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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.

Introduction: Breast cancer (BC) during pregnancy (PrBC) is an uncommon malignancy characterized by a more aggressive clinical course compared to pregnancy-unrelated BC. Specific patterns of tumor-infiltrating lymphocytes (TILs) subpopulations have been observed in these patients, with significant prognostic roles. Previous studies demonstrated the varying histopathologic and prognostic profiles of PrBC by gestational age. However, the underlying immune landscape dynamics has never been investigated. Here, we sought to provide comprehensive insights into the association between gestational age at breast cancer diagnosis and tumor immune microenvironment (TIME) composition. Materials and Methods: A total of 110 PrBC were selected from our Institutional registry and categorized based on the trimester in which they were diagnosed. All cases were subjected to TILs profiling according to the International TILs Working Group recommendations. Immunohistochemistry for CD4, CD8, forkhead box P3 (FOXP3), and PD-L1 (clone 22C3) on a Dako Omnis platform was performed. Fisher’s and Chi-squared tests, multinomial logistic regression models, ROC curve, and survival analyses were performed. Results: The proportion of patients with high histologic grades incremented with the increase in gestational age (1st, n=24, 53%; 2nd, n=27, 69.2%; 3rd trimester, n=20, 87.0%; p=0.02). Neither breast cancer subtypes nor the hormone receptor (HR) and HER2 status changed significantly according to the pregnancy trimester. In HR+/HER2- subtype, the proportion of TILs+ tumors were higher in the early phases of pregnancy (1st, n=29, 100%; 2nd, n=17, 89.5%; 3rd trimester, n=9, 81.8%; p=0.04) imprinted by FOXP3 positivity where more FOXP3+ TILs were seen in the first months and decreased progressively (1st, n=10, 55.6%; 2nd, n=2, 11.8%; 3rd trimester, n=0, 0%; p< 0.01)). While in the triple negative breast cancer (TNBC) population, the proportion of PD-L1+ tumors (i.e. CPS>1) was significantly higher in the later stages of pregnancy (1st, n=2, 16.7%; 2nd, n=2, 18.2%; 3rd trimester, n=5, 71.4%; p=0.03). Patients who relapsed after a BC diagnosis during the 1st and 2nd trimesters lacked more frequently FOXP3+ and CD8+ cells, unlike those with no disease recurrence (n=21, 77.8% vs. n=17, 48.6%; p=0.02 and n=18, 66.7% vs. n=10, 28.6%; p< 0.01, respectively). Conclusions: TIME dynamics of PrBC are different according to the gestational age in both HR+ and TNBC PrBC. Our results suggest that immune tolerance events are likely to involve PrBC at later gestational age. Specific escape mechanisms (i.e., TILs and FOXP3 decrease in HR+ and PD-L1 expression in TNBC) might explain the aggressiveness of PrBC diagnosed during the later gestational age. Citation Format: Elham Sajjadi, Konstantinos Venetis, Mariia Ivanova, Marianna Noale, Concetta Blundo, Giovanna Scarfone, Eugenia Di Loreto, Stefano Ferrero, Stefania Maggi, Paolo Veronesi, Viviana Enrica Galimbreti, Giuseppe Viale, Fedro Alessandro A. Peccatori, Elena Guerini-Rocco, Nicola Fusco. The tumor immune microenvironment composition and prognostic value in breast cancer during pregnancy is dynamic during the gestation period [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 P2-21-04.

