Carey A. Eberle

A Phase II Trial of Erlotinib in Combination with Bevacizumab in Patients with Metastatic Breast Cancer

Purpose: To evaluate the efficacy and toxicity of erlotinib plus bevacizumab in patients with metastatic breast cancer (MBC), targeting the epidermal growth factor receptor (EGFR/HER1) and the vascular endothelial growth factor (VEGF) pathway. Experimental Design: Thirty-eight patients with MBC were enrolled and treated at two institutions with erlotinib, a small molecule EGFR tyrosine kinase inhibitor (150 mg p.o. daily) plus bevacizumab, an anti-VEGF antibody (15 mg/kg i.v. every 3 weeks). Patients had one to two prior chemotherapy regimens for metastatic disease. The primary end point was response rate by Response Evaluation Criteria in Solid Tumors criteria using a Simon 2-stage design. Secondary end points included toxicity, time to progression, response duration, and stabilization of disease of ≥26 weeks. Correlative studies were done on tumor tissue, including EGFR expression and mutation analysis. Results: One patient achieved a partial response for 52+ months. Fifteen patients had stable disease at first evaluation at 9 weeks; 4 of these patients had stable disease beyond 26 weeks. Median time to progression was 11 weeks (95% confidence interval, 8-18 weeks). Diarrhea of any grade was observed in 84% of patients (grade 3 in 3%); 76% experienced grade 1 or 2 skin rash, and 18% developed hypertension (grade 3 in 11%). The level of EGFR expression was not predictive of response to therapy. Conclusions: The combination of erlotinib and bevacizumab was well-tolerated but had limited activity in unselected patients with previously treated MBC. Biomarkers are needed to identify those MBC patients likely to respond to anti-EGFR/HER1 plus anti-VEGF therapy.

The Genomic Landscape of Male Breast Cancers

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

Metaplastic breast carcinoma: more than a special type

Metaplastic breast carcinoma (MBC) is a special histological type of invasive breast cancer. MBC is a descriptive and operational term for a heterogeneous collection of tumours with distinct histologies, clinical behaviours and potentially responses to therapy.

The Landscape of Somatic Genetic Alterations in Metaplastic Breast Carcinomas

Purpose: Metaplastic breast carcinoma (MBC) is a rare and aggressive histologic type of breast cancer, predominantly of triple-negative phenotype, and characterized by the presence of malignant cells showing squamous and/or mesenchymal differentiation. We sought to define the repertoire of somatic genetic alterations and the mutational signatures of MBCs. Experimental Design: Whole-exome sequencing was performed in 35 MBCs, with 16, 10, and 9 classified as harboring chondroid, spindle, and squamous metaplasia as the predominant metaplastic component. The genomic landscape of MBCs was compared with that of triple-negative invasive ductal carcinomas of no special type (IDC-NST) from The Cancer Genome Atlas. Wnt and PI3K/AKT/mTOR pathway activity was assessed using a qPCR assay. Results: MBCs harbored complex genomes with frequent TP53 (69%) mutations. In contrast to triple-negative IDC-NSTs, MBCs more frequently harbored mutations in PIK3CA (29%), PIK3R1 (11%), ARID1A (11%), FAT1 (11%), and PTEN (11%). PIK3CA mutations were not found in MBCs with chondroid metaplasia. Compared with triple-negative IDC-NSTs, MBCs significantly more frequently harbored mutations in PI3K/AKT/mTOR pathway–related (57% vs. 22%) and canonical Wnt pathway–related (51% vs. 28%) genes. MBCs with somatic mutations in PI3K/AKT/mTOR or Wnt pathway–related genes displayed increased activity of the respective pathway. Conclusions: MBCs are genetically complex and heterogeneous, and are driven by a repertoire of somatic mutations distinct from that of triple-negative IDC-NSTs. Our study highlights the genetic basis and the importance of PI3K/AKT/mTOR and Wnt pathway dysregulation in MBCs and provides a rationale for the metaplastic phenotype and the reported responses to PI3K/AKT/mTOR inhibitors in these tumors. Clin Cancer Res; 23(14); 3859–70. ©2017 AACR.

