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Purpose: Malignant phyllodes tumors (MPT) are exceptionally rare, and the genomic drivers of these tumors are still being elucidated. We performed comprehensive genomic profiling (CGP) of MPT to identify genomic alterations that will inform approaches to targeted therapy for patients with MPT, including relapsed, refractory, and metastatic disease., Methods: DNA was extracted from formalin-fixed, paraffin-embedded samples from 24 consecutive patient cases of MPT. CGP was performed using a hybrid capture, adaptor ligation-based next generation sequencing assay to a high, uniform coverage (mean, 582×). Tumor mutational burden (TMB) was calculated from a minimum of 1.14 Mb of sequenced DNA as previously described and reported as mutations/Mb. The results were analyzed for all classes of genomic alterations, including short variants (SV; base substitutions, small insertions, and deletions), rearrangements, and copy number changes, including amplifications and homozygous deletions., Results: The 24 cases of MPT included 15 patients with localized and 9 with metastatic disease. The median TMB was 2.7 mut/Mb, and no cases had a TMB > 10 mut/Mb. 20 out of 24 cases were evaluable for microsatellite status, and all were microsatellite stable. The most commonly mutated genes were TP53 (58.3%), TERT-promoter (57.9%), NF1 (45.8%), MED12 (45.8%), CDKN2A/B (33.3%), and MLL2 (33.3%). Targetable kinase fusions including KIAA1549-BRAF or FGFR3-TACC3 were identified in 2/24 (8.3%) tumors., Conclusions: This study identifies clinically relevant genomic alterations that suggest novel targeted therapy approaches for patients with MPT.

Context: Metastatic metaplastic breast carcinoma (MPBC) is an uncommon, but aggressive, tumor resistant to conventional chemotherapy., Objective: To learn whether next-generation sequencing could identify potential targets of therapy for patients with relapsed and metastatic MPBC., Design: Hybridization capture of 3769 exons from 236 cancer-related genes and 47 introns of 19 genes commonly rearranged in cancer was applied to a minimum of 50 ng of DNA extracted from 20 MPBC formalin-fixed, paraffin-embedded specimens and sequenced to high uniform coverage., Results: The 20 patients with MPBC had a median age of 62 years (range, 42-86 years). There were 9 squamous (45%), 9 chondroid (45%), and 2 spindle cell (10%) MPBCs, all of which were high grade. Ninety-three genomic alterations were identified, (range, 1-11) with 19 of the 20 cases (95%) harboring an alteration that could potentially lead to a targeted treatment option. The most-common alterations were in TP53 (n = 69; 75%), PIK3CA (n = 37; 40%), MYC (n = 28; 30%), MLL2 (n = 28; 30%), PTEN (n = 23; 25%), CDKN2A/B (n = 19; 20%), CCND3 (n = 14; 15%), CCNE1 (n = 9; 10%), EGFR (n = 9; 10%), and KDM6A (n = 9; 10%); AKT3, CCND1, CCND2, CDK4, FBXW7, FGFR1, HRAS, NF1, PIK3R1, and SRC were each altered in a single case. All 16 MPBCs (100%) that were negative for ERBB2 (HER2) overexpression by immunohistochemistry and/or ERBB2 (HER2) amplification by fluorescence in situ hybridization were also uniformly (100%) negative for ERBB2 amplification by next-generation sequencing-based copy-number assessment., Conclusions: Our results indicate that genomic profiling using next-generation sequencing can identify clinically meaningful alterations that have the potential to guide targeted treatment decisions in most patients with metastatic MPBC.

