: The repertoire of somatic mutations in MCBs is distinct from that of common forms of ER+/HER2- IBCs. These differences include the lack of concurrent 1q gains/16q losses, a lower frequency of PIK3CA mutations and a higher frequency of GATA3 mutations in pure MCBs. Citation Format: Pareja F, Geyer FC, Piscuoglio S, Selenica P, Kumar R, Lim RS, Guerini-Rocco E, Marchio C, Mariani O, Ng CKY, Brogi E, Norton L, Vincent-Salomon A, Weigelt B, Reis-Filho JS. Mucinous breast carcinomas: A genomically distinct subtype of estrogen receptor-positive invasive breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-05-08.

Programmed cell death protein 1 (PD-1) and its ligand programmed cell death-ligand 1 (PD-L1) are overexpressed in a number of human malignancies. More interestingly, their expression has been associated with patient survival in non-small cell lung cancer (NSCLC), melanoma, renal cell carcinoma, esophageal, pancreatic and colorectal carcinoma, with the data commonly suggesting a negative prognostic role. In this review, we summarize the pros and cons regarding the predictive role of PD-L1 expression in candidate patients for checkpoint inhibitors. Furthermore, we discuss the potential predictive role of other biomarkers, such as tumor mutational burden, microsatellite instability, mismatch repair deficiency and tumor infiltrating lymphocytes. We conclude that PD-L1 testing probably represents simply a “snapshot” of an intricate, fluctuating and dynamic process, that in turn represents the interplay between the immune system and cancer. The PD-L1 assay can be considered more useful for response stratification than in patient selection.

Purpose Crizotinib, a mesenchymal-epithelial transition/anaplastic lymphoma kinase/c-ros oncogene 1 (ROS1) inhibitor, has recently been approved by the US Food and Drug Administration for the treatment of patients with advanced ROS1-positive non–small-cell lung cancer (NSCLC). Therefore, interest in ROS1 testing is growing. ROS1 gene fusions affect approximately 0.5% to 2% of unselected NSCLCs. Limited data are available on the prevalence and distribution of ROS1 fusions in patients with advanced-stage NSCLC. Material and Methods A series of 727 lung adenocarcinomas from patients with stage IV disease, negative for epidermal growth factor receptor and anaplastic lymphoma kinase alterations, were tested for ROS1 fusions by fluorescent in situ hybridization analysis, with confirmation by immunohistochemistry. Results were correlated with clinicopathologic parameters and compared with data from the literature. Results ROS1 fusions were detected in 29 patients (4%), including 27 of 266 females (10.2%) and two of 461 males (0.4%; P = 1.2E-10). The mean age of patients with ROS1-positive disease was lower than that of patients with ROS1-negative disease (49.21 v 62.96 years, respectively; P = 1.1E-10). Eleven of 583 smokers (1.9%) and 18 of 144 nonsmokers (12.5%) showed ROS1 rearrangement ( P = 4.05E-7). By logistic regression analysis, ROS1 fusions were independently associated with female sex, younger age at diagnosis, and absence of smoking history, (odds ratios, 12.4, 7.9, and 3.6, respectively). These data, integrated with those reported in the literature, indicate that the prevalence of ROS1 fusions in females and in nonsmokers was higher in patients with advanced disease than in patients with operable disease (11.2% v 3.1%, P < .001; 11.6% v 2.8%, P < .001, respectively). The mean age at diagnosis was significantly lower in patients with advanced disease (49.8 years) than in patients with operable disease (55.6 years; P < .001). Conclusion Our data indicate that ROS1 fusions in patients with advanced-stage lung adenocarcinoma are more frequent in females, particularly if young and nonsmokers. A diagnostic algorithm for an accurate screening of ROS1 alterations was elaborated.