Integrative genomic and transcriptomic characterization of papillary carcinomas of the breast

Papillary carcinoma (PC) is a rare type of breast cancer, which comprises three histologic subtypes: encapsulated PC (EPC), solid PC (SPC) and invasive PC (IPC). Microarray‐based gene expression and Affymetrix SNP 6.0 gene copy number profiling, and RNA‐sequencing revealed that PCs are luminal breast cancers that display transcriptomic profiles distinct from those of grade‐ and estrogen receptor (ER)‐matched invasive ductal carcinomas of no special type (IDC‐NSTs), and that the papillary histologic pattern is unlikely to be underpinned by a highly recurrent expressed fusion gene or a highly recurrent expressed mutation. Despite displaying similar patterns of gene copy number alterations, significant differences in the transcriptomic profiles of EPCs, SPCs and IPCs were found, and may account for their different histologic features.

Microglandular adenosis associated with triple-negative breast cancer is a neoplastic lesion of triple-negative phenotype harbouring TP53 somatic mutations

Microglandular adenosis (MGA) is a rare proliferative lesion of the breast composed of small glands lacking myoepithelial cells and lined by S100‐positive, oestrogen receptor (ER)‐negative, progesterone receptor (PR)‐negative, and HER2‐negative epithelial cells. There is evidence to suggest that MGA may constitute a non‐obligate precursor of triple‐negative breast cancer (TNBC). We sought to define the genomic landscape of pure MGA and of MGA, atypical MGA (AMGA) and associated TNBCs, and to determine whether synchronous MGA, AMGA, and TNBCs would be clonally related. Two pure MGAs and eight cases of MGA and/or AMGA associated with in situ or invasive TNBC were collected, microdissected, and subjected to massively parallel sequencing targeting all coding regions of 236 genes recurrently mutated in breast cancer or related to DNA repair. Pure MGAs lacked clonal non‐synonymous somatic mutations and displayed limited copy number alterations (CNAs); conversely, all MGAs (n = 7) and AMGAs (n = 3) associated with TNBC harboured at least one somatic non‐synonymous mutation (range 3–14 and 1–10, respectively). In all cases where TNBCs were analyzed, identical TP53 mutations and similar patterns of gene CNAs were found in the MGA and/or AMGA and in the associated TNBC. In the MGA/AMGA associated with TNBC lacking TP53 mutations, somatic mutations affecting PI3K pathway‐related genes (eg PTEN, PIK3CA, and INPP4B) and tyrosine kinase receptor signalling‐related genes (eg ERBB3 and FGFR2) were identified. At diagnosis, MGAs associated with TNBC were found to display subclonal populations, and clonal shifts in the progression from MGA to AMGA and/or to TNBC were observed. Our results demonstrate the heterogeneity of MGAs, and that MGAs associated with TNBC, but not necessarily pure MGAs, are genetically advanced, clonal, and neoplastic lesions harbouring recurrent mutations in TP53 and/or other cancer genes, supporting the notion that a subset of MGAs and AMGAs may constitute non‐obligate precursors of TNBCs. Copyright

Infiltrating epitheliosis of the breast: characterization of histological features, immunophenotype and genomic profile

Infiltrating epitheliosis is a rare complex sclerosing lesion (CSL) of the breast, characterized by infiltrating ducts immersed in a scleroelastotic stroma and filled with cells having architectural and cytological patterns reminiscent of those of usual ductal hyperplasia. In this study we sought to define the molecular characteristics of infiltrating epitheliosis.

Resolution of recurrent fusarium arthritis after prolonged antifungal therapy.