Purpose: We queried whether comprehensive genomic profiling using a next-generation sequencing-based assay could identify novel and unanticipated targets of therapy for patients with relapsed invasive lobular carcinoma (ILC)., Experimental Design: DNA sequencing (Illumina HiSeq 2000) was conducted for 3,320 exons of 182 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer on indexed, adaptor-ligated, hybridization-captured libraries using DNA isolated from formalin-fixed paraffin-embedded sections from 22 histologically verified ILC., Results: A total of 75 genomic alterations were identified with an average of 3.4 alterations per tumor (range, 1-6), of which 35 were actionable for an average of 1.59 actionable alterations per patient (range, 0-3). Nineteen of 22 (86%) of the ILC samples harbored at least one actionable alteration. Six (27%) cases featured alterations in ERRB2 including 4 (18%) with ERBB2 mutation, 1 (5%) with an ERBB2 gene fusion, and 1 (5%) with an ERBB2 copy number gain (amplification). The enrichment of ERBB2 mutations/fusion in CDH1-mutated ILC (5 of 22, 23%) compared with the 5 ERBB2 mutations in a series of 286 non-CDH1-mutated breast cancers from which the ILC cases were obtained (5 of 286, 2%) was significant (P = 0.0006)., Conclusions: Comprehensive genomic profiling of relapsed CDH1-mutated ILC revealed actionable genomic alterations in 86% of cases, featured a high incidence of ERBB2 alterations, and can reveal actionable alterations that can inform treatment decisions for patients with ILC., (©2013 AACR)

Objective: To examine fluorescence in situ hybridization (FISH) in HER2 amplification in response rates to trastuzumab therapy and both taxane and anthracycline-based chemotherapy regimens., Study Design: A total of 400 tumor sections were analyzed over an 8-month period. The sections were hybridized with probes for the HER2 gene and chromosome 17 centromere using standard FISH methods and analyzed on an automated fluorescence microscopy system., Results: Reliable and valid methods for identification of the patients that will respond to treatment with trastuzumab are needed in order to achieve maximum therapy efficacy and maintain cost efficiency. FISH-based analysis is potentially an objective and reproducible approach to determination of HER2 gene status; however, manual FISH counting is a laborious task and subject to inter and intraobserver variability., Conclusion: The system described in this paper is a valuable tool in providing a consistent approach to the interpretation of breast tumor tissue analyzed by FISH analysis. In addition to consistency, an automated system provides a record of the images produced that can be of immediate benefit in multiple review of a difficult or equivocal case and long-term benefit in terms of providing a permanent case record.

Purpose: BLID is a BH3-like motif containing apoptotic member of the Bcl-2 family of proteins. This study was designed to investigate the mechanism of BLID-induced apoptosis and to assess the significance of BLID expression in breast cancer., Experimental Design: The interaction between BLID and Bcl-X(L) was examined using in vitro transcription/translation, coimmunoprecipitation, and immunoflourescence assays. The relationship between BLID mRNA expression and pathologic measures in breast cancer specimens (n = 55) was examined using the publicly available ONCOMINE microarray database. Immunohistochemistry was done using formalin-fixed, paraffin-embedded sections of 148 cases of invasive ductal breast carcinomas (IDC) and 58 cases of invasive lobular breast carcinomas, and breast tissue microarrays representing additional 437 cases (>85% IDC) with associated clinicopathologic database and long-term clinical follow-up (median 7 years)., Results: BLID was found to interact with Bcl-X(L), and the binding was enhanced in cancer cells exposed to doxorubicin or cisplatin. Exogenous expression of BLID correlated with activation of Bax and an increase in cytosolic cytochrome c. BLID mRNA expression was significantly reduced in grade 3 relative to grade 1 and 2 breast cancer (P = 0.023). Cytoplasmic BLID immunoreactivity was absent in IDC compared with invasive lobular breast carcinoma (P < 0.001). Lack of BLID expression was associated with younger age (median 40 years), African American ethnicity, tumor size, and triple-negative breast cancer (estrogen receptor negative, progesterone receptor negative, and human epidermal growth factor receptor 2 negative; all P < 0.005). Significant correlations were observed between BLID negativity and declines in overall, cause-specific, and local relapse-free survival (all P < 0.03). Multivariate analysis indicated that BLID is an independent prognostic factor of distant metastasis-free survival (hazard ratio, 0.302; 95% confidence interval, 0.160-0.570, P = 0.0002)., Conclusion: BLID is a new binding partner of Bcl-X(L) and a significant prognostic factor in breast cancer., (Copyright 2010 AACR.)