The advances in understanding the inherited biological mechanisms of non-small cell lung cancer harbouring epidermal growth factor receptor (EGFR) mutations led to a significant improvement in the outcomes of patients treated with EGFR tyrosine kinase inhibitors. Despite these clinically impressive results, clinical results are not always uniform, suggesting the need for deepening the molecular heterogeneity of this molecularly defined subgroup of patients beyond the clinical and biological surface. The availability of tissue and blood-based tumour genotyping allows us to improve the understanding of molecular and genetic intratumor heterogeneity, driving the measurement of clonal evaluation in patients with lung cancer carrying EGFR mutations. Genetic diversification, clonal expansion and selection are highly variable patterns of genetic diversity, resulting in different biological entities, also a prerequisite for Darwinian selection and therapeutic failure. Such emerging pieces of evidence on the genetic diversity, including adaptive and immunomodulated aspects, provide further evidence for the role of the tumour microenvironment (TME) in drug-resistance and immune-mediated mechanisms. Matching in daily clinical practice, the detailed genomic profile of lung cancer disease and tracking the clonal evolution could be the way to individualise the further target treatments in EGFR-positive disease. Characterising the tumour and immune microenvironment during the time of the cancer evaluation could be the way forward for the qualitative leap needed from bench to bedside. Such a daring approach, aiming at personalising treatment selection in order to exploit the TME properties and weaken tumour adaptivity, should be integrated into clinical trial design to optimise patient outcome.

BackgroundWith the approval of the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib for newly diagnosed, breast cancer gene (BRCA)1/2 mutated, ovarian cancer women, the assessment of BRCA1/2 tumour status will be shortly required at the time of diagnosis.AimTo investigate the feasibility of next-generation sequencing (NGS)-based BRCA tumour test on cytological specimens from ovarian cancer ascites.MethodsWe evaluated the BRCA1/2 status on neoplastic ascites and corresponding tumour tissue of 11 patients with ovarian cancer, using the NGS ‘Oncomine BRCA Research Assay’.ResultsThe NGS-based BRCA test on cytological samples had a success rate of 100%, with 11 of 11 concordant BRCA1/2 results between ascites and tumour tissues analyses, including two wild type samples and nine cases harbouring somatic or germline variants.ConclusionBRCA test may be performed on ovarian cancer ascites, reproducing BRCA1/2 tumour status and representing a useful tool for clinical decision-making.

Broad-based genomic sequencing is being used more frequently for patients with advanced non-small cell lung cancer (NSCLC). However, little is known about the association between broad-based genomic sequencing and treatment selection or survival among patients with advanced NSCLC in a community oncology setting.To compare clinical outcomes between patients with advanced NSCLC who received broad-based genomic sequencing vs a control group of patients who received routine testing for EGFR mutations and/or ALK rearrangements alone.Retrospective cohort study of patients with chart-confirmed advanced NSCLC between January 1, 2011, and July 31, 2016, and who received care at 1 of 191 oncology practices across the United States using the Flatiron Health Database. Patients were diagnosed with stage IIIB/IV or unresectable nonsquamous NSCLC who received at least 1 line of antineoplastic treatment.Receipt of either broad-based genomic sequencing or routine testing (EGFR and/or ALK only). Broad-based genomic sequencing included any multigene panel sequencing assay examining more than 30 genes prior to third-line treatment.Primary outcomes were 12-month mortality and overall survival from the start of first-line treatment. Secondary outcomes included frequency of genetic alterations and treatments received.Among 5688 individuals with advanced NSCLC (median age, 67 years [interquartile range, 41-85], 63.6% white, 80% with a history of smoking); 875 (15.4%) received broad-based genomic sequencing and 4813 (84.6%) received routine testing. Among patients who received broad-based genomic sequencing, 4.5% received targeted treatment based on testing results, 9.8% received routine EGFR/ALK targeted treatment, and 85.1% received no targeted treatment. Unadjusted mortality rates at 12 months were 49.2% for patients undergoing broad-based genomic sequencing and 35.9% for patients undergoing routine testing. Using an instrumental variable analysis, there was no significant association between broad-based genomic sequencing and 12-month mortality (predicted probability of death at 12 months, 41.1% for broad-based genomic sequencing vs 44.4% for routine testing; difference -3.6% [95% CI, -18.4% to 11.1%]; P = .63). The results were consistent in the propensity score-matched survival analysis (42.0% vs 45.1%; hazard ratio, 0.92 [95% CI, 0.73 to 1.11]; P = .40) vs unmatched cohort (hazard ratio, 0.69 [95% CI, 0.62 to 0.77]; log-rank P .001).Among patients with advanced non-small cell lung cancer receiving care in the community oncology setting, broad-based genomic sequencing directly informed treatment in a minority of patients and was not independently associated with better survival.