S everal recent reports have highlighted the emerging importance of Fusarium as a pathogen in immunosuppressed patients. Fusarium is a genus of filamentous molds occurring abundantly in soil and plants. Fusarium does not usually cause serious disease in humans with intact immunity. When infection does occur, nails and corneas are the commonest sites. In immunocompromised patients, however, invasive or disseminated infection can occur, and mortality rates as high as 75% have been described. Hematologic malignancy and severe neutropenia appear to represent special risk factors for dissemination, most commonly to blood, skin, sinuses, and lungs. Bone and joint infections are less common but have been described; in one 10-year review, Fusarium accounted for some 20% of fungal osteoarticular infections in patients with hematologic malignancy. In this report, we describe a case of joint infection with Fusarium, a mold of emerging importance in immunocompromised individuals. The patient was a 62-year-old man with acute myeloid leukemia (AML), presenting 5 weeks after induction chemotherapy with Ara-C and daunomycin. He complained of 1 week of left ankle pain and swelling, fevers, and drenching night sweats. On examination, his left ankle was warm, and an effusion was present. His left second metacarpophalangeal and proximal interphalangeal joints were swollen and tender. M4/M5 AML had been diagnosed 2 months previously. Induction chemotherapy was complicated by febrile neutropenia and erythematous skin nodules. Skin biopsy showed fungal hyphae (Fig. A), and culture of skin and blood grew Fusarium species. He was treated with intravenous voriconazole with resolution of fever and rash and discharged on oral voriconazole. At the onset of current joint symptoms, he continued on oral voriconazole and was not neutropenic. Analysis of synovial fluid from the left ankle showed a white cell count of 22,000 (75% neutrophils, 12% lymphocytes), with negative Gram stain and no crystals. Radiographs of the left ankle and hand showed soft-tissue swelling with preserved joint space. He was admitted for presumed polyarticular septic arthritis. Ceftriaxone and vancomycin were added, and his ankle effusion was aspirated daily. Initial blood and synovial fluid cultures were negative. On hospital day 8, the second of 5 synovial fluid cultures grew fungus, later confirmed to be Fusarium. Voriconazole and antibiotics were discontinued, and liposomal amphotericin (AmBisome) was started with azithromycin for synergy. The effusion resolved, but new skin lesions consistent with Fusarium subsequently appeared. Sensitivities requested during his initial fungemia returned, showing intermediate sensitivity (minimal inhibitory concentration, 2) to amphotericin B and voriconazole and sensitivity to caspofungin (minimal inhibitory concentration, 1.5). Caspofungin was added, his skin lesions resolved, and hewas discharged on azithromycin, AmBisome, and caspofungin. He was readmitted for consolidation chemotherapy with Ara-C and daunomycin 5 weeks after initiation of AmBisome. Despite ongoing AmBisome/azithromycin and caspofungin, his left ankle effusion recurred after onset of neutropenia, highlighting the critical role of neutrophils in Fusarium infection. A left ankle magnetic resonance imaging (MRI) showed a loculated joint effusion and a lesion consistent with osteomyelitis (Figs. AYC). Voriconazole and broad-spectrum antibiotics were added without improvement. When neutropenia resolved 8 days later, he defervesced, and ankle swelling improved. He received a total of 2.5 months of AmBisome/azithromycin, 6 months of caspofungin, and 8 months of oral voriconazole. Subsequent MRI showed resolution of the ankle effusion and osteomyelitis. Four years later, he remains in remission from his AML and has had an excellent functional recovery from his ankle arthritis. Early diagnosis and treatment of invasive fusariosis are critically important. In contrast to some fungal pathogens, blood cultures are frequently positive. Biopsy of affected tissue can permit rapid identification of hyphae, while culture is awaited. In our case, only 1 of 5 synovial cultures grew Fusarium, emphasizing that multiple arthrocenteses may be needed for diagnosis. Voriconazole, amphotericin, or posaconazole forms the mainstay of empiric therapy. Local resistance trends should assist selection of empiric agents in individual cases. Although susceptibility testing methods are not standardized, sensitivity testing remains crucial. In the present case, synovial fluid isolates were sensitive to caspofungin, despite the fact that Fusarium is generally considered resistant to echinocandins. Insufficient data exist, at present, to recommend firmly between the use of single agents or combination therapy, and duration of therapy is also unclear. Surgical resection of heavily infected tissue, removal of conduits such as potentially infected lines, and the use of colonystimulating factors to hasten neutrophil reconstitution should also be considered as adjunctive measures. In this case, surgical debridement was considered, but deferred pending reconstitution of his bone marrow. Recovery from neutropenia represents a particularly important prognostic factor in invasive fusariosis and often coincides with clinical improvement, as demonstrated dramatically in this case. However, as our case also illustrates, antifungal therapy should be continued while any clinical or radiographic features of active disease persist, and at least until all cultures are negative. It is hoped that the present report will serve to inform clinicians of this invasive organism’s potential to cause joint and bone infection in immunocompromised patients, CONCISE REPORT

Abstract S6-06: The genomic landscape of male breast cancers

Background: Male breast cancer (MaBC) accounts for Methods: Subtyping of the 64 MaBCs included in this study was performed by means of immunohistochemistry using the definitions described in the latest St. Gallen’s consensus report. DNA extracted from microdissected tumor and adjacent normal tissue were subjected to massively parallel targeting sequencing of all exons of 273 genes most frequently mutated in FBCs or directly related to DNA repair. Somatic mutations were defined using a combination of MuTect, SomaticSniper, MutationSeq and Haplotype Caller. Selected mutations were validated with Sequenom MassARRAY. CNAs were identