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Metastasis is the cause of over 90% of all deaths associated with breast cancer, yet the strategies to predict cancer spreading based on primary tumor profiles and therefore prevent metastasis are egregiously limited. As rare precursor cells to metastasis, circulating tumor cells (CTCs) in multicellular clusters in the blood are 20-50 times more likely to produce viable metastasis than single CTCs. However, the molecular mechanisms underlying various CTC clusters, such as homotypic tumor cell clusters and heterotypic tumor-immune cell clusters, are yet to be fully elucidated. Combining machine learning-assisted computational ranking with experimental demonstration to assess cell adhesion candidates, we identified a transmembrane protein Plexin- B2 (PB2) as a new therapeutic target that drives the formation of both homotypic and heterotypic CTC clusters. High PB2 expression in human primary tumors predicts an unfavorable distant metastasis-free survival and is enriched in CTC clusters compared to single CTCs in advanced breast cancers. Loss of PB2 reduces formation of homotypic tumor cell clusters as well as heterotypic tumor-myeloid cell clusters in triple-negative breast cancer. Interactions between PB2 and its ligand Sema4C on tumor cells promote homotypic cluster formation, and PB2 binding with Sema4A on myeloid cells (monocytes) drives heterotypic CTC cluster formation, suggesting that metastasizing tumor cells hijack the PB2/Sema family axis to promote lung metastasis in breast cancer. Additionally, using a global proteomic analysis, we identified novel downstream effectors of the PB2 pathway associated with cancer stemness, cell cycling, and tumor cell clustering in breast cancer. Thus, PB2 is a novel therapeutic target for preventing new metastasis.

Supplemental Table 1: Treatments by subgroup. Supplemental Figure 1A-D: Association of baseline MAF and NOA with survival

Solid papillary carcinoma with reverse polarity (SPCRP) is a rare breast cancer subtype with an obscure etiology. In this study, we sought to describe its unique histopathologic features and to identify the genetic alterations that underpin SPCRP using massively parallel whole-exome and targeted sequencing. The morphologic and immunohistochemical features of SPCRP support the invasive nature of this subtype. Ten of 13 (77%) SPCRPs harbored hotspot mutations at R172 of the isocitrate dehydrogenase IDH2, of which 8 of 10 displayed concurrent pathogenic mutations affecting PIK3CA or PIK3R1. One of the IDH2 wild-type SPCRPs harbored a TET2 Q548* truncating mutation coupled with a PIK3CA H1047R hotspot mutation. Functional studies demonstrated that IDH2 and PIK3CA hotspot mutations are likely drivers of SPCRP, resulting in its reversed nuclear polarization phenotype. Our results offer a molecular definition of SPCRP as a distinct breast cancer subtype. Concurrent IDH2 and PIK3CA mutations may help diagnose SPCRP and possibly direct effective treatment. Cancer Res; 76(24); 7118–29. ©2016 AACR.

Document containing methods on whole exome and targeted massively parallel sequencing analysis, 2-hydroxyglutarate (2HG) assay of cell lines, methylation profiling, and supplementary references.

Document containing figures illustrating results of Sanger sequencing, DNA methylation profiling of SPCRPs, the analysis of IDH2 expression in non-malignant breast epithelial cells and its downstream effects, and tables describing the antibodies used for immunohistochemical analysis, the alterations surveyed by the SNaPshot genotyping assay, Sanger sequencing primers, and statistics and results of the massively parallel sequencing analyses.

Legends for Supplementary Figures S1-S5

Purpose:Circulating tumor DNA (ctDNA) is a promising tool for noninvasive longitudinal monitoring of genomic alterations. We analyzed serial ctDNA to characterize genomic evolution in progressive metastatic breast cancer.Experimental Design:This was a retrospective cohort between 2015 and 2019 obtained under an Institutional Review Board–approved protocol at Northwestern University (Chicago, IL). ctDNA samples were analyzed with Guardant360 next-generation sequencing (NGS) assay. A total of 86 patients had at least two serial ctDNA collections with the second drawn at first post-NGS progression (PN1) by imaging and clinical assessment. A total of 27 participants had ctDNA drawn at second post-NGS clinical progression (PN2). We analyzed alterations, mutant allele frequency (MAF), number of alterations (NOA), and sites of disease on imaging in close proximity to ctDNA evaluation. Matched pairs' variations in MAF, NOA, and alterations at progression were tested through Wilcoxon test. We identified an independent control cohort at Massachusetts General Hospital (Boston, MA) of 63 patients with serial ctDNA sampling and no evidence of progression.Results:We identified 44 hormone receptor–positive, 20 HER2+, and 22 triple-negative breast cancer cases. The significant alterations observed between baseline and PN1 were TP53 (P < 0.0075), PIK3CA (P < 0.0126), AR (P < 0.0126), FGFR1 (P < 0.0455), and ESR1 (P < 0.0143). Paired analyses revealed increased MAF and NOA from baseline to PN1 (P = 0.0026, and P < 0.0001, respectively). When compared with controls without progression, patients with ctDNA collection at times of progression were associated with increased MAF and NOA (P = 0.0042 and P < 0.0001, respectively).Conclusions:Serial ctDNA testing identified resistance alterations and increased NOA and MAF were associated with disease progression. Prospective longitudinal ctDNA evaluation could potentially monitor tumor genomic evolution.

New assays are developed regularly to improve health care for patients. It is important to ensure that assays are performed correctly. Therefore, it is advised to participate in training and proficiency (competence assessment) programs. Tumor infiltrating lymphocytes (TILs) might improve the estimates of response to therapy and prognosis. Herewith, we propose a new training and proficiency program in which each pathologist can train and test themselves regarding TILs (and PDL1) scoring.

Purpose: Circulating tumor DNA (ctDNA) is a promising tool for noninvasive longitudinal monitoring of genomic alterations. We analyzed serial ctDNA to characterize genomic evolution in progressive metastatic breast cancer. Experimental Design: This was a retrospective cohort between 2015 and 2019 obtained under an Institutional Review Board–approved protocol at Northwestern University (Chicago, IL). ctDNA samples were analyzed with Guardant360 next-generation sequencing (NGS) assay. A total of 86 patients had at least two serial ctDNA collections with the second drawn at first post-NGS progression (PN1) by imaging and clinical assessment. A total of 27 participants had ctDNA drawn at second post-NGS clinical progression (PN2). We analyzed alterations, mutant allele frequency (MAF), number of alterations (NOA), and sites of disease on imaging in close proximity to ctDNA evaluation. Matched pairs' variations in MAF, NOA, and alterations at progression were tested through Wilcoxon test. We identified an independent control cohort at Massachusetts General Hospital (Boston, MA) of 63 patients with serial ctDNA sampling and no evidence of progression. Results: We identified 44 hormone receptor–positive, 20 HER2+, and 22 triple-negative breast cancer cases. The significant alterations observed between baseline and PN1 were TP53 (P < 0.0075), PIK3CA (P < 0.0126), AR (P < 0.0126), FGFR1 (P < 0.0455), and ESR1 (P < 0.0143). Paired analyses revealed increased MAF and NOA from baseline to PN1 (P = 0.0026, and P < 0.0001, respectively). When compared with controls without progression, patients with ctDNA collection at times of progression were associated with increased MAF and NOA (P = 0.0042 and P < 0.0001, respectively). Conclusions: Serial ctDNA testing identified resistance alterations and increased NOA and MAF were associated with disease progression. Prospective longitudinal ctDNA evaluation could potentially monitor tumor genomic evolution.

Background: The development of cancer is understood to be a series of molecular events causing uncontrollable cell growth and malignant behavior. Primary breast carcinoma is associated with a number of molecular alterations that may also be implicated in the molecular deregulation underlying other malignancies. In addition, underlying germline genetic mutations have been clearly associated with the development of breast carcinoma (BC) in young patients. Increased risks for the development of additional malignancies following or preceding breast cancer have been reported, however, specific associations and genetic mutations remain unclear, especially in young patients.Materials and methods: Our patient cohort consisted of young women ( Table 1: Summary of clinico-pathologic characteristics of BC with second malignanciesNo.Type of carcinomaGradePrognostic markersAssociated in-situ carcinomaAge at diagnosisSecondary diagnosisTimeline of second diagnosisInitial management of BCLymph node statusGenetic testing results1.Invasive lobular carcinoma1UnknownLobular carcinoma in situ39Malignant phyllodes tumorConcurrentMastectomyNegativeUnknown2.Invasive ductal carcinoma3Triple negativeDuctal carcinoma in situ36Squamous cell carcinoma, esophagus7 years laterMastectomyNegativeBRCA1+3.Invasive ductal carcinoma3Triple negativeNone38High grade serous carcinoma, ovary5 years laterMastectomyOne lymph node positiveBRCA1+4.Invasive ductal carcinoma with mucinous features1Estrogen receptor+/Progesterone receptor+/HER2-Ductal carcinoma in situ33Gastrointestinal stromal tumor, jejunum7 years laterMastectomyNegativeNegative5.Invasive ductal carcinoma2Estrogen receptor+/Progesterone receptor+/HER2-Lobular carcinoma in situ31Malignant phyllodes tumor7 years earlierMastectomyNegativeNegative6.Invasive ductal carcinoma2Estrogen receptor+/Progesterone receptor-/HER2-Ductal carcinoma in situ37Complex endometrial hyperplasia with atypia and leiomyoma with Fumarate hydratase features6 years laterMastectomyNot performedNegative7.Invasive ductal carcinoma2Estrogen receptor+/Progesterone receptor+/HER2-None36Hodgkin disease20 years earlierMastectomyNegativeNegative8.Invasive ductal carcinoma3Estrogen receptor+/Progesterone receptor+/HER2-Ductal carcinoma in situ29High grade squamous intraepithelial lesion1 year earlierMastectomyOne lymph node positiveNegative9.Invasive ductal carcinoma2Estrogen receptor+/Progesterone receptor+/HER2-Ductal carcinoma in situ36High grade squamous intraepithelial lesion1.5 year laterMastectomyOne lymph node positiveNegative10.Invasive ductal carcinoma2Estrogen receptor+/Progesterone receptor+/HER2-Ductal carcinoma in situ36High grade squamous intraepithelial lesion1 month earlierLumpectomyNegativeNegative11.Invasive ductal carcinoma1Estrogen receptor+/Progesterone receptor+/HER2-Ductal carcinoma in situ38Endometrioid adenocarcinoma10 months laterLumpectomyNot performedCowden syndrome (PTEN gene)12.Invasive mucinous carcinoma1Estrogen receptor+/Progesterone receptor+/HER2-Ductal carcinoma in situ41Papillary thyroid carcinoma14 years earlierLumpectomyNot performedNegative13.Invasive tubular carcinoma1Estrogen receptor+/Progesterone receptor+/HER2-Ductal carcinoma in situ37Malignant phyllodes tumorConcurrentMastectomyNegativeNegative Citation Format: Indu Agarwal, Brian Finkelman, Luis Z. Blanco, Amir Behdad, Kalliopi Siziopikou. Associations and spectrum of genetic mutations in younger patients with breast carcinoma and additional malignancies [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-74.

Background: Circulating tumor DNA (ctDNA) has emerged as a potential tool for detecting disease recurrence, monitoring response to therapy, and identifying resistance mutations in the peripheral blood. With increased frequency of testing, there is an unmet need to recognize putative germline variants in ctDNA, and the probability that these variants are associated with inherited conditions. Here, we evaluated a large cohort of breast cancer patients who underwent ctDNA evaluation to determine the type and frequency of ctDNA mutations identified with confirmed germline testing. Methods: We reviewed ctDNA testing from a single institution (Northwestern University). All breast cancer patients who had next-generation sequencing (NGS) performed by Guardant Health (Redwood City, CA) from 2015-2020 were included in this retrospective study. An allele frequency cutoff of 30% was pre-established as a threshold to review patient charts to determine whether genetic counseling and germline testing were performed, along with the timeframe of this testing (e.g. before or after ctDNA evaluation). Clinical information including demographics, pathology, tissue NGS testing, and germline testing were collected. Descriptive analyses and statistical associations were performed using STATA. Results: The initial cohort consisted of 520 patients with breast cancer who underwent ctDNA testing. From this, we identified 84 patients (16.2%) who had at least one variant with allele frequency ≥30%. The most common variants identified were the following: TP53 (34%), PIK3CA (27%), BRCA1 (9%), BRCA2 (8%), and AKT1 (4%). Guardant360 classified 99% of these variants as pathogenic and 1% as a variant of unknown significance. Germline positivity using a separate CLIA-approved test for this indication was confirmed at the following frequencies: BRCA1 (2 of 8 positive, 25%), BRCA2 (2 of 5 positive, 40%), PIK3CA (0 of 5 positive), and TP53 (0 of 26 positive). In total, 14% of patients with ctDNA allele frequency ≥30% had a confirmed germline mutation. Lower age at breast cancer diagnosis was significantly associated with the probability of germline testing prior to ctDNA evaluation (P=0.0001). For patients who had a variant with allele frequency ≥30%, 24.3% never received genetic counseling or germline testing. Conclusion: High allele frequency ctDNA variants (≥30%) were present in 16% of patients who underwent ctDNA evaluation with 14% of these variants confirmed as true germline variants. Consenting patients for ctDNA testing should include the possibility of identifying putative germline variants, and criteria should be established to refer patients for subsequent genetic counseling and germline testing, given the potential implications for patients and their family members. Citation Format: Andrew A Davis, Michael C Burns, Lorenzo Gerratana, Paolo D'Amico, Saya Jacob, Ami Shah, Neelima Katam, Firas Wehbe, Qiang Zhang, Elena Vagia, Lisa Flaum, Kalliopi P. Siziopikou, Leonidas C Platanias, Amir Behdad, William J Gradishar, Massimo Cristofanilli. Identification of incidental putative germline variants in circulating tumor DNA [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-08.

Background: As the role of precision medicine in metastatic breast cancer (MBC) expands, there is an increasing emphasis on diagnostic tools to characterize both somatic and germline alterations that drive carcinogenesis. Circulating tumor DNA (ctDNA) has provided an important means for real-time, non-invasive detection and monitoring of tumor somatic alterations without standard paired white blood cell testing. However, its application for the detection of germline mutations remains relatively understudied. Here, we characterize the role of BRCA1&2 ctDNA mutant allele frequency (MAF) to identify underlying putative germline BRCA1&2 mutations. Methods: Patient data were retrospectively obtained under an IRB-approved protocol to review ctDNA data at Northwestern University between 2015 and 2020. All ctDNA samples were analyzed using the Guardant360 next-generation sequencing (NGS) assay (Guardant Health). Patients with ctDNA alterations in the BRCA genes were identified at all mutant allele frequencies (MAF). Clinical data were collected including breast cancer subtype, prior lines of therapy, tissue-based NGS and germline testing information. Separate CLIA-approved testing performed per standard of care was used as the gold standard for germline testing. Statistical analysis was used to test the association of MAF cut-offs with the presence or absence of a germline alteration. Receiver operating characteristic (ROC) analysis was performed. Results: We identified 127 patients with breast cancer who underwent ctDNA collection. There were 69 HR+ HER2-, 24 HER2+, and 34 triple negative breast cancer (TN) patients. Of these, 8 patients had known BRCA1 germline mutations, as confirmed with separate germline testing, and 9 patients had known BRCA2 germline mutations. BRCA1 germline mutations were significantly association with age < 40 (p=0.016) and triple negative subtype (p=0.042). Mean ctDNA BRCA1 MAF was 2.27% (Standard Deviation {SD} 10.19%) and mean BRCA2 MAF was 2.04% (SD 9.51%). BRCA1/2 MAF was analyzed with respect to germline mutations through ROC analysis. For BRCA1 ctDNA MAF cut-off 32.4% was associated with confirmed putative germline mutation. This cutoff demonstrated sensitivity of 1 and specificity of 0.99. Area under the curve (AUC) was 1.00 at this cut-off. For BRCA2, ctDNA MAF cutoff of 28.5% was associated with confirmed putative germline mutations. This cutoff demonstrated sensitivity of 0.86, specificity of 0.99 and AUC of 0.93. Discussion: ctDNA alterations of BRCA1 with MAF>32.4% and BRCA2 with MAF>28.5% in clinical ctDNA testing were useful criterion to identify germline BRCA mutations. The data suggest the potential to utilize ctDNA as a diagnostic tool to predict germline mutations based on MAF thresholds. Future prospective studies are needed to determine how ctDNA testing may be incorporated into existing clinical algorithms to refer patients for germline testing. Citation Format: Saya Jacob, Andrew Davis, Paolo D'Amico, Lorenzo Gerratana, Michael Burns, Ami Shah, Neelima Katam, Firas Wehbe, Qiang Zhang, Elena Vagia, Lisa Flaum, Kalliopi Siziopikou, Leonidas Platanias, Amir Behdad, William Gradishar, Massimo Cristofanilli. Circulating tumor DNA (ctDNA) as a diagnostic tool to identify putative germline BRCA mutations [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-10.

Background: Human epidermal growth factor receptor 2 (HER2)-positive breast cancer accounts for 20-30% of breast carcinomas and is associated with a poor prognosis. While anti-HER2 targeted therapies have improved outcomes within this group, a subset of these tumors eventually develop resistance urgently requiring novel therapeutic options. In addition to classic HER2+ subtype, estrogen receptor (ER)-positive and HER2-positive tumors (luminal B) are another subtype of HER2 expressing breast carcinomas which could also benefit from additional therapies. Targeting the androgen receptor (AR) pathway and the PD-L1 axis may represent potential new therapeutic strategies in breast cancer. Our group previously reported that AR expression is seen in ¾ of HER2+ carcinomas and is more common in luminal B compared to classic HER2+ tumors. In this report, we evaluated expression of PD-L1 in tumor infiltrating lymphocytes (TILs) in both classic HER2 + and Luminal B subtypes of breast carcinomas and correlated the expression of PD-L1 with AR expression in these tumors. Methods: The study population consisted of 114 patients with HER2-positive invasive breast carcinoma diagnosed from 2009-2014 at Northwestern Memorial Hospital. Electronic medical records were reviewed for patient demographics (mean age 54, range 23-80). Pathologic tumor characteristics (histologic type, size, and grade) and tumor marker profile (ER and HER2) were evaluated. Tissue microarrays were constructed (3 cores from each case to account for tumor heterogeneity) for immunohistochemical evaluation of AR and PD-L1. AR was graded as positive when at least 1% of tumor cells showed nuclear immunoreactivity. For PD-L1, the extent of staining (0%=0, 1-10%=1, 11-50%=2, and 51-100%=3) and staining intensity (0, 1+, 2+, 3+) were assessed in TILs. Then a composite score (CS) was calculated by multiplying extent and intensity results (range 0-9; 0-3=negative, 4-9=positive). Results: Of the 114 studied breast carcinoma cases, 54 were classic HER2+ tumors (47%) and 60 were luminal B (53%) tumors. Approximately 69% of classic HER2+ and 87% of luminal B cases were positive for AR. Of interest, in classic HER2+ cases, the expression of PD-L1 in TILs was inversely correlated with the expression of AR, with 82% (14/17) of AR- tumors being positive for PD-L1 in TILs compared to only 46% (17/37) of AR+ tumors with PD-L1 expression in TILs (p Citation Format: Tiansheng Shen, Jennifer L. Pincus, Kalliopi P. Siziopikou. PD-L1 expression in tumor infiltrating lymphocytes (TILs) inversely correlates with androgen receptor (AR) expression in HER2-positive breast cancer [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 P4-10-30.

Simple Summary: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that lacks significant expression of estrogen receptor, progesterone receptor, and HER2. Patients with locally advanced or metastatic TNBC benefit from treatment with atezolizumab, a humanized monoclonal antibody that blocks the PD-L1 protein. Immunohistochemical analysis of the tumor microenvironment is essential to determine the amount of tumor-infiltrating PD-L1-positive immune cells. The PD-L1/SP142 clone is the companion diagnostic for atezolizumab. Here we investigate the degree of interobserver agreement among ten breast pathologists in the assessment of PD-L1/SP142 immunohistochemistry, as well as the assessment of tumor-infiltrating lymphocytes (TILs) in 49 metastatic TNBCs. This multicenter study shows that both PD-L1 assessment and TILs assessment are robust markers at the group level, but the observed interobserver variability likely affects treatment decisions for individual patients.Patients with advanced triple-negative breast cancer (TNBC) benefit from treatment with atezolizumab, provided that the tumor contains >= 1% of PD-L1/SP142-positive immune cells. Numbers of tumor-infiltrating lymphocytes (TILs) vary strongly according to the anatomic localization of TNBC metastases. We investigated inter-pathologist agreement in the assessment of PD-L1/SP142 immunohistochemistry and TILs. Ten pathologists evaluated PD-L1/SP142 expression in a proficiency test comprising 28 primary TNBCs, as well as PD-L1/SP142 expression and levels of TILs in 49 distant TNBC metastases with various localizations. Interobserver agreement for PD-L1 status (positive vs. negative) was high in the proficiency test: the corresponding scores as percentages showed good agreement with the consensus diagnosis. In TNBC metastases, there was substantial variability in PD-L1 status at the individual patient level. For one in five patients, the chance of treatment was essentially random, with half of the pathologists designating them as positive and half negative. Assessment of PD-L1/SP142 and TILs as percentages in TNBC metastases showed poor and moderate agreement, respectively. Additional training for metastatic TNBC is required to enhance interobserver agreement. Such training, focusing on metastatic specimens, seems worthwhile, since the same pathologists obtained high percentages of concordance (ranging from 93% to 100%) on the PD-L1 status of primary TNBCs.

Triple-negative breast cancer (TNBC) accounts for approximately 12% to 17% of all breast cancers and has an aggressive clinical behavior. Increased tumor-infiltrating lymphocyte counts are prognostic for survival in TNBC, making this disease a potential target for cancer immunotherapy. Research on immunophenotyping of tumor-infiltrating lymphocytes is revealing molecular and structural organization in the tumor microenvironment that may predict patient prognosis. The anti–programmed death-ligand 1 (PD-L1) antibody atezolizumab plus nab-paclitaxel was the first cancer immunotherapy combination to demonstrate progression-free survival benefit and clinically meaningful overall survival benefit in the first-line treatment of metastatic TNBC (mTNBC) in patients with PD-L1–expressing tumor-infiltrating immune cells in 1% or more of the tumor area. This led to its United States and European Union approval for mTNBC and US approval of the VENTANA PD-L1 (SP142) assay as a companion diagnostic immunohistochemistry assay. Subsequently, the anti–programmed death-1 (PD-1 ) antibody pembrolizumab plus chemotherapy was approved by the US Food and Drug Administration for mTNBC based on progression-free survival benefit in patients with a combined positive score of at least 10 by its concurrently approved 22C3 companion diagnostic assay. Treatment guidelines now recommend PD-L1 testing for patients with mTNBC, and the testing landscape will likely become increasingly complex as new anti–PD-L1 and anti–PD-1 agents and diagnostics are approved for TNBC. Integrating PD-L1 testing into current diagnostic workflows for mTNBC may provide more treatment options for these patients. Therefore, it is critical for medical oncologists and pathologists to understand the available assays and their relevance to therapeutic options to develop an appropriate workflow for immunohistochemistry testing.

Background: With the recent FDA approvals of atezolizumab and pembrolizumab for the treatment of unresectable locally advanced or metastatic triple-negative breast cancer, immuno-oncology (IO) therapy has become feasible for the first time in breast cancer, and PD-L1 immunohistochemistry (IHC) has become an essential assay in breast cancer diagnostics. However, relatively little is known about the molecular profile of PD-L1+ breast cancers compared to other tumors, such as lung cancer. Furthermore, although the initial data have shown the most benefit in triple-negative patients, it is unclear whether additional subgroups of patients with advanced breast cancer enriched for PD-L1 expression can be identified, and, therefore, might benefit from IO therapy. Methods: We identified 60 women with locally advanced or metastatic breast cancer who underwent next generation sequencing (NGS) of tumor samples obtained at our institution as part of their clinical care and performed PD-L1 testing using the Ventana SP142 platform. Samples were considered positive for PD-L1 if ≥1%. The NGS assay was designed to detect single nucleotide variants, insertions and deletions, copy number variants, and specific translocations in approximately 600 genes. Associations between PD-L1 status, molecular alterations, and tumor histopathologic features were assessed, with P < 0.05 considered statistically significant. Results: Our cohort consisted of 60 women at a median age of 56 years (range 31-81). 50% of samples were from primary tumors within the breast, 15% were from regional lymph node metastases, and 35% were from distant metastatic sites. Most tumors showed ductal histology (80%) and were either grade 2 (38%) or grade 3 (57%), while half showed focal (27%) or extensive (23%) necrosis. Based on IHC, most tumors were Luminal B (54%), followed by triple-negative (25%), Luminal A (13%), and HER2-enriched (8%) tumors. About one third of tumors had either moderate (25%) or marked (8%) TILs, and about half showed positivity for PD-L1 (48%). Most tumors had a low tumor mutational burden (TMB), with 83% having Citation Format: Brian S Finkelman, Massimo Cristofanilli, Luis Z Blanco, Jr, Amir Behdad, Leonidas C Platanias, William J Gradishar, Kalliopi P Siziopikou. Somatic alterations and PD-L1 positivity in advanced breast cancer [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 P1-02-11.

ObjectivesThe aims were to evaluate the programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) axis in ductal carcinoma in situ (DCIS) of the breast.MethodsWe reviewed 85 pure DCIS cases treated with surgical excision at our institution, including 51 luminal A (estrogen receptor [ER] positive/human epidermal growth factor 2 [HER2] negative), 15 luminal B (ER+/HER2+), 13 HER2 (ER–/HER2+), and six basal-like (ER–/HER2–/CK5/6+). The extent and intensity of PD-1 and PD-L1 immunohistochemical staining in the tumor-infiltrating lymphocytes (TILs) and in the tumor cells were recorded.ResultsOur study found that moderate/severe inflammation around DCIS correlated with HER2 expression (20/28 HER2+ cases [71%] vs 21/57 HER2– cases [37%], P = .005). Of interest, over half of the TILs around the HER2 subtype expressed PD-L1 (7/13, 54%). In addition, about one-third of TILs around the HER2 subtype expressed PD-1 (4/13, 31%).ConclusionsThese findings suggest that immune-based therapeutic strategies may be used as a potential therapy in DCIS cases with PD-L1 overexpression, especially those of the HER2 molecular subtype.

PURPOSE Preclinical studies report that trastuzumab (T) can boost radiotherapy (RT) effectiveness. The primary aim of the B-43 trial was to assess the efficacy of RT alone vs concurrent RT plus T in preventing recurrence of ipsilateral breast cancer (IBTR) in women with ductal carcinoma in situ (DCIS). PATIENTS AND METHODS Eligibility: Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1, DCIS resected by lumpectomy, known estrogen receptor (ER) and/or progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2) status by centralized testing. Whole-breast RT was given concurrently with T. Stratification was by menopausal status, adjuvant endocrine therapy plan, and nuclear grade. Definitive intent-to-treat primary analysis was to be conducted when either 163 IBTR events occurred or all accrued patients were on study ≥ 5 years. RESULTS There were 2,014 participants who were randomly assigned. Median follow-up time as of December 31, 2019, was 79.2 months. At primary definitive analysis, 114 IBTR events occurred: RT arm, 63 and RT plus T arm, 51 (hazard ratio [HR], 0.81; 95% CI, 0.56 to 1.17; P value = .26). There were 34 who were invasive: RT arm, 18 and RT plus T arm, 20 (HR, 1.11; 95% CI, 0.59 to 2.10; P value = .71). Seventy-six were DCIS: RT arm, 45 and RT plus T arm, 31 (HR, 0.68; 95% CI, 0.43 to 1.08; P value = .11). Annual IBTR event rates were: RT arm, 0.99%/y and RT plus T arm, 0.79%/y. The study did not reach the 163 protocol-specified events, so the definitive analysis was triggered by all patients having been on study for ≥ 5 years. CONCLUSION Addition of T to RT did not achieve the objective of 36% reduction in IBTR rate but did achieve a modest but statistically nonsignificant reduction of 19%. Nonetheless, this trial had negative results. Further exploration of RT plus T is needed in HER2-positive DCIS before its routine delivery in patients with DCIS resected by lumpectomy.