The PARP inhibitor olaparib has been approved in the maintenance setting of platinum-sensitive epithelial ovarian cancer patients with germline or somatic BRCA1/2 mutation. Therefore, the availability of a tumor BRCA test has become a clinical need. We report the results of the clinical implementation of a tumor BRCA test within the frame of an institutional workflow for the management of patients with nonmucinous and nonborderline epithelial ovarian cancer. In total, 223 patients with epithelial ovarian cancer were prospectively analyzed. BRCA1/2 status was evaluated on formalin-fixed, paraffin-embedded tumor specimens using next-generation sequencing technology. The tumor BRCA test had a success rate of 99.1% (221 of 223 successfully analyzed cases) and a median turnaround time of 17 calendar days. Among the 221 cases, BRCA1 or BRCA2 pathogenic/likely pathogenic mutations were found in 62 (28.1%) cases and variants of uncertain significance in 25 (11.3%) cases. The concordance rate between tumor BRCA test results and germline BRCA1/2 status was 87%, with five cases harboring pathogenic/likely pathogenic somatic-only mutations. The next-generation, sequencing-based tumor BRCA test showed a high success rate and a turnaround time compatible with clinical purposes. The tumor BRCA test could be implemented in a molecular diagnostic setting and it may guide the clinical management of patients with epithelial ovarian cancer.

Neuroendocrine breast carcinomas (NBCs) account for 2-5% of all invasive breast cancers, and are histologically similar to neuroendocrine tumours from other sites. They typically express oestrogen receptor (ER), and are HER2-negative and of luminal 'intrinsic' subtype. Here, we sought to define the mutational profile of NBCs, and to investigate whether NBCs and common forms of luminal (ER+ /HER2- ) breast carcinoma show distinct repertoires of somatic mutations. Eighteen ER+ /HER2- NBCs, defined as harbouring >50% of tumour cells expressing chromogranin A and/or synaptophysin, and matched normal tissues were microdissected and subjected to massively parallel sequencing targeting all exons of 254 genes most frequently mutated in breast carcinomas and/or related to DNA repair. Their mutational repertoire was compared with that of ER+ /HER2- breast carcinomas (n = 240), PAM50-defined luminal breast carcinomas (luminal A, n = 209; luminal B, n = 111) and invasive lobular carcinomas (n = 127) from The Cancer Genome Atlas. NBCs were found to harbour a median of 4.5 (range 1-11) somatic mutations, similar to that of luminal B breast carcinomas (median = 3, range 0-17) but significantly higher than that of luminal A breast carcinomas (median = 3, range 0-18, p = 0.02). The most frequently mutated genes were GATA3, FOXA1, TBX3, and ARID1A (3/18, 17%), and PIK3CA, AKT1, and CDH1 (2/18, 11%). NBCs less frequently harboured PIK3CA mutations than common forms of ER+ /HER2- , luminal A and invasive lobular carcinomas (p

Adenoid cystic carcinoma of the breast is a rare histological type of triple-negative breast cancer with an indolent clinical behavior, often driven by the MYB-NFIB fusion gene. Here we sought to define the repertoire of somatic genetic alterations in two adenoid cystic carcinomas associated with high-grade triple-negative breast cancer. The different components of each case were subjected to copy number profiling and massively parallel sequencing targeting all exons and selected regulatory and intronic regions of 488 genes. Reverse transcription PCR and fluorescence in situ hybridization were employed to investigate the presence of the MYB-NFIB translocation. The MYB-NFIB fusion gene was detected in both adenoid cystic carcinomas and their associated high-grade triple-negative breast cancer components. Although the distinct components of both cases displayed similar patterns of gene copy number alterations, massively parallel sequencing analysis revealed intratumor genetic heterogeneity. In case 1, progression from the trabecular adenoid cystic carcinoma to the high-grade triple-negative breast cancer was found to involve clonal shifts with enrichment of mutations affecting EP300, NOTCH1, ERBB2 and FGFR1 in the high-grade triple-negative breast cancer. In case 2, a clonal KMT2C mutation was present in the cribriform adenoid cystic carcinoma, solid adenoid cystic carcinoma and high-grade triple-negative breast cancer components, whereas a mutation affecting MYB was present only in the solid and high-grade triple-negative breast cancer areas and additional three mutations targeting STAG2, KDM6A and CDK12 were restricted to the high-grade triple-negative breast cancer. In conclusion, adenoid cystic carcinomas of the breast with high-grade transformation are underpinned by the MYB-NFIB fusion gene and, akin to other forms of cancer, may be constituted by a mosaic of cancer cell clones at diagnosis. The progression from adenoid cystic carcinoma to high-grade triple-negative breast cancer of no special type may involve the selection of neoplastic clones and/or the acquisition of additional genetic alterations.

HER2 mutations are uncommon molecular mutations in nonesmall-cell lung cancer (NSCLC) (4%). Tyrosine kinase inhibitors, such as erlotinib or gefitinib, have demonstrated promising results in patients with advanced NSCLC who harbor epidermal growth factor receptor mutations. Human epidermal growth factor 2 (HER2/ERBB2/neu) is a member of the ERBB family of tyrosine kinase receptors, and is activated by homodimerisation or heterodimerisation with other ERBB receptors. It is possible that NSCLC patients with HER2 mutations could benefit from targeted therapy. There are some data on afatinib and trastuzumab activity in this subgroup of patients. There are no data on the efficacy of immunotherapy in patients harboring epidermal growth factor receptor or ERB2 mutations. The results of CheckMate 057 suggested that, among patients harboring druggable mutations, better outcomes were achieved with docetaxel treatment than with nivolumab (84 patients). We report the first case of a patient with a HER2 mutation treated with nivolumab: this case suggests that nivolumab has strong activity even in patients with targeting biomarkers, in particular with advanced HER2-positive lung cancer.

Metastatic involvement of the prostate from noncontiguous solid tumors is a rare event occurring by means of vascular dissemination. The reported cases of biopsy and surgical samples with metastatic involvement have increased; however, a comprehensive understanding of secondary tumors of the prostate is currently missing. Metastases to the prostate carry a dismal prognosis and may pose serious diagnostic challenges to both clinicians and pathologists, with crucial therapeutic implications. Secondary tumors of the prostate spread more frequently from the digestive tract, the lung, and the kidney. The integration of clinicoradiologic data with appropriate pathologic and immunohistochemical analyses is essential for the identification and the characterization of secondary tumors of the prostate, whereas molecular analyses could provide additional and complementary information, enabling precise diagnosis and appropriate clinical management. Patients with solitary metastases could benefit from prostatic resection and adjuvant therapy, whereas in cases of disseminated diseases, symptom control may be obtained with palliative procedures. The purpose of this review was to assess the current state of knowledge of secondary tumors involving the prostate gland and to discuss short-term future perspectives, while providing a practical approach to these uncommon conditions for pathologists and oncologists.

Personalized cancer therapy matches the plan of treatment with specific molecular alterations [...]