WWP1, a HECT type E3 ubiquitin ligase frequently amplified and overexpressed in breast cancer, has the potential to become a useful clinical biomarker and therapeutic target in breast cancer. Here, we performed immunohistochemical staining in formalin-fixed and paraffin-embedded tissue sections from 187 cases of primary invasive mammary carcinoma [137 ductal carcinomas (IDC) and 50 lobular carcinomas (ILC)] by using a monoclonal anti-WWP1 antibody. The normal breast epithelium and adjacent benign epithelium are essentially negative for WWP1. Cytoplasmic WWP1 immunoreactivity was observed in 76/187 (40.6%) tumors and showed a positive correlation with ERalpha (p = 0.05) and IGF-1R proteins (p = 0.001) in this cohort. The positive correlations between WWP1 and ER/IGF-1R were also observed in a panel of 12 breast cancer cell lines by Western blot. Interestingly, the ER levels are decreased when WWP1 is silenced in ER positive MCF7 and T47D breast cancer cell lines. Finally, WWP1 ablation collectively inhibits cell proliferation with tamoxifen in MCF7 and T47D, as measured by (3)H-thymidine incorporation assays. These findings suggest that WWP1 may play an important role in ER positive breast cancer., (Copyright 2008 UICC.)

Background: Abnormal expression of the HER-2/neu oncogene, a tyrosine kinase-type transmembrane growth factor receptor localized to chromosome 17q, has been associated with poor prognosis and the prediction of therapy response in invasive breast cancer. The comparative incidence and significance of HER-2/neu gene amplification for lobular and ductal breast cancer have not been previously characterized., Design: Formalin-fixed, paraffin-embedded primary breast cancer tissue sections from 71 women diagnosed with invasive lobular carcinoma were tested for HER-2/neu gene amplification by fluorescence in situ hybridization (FISH) method using the Ventana unique sequence probe (Ventana Medical Systems, Tucson, AZ). A series of 106 cases of invasive ductal carcinoma was similarly processed and tested. Lymph node status was available for 155 (88%) of the 177 cases and 82 (46%) were lymph node-negative (LN-) and 73 (41%) were lymph node-positive (LN+). Patients were treated for a mean of 65 months (range 1-169 months)., Results: 9 of 71 (13%) cases of lobular cancer featured HER-2/neu gene amplification, whereas 51 (48%) of 106 cases of ductal cancer showed amplification (P < 0.0001). On univariate analysis of combined lobular and ductal cases, HER-2/neu gene amplification detected by FISH predicted disease-related death (P < 0.0001). HER-2/neu gene amplification also predicted disease-related death in lobular cases alone (P = 0.003), LN+ lobular cases separately (P = 0.019), and LN- and LN+ ductal cases separately and alone (P < 0.0001). Multivariate analysis of the lobular group alone revealed that LN+ status (P = 0.015) and stage (P = 0.01) were independent predictors of disease-related death, and HER-2/neu gene amplification reached near significance (P = 0.086). In the ductal carcinoma group alone, HER-2/neu gene amplification (P = 0.03), lymph node status (P = 0.0001), tumor stage (P = 0.0001), and tumor grade (P = 0.044) were independent predictors of overall disease survival., Conclusions: HER-2/neu gene amplification detected by FISH was identified at a significantly lower rate in lobular compared with ductal breast cancer. HER-2/neu gene amplification when present in lobular breast cancer is a significant adverse prognostic factor.

The HER-2/neu oncogene, localized to chromosome 17q, shares substantial homology with the epidermal growth factor receptor. HER-2/neu gene amplification and protein overexpression have been associated with poor prognosis in breast cancer. Formalin-fixed paraffin-embedded primary invasive breast cancer tissues from 135 women were tested for HER-2/neu gene amplification by automated fluorescence in situ hybridization (FISH) using a sequence probe. The tumors also were evaluated by immunohistochemistry for proliferation markers Ki 67 (MIB1) and p34cdc2 cyclin-dependent kinase. Patients were followed up for a mean of 61 months. There were 70 node-negative and 65 node-positive cases. Ki 67 and p34cdc2 proliferation marker overexpression, HER-2/neu oncogene amplification, large tumor size, high tumor grade, advanced tumor stage, positive lymph node status, and distant metastasis at the time of diagnosis predicted disease-related death in combined node-negative and node-positive breast cancer. HER-2/neu gene amplification, tumor stage, lymph node metastasis, tumor grade, and distant metastasis at the time of diagnosis independently predicted disease outcome. HER-2/neu amplification detected by FISH also predicted disease-related death independent of lymph node status, tumor grade, and distant metastasis in breast cancer patients who received adjuvant therapy.