Background: We describe the genomic landscape of circulating tumour DNA (ctDNA) across pathological subtypes of metastatic breast cancer. Methods: 255 clinically annotated patients with ctDNA testing by Guardant360 were stratified into HR+, HER2+, and TNBC cohorts. Frequency and heterogeneity of alterations were reported. Paired ctDNA and tissue sequencing were compared for a subset of patients. The association of ctDNA and metastatic sites of disease on imaging was also assessed. Findings: 89% of patients had at least one ctDNA alteration detected. The most common single nucleotide variants (SNVs) for HR+ patients were PIK3CA, ESR1, and TP53. For HER2+, these were TP53, PIK3CA, and ERBB2 with ERBB2 as the most frequent copy number variant (CNV). For TNBC, the most common SNVs were TP53 and PIK3CA, and the most frequent CNVs were MYC, CCNE1, and PIK3CA. TNBC patients had a significantly higher mutant allele frequency (MAF) of the highest variant compared to HR+ or HER2+ patients (P

Context.—HER2 status is a prognostic factor and therapeutic target in invasive breast carcinomas. Reflex testing using an alternate method is recommended on equivocal cases via immunohistochemistry or fluorescence in situ hybridization (FISH). Therapeutic dilemmas arise when both tests are equivocal. The standard chromosome 17 centromere reference probe (CEP17) is in close proximity to the HER2 locus and may be coamplified, leading to equivocal results. Alternate chromosome 17 reference probes may aid in establishing the true HER2 status.Objective.—To describe our institutional experience using D17S122 probe for reflex FISH testing on double-equivocal invasive breast carcinomas and review the literature on alternate reference probes.Data Sources.—Twenty-two patients with double-equivocal invasive breast carcinomas, defined as HER2 immunohistochemistry score 2+ and FISH equivocal per the 2013 guidelines, were reviewed. Reflex FISH was performed with alternate probe D17S122 and the HER2 status classified for 11 cases by using a revised HER2:D17S122 ratio. Seven of 11 cases (63.6%) were ultimately classified as HER2 positive, while 4 cases (36.4%) remained equivocal. The 7 positive cases showed a HER2:D17S122 greater than 2.0.Conclusions.—Alternate probe D17S122 reclassified more than half of our cases as HER2 positive. Alternate probes may establish true HER2 status and direct proper management, as evidenced by our experience and the literature. Additional investigation is needed to determine which alternate probe(s) is(are) best for reflex testing. Finally, the American Society of Clinical Oncology/College of American Pathologists guidelines may need to be updated to reflect more specific recommendations for the utilization of appropriate probes in double-equivocal HER2 cases.

Solid papillary carcinoma with reverse polarity (SPCRP) is a rare breast cancer subtype with an obscure etiology. In this study, we sought to describe its unique histopathologic features and to identify the genetic alterations that underpin SPCRP using massively parallel whole-exome and targeted sequencing. The morphologic and immunohistochemical features of SPCRP support the invasive nature of this subtype. Ten of 13 (77%) SPCRPs harbored hotspot mutations at R172 of the isocitrate dehydrogenase IDH2, of which 8 of 10 displayed concurrent pathogenic mutations affecting PIK3CA or PIK3R1. One of the IDH2 wild-type SPCRPs harbored a TET2 Q548* truncating mutation coupled with a PIK3CA H1047R hotspot mutation. Functional studies demonstrated that IDH2 and PIK3CA hotspot mutations are likely drivers of SPCRP, resulting in its reversed nuclear polarization phenotype. Our results offer a molecular definition of SPCRP as a distinct breast cancer subtype. Concurrent IDH2 and PIK3CA mutations may help diagnose SPCRP and possibly direct effective treatment. Cancer Res; 76(24); 7118–29. ©2016 AACR.

Background: Pregnancy associated breast cancer (PABC) arises during or after pregnancy, is typically triple negative, and is associated with a poor prognosis. Enhancer of Zeste Homolog 2 (EZH2), a core protein of the polycomb-repressive complex, plays a vital role in the epigenetic maintenance of histone H3 lysine (H3K27) repressive chromatin mark and has been found to be aberrantly expressed in various cancers including the breast. EZH2 is involved in stem cell renewal and is involved the initiation, progression and maintenance of cancer cells. EZH2 has been reported to be highly expressed in triple negative breast cancers and to be associated with poor prognosis. In this study, we assessed expression of EZH2 and its downstream markers phosphorylated EZH2 (pEZH2) and H3K27 and correlated their expression with clinicopathologic characteristics in this aggressive type of breast carcinoma. Design: 23 patients diagnosed with PABC within 2 years of pregnancy (mean age=35.8, range=26-48) and control age-/stage-matched nulliparous women (mean age=37.5, range=29-51) were evaluated. Slides were reviewed and pathologic tumor characteristics were noted. Immunohistochemical stains for EZH2, pEZH2, H3K27 and the cancer stem cell marker ALDH1 were performed on 23 PABC and 15 control cases. Extent (1=1-25% positive tumor cells, 2=26-50%, 3=51-75% or 4=76-100%) and intensity (1=weak, 2=moderate or 3=strong) of staining were assessed. A composite score (CS) was calculated by multiplying the extent by intensity (range=0-9; weak=1-3; moderate=4-6 and strong=7-9). Results: PABCs were more likely than controls to have moderate to strong immunoreactivity for EZH2 (69.6% vs. 33.3%, p=0.04). All PABC and control cases expressed EZH2 and pEZH2, while 60.9% of PABC and 40% of control cases expressed H3K27. Within the PABC group, moderate to strong EZH2 expression was seen more frequently in grade 3 tumors (86.7% in grade 3 tumors vs. 37.5% in grades 1-2, p=0.03). Of interest, all triple negative (TN) PABC cases had moderate to strong EZH2 (100% vs. 43.8%, p=0.02). All five PABC cases with the strongest EZH2 (CS=9) were grade 3 tumors (3 TN, 1 HER2+ and 1 luminal B), 60% of which had positive lymph node metastasis. Although 83.3% of ALDH1+ cases also had moderate to strong EZH2, no significant correlation was observed. Conclusions: 1. PABCs express EZH2 and pEZH2 consistently. 2. PABCs are more likely than controls to have moderate to strong immunoreactivity for EZH2. 3. Within the PABC group, all triple negative tumors and the majority of grade 3 tumors have high EZH2 expression. 4. PABC cases with the strongest EZH2 are grade 3 tumors that have positive lymph nodes. Increased expression of EZH2 in PABC may contribute to the poor prognosis in these cases. Our findings add to the understanding of the molecular pathways that operate in different subtypes of breast carcinomas. Citation Format: Blanco Jr LZ, Parini V, Dhamne SA, Siziopikou KP. Expression of EZH2 and its downstream effectors pEZH2 and H3K27 in pregnancy associated breast cancer (PABC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-21.

Background: The clinical utility of routine clinical assessment of ctDNA in metastatic breast cancer (MBC) has not been clearly established. Recently, the Food and Drug Administration approved alpelisib (Piqray®) with a ctDNA-based companion diagnostic. Therefore, there is significant interest in describing the landscape of ctDNA alterations and establishing further clinical utility of this assessment. Methods: We retrospectively identified ctDNA data from a single institution (Northwestern University). All patients had next-generation sequencing performed by Guardant Health (Redwood City, CA) from 2015-2019 for clinical evaluation, and patients were consented for participation in a prospective registry study. Reports were evaluated for type of genomic alterations, number of alterations, and mutant allele frequency (MAF) of the dominant clone. Clinical information including demographics, pathology, imaging, treatment, and response data were collected via patient chart review. Associations were tested through Fisher’s exact test or Kruskal-Wallis test as statistically appropriate. Matched paired samples were investigated through McNemar test. Results: The cohort consisted of 255 patients with MBC. In total, 371 ctDNA reports were reviewed, including 116 patients with serial assessments. Baseline ctDNA evaluation occurred at a median of 3 lines of therapy (interquartile range (IQR): 1 - 5). Median number of alterations was 4 (IQR: 2-7), while median MAF was 4% (IQR: 0.6% - 17.9%). Across the entire cohort, PIK3CA and TP53 were the most commonly detected mutations, while MYC, FGFR1, PIK3CA, and ERBB2 were the most commonly detected amplifications. 40% of patients had PIK3CA aberrations observed in the analyzed plasma samples (28% amplification, 22% H1047R, 11% E542K), which was independent of breast cancer subtype. In the subset of patients with hormone-receptor positive (HR+) breast cancer, 30% of patients had ESR1 mutations (27% D538G, 19% Y537S, 12% Y537N, 11% E380Q). Patients with triple negative breast cancer (TNBC) had statistically higher MAF compared to HR+ or HER2+ breast cancer (P Conclusion: Liquid biopsy evaluation using ctDNA identified clinically meaningful genomic alterations at high frequency. In addition, our data indicate the potential clinical utility of ctDNA as a surrogate for tumor burden on imaging based on the observed correlation with lines of therapy received and sites of disease. Serial assessment of ctDNA demonstrated clonal evolution, particularly in the HR+ cohort, and identified resistant clones. Validation with an independent cohort is warranted. Citation Format: Andrew A Davis, Saya Jacob, Lorenzo Gerratana, Ami N Shah, Firas Wehbe, Neelima Katam, Qiang Zhang, Lisa Flaum, Kalliopi Siziopikou, Leonidas C Platanias, William J Gradishar, Amir Behdad, Massimo Cristofanilli. Landscape of circulating tumor DNA (ctDNA) in metastatic breast cancer [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 P5-01-08.

Phyllodes tumours constitute an uncommon but complex group of mammary fibroepithelial lesions. Accurate and reproducible grading of these tumours has long been challenging, owing to the need to assess multiple stratified histological parameters, which may be weighted differently by individual pathologists. Distinction of benign phyllodes tumours from cellular fibroadenomas is fraught with difficulty, due to overlapping microscopic features. Similarly, separation of the malignant phyllodes tumour from spindle cell metaplastic carcinoma and primary breast sarcoma can be problematic. Phyllodes tumours are treated by surgical excision. However, there is no consensus on the definition of an appropriate surgical margin to ensure completeness of excision and reduction of recurrence risk. Interpretive subjectivity, overlapping histological diagnostic criteria, suboptimal correlation between histological classification and clinical behaviour and the lack of robust molecular predictors of outcome make further investigation of the pathogenesis of these fascinating tumours a matter of active research. This review consolidates the current understanding of their pathobiology and clinical behaviour, and includes proposals for a rational approach to the classification and management of phyllodes tumours.

Background: While breast cancer risk increases with age, about 7% of breast cancer cases in the US are diagnosed in women Methods: Our patient population consisted of all cases of invasive breast carcinoma at Northwestern Memorial Hospital in women Results: A total of 301 invasive breast carcinoma cases were identified (age range 18-39). 40 cases of very young (age ≤30) women with breast cancer (13%) were identified. Nearly all very young patients were diagnosed with tumors of ductal histology (39/40, 98%). Just under half of these patients (19/40, 48%) had a T1 (5 cm) tumor at the time of surgery. The majority of these carcinomas were grade 3 (25/39, 64%), 14/39 (36%) were grade 2, and none were grade 1 tumors. Over half of the tumors were highly proliferative with a high Ki-67 count of >20% (17/31, 55%). ER was expressed in 34/40 (85%), PR in 25/40 (63%) and HER2 in 9/40 (23%). LVI was present in half of the cases (20/40, 50%), and positive lymph nodes were identified in over a third (15/40, 38%) of the cases. Neoadjuvant chemotherapy was used in 9/40 (23%). Three patients (8%) had no residual carcinoma at the time of surgery post chemotherapy. Most of these very young patients chose a mastectomy (28/40, 70%), and 19/40 (48%) also underwent a prophylactic mastectomy on the contralateral side. Conclusions: Our study is one of the largest to date to describe the characteristics of breast carcinomas in very young women (age ≤30). Poor pathologic features such as high tumor grade, high proliferation rate, and presence of LVI were seen in half or more of these very young patients. A high rate of positive lymph nodes was also seen. Mastectomy, often with contralateral prophylactic surgery, was the procedure of choice. Further studies evaluating the molecular characteristics of these tumors and the prevalence of underlying genetic mutations are under way in this unique population of very young breast cancer patients. Citation Format: Finkelman BS, Blanco LZ, Siziopikou KP. Clinical and histopathologic characteristics of breast cancer in very young patients [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 P4-02-09.

Background: Several neoadjuvant trials have been conducted directed at treating triple negative breast cancer (TNBC) patients with platinum agents with pathologic complete response (pCR) rates ranging from 16%-32%. Eribulin mesylate, a nontaxane microtubule dynamics inhibitor with a novel mechanism of action, has clinical activity as monotherapy in breast cancer and other solid tumors. A recent phase I trial found that the combination of eribulin mesylate with carboplatin was well tolerated and showed activity in advanced solid tumors. The recommended dose for future trials was eribulin mesylate 1.1 mg/m2 and carboplatin AUC6. We proposed a neoadjuvant phase II trial with the combination of carboplatin and eribulin in patients with TNBC. Methods: 30 patients were enrolled between November 2011 and February 2013. Patients received eribulin at 1.4 mg/m2 (intravenously over 2-5 minutes) on days 1 and 8 followed by carboplatin AUC = 6 (intravenously over 30 minutes) on day 1 every 21 days for a total of 4 cycles. Definitive surgery was performed 3-8 weeks after completion of therapy. Our primary endpoint was to determine the pCR in TNBC patients treated with the combination of carboplatin and eribulin. Secondary objectives included determination of the clinical response rate, residual cancer burden (RCB), toxicity evaluation, in addition to correlative markers including TLE3, Smad3, cyclins/CDKs, and PIN1 and the Homologous Recombination Deficiency Assay (HRD). Results: There was an initial safety run-in to evaluate the appropriate dose of eribulin in the study population. After the 10th patient, the study was temporarily suspended; toxicity was assessed for the first 10 patients (cycle 1 only) to assess whether eribulin at 1.4mg/m2 or a dose reduction to 1.1 mg/m2 would be required for the remaining patients. Of the first 10 patients, only 2 of 10 experienced grade 3 or 4 neutropenia, and 0 of 10 patients experienced grade 3 or 4 peripheral neuropathy. Therefore, the study was continued for the remaining 20 patients with eribulin dosed at 1.4 mg/m2 and carboplatin AUC = 6. Thirty of the planned 30 patients have been enrolled to date. Of the 30 patients, 24 have completed therapy and 6 are currently on study. Of the 24 patients who have completed therapy, 11 have achieved a pCR (45.83%). As for clinical response rates, 5 have had stable disease (20.8%), 5 have had a complete response (20.8%), and 13 have had a partial response (54.2%). Final results will be presented at the time of the meeting including pCR, RCB, toxicity and correlative studies. The combination of eribulin and carboplatin was well tolerated with a predictable side effect profile. Of the 24 patients who have completed therapy, 8 (33%) required a dose reduction in eribulin for grade 3 or 4 neutropenia. One patient had neutropenic fever. There were no dose reductions for thrombocytopenia thus far. Correlative studies are in progress. Conclusion: The combination of carboplatin and eribulin in the neoadjuvant setting appears to be safe and efficacious in patients with TNBC. Statistical analysis on outcomes for the entire study population and correlative study results on pre- and post-treatment tissues are forthcoming. Future randomized clinical trials should evaluate further this regimen. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-14-14.

Kras-induced non-small-cell lung adenocarcinoma is the major subtype of lung cancers and is associated with poor prognosis. Using a lung cancer mouse model that expresses a cre-mediated KrasG12D mutant, we identified a critical role for the cell surface molecule CD44 in mediating cell proliferation downstream of oncogenic Kras signaling. The deletion of CD44 attenuates lung adenocarcinoma formation and prolongs the survival of these mice. Mechanistically, CD44 is required for the activation of Kras-mediated signaling through the mitogen-activated protein kinase (MAPK) pathway and thus promotes tumor cell proliferation. Together, these results reveal an unrecognized role for CD44 in oncogenic Kras-induced lung adenocarcinoma and suggest that targeting CD44 could be an effective strategy for halting Kras-dependent carcinomas.

Mucocele-like lesion (MLL) is a rare mucinous lesion of the breast with highly variable upgrade rates to atypia or malignancy on excision. This spectrum of data has led to differing opinions on the need for surgical excision. We evaluated 50 core biopsy specimens diagnosed as having MLLs and correlated the findings with those of excision pathology. Thirty-eight patients underwent surgical excision and 29 were benign (76%), 4 had atypical ductal hyperplasia (11%), and 5 had ductal carcinoma in situ (13%), with an overall upgrade rate of 13%. However, the risk of upgrade was exclusively associated with the presence of atypia as seen on the needle core biopsy. All 22 MLLs without atypia had benign excisions, while 5 (31%) of the 16 patients with MLLs with atypia were upgraded to ductal carcinoma in situ on excision. No invasive carcinoma was identified. We believe it is reasonable that women with the core biopsy diagnosis of MLL without atypia and no associated mass be offered close clinical follow-up as an alternative to surgery.

Triple negative breast carcinomas (TNBC), are defined by the absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression. The majority of TNBC are “basal-like”, a group originally defined by studies of mRNA gene expression profiles, but increasingly defined in the clinic by using surrogate markers such as CK 5/6. However, not all TNBC are basal-like. It is postulated that these subcategories of TNBC have distinct underlying biologies that drive their ultimate behavior, and response to treatment, with important implications for designing appropriate targeted therapies. In this study we report that within our cohort of 197 TNBC, distinct groups were identified that varied by CK 5/6 and p53 expression based on age and race. We propose that awareness of CK5/6 and p53 expression in younger and AA TNBC patients may facilitate identifying patients with unique tumor subtypes and may lead to better use of targeted therapies in this group of aggressive breast cancers.