Background: Patients with advanced type B3 thymoma and thymic carcinoma resistant to chemotherapy have few treatment options. We report the efficacy and safety results of the combination of the anti-PD-L1 inhibitor avelumab with the anti-angiogenesis drug axitinib in patients with advanced type B3 thymoma and thymic carcinoma. Methods: CAVEATT was a single-arm, multicentre, phase 2 trial, conducted in two Italian centres (the European Instituteof Oncology and the Humanitas Institute, Milan) in patients with histologically confirmed type B3 thymoma or thymic carcinoma, with advanced stage of disease who had progressed after at least one line of platinum-based chemotherapy. Previous treatment with an anti-angiogenesis drug was allowed but not with immune checkpoint inhibitors. Other inclusion criteria were age 18 years or older, an Eastern Cooperative Oncology Group performance status of 0–2, progressive disease, and presence of measurable disease according to Response Evaluation Criteria In Solid Tumours (RECIST) version 1.1. Patients received avelumab 10 mg/kg intravenously every 2 weeks and axitinib 5 mg orally twice daily until disease progression or unacceptable toxicity. The primary endpoint was the centrally assessed overall response rate according to RECIST version 1.1. Patients who received at least one cycle of treatment and had at least one CT scan after treatment start at scheduled time point by protocol were judged assessable for response and were included in efficacy and safety analyses. This study is registered with EUDRACT, 2017–004048–38; enrolment is completed and follow-up is ongoing. Findings: Between April 22, 2019, and June 30, 2021, 32 patients were enrolled. 27 patients had a thymic carcinoma, three a type B3 thymoma, and two a mixed type B3 thymoma and thymic carcinoma. 29 (91%) of 32 patients had stage IVB disease and 13 (41%) of 32 had been pretreated with an anti-angiogenesis drug. 11 of 32 patients had an overall response; thus the overall response rate was 34% (90% CI 21–50); no patients had a complete response, 11 (34%) had a partial response, 18 (56%) had stable disease, and in two patients (6%) progressive disease was the best response. The most common grade 3 or 4 adverse event was hypertension (grade 3 in six [19%] of 32 patients). Four (12%) of 32 patients developed serious adverse events that were new-onset immune-related adverse events, including one grade 3 interstitial pneumonitis, one grade 4 polymyositis, and two grade 3 polymyositis. There were no treatment-related deaths. Interpretation: Avelumab combined with axitinib has promising anti-tumour activity and acceptable toxicity in patients with advanced type B3 thymoma and thymic carcinoma progressing after chemotherapy, and could emerge as a new standard treatment option in this setting. Funding: Pfizer.

Background. Homologous recombination repair (HRR) is the main mechanism of repair of DNA double-strand breaks. Its deficiency (HRD) is a common feature of epithelial ovarian cancers (EOCs). BRCA1/2 mutations and/or other aberrations in genes of HRR are well known causes of HRD and genomic instability. Poly ADP-ribose polymerase inhibitors (PARPi) have revolutionized the management of BRCA mutant EOCs and demonstrated activity in HRD tumor cells. Determining HRD status can provide informations on the magnitude of benefit for PARPi therapy. Myriad MyChoice CDx is a next generation sequencing- based in vitro diagnostic test that assesses the Genomic Instability Score (GIS) which is an algorithmic measurement of loss of heterozygosity, telomeric allelic imbalance, and large-scale state transitions using DNA isolated from formalin-fixed paraffin embedded tumor tissue specimens. However Myriad MyChoice CDx, is a centrally performed and costly assay, with no reimbursement scheduled, at least in Italy. Methods. In this report, we described our experience in performing the HRD Focus AmoyDx (Amoy Diagnostics Ltd, Xiamen, Fujian, China) on the same samples of EOCs evaluated with Myriad MyChoiceCDx assay. Results. The overall percent agreement between AmoyDx and Myriad was 87.8% (65 of 74 tumors tested). All the 36 AmoyDx negative cases were confirmed to be negative by Myriad (negative predictive value, 100%). Conclusions. The concordance of the results with the gold standard Myriad MyChoice CDx assay suggest the feasibility and reliability of HRD testing in diagnostic laboratories with high-throughput NGS platforms and qualified personnel.

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.