Introduction. DCIS is a potential precursor of invasive breast cancer (IBC). Immune escape might play a critical role in the transition from DCIS to IBC. We aim to identify an immune-related gene expression signature of DCIS to distinguish hazardous lesions, and to avoid the burden of overtreatment in women with less harmful DCIS. Since tumor-infiltrating lymphocytes (TILs) occur more frequently in hormone receptor-negative, Her-2 positive DCIS, we focused the analysis on this subtype. Methods. We performed a prospective nested case-control study involving women with pure DCIS (hormone receptor-negative, Her-2 positive, TILs ≥ 20%), treated with breast conservative surgery with or without radiotherapy. Cases were defined as women with DCIS developing subsequent ipsilateral breast event (IBE), controls as women with DCIS without IBE at 8 years of follow-up. Next-generation sequencing gene expression assay (Oncomine™ Immune Response Research Assay) targeting 395 genes associated with tumor-immune systems interactions was performed on RNA extracted from DCIS samples of 25 cases and 25 age- and radiotherapy-matched controls. In order to obtain reliable results, we considered adequate samples with mapped reads > 1 million and valid reads > 800.000. Normalized gene-level count data generated from the run were further processed with Affymetrix™. A Volcano plot was also employed in order to distinguish genes differently expressed between cases and controls according to the log-gene-level fold change and the ANOVA test (p values < 0.05). Univariate (Log-rank) and multivariate Cox proportional hazard models identified genes associated with the disease free survival. Results. Thirty-one samples (15 cases; 16 controls) reached the quality parameters. The analysis identified 11 genes with a significant different expression between cases and controls. The upregulation of three immune-related genes (VTCN1, NT5E and CMKLR1) remained significantly associated with disease free survival. Notably, VTCN1 and NT5E expressions were significantly associated with the risk of invasive local recurrence. Conclusions. This exploratory analysis of hormone receptor-negative, Her-2 positive DCIS with TILs ≥ 20% showed significant differences in immune-related gene expression profiles between women with subsequent IBE and controls. VTCN1 and NT5E may play an important role in immune surveillance mechanisms of DCIS and may be targeted by immune-based therapy to slow or prevent DCIS progression. These results are well worth further validation on future studies. Citation Format: Matteo Lazzeroni, Caternia Fumagalli, Federica Corso, Alberto Ranghiero, Debora Macis, Elena Guerini Rocco, Valentina Aristarco, Harriet Johansson, Aliana Guerrieri-Gonzaga, Davide Serrano, Andrea DeCensi, Sara Gandini, Massimo Barberis, Bernardo Bonanni. Identification of an immune gene expression signature for progression of breast ductal carcinoma in situ (dcis) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1705.

Genetic alterations of leucine-rich repeat kinase 2 (LRRK2), one of the most important contributors to familial Parkinson&rsquo, s disease (PD), have been hypothesized to play a role in cancer development due to demographical and preclinical data. Here, we sought to define the prevalence and prognostic significance of LRRK2 somatic mutations across all types of human malignancies by querying the publicly available online genomic database cBioPortal. Ninety-six different studies with 14,041 cases were included in the analysis, and 761/14,041 (5.4%) showed genetic alterations in LRRK2. Among these, 585 (76.9%) were point mutations, indels or fusions, 168 (22.1%) were copy number variations (CNVs), and 8 (1.0%) showed both types of alterations. One case showed the somatic mutation R1441C. A significant difference in terms of overall survival (OS) was noted between cases harboring somatic LRRK2 whole deletions, amplifications, and CNV-unaltered cases (median OS: 20.09, 57.40, and 106.57 months, respectively, p = 0.0008). These results suggest that both LRRK2 amplifications and whole gene deletions could play a role in cancer development, paving the way for future research in terms of potential treatment with LRRK2 small molecule inhibitors for LRRK2-amplified cases.