Background: Pregnancy associated breast cancer (PABC), diagnosed during or after gestation, is typically triple negative, and is associated with a poor prognosis. Those diagnosed within two years have an even worse outcome. We previously assessed the immune microenvironment of invasive breast carcinomas in young women and reported that tumor infiltrating lymphocytes (TILs) were more prominent in PABC. Programmed cell death protein 1 (PD-1) is upregulated following activation of lymphocytes, while programmed death ligand 1 (PD-L1) is one of the primary ligands that it interacts with to inhibit T-cell activation and proliferation. PD-L1 may also be constitutively expressed on tumor cells as a result of oncogenic signaling or epithelial-mesenchymal transition. Emerging evidence suggests that the effect of the local immune system, particularly the interactions between PD-1 and PD-L1, is also key in breast cancer progression and in breast tumor responses to chemotherapy and targeted therapy. In this study, we assessed expression of PD-1 and PD-L1 in both TILs and tumor cells in PABC and in age-/stage-/grade-matched nulliparous women, and correlated their expression with clinicopathologic characteristics in this aggressive type of breast carcinomas. Design: 21 patients diagnosed with PABC within two years of pregnancy (mean age=35.7, range=26-48) and 15 matched controls (mean age=37.5, range=29-51) were evaluated. Slides were reviewed and pathologic tumor characteristics, including TILs, were noted. Immunohistochemical stains for PD-1 and PD-L1 were performed. Extent (1=1-25% positive tumor cells, 2=26-50%, 3=51-75%, 4=76-100%) and intensity (1=weak, 2=moderate or 3=strong) of staining were assessed. A composite score (CS) was calculated by multiplying the extent by intensity (range=0-12; weak=1-3; moderate=4-8 and strong=9-12). Results: The mean CS for PD-L1 in TILs was significantly higher in PABC (5.86) compared to controls (3.07), p=0.03. Further, strong expression of PD-L1 in TILs was only observed in PABC (9/21, 42.9%); none of the controls had strong PD-L1, p=0.01. The high expression of PD-L1 in PABC TILs was independent of tumor grade, hormone receptor and HER2 status, and other histologic features including lymph node metastasis. Expression of PD-1 in TILs was similar in both PABC and controls (mean CS 6.81 and 5.36, respectively). Immunoreactivity in the tumor cells themselves was rare with only two PABC and four control cases expressing PD-1 and PD-L1. Conclusion: 1. TILs in PABC have significantly higher PD-L1 expression. 2. Strong expression of PD-L1 in TILs was only observed in PABC. 3. High PD-L1 expression in TILs was independent of the tumor characteristics in this series. 4. PD-1 is expressed similarly in TILs in both PABC and controls. 5. Rare cases may have PD-1 and PD-L1 expression in the tumor cells themselves. The results of our study showing significant expression of PD-L1 in PABC TILs add to the understanding of the role of the microenvironment in breast cancer progression. These complex interactions between tumors cells and the local immune system may predict response to therapy and investigation into the role of immune based therapies is under way in these aggressive breast carcinomas that affect young women. Citation Format: Blanco Jr LZ, Pincus JL, Siziopikou KP. PD-L1 is highly expressed in tumor infiltrating lymphocytes in pregnancy associated breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD6-07.

Background: Ductal carcinoma in situ (DCIS) consists of a heterogeneous group that can be subclassified by molecular signatures. High grade DCIS is commonly associated with periductal inflammation, a feature associated with increased risk for microinvasion. We previously assessed the immune microenvironment of DCIS and reported that HER2+ DCIS has increased numbers of tumor infiltrating lymphocytes (TILs), with significant expression of PD-L1. CTLA-4 is a supplementary immunomodulatory marker, which serves as a negative regulator of T-cell response. Targeted therapy is currently available against both PD-L1 and CTLA-4, and studies suggest an enhanced response with combination therapy as these therapies act though different mechanisms. In this study we evaluated the expression of CTLA-4 in both TILs and tumor cells in DCIS. Design: The study population consisted of 74 cases of DCIS treated with surgical excision from 2008-2012. The molecular subtypes of DCIS were determined based on ER and HER2 expression. Tissue microarrays were constructed (3 cores/case) to account for tumor heterogeneity. The TMAs were immunostained for CTLA-4 and the expression CTLA-4 was assessed. Results: Of the 74 cases, 41 were classified as Luminal A (ER+/HER2-), 17 as Luminal B (4 as Luminal B HER2- [ER+/HER2-/high Ki-67] and 13 as Luminal B HER2+ [ER+/HER2+]), 11 as HER2+ (ER-/HER2+) and 5 as basal (ER-/HER2-). Overall, the DCIS tumor cells expressed CTLA-4 in 58% of Luminal B (HER2+ 53% [7/13]; HER2- 75% [3/4]) and 36% of HER2+ (4/11), while only 39% of Luminal A (16/41), and one basal DCIS (1/5) did. In addition, the presence of CTLA-4 positive TILs was strongly associated with HER2+ expression (Luminal B HER2+=21.8%, HER2+=13.4%), compared to HER2- DCIS (Luminal B HER2-=8.5%, Luminal A=7.3%, Basal=6.4%) (p=0.026). Conclusion: 1. CTLA-4 expression in TILs is increased in HER2+ DCIS. 2. A significant percent of tumor cells express CTLA-4 in all DCIS subtypes. Our results further advance our understanding of the role of the immune microenvironment in DCIS. Additional studies are needed to further elucidate the complex interplay between the host immune system and DCIS, which may offer insights into breast carcinoma progression and possible use of novel immune-based therapies. Citation Format: Jorge E. Novo, Lauren Rosen, Julianne M. Ubago, Jennifer Pincus, Luis Blanco, Kalliopi P. Siziopikou. Cytotoxic T lymphocyte antigen 4 (CTLA-4) expression in ductal carcinoma in situ (DCIS) of the breast [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 625.

A number of breast lesions diagnosed by pathologic examination of breast tissue are known to confer an increased risk for future development of breast cancer. Atypical ductal hyperplasia (ADH) and atypical lobular hyperplasia (ALH) are lesions conferring a moderately increased risk of 4-5x over baseline. Lobular carcinoma in situ (LCIS) and variants confer a markedly increased risk of 8-10x over the baseline. The magnitude of the risk, the effect of the patient's age and family history, the laterality of the future malignancy and the type of subsequent cancer development are different between all these high risk proliferative breast lesions. In addition, the spectrum of columnar cell lesions of the breast are thought to represent an early non-obligate precursor in the development of the low grade neoplasia of the breast. Of interest, many of these lesions share common genetic abnormalities that are different than those seen in high grade in situ and invasive carcinomas of the breast. These observations have implications for our current understanding of the breast cancer progression model and our recommendations for the therapeutic management of patients with high risk proliferative lesions of the breast. Citation Format: Siziopikou KP. The pathology of high risk proliferative lesions of the breast [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 ES2-1.

Mutations of the BRCA1/2 genes have been described in association with a number of malignancies including cancers of the breast, ovary, prostate and stomach, but have never been described in relation to gastrointestinal stromal tumours (GIST). We describe a patient with a BRCA2 8642del3insC mutation who developed prostate cancer, breast cancer and GIST. GIST has been shown to be associated with a number of malignancies, including some of the common BRCA1/2-related cancers, but it has never been associated with BRCA1/2 gene mutations. This report highlights the potential association between BRCA1/2 mutations and GIST, and aims to raise awareness for further genetic screening in GIST patients.

Truncated Notch receptors have transforming activity in vitro and in vivo. However, the role of wild-type Notch signaling in neoplastic transformation remains unclear. Ras signaling is deregulated in a large fraction of human malignancies and is a major target for the development of novel cancer treatments. We show that oncogenic Ras activates Notch signaling and that wild-type Notch-1 is necessary to maintain the neoplastic phenotype in Ras-transformed human cells in vitro and in vivo. Oncogenic Ras increases levels and activity of the intracellular form of wild-type Notch-1, and upregulates Notch ligand Delta-1 and also presenilin-1, a protein involved in Notch processing, through a p38-mediated pathway. These observations place Notch signaling among key downstream effectors of oncogenic Ras and suggest that it might be a novel therapeutic target.

Molecular biomarkers, a cornerstone of precision oncology, are critical in breast, gastroesophageal, and non-small cell lung cancer management (BC, GEC, NSCLC). Testing practices are intensely debated, impacting diagnostic quality and affecting pathologists, oncologists and patients. However, little is known about testing approaches used in practice. Our study described biomarker practices in BC, GEC, and NSCLC at the leading US cancer centers.We conducted a survey of the National Cancer Institute (NCI) designated centers on BC, GEC, and NSCLC biomarker testing. We used simple frequencies to describe practices, two-sided Fisher's exact test and two-sided McNemar's test for cross-cancer comparison. All statistical tests were two-sided.For BC human epidermal growth factor receptor 2 (HER2), 39% of centers combine guidelines by using in situ hybridization (ISH) and immunohistochemistry (IHC) concurrently, and 21% reflex-test beyond guideline-recommended IHC2+. For GEC HER2, 44% use ISH and IHC concurrently, and 28% reflex-test beyond IHC2+. In NSCLC, the use of IHC is limited to 4% for epidermal growth factor receptor (EGFR) and 7% for anaplastic lymphoma kinase (ALK). 43.5% test NSCLC biomarkers on oncologist order; 34.5% run all biomarkers upfront, and 22% use a sequential protocol. NSCLC external testing is statistically significantly higher than BC (P.0001) and GEC (P.0001). NSCLC internally developed tests are statistically significantly more common than BC (P.0001) and GEC (P.0001).At the NCI cancer centers, biomarker testing practices vary, but exceeding guidelines is a common practice for established biomarkers and emerging practice for newer biomarkers. Use of internally developed tests declines as biomarkers mature. Implementation of multibiomarker protocols is lagging. Our study represents a step toward developing a biomarker testing practice landscape.

Tumor metastasis remains the major cause of cancer-related death, but its molecular basis is still not well understood. Here we uncovered a splicing-mediated pathway that is essential for breast cancer metastasis. We show that the RNA-binding protein heterogeneous nuclear ribonucleoprotein M (hnRNPM) promotes breast cancer metastasis by activating the switch of alternative splicing that occurs during epithelial–mesenchymal transition (EMT). Genome-wide deep sequencing analysis suggests that hnRNPM potentiates TGFβ signaling and identifies CD44 as a key downstream target of hnRNPM. hnRNPM ablation prevents TGFβ-induced EMT and inhibits breast cancer metastasis in mice, whereas enforced expression of the specific CD44 standard (CD44s) splice isoform overrides the loss of hnRNPM and permits EMT and metastasis. Mechanistically, we demonstrate that the ubiquitously expressed hnRNPM acts in a mesenchymal-specific manner to precisely control CD44 splice isoform switching during EMT. This restricted cell-type activity of hnRNPM is achieved by competition with ESRP1, an epithelial splicing regulator that binds to the same cis-regulatory RNA elements as hnRNPM and is repressed during EMT. Importantly, hnRNPM is associated with aggressive breast cancer and correlates with increased CD44s in patient specimens. These findings demonstrate a novel molecular mechanism through which tumor metastasis is endowed by the hnRNPM-mediated splicing program.

Tumor growth is the net result of cell proliferation and cell death. To investigate the relationship between these phenomena in ductal carcinoma in situ (DCIS), we studied proliferation index (PI) in DCIS in relation to the expression of two proteins involved in the regulation of cell death, bcl-2 and p53. Thirty-nine consecutive cases of DCIS were studied. PI was determined using immunolabeling with the monoclonal antibody MIB-1. The proportion of MIB-1-positive nuclei among 500 tumor cell nuclei was determined for each case and constituted the PI. All cases were also assessed immunohistochemically for bcl-2 and p53 protein expression. DCIS cases were graded using the criteria of Holland et al. PI ranged from 0 to 57% (mean 11.2%, median 4.6%). PI was significantly lower in well-differentiated and intermediately differentiated DCIS cases (mean 7.3% and 4.8%, respectively) than in poorly differentiated lesions (mean 24%, p = 0.01). PI was significantly lower in bcl-2-positive cases than in bcl-2-negative cases (mean PI for bcl-2-positive cases 6% and for bcl-2-negative cases 26%, p = 0.01). PI was higher in lesions expressing the p53 protein than in p53 negative cases (19% versus 8.3%), but this difference did not reach statistical significance. PI was also examined in relation to combinations of bcl-2 and p53 expression. Twenty-five of the DCIS lesions (64%) showed the bcl-2-positive/p53-negative phenotype which is similar to that seen in normal breast tissue and benign lesions and can be considered the “physiologic” combination. Among these cases the mean PI was 6.4%. In contrast, 14 cases showed “aberrant” combinations of bcl-2 and p53 expression suggesting dysregulated control of apoptosis. Among these cases the mean PI was 19.6% (p = 0.03). The highest mean PI was in cases with the bcl-2-negative/p53-positive phenotype (PI = 29.7%). DCIS lesions with the physiologic bcl-2-positive/p53-negative phenotype have low PI. In contrast, DCIS lesions with “aberrant” bcl-2/p53 phenotypes have high PI. This combination may favor tumor growth and progression in DCIS.

While epidermal growth factor receptor (EGFR)-targeted therapy has been very promising in a number of human malignancies, to date these targeted biologic agents have not proven effective in breast cancer. However, the EGFR tyrosinase inhibitors have been used indiscriminately against all types of breast tumors, perhaps missing a subpopulation of patients who may be prime candidates for EGFR-targeted therapy. In this communication we propose that patients with estrogen receptor (ER)-negative/progesterone receptor (PR)-negative/HER-2-negative tumors, which currently present a therapeutic challenge for the oncologist, may be the subgroup of breast cancer patients that might benefit from specific EGFR-targeted therapies.

Context.—In situ carcinomas of the breast constitute 15% to 30% of all newly diagnosed breast cancer cases; 80% of these in situ lesions belong to the ductal carcinoma in situ (DCIS) category. Similar to invasive breast carcinomas, DCIS is not a single disease but rather many distinct diseases with different histopathologic and molecular characteristics, a propensity to progress to invasive disease, and differential response to treatment.Objective.—To review the classic pathologic parameters of clinical significance and the differential diagnosis of the DCIS lesions, present our new understanding of the importance of biomarkers, and discuss innovative approaches for targeted therapy in DCIS.Data Sources.—Extensive review of the relevant peer-reviewed literature.Conclusions.—In DCIS, improved understanding of the underlying biologic pathways of tumor progression is expected to lead to more accurate classification and innovative targeted treatment approaches for the management of these lesions.

Background: HER2-positive breast cancers comprise 20-30% of all breast cancers; amplification of HER2 confers a poorer prognosis. While treatment with anti-HER2 targeted therapies has dramatically improved outcomes in this group, a significant subset of patients develops resistance, highlighting the need for novel therapeutic options. In addition, like triple negative breast carcinomas, HER2-positive invasive carcinomas also consist of different intrinsic subtypes with different responses to therapy and different overall prognosis. Finally, recent reports suggest that bcl-2 expression contributes to resistance to anti-HER2 therapies. In this study, we evaluated expression of bcl-2 in HER2-positive breast tumors and correlated the expression of bcl-2 with HER2 subtype, patient demographics and histologic and molecular characteristics. Design: The study population consisted of 114 patients with HER2-positive invasive breast carcinoma treated with surgical excision or mastectomy at Northwestern Memorial Hospital (2009-14) (mean age 53, range 18-80). Electronic medical records were reviewed for patient demographics. Pathologic tumor characteristics (histologic type, size, grade, lymph node status) and tumor marker profile (ER, PR, p53 and Ki-67) were evaluated. Tissue microarrays were constructed (3 cores from each case to account for tumor heterogeneity) for immunohistochemical evaluation of bcl-2 (Dako, clone 124). Results: Overall, these HER2-pos breast carcinomas were almost exclusively of ductal histologic type (101/114, 88.6%). Almost ¾ of the cases were poorly differentiated, grade 3 carcinomas (83/114, 73%), while the other 31 were grade 2 tumors. No grade 1 tumors were seen. bcl-2 was expressed in 64/114 (56%) of the HER2-pos carcinomas. No association of the bcl-2 expression with age, race, tumor size, lymph node status, p53 expression, or Ki-67 proliferation index was seen. Of interest, this sample of HER2-pos carcinomas was almost equally balanced between ER-neg/HER2-pos (HER2 subtype) and ER-pos/HER2-pos (Luminal B subtype), 54 vs 60 cases respectively. bcl-2 was differentially expressed in these two HER2-pos tumor subtypes: only 9/54 (14%) of the HER2 subtype expressed bcl-2 compared to 55/64 (85.9%) of the Luminal B subtype (p Conclusions: 1. bcl-2 is expressed in just over half of the HER2-pos carcinomas. 2. bcl-2 expression is far more common in Luminal B compared to the HER2-pos subtype of breast carcinomas. 3. bcl-2 expression does not appear to have a differential expression between age or racial groups and does not correlate with tumor size or lymph node status. Our findings add to the understanding of the molecular mechanisms that drive the different subtypes of HER2-pos breast carcinomas and raise the possibility that bcl-2 targeted therapies may lead to novel therapeutic approaches for the management of subgroups of patients with HER2-pos breast tumors. Citation Format: Kalliopi P. Siziopikou, Alexandra Larson, Luis Z. Blanco, Virginia Kaklamani. bcl-2 expression in HER2-positive breast carcinoma subtypes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2271.