Background: Breast cancer is the most common malignancy occurring during gestation and should be managed with the same protocols as for young non-pregnant women. In early-stage breast cancer during pregnancy (PrBC), breast-conserving surgery (BCS) with delayed radiation therapy (RT) is a reasonable alternative to radical mastectomy. Regrettably, no specific and widely adopted 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. Method: The first trimester was defined as 12 weeks and 6 days after the first day of the last menstruation. As for internal protocols, the surgical strategy for PrBC followed the same conservative-oriented schemes applied for nonpregnant patients. Only women with early-stage PrBC treated with BCS during pregnancy followed by planned RT to the whole breast after delivery were included. Exclusion criteria were i) clinical diagnosis or suspicion of inflammatory breast cancer; ii) multicentric disease; iii) diffuse malignant microcalcifications on mammography; iv) previous; v) connective tissue disease, and vi) syndromic breast cancers. All cases underwent central pathological review at the Pathology Department of the European Institute of Oncology. The primary outcome was the isolated local recurrence (ILR). Results: 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. None of the patients included in this study experienced perioperative surgical complications. No ILR was observed within three months (n=30), 1 year (n=27), and 5 years (n=18) after surgery. The 5-year overall survival rate for all patients was 97% (n=29/30). Four patients experienced ILR or new carcinomas after 6-13 years. These patients are still alive and disease-free after 14-17 years of follow-up. Discussion: In our study, both adjuvant endocrine therapy and anti-HER2 treatment were postponed after delivery. Local recurrences/second primary tumors were observed in 4 out of 30 patients treated with BCS. Given that patient #4 did not receive postoperative RT, but an after-delivery mastectomy for preoperative diagnostic underestimation during pregnancy, this case does not represent a post-BCS recurrence. On the other hand, cases #1-3 could be considered real relapses. Two of these tumors occurred in patients that underwent CT during pregnancy, in which the interval between the end of systemic therapies and the onset of RT was not influenced by the pregnancy. In a single patient (not eligible for systemic treatment in pregnancy), the RT was performed with a longer interval than the usual one of the non-pregnant patients. Survival was not affected by local relapse, underlining the efficacy of salvage treatment.Conclusion: The findings of this study suggest that BCS in the first trimester of pregnancy can be considered reasonably safe for both mother and the child. Mammography should be mandatory in all patients for whom conservative surgery is recommended. Given that the availability of data concerning PrBC and its treatment is scarce and heterogeneous, multicentric studies are warranted. Citation Format: Nicola Fusco, Concetta Blundo, Massimo Giroda, Elham Sajjadi, Konstantinos Venetis, M. Cristina Leonardi, Elisa Vicini, Luca Despini, Claudia F. Rossi, Letterio Runza, Eugenia Di Loreto, Giovanna Scarfone, Elena Guerini-Rocco, Giuseppe Viale, Paolo Veronesi, Barbara Buonomo, Fedro A. Peccatori, Viviana E. Galimberti. Feasibility and safety of breast-conserving surgery in early-stage breast cancers during the first trimester of pregnancy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-18-08.

Background. Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer (IBC). High variability in clinical outcomes and propensity for invasion among DCIS is reported, but identifying high-risk DCIS remains a major clinical challenge. Therefore, biomarkers to differentiate patients with indolent DCIS from those who would benefit from therapy are warranted. There are recent data on the role of the tumor immune microenvironment in the progression from DCIS to IBC and the risk of recurrences. However, no comprehensive information on the clinical actionability of DCIS immune-biology is available. We hypothesize that immune escape mechanisms might play a critical role in the transition from DCIS to IBC. Here, we sought to establish whether immune-related gene expression signatures of DCIS might identify women at high risk of disease recurrence and/or progression. Methods. We performed a retrospective nested case-control study including women with pure DCIS, diagnosed between 2009 and 2015 at European Institute Oncology (median follow-up 39,5 months) treated with conserving surgery +/- adjuvant therapy (endocrine therapy or radiotherapy). The study group (cases) was composed of women with DCIS and subsequent ipsilateral breast events (IBE, in situ or invasive). Controls were selected in a 1.1 ratio among DCIS without IBE, matched for age, tumor size, treatment, and hormone receptors (HR), and HER2 status. Stromal tumor-infiltrating lymphocytes (sTILs) were assessed according to the International Immuno-Oncology Biomarker Working Group on Breast Cancer guidelines. RNA extracted from formalin-fixed paraffin-embedded blocks was subjected to gene expression analysis using a next-generation sequencing assay (Oncomine™ Immune Response Research Assay) targeting 395 immune-related genes. Samples that achieve run quality parameters (mapped reads >1 million, valid reads >800.000) were further processed with the Affymetrix Transcriptome Analysis Console software to compare the gene expression between cases and controls (ANOVA, gene-level fold change 2).Results. A total of 116 patients were included, 58 cases and 58 controls. High sTILs count was significantly associated with high-grade DCIS (p= Citation Format: Elena Guerini Rocco, Caterina Fumagalli, Alberto Concardi, Sergio V. Taormina, Aliana Guerrieri-Gonzaga, Federica Corso, Sara Gandini, Bernardo Bonanni, Giuseppe Viale, Massimo Barberis, Nicola Fusco, Lazzeroni Matteo. Expression of immune-related genes and breast cancer recurrence in women with ductal carcinoma in situ [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-22-03.

Introduction: Breast cancer during pregnancy (PrBC) accounts for ~4% of breast cancer cases in young women and its intrinsic biology is still largely undetermined. Tumor microenvironment (TME) of PrBC has been recently characterized with low levels of stromal tumor-infiltrating lymphocytes (TILs) and high relative expression of programmed death-ligand 1 (PD-L1), suggesting an increased immune evasion. The underlying immune landscape, however, has not been unveiled. Given the significant alterations of the immune system during gestation, we hypothesized that the TME of PrBC might have distinct biological traits. Here, we sought to evaluate the outcome of PrBC according to the TME characteristics and to assess whether pathogenic mechanisms of immune evasion are involved. Methods: Representative formalin-fixed paraffin-embedded tissue blocks of 83 consecutive PrBC and 89 age-matched early-onset pregnancy-unrelated breast cancers (controls) were subjected to immunohistochemistry (IHC) using antibodies against CD4, CD8, forkhead box P3 (FOXP3), and PD-L1 (clone 22C3) on a Dako Omnis platform. For all cases, TILs were evaluated according to the International TILs Working Group recommendations. Next-generation sequencing gene expression of 395 genes involved in tumor-immune interactions (Oncomine™ Immune Response Research Assay) was performed on RNA extracted from PrBC (n=20) and controls (n=16). Samples with mapped reads >1,000,000 and valid reads >800,000 were considered adequate. Fisher’s and Chi-squared tests, multinomial logistic regression models, ROC curve, and survival analyses were performed. Results: The fraction of tumors with CD8+TILs was significantly higher in PrBC than in the controls (n=71(85.0%) vs. n=61(68.5%); p=0.02), being mirrored by less cases with CD4+TILs (n=27 (32.5%) vs. n=43 (48.3%); p=0.03). Even higher differences were observed in hormone receptor (HR)+/HER2-negative tumors (CD8: n=39 (88.6%) vs. n=39 (66.1%); p=0.01). After a median follow-up of 78 (range, 1-247), 66/83 women (79.5%) with PrBC were alive and 53/83 (63.8%) relapse-free. Overall, PrBC with CD8+TILs had a better outcome compared to CD8-negative PrBC (OS 81 vs 69 months p=0.05) and CD8 expression was associated with better outcomes in HR+/HER2-negative tumors (OS p=0.02; DFS p=0.04). The overall comparison of immune-related genes in the 34 cases (PrBC, n=18; controls, n=16) that reached the quality parameters revealed significant differences in the expression of 63 immune-related genes. Of these, 4 genes (IFNA17, IFNB1, FUT4, and PECAM1) were upregulated, while 59 genes were downregulated in PrBC compared to the controls. Interestingly, IFNA17, IFNB1, and FUT4 remained upregulated in HR+ PrBC, where a slightly reduced number of differentially expressed genes was observed (n=60). In HR-/HER2- PrBC, only 25 genes were differentially expressed, of which 9, including IFNA17 and PECAM1, were significantly upregulated. Discussion: These data have the potential of improving our knowledge of the immunobiology that characterizes PrBC, suggesting that in these tumors the higher frequency of CD8+TILs might be related to an enhanced anti-tumor immune response, as CD8 expression was associated with better outcomes in PrBC. On the other hand, given that interferons (IFNs) may also trigger immune suppressive mechanisms in cancer cells, the activation of type I IFNs encoded by IFNA17 and IFNB1 seen in our RNA-seq analysis, combined with the lower frequency of CD4+TILs observed, suggest CD4+ cell suppression as a possible mechanism of immune evasion. Conclusion: PrBC TME is characterized by specific patterns of TILs subpopulations due to the possible activation of type I IFNs and its assessment might help in identifying women at high risk of death and recurrence. Citation Format: Nicola Fusco, Elham Sajjadi, Konstantinos Venetis, Barbara Buonomo, Concetta Blundo, Massimo Giroda, Eugenia Di Loreto, Giovanna Scarfone, Stefano Ferrero, Paolo Veronesi, Viviana E. Galimberti, Massimo Barberis, Giuseppe Viale, Elena Guerini-Rocco, Fedro A. Peccatori. Tumor microenvironment characteristics and prognosis in breast cancer during pregnancy: The role of differentially expressed immune-related genes [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-07-02.

Background: The activity impairment of Phosphatase and tensin homolog (PTEN), a tumor suppressor and negative regulator of the PI3K/Akt pathway, 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 for the identification of high-risk breast cancer is improved by the analysis of PTEN status.Methods: The study group consisted of 608 breast cancers (group A) extracted from our tissue microarray biobank and a total of 4,265 patients (group B) from the METABRIC and MSK studies available at cBioPortal. Cases from the group A were classified as PTEN-low (PTEN-L) or PTEN-retained (PTEN-WT) according to the PTEN expression levels by immunohistochemistry (IHC) and scored based on the ratio between normal and tumor tissue. Clinical and genomic data from group B were also analyzed, and, together with those from group A, relationships between PTEN and the clinicopathologic and molecular features were assessed using Fisher’s exact test, Chi-squared test, or Shapiro-Wilk test for continuous variables. Odds ratio (OR) and corresponding 95% confidence interval (CI) were calculated for each variable. To identify factors associated with PTEN expression, multinomial logistic regression models were defined considering a stepwise selection procedure. Survival curves were built according to the Kaplan-Meier method and compared using the log-rank test.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 group A, while genetic aberrations in only 315/4,265 (7.4%) cases of group B. 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 but not disease-free survival in PTEN-L but not in PTEN-WT breast cancers (p Citation Format: Nicola Fusco, Elham Sajjadi, Konstantinos Venetis, Marco Invernizzi, Donatella Gambini, Letterio Runza, Stefano Ferrero, Elena Guerini-Rocco. Combined analysis of PTEN and routinely assessed biomarkers reveals new high-risk subgroups of HR- or HER2+ breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-10-23.

Constitutively active estrogen receptor α (ER/ESR1) mutations have been identified in approximately one-third of ER+ metastatic breast cancers. Although these mutations are known as mediators of endocrine resistance, their potential role in promoting metastatic disease has not yet been mechanistically addressed. In this study, we show the presence of ESR1 mutations exclusively in distant but not local recurrences in five independent breast cancer cohorts. In concordance with transcriptomic profiling of ESR1-mutant tumors, genome-edited ESR1 Y537S and D538G-mutant cell models exhibited a reprogrammed cell adhesive gene network via alterations in desmosome/gap junction genes and the TIMP3/MMP axis, which functionally conferred enhanced cell–cell contacts while decreasing cell-extracellular matrix adhesion. In vivo studies showed ESR1-mutant cells were associated with larger multicellular circulating tumor cell (CTC) clusters with increased compactness compared with ESR1 wild-type CTCs. These preclinical findings translated to clinical observations, where CTC clusters were enriched in patients with ESR1-mutated metastatic breast cancer. Conversely, context-dependent migratory phenotypes revealed cotargeting of Wnt and ER as a vulnerability in a D538G cell model. Mechanistically, mutant ESR1 exhibited noncanonical regulation of several metastatic pathways, including secondary transcriptional regulation and de novo FOXA1-driven chromatin remodeling. Collectively, these data provide evidence for ESR1 mutation–modulated metastasis and suggest future therapeutic strategies for targeting ESR1-mutant breast cancer. Significance: Context- and allele-dependent transcriptome and cistrome reprogramming in mutant ESR1 cell models elicit diverse metastatic phenotypes related to cell adhesion and migration, which can be pharmacologically targeted in metastatic breast cancer.

Supplementary Table S1: Histologic and immunohistochemical profiling and molecular classification of 59 male breast cancers. Supplementary Table S2: Antibody clones and amplification primers used. Supplementary Table S3: List of 241 genes included in the targeted capture massively parallel sequencing platform.

Clinico-pathologic characteristics of patients according to NGS results (success/failure).

Supplementary Methods detailing the methods employed for Copy number alteration (CNA) analysis, Sanger sequencing and supplementary references.

Weighted Kaplan-Meier (KM) estimates of breast cancer-free interval (BCFI) and distant recurrence-free interval (DRFI) according to tumor subtype, separately by PIK3CA mutational status (mutated, MUT vs wild-type, WT). The 5-year event-free estimates (95% CI) are provided. Hazard ratio (HR) estimates (95% CI) and log-rank tests from weighted univariate models.