Immunotherapy is a new standard first-line treatment for non-small cell lung cancers (NSCLC) with high programmed cell death-ligand 1 (PD-L1) expression (≥ 50%) and second-line treatment regardless of PD-L1 status, though not all patients benefit from this approach. Much effort is ongoing to identify robust prognostic and predictive biomarkers of response to immune checkpoint inhibitors, overcoming PD-L1 that appears limited in its ability to discriminate patient candidates to this new class of anticancer agents. The purpose of this research study is to identify potential new biomarkers for immunotherapy in lung cancer.Fifty-three consecutive patients with advanced NSCLC treated with nivolumab were enrolled in the study. All the patients received a blood analysis looking for the relationship between different populations of baseline white blood cells and granulocytic myeloid-derived suppressor cells (Gr-MDSC) detected by flow cytometry, to identify and characterize patients with poor likelihood of benefit from nivolumab in NSCLC second-line setting, regardless of clinical feature and PDL1 expression.Univariate analysis showed that high baseline levels of Gr-MDSC and low baseline CD8/Gr-MDSC ratio are associated with significantly better (P = 0.02) response to immunotherapy treatment. Log-rank tests suggested a significant improvement in OS and PFS with high baseline levels of Gr-MDSC levels (≥ 6 cell/μl), low absolute neutrophil count ( 5840/μl), high eosinophil count ( 90 /μl), and NLR 3. The multivariate analysis showed a statistically significant improvement for PFS (P = 0.003) and OS (P = 0.05) in favour of the identified good prognostic Gr-MDSC-linked asset group, compared with the poor prognosis group.The role of Gr-MDSC appears interesting as a potential biomarker in NSCLC patients receiving immune-checkpoint inhibitors. Further analyses are needed to confirmed and study in deep the role of these particular cells and their role in cancer response and progression during ICI therapy.

e12570 Background: Hormone receptors and HER2 negative (triple negative, TN) invasive lobular carcinomas (ILCs) are very rare, accounting for 1-2% of all TN breast cancers (BCs). Methods: We extracted data from our prospectively collected institutional database on all consecutive patients (pts) with early stage TN ILC operated at the European Institute of Oncology (IEO) between June 1994 and December 2012. Invasive disease-free survival (iDFS) and cumulative incidence of distant metastases (CI-DM) were calculated. Biological features of these tumors, including molecular intrinsic subtypes based on PAM50 assay, expression of androgen receptor (AR) and mutational status of c-erbB2 gene were also evaluated. Additionally, NGS data of 45 TN ILCs were obtained from 3 large public databases, to confirm mutations in c-erbB2 gene and to identify other recurrently mutated genes. Results: Among 2952 ILCs treated at IEO, 44 (1.5%) were TN and were included in the analysis. All pts received adjuvant chemotherapy. The iDFS rates at 5 and 10 years of follow-up were 47.4% (95% CI, 31.1-62.0) and 29.5% (95% CI, 14.8-45.8), respectively. The corresponding CI-DM percentages were 17.6% (95% CI, 7.6-31.2) and 20.8% (95% CI, 9.5-35.1). The molecular intrinsic subtype was defined through PAM50 for 31 out of 44 TN ILCs: 48% were classified as luminal A, 3% luminal B, 32% HER2-enriched, and only 16% basal-like. The group of pts with luminal A or B tumors had a significantly better CI-DM as compared with pts with non-luminal tumors (i.e. HER2-enriched and basal-like; p=0.003). Luminal tumors expressed AR more frequently than non-luminal tumors (AR≥1% in 96% of luminal tumors versus 53% in non-luminal tumors; p=0.001), and at significantly higher levels (median percentage of AR-positive cells was 80% in luminal tumors versus 15% in non-luminal tumors; p=0.01). Higher AR expression was associated with significantly better iDFS in the whole cohort of TN ILCs (p=0.01), as well as in the group of luminal tumors (p=0.05). 27 TN ILCs of the IEO cohort were analyzed for mutations in c-erbB2 gene, and 9 (33%) harbored mutations. Analysis of the 3 public databases, confirmed c-erbB2 mutations in 9 out of 45 (20%) TN ILCs. All the c-erbB2 mutations found were previously reported to be pathogenetic in BCs and to predict response to neratinib. ErbB signaling and DNA damage response were among the top 10 pathways significantly enriched for mutated genes in TN ILCs. Conclusions: TN ILCs are rare tumors with dire prognosis. Their specific biological features require newly defined targeted therapeutic strategies.