Background: Solid papillary carcinoma with reverse polarization (SPCRP) is a rare breast cancer subtype, whose most striking morphologic feature is the presence of back-to-back columnar epithelial cells with nuclei situated in an apical rather than in the normal basal location. We sought to characterize the morphologic and genetic landscape of SPCRP and determine whether it represents a distinct subtype of breast cancer underpinned by highly recurrent disease-specific genetic alterations. Material and Methods: Archival sections of 13 SPCRPs were subjected to microdissection. DNA samples extracted from microdissected tumor and matched normal samples were subjected to whole exome sequencing (n = 2) on an Illumina HiSeq2000, and DNA samples from tumors were further subjected to targeted massively parallel sequencing (n = 1), SNaPshot profiling (n = 7) and/or Sanger sequencing. State-of-the-art bioinformatics methods were used to define somatic mutations. Non-malignant breast epithelial MCF10A cells with and without somatic PIK3CA H1047R knock-in were used to assess the functional impact of the mutations identified by sequencing of SPCRPs in two- and three-dimensional cell culture systems. Results: Ten of 13 (77%) SPCRPs were found to harbor IDH2 R172 hotspot mutations, and eight of these cases had concurrent pathogenic mutations affecting PIK3CA or PIK3R1, in particular PIK3CA H1047R hotspot mutations. Functional monolayer and three-dimensional cell culture studies demonstrated that IDH2 R172 and PIK3CA mutations constitute likely drivers of this tumor and contribute to its unusual nuclear polarization phenotype. Conclusions: Our results suggest that SPCRP is a distinct subtype of breast cancer underpinned by IDH2 hotspot mutations in conjunction with mutations affecting canonical genes of the PI3K pathway. Given that IDH2 hotspot mutations have not been described in breast cancer to date, these may be used as a diagnostic ancillary marker for SPCRPs. Furthermore, IDH2 mutations may serve as a potential therapeutic target in SPCRP patients with disseminated disease. Citation Format: Sarah Chiang, Britta Weigelt, Huei-Chi Wen, Salvatore Piscuoglio, Luciano G. Martelotto, Charlotte K Y Ng, Jörg Balss, Gabrielle Baker, Kimberly S. Cole, Andreas von Deimling, Julie M. Batten, Jonathan D. Marotti, Hwei-Choo Soh, Benjamin L. McCalip, Raymond S. Lim, Kalliopi P. Siziopikou, Randi Burke, Song Lu, Xiaolong Liu, Tarek Hammour, Edi Brogi, A John Iafrate, Jorge S. Reis-Filho, Stuart J. Schnitt. Solid papillary carcinoma with reverse polarization are driven by IDH2 and PI3K pathway mutations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 138.

Recent studies have identified a small population of highly tumorigenic cells with stem cell properties in human breast and other solid tumors that are considered to be the source of tumor initiation and maintenance; these cells are referred to as cancer stem cells (CSCs). Preclinical data suggest that current breast cancer treatment strategies lead to CSC enrichment, contributing to chemotherapy and radiotherapy resistance, although a strong correlation with clinical parameters and prognosis is yet to be established. Importantly, overcoming treatment failure by effective targeting of CSCs may be an appealing approach, potentially leading to improved clinical outcomes for patients with breast cancer. Several preclinical studies provide promising results that support this hypothesis. The purpose of this review is to summarize the role of CSCs in breast cancer recurrence and resistance and to discuss current attempts of CSC targeting.

Background Obesity has been shown to increase breast cancer risk. FTO is a novel gene which has been identified through genome wide association studies (GWAS) to be related to obesity. Our objective was to evaluate tissue expression of FTO in breast and the role of FTO SNPs in predicting breast cancer risk. Methods We performed a case-control study of 354 breast cancer cases and 364 controls. This study was conducted at Northwestern University. We examined the role of single nucleotide polymorphisms (SNPs) of intron 1 of FTO in breast cancer risk. We genotyped cases and controls for four SNPs: rs7206790, rs8047395, rs9939609 and rs1477196. We also evaluated tissue expression of FTO in normal and malignant breast tissue. Results We found that all SNPs were significantly associated with breast cancer risk with rs1477196 showing the strongest association. We showed that FTO is expressed both in normal and malignant breast tissue. We found that FTO genotypes provided powerful classifiers to predict breast cancer risk and a model with epistatic interactions further improved the prediction accuracy with a receiver operating characteristic (ROC) curves of 0.68. Conclusion In conclusion we have shown a significant expression of FTO in malignant and normal breast tissue and that FTO SNPs in intron 1 are significantly associated with breast cancer risk. Furthermore, these FTO SNPs are powerful classifiers in predicting breast cancer risk.

The role of MRI in the management of breast carcinoma is rapidly evolving from its initial use for specific indications only to a more widespread use on all women with newly diagnosed early stage breast cancer. However, there are many concerns that such widespread use is premature since detailed correlation of MRI findings with the underlying histopathology of the breast lesions is still evolving and clear evidence for improvements in management and overall prognosis of breast cancer patients evaluated by breast MRI after their initial cancer diagnosis is lacking. In this paper, we would like to bring attention to a benign lesion that is frequently present on MRI-guided breast biopsies performed on suspicious MRI findings in the affected breast of patients with a new diagnosis of breast carcinoma.

Partial breast irradiation post-lumpectomy, with a balloon bearing a radioactive source in its center, is practiced as an alternative to whole breast irradiation in the treatment of breast cancer. The goal is to ablate residual malignant cells within 1 cm radius of the resected lumpectomy margin. We hypothesize that this goal may be achieved with a fluid-filled heated balloon.Nubian-cross goats were treated under general anesthesia. The two mammary glands were sequentially bisected and a non-inflated balloon with a heating element was placed in the center of the gland which was re-sutured. Two series of experiments were conducted. In the first 22 goats (44 glands), the balloon was inflated with 5% dextrose to a pressure of 150 mmHg and heated at 87 degrees C over selected time intervals of 1-24 minutes. In the second series (16 glands), the re-programmed device operated at 50-80 mmHg over selected time intervals of 5-20 minutes. The depth of necrosis was histologically determined after sacrificing the goats and excising the glands.In the first series, glandular necrosis was noted to extend to a depth of 3.2-9.6 mm for the above heating cycles. Corresponding figures for the second series ranged from 4.7-8.6 mm for treatment times of one minute 'warm up' to 20 minutes of heating at 90 degrees C. The animals exhibited no systemic side effects post-treatment.An experimental model describing a thermal technique causing necrosis of the goat mammary gland is described.

The capacity of alveolar macrophages and peripheral blood monocytes from patients with non-small cell lung cancer to develop tumoricidal function after in vitro stimulation with different macrophage activators was investigated. Alveolar macrophages were found to be impaired in their ability to develop cytotoxic activity compared with either the peripheral blood monocytes from the same patients or alveolar macrophages from patients with nonmalignant lung disorders. This result was observed consistently under diverse culture conditions and with different macrophage activators including gamma-interferon (gamma-IFN), granulocyte-macrophage colony-stimulating factor (GM-CSF), phorbol myristate acetate, or endotoxin. The impairment in tumoricidal function observed in alveolar macrophages was not associated with reduced target cell binding compared to peripheral blood monocytes. Alveolar macrophages from patients with lung cancer were found to secrete significantly greater amounts of tumor necrosis factor (TNF) and interleukin-1 (IL-1) than either peripheral blood monocytes from the same patients or alveolar macrophages from the patients with nonmalignant disorders. These results are consistent with either different regulatory pathways for cytotoxicity and cytokine secretion in the alveolar macrophages of patients with lung cancer or diversity in the subpopulations of cells responsible for these functions.

Few individual cases of invasive cystic hypersecretory ductal carcinoma of the breast have been described. Review of 33 cases of cystic hypersecretory carcinoma, including the current case, indicate that only 6 cases presented with invasive disease. Two of these cases had positive nodes and 2 had distal metastases. The case presented here is unique in an additional aspect: the contralateral breast harbored lobular breast carcinoma 10 years after mastectomy of the first malignancy. Bilateral breast disease resulting in bilateral mastectomies over long-term follow-up, as in the case presented here, was reported in 3 of 33 cases.

High expression of Notch-1 and Jagged-1 mRNA correlates with poor prognosis in breast cancer. Elucidating the cross-talk between Notch and other major breast cancer pathways is necessary to determine which patients may benefit from Notch inhibitors, which agents should be combined with them, and which biomarkers indicate Notch activity in vivo. We explored expression of Notch receptors and ligands in clinical specimens, as well as activity, regulation, and effectors of Notch signaling using cell lines and xenografts. Ductal and lobular carcinomas commonly expressed Notch-1, Notch-4, and Jagged-1 at variable levels. However, in breast cancer cell lines, Notch-induced transcriptional activity did not correlate with Notch receptor levels and was highest in estrogen receptor α–negative (ERα−), Her2/Neu nonoverexpressing cells. In ERα+ cells, estradiol inhibited Notch activity and Notch-1IC nuclear levels and affected Notch-1 cellular distribution. Tamoxifen and raloxifene blocked this effect, reactivating Notch. Notch-1 induced Notch-4. Notch-4 expression in clinical specimens correlated with proliferation (Ki67). In MDA-MB231 (ERα−) cells, Notch-1 knockdown or γ-secretase inhibition decreased cyclins A and B1, causing G2 arrest, p53-independent induction of NOXA, and death. In T47D:A18 (ERα+) cells, the same targets were affected, and Notch inhibition potentiated the effects of tamoxifen. In vivo, γ-secretase inhibitor treatment arrested the growth of MDA-MB231 tumors and, in combination with tamoxifen, caused regression of T47D:A18 tumors. Our data indicate that combinations of antiestrogens and Notch inhibitors may be effective in ERα+ breast cancers and that Notch signaling is a potential therapeutic target in ERα− breast cancers. [Cancer Res 2008;68(13):5226–35]

Extrapulmonary neuroendocrine carcinoma is uncommon. Cases of primary neuroendocrine carcinoma of the breast have been reported, though rare. We report the case of a 53-year-old woman who underwent a mastectomy for breast carcinoma and presented three years later with synchronous masses in the head of the pancreas and liver. Fine-needle aspiration of both organs revealed a neuroendocrine carcinoma. The original breast tumor was reviewed and found to express neuroendocrine markers. A diagnosis of a primary neuroendocrine carcinoma of the breast was rendered.