PDF file - 226KB, Representative micrographs of DCIS and invasive breast cancers displaying PTEN expression (A, left) or loss (A, right), INPP4B expression (B, left) or loss (B right), pAKT expression lack of expression (C, left) and pAKT expression in neoplastic cells (C, right), pS6 lack of expression (D, left) and pS6 expression in neoplastic cells (D, right). Please note that in A and B, the expression of PTEN and INPP4B in stromal cells and/or normal breast epithelium was employed as an internal positive control in cases lacking PTEN and INPP4B expression in neoplastic cells, respectively.

Supplementary Data from Hotspot ESR1 Mutations Are Multimodal and Contextual Modulators of Breast Cancer Metastasis

PDF file - 23KB, Characteristics of patients with pure high-grade DCIS and high-grade DCIS associated with IBC included in this study.

Purpose:Women with hormone receptor–positive early breast cancers have a persistent risk of relapse and biomarkers for late recurrence are needed. We sought to identify tumor genomic aberrations associated with increased late-recurrence risk.Experimental Design:In a secondary analysis of Study of Letrozole Extension trial, a case-cohort–like sampling selected 598 primary breast cancers for targeted next-generation sequencing analysis of gene mutations and copy-number gains (CNGs). Correlations of genomic aberrations with clinicopathologic factors and breast and distant recurrence-free intervals (BCFIs and DRFIs) were analyzed using weighted Cox models.Results:Analysis of mutations and CNGs was successfully performed for 403 and 350 samples, including 148 and 134 patients with breast cancer recurrences (median follow-up time, 5.2 years), respectively. The most frequent alterations were PIK3CA mutations (42%) and CNGs of CCND1 (15%), ERBB2 (10%), FGFR1 (8%), and MYC (8%). PIK3CA mutations and MYC CNGs were associated with lower (P = 0.03) and higher (P = 0.004) tumor grade, respectively; a higher Ki-67 was seen in tumor with CCND1, ERBB2, and MYC CNGs (P = 0.01, P < 0.001, and P = 0.03, respectively). FGFR1 CNG was associated with an increased risk of late events in univariate analyses [17/29 patients; BCFI: HR, 3.2; 95% confidence interval (CI), 1.48–6.92; P = 0.003 and DRFI: HR, 3.5; 95% CI, 1.61–7.75; P = 0.002) and in multivariable models adjusted for clinicopathologic factors.Conclusions:Postmenopausal women with hormone receptor–positive early breast cancer harboring FGFR1 CNG had an increased risk of late recurrence despite extended therapy. FGFR1 CNG may represent a useful prognostic biomarker for late recurrence and a therapeutic target.

Supplementary Figure S4: Morphologic and immunophenotypic features of case MB-T88. Supplementary Figure S5: GATA3 mutational spectrum in male breast cancer and female breast cancer and the impact of selected mutations on the survival of male breast cancer patients.

Constitutively active estrogen receptor α (ER/ESR1) mutations have been identified in approximately one-third of ER+ metastatic breast cancers. Although these mutations are known as mediators of endocrine resistance, their potential role in promoting metastatic disease has not yet been mechanistically addressed. In this study, we show the presence of ESR1 mutations exclusively in distant but not local recurrences in five independent breast cancer cohorts. In concordance with transcriptomic profiling of ESR1-mutant tumors, genome-edited ESR1 Y537S and D538G-mutant cell models exhibited a reprogrammed cell adhesive gene network via alterations in desmosome/gap junction genes and the TIMP3/MMP axis, which functionally conferred enhanced cell–cell contacts while decreasing cell-extracellular matrix adhesion. In vivo studies showed ESR1-mutant cells were associated with larger multicellular circulating tumor cell (CTC) clusters with increased compactness compared with ESR1 wild-type CTCs. These preclinical findings translated to clinical observations, where CTC clusters were enriched in patients with ESR1-mutated metastatic breast cancer. Conversely, context-dependent migratory phenotypes revealed cotargeting of Wnt and ER as a vulnerability in a D538G cell model. Mechanistically, mutant ESR1 exhibited noncanonical regulation of several metastatic pathways, including secondary transcriptional regulation and de novo FOXA1-driven chromatin remodeling. Collectively, these data provide evidence for ESR1 mutation–modulated metastasis and suggest future therapeutic strategies for targeting ESR1-mutant breast cancer.Significance:Context- and allele-dependent transcriptome and cistrome reprogramming in mutant ESR1 cell models elicit diverse metastatic phenotypes related to cell adhesion and migration, which can be pharmacologically targeted in metastatic breast cancer.

Purpose: To assess the prevalence of phosphoinositide 3-kinase (PI3K) pathway alterations in pure high-grade ductal carcinoma in situ (DCIS) and DCIS associated with invasive breast cancer (IBC), and to determine whether DCIS and adjacent IBCs harbor distinct PI3K pathway aberrations.Experimental Design: Eighty-nine cases of pure high-grade DCIS and 119 cases of high-grade DCIS associated with IBC were characterized according to estrogen receptor (ER) and HER2 status, subjected to immunohistochemical analysis of PTEN, INPP4B, phosphorylated (p)AKT and pS6 expression, and to microdissection followed by Sequenom genotyping of PIK3CA and AKT1 hotspot mutations.Results: Alterations affecting the PI3K pathway were found in a subset of pure DCIS and DCIS adjacent to IBC. A subtype-matched comparison of pure DCIS and DCIS adjacent to IBC revealed that PIK3CA hotspot mutations and pAKT expression were significantly more prevalent in ER-positive/HER2-negative DCIS adjacent to IBC (P values, 0.005 and 0.043, respectively), and that in ER-negative/HER2-positive cases INPP4B loss of expression was more frequently observed in pure DCIS (a P value of 0.013). No differences in the parameters analyzed were observed in a pairwise comparison of the in situ and invasive components of cases of DCIS and adjacent IBC. Analysis of the PIK3CA-mutant allelic frequencies in DCIS and synchronous IBC revealed cases in which PIK3CA mutations were either restricted to the DCIS or to the invasive components.Conclusion: Molecular aberrations affecting the PI3K pathway may play a role in the progression from high-grade DCIS to IBC in a subset of cases (e.g., a subgroup of ER-positive/HER2-negative lesions). Clin Cancer Res; 20(9); 2326–37. ©2014 AACR.

Supplementary Table S11: Comparative analysis of the frequencies of mutations in luminal A-like male breast cancers and subsets of luminal A female breast cancers from the TCGA study. Supplementary Table S12: Comparative analysis of the frequencies of mutations in luminal B-like male breast cancers and subsets of luminal B female breast cancers from the TCGA study.

Purpose: Male breast cancer is rare, and its genomic landscape has yet to be fully characterized. Lacking studies in men, treatment of males with breast cancer is extrapolated from results in females with breast cancer. We sought to define whether male breast cancers harbor somatic genetic alterations in genes frequently altered in female breast cancers.Experimental Design: All male breast cancers were estrogen receptor–positive, and all but two were HER2-negative. Fifty-nine male breast cancers were subtyped by immunohistochemistry, and tumor–normal pairs were microdissected and subjected to massively parallel sequencing targeting all exons of 241 genes frequently mutated in female breast cancers or DNA-repair related. The repertoires of somatic mutations and copy number alterations of male breast cancers were compared with that of subtype-matched female breast cancers.Results: Twenty-nine percent and 71% of male breast cancers were immunohistochemically classified as luminal A–like or luminal B–like, respectively. Male breast cancers displayed a heterogeneous repertoire of somatic genetic alterations that to some extent recapitulated that of estrogen receptor (ER)-positive/HER2-negative female breast cancers, including recurrent mutations affecting PIK3CA (20%) and GATA3 (15%). ER-positive/HER2-negative male breast cancers, however, less frequently harbored 16q losses, and PIK3CA and TP53 mutations than ER-positive/HER2-negative female breast cancers. In addition, male breast cancers were found to be significantly enriched for mutations affecting DNA repair–related genes.Conclusions: Male breast cancers less frequently harbor somatic genetic alterations typical of ER-positive/HER2-negative female breast cancers, such as PIK3CA and TP53 mutations and losses of 16q, suggesting that at least a subset of male breast cancers are driven by a distinct repertoire of somatic changes. Given the genomic differences, caution may be needed in the application of biologic and therapeutic findings from studies of female breast cancers to male breast cancers. Clin Cancer Res; 22(16); 4045–56. ©2016 AACR.

Supplementary Figure 1: Validation of somatic mutations in orthogonal methods. Supplementary Figure S2: Mutations and copy number alterations in ER-positive/HER2-negative male and female breast cancers. Supplementary Figure S3: Cancer cell fraction and clonality of non-synonymous mutations in male breast cancer.

PDF file - 39KB, PI3K pathway alterations in pure high-grade DCIS, high-grade DCIS components adjacent to IBC, and in synchronous IBC components.