Immunotherapy has become the standard-of-care in many solid tumors. Despite the significant recent achievements in the diagnosis and treatment of cancer, several issues related to patients’ selection for immunotherapy remain unsolved. Multiple lines of evidence suggest that, in this setting, the vision of a single biomarker is somewhat naïve and imprecise, given that immunotherapy does not follow the rules that we have experienced in the past for targeted therapies. On the other hand, additional immune-related biomarkers that are reliable in real-life clinical practice remain to be identified. Recently, the immune-checkpoint blockade has been approved in the US irrespective of the tumor site of origin. Further histology-agnostic approvals, coupled with with tumor-specific companion diagnostics and guidelines, are expected in this field. In addition, immune-related biomarkers can also have a significant prognostic value. In this review, we provide an overview of the role of these biomarkers and their characterization in the management of lung cancer, melanoma, colorectal cancer, gastric cancer, head and neck cancer, renal cell carcinoma, urothelial cancers, and breast cancer.

Introduction: Acinic cell carcinoma of the breast (ACC) is an exceedingly rare histologic form of triple-negative breast cancer (TNBC) with a generally good clinical behavior. Despite its unique histology, targeted sequencing analysis of ACC has failed to identify recurrent genetic alterations other than those found in common forms of TNBC. Here, we subjected three breast ACCs to whole-exome and RNA-sequencing, seeking to define whether they would be underpinned by a pathognomonic genetic alteration that would distinguish them from common forms of TNBC. Materials and methods: Three breast ACCs were subjected to whole-exome sequencing (WES) and RNA-sequencing. Somatic mutations, copy number alterations, mutational signatures and fusion genes were determined using a validated bioinformatics pipeline. We also estimated DNA-based measures of genomic instability including mutational signatures (using SigMA), large-scale state transition (LST) and telomeric allelic imbalance (NtAI). Results: No oncogenic in-frame fusion transcript was identified in the breast ACCs analyzed. ACCs displayed a median of 173 (range, 92-230) non-synonymous somatic mutations as defined by WES. We detected clonal TP53 hotspot mutations associated with loss of heterozygosity (LOH) of the wild-type allele in two ACCs (ACC1 and ACC18). In contrast, the TP53 wild-type ACC (ACC12) harbored a pathogenic MLH1 germline mutation (p.Glu227_Ser295del) and a clonal hotspot mutation in CTNNB1 (p.Lys335Ile). No cancer gene other than TP53 was found to be mutated in >1 case. Copy number analysis revealed that ACC18 harbored a BRCA1 homozygous deletion and focal amplification of 20p12.3 encompassing PCNA. Moreover, the TP53-mutant cases, ACC1 and ACC18, displayed complex copy number profiles, with multiple gains and losses and focal high-level amplifications. Consistent with the BRCA1 homozygous deletion, ACC18 displayed genomic features of homologous recombination DNA repair deficiency (HRD), such as a dominant mutational signature 3, a high LST score (24), a high NtAI score (23) and a high number of ‘small deletions’ >5bps. In contrast, and consistent with its DNA mismatch repair (MMR) deficiency, ACC12 had a simple genome without detectable copy number alterations, displayed a dominant mutational signature 6 associated with defective MMR, microsatellite instability (MSI) as determined by MSIsensor and loss of MLH1 expression by immunohistochemical analysis. Conclusions: Our findings lend further support to the notion that ACCs of the breast are genetically heterogeneous and display genomic features overlapping with those of common forms of TNBCs. Although no pathognomonic genetic alterations were detected in these cases, our data suggest that at least some ACCs of the breast may arise in the setting of HRD or MSI through distinct molecular mechanisms. Our data also suggest the possibility that both BRCA1 and TP53 loss of function may not be sufficient for a TNBC to display high-grade features and that inactivation of these two genes may not necessarily result in the development of high-grade TNBC. Citation Format: Francisco Beca, Simon SK Lee, Fresia Pareja, Arnaud Da Cruz Paula, Pier Selenica, Lorenzo Ferrando, Hannah Y Wen, Hong Zhang, Elena Guerini-Rocco, Emad A Rakha, Britta Weigelt, Jorge S Reis-Filho. Whole-exome and RNA-sequencing analyses of acinic cell carcinomas of the breast [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-07.