PDF file - 58KB, PIK3CA mutations and mutation frequencies in pure high-grade DCIS and high-grade DCIS associated with IBC.

Metaplastic breast cancer (MpBC) is a rare, aggressive type of breast cancer, often classified as triple negative (TN). Scarce information is available about germline testing in MpBC. We retrospectively reviewed MpBC patients counseled at our Institute and found to harbor germline pathogenic variants (PVs), and we revised literature data. We identified a germline PV in 15 MpBC patients: 13 in BRCA1 (86.7%), one in TP53 (6.7%), one in MLH1 (6.7%) genes. Eight MpBC PV carriers in BRCA1 have been previously described, including a patient with a PV in both BRCA1 and TP53. MpBC histological subtype in PV carriers was heterogeneous. All MpBCs were TN but 13.3% in our series showed HER2 overexpression. We described the largest series of MpBCs with germline PVs. As previously reported, we observed that BRCA1 is the mainly involved gene in MpBC patients who underwent germline testing according to specific selection criteria. Additional studies on unselected patients are required to assess the authentic role of germline BRCA1 PVs in MpBCs and to explore the possible involvement of other genes in MpBC predisposition. Unraveling a specific MpBC molecular landscape is a starting point for the definition of new therapeutic strategies, since these tumors have a poor prognosis.

AimsPoly (ADP-ribose) polymerase (PARP) inhibitors (PARPIs) represent a standard of care for the clinical management of high-grade serous ovarian cancer (HGSOC). The recognition of homologous recombination deficiency (HRD) has emerged as a predictive biomarker of response for first-line PARPIs treatment in patients with HGOSC. On the other hand, this test is extremely complex and therefore it is often externalised. Regrettably, the reliability of outsourced HRD testing can be troubled by inconclusive results and high rejection rates. In this methodological study, we assessed the technical feasibility, interassay and interlaboratory reproducibility of in-house HRD testing using three different commercially available next-generation sequencing assays.MethodsA total of n=20 epithelial ovarian cancer samples previously analysed with MyChoice CDx were subjected to HRD retesting using three different platforms in three different major pathology laboratories, that is, SOPHiA DDM HRD Solution, HRD focus and Oncomine homologous recombination repair pathway predesigned panel. Concordance was calculated by Cohen’s (dual) and Fleiss (triple) κ coefficients.ResultsIn-houseBRCA1/2molecular testing yielded a concordance rate >90.0% among all participating centres. HRD scores were successfully calculated by each institution with a concordance rate of 76.5%. Concerning the external gold standard test, the overall percentage of agreement ranged from 80.0% to 90.0% with a positive percentage agreement ranging from 75.0% to 80.0% and a negative percentage agreement ranging from 80.0% to 100%.ConclusionsIn-house testing for HRD can be reliably performed with commercially available next-generation sequencing assays.

We present a case of a woman with epidermal growth factor receptor (EGFR)-mutated lung adenocarcinoma who received gefitinib for 2 years and obtained a partial response. The patient then developed liver metastasis and a breast lesion, displaying high estrogen receptor (ER) expression and harboring the same EGFR mutation. From the radiological studies, it was not possible to make a differential diagnosis between primary breast cancer and breast metastasis from lung cancer. After the removal of the breast nodule, thanks to the clinical history, radiology, and above all, molecular and immunohistochemical investigations, a diagnosis of breast metastasis from lung adenocarcinoma was made. This case emphasizes the importance of a comprehensive clinical, pathological, and molecular analysis in the differential diagnosis between primary breast cancer and metastases from extramammary tumor to guide adequate treatment decision making.

Somatic mutations in

HER2 is an emerging biomarker in colorectal cancer (CRC). This oncogene plays an essential role in regulating cell proliferation, differentiation, migration, and, more in general, tumorigenesis and tumor progression. The most frequent types of HER2 alterations in CRC include gene amplification and missense mutations in 7–8% of CRC, often being mirrored by HER2 protein overexpression, representing founder events in solid tumors, including CRC. There are currently no approved HER2-targeted therapy guidelines for CRC; however, several studies have shown that HER2 can be effectively targeted in meta-static CRC settings. In this review, we discuss the current knowledge of HER2 testing in CRC and the immediate future perspectives for HER2 targeting in the metastatic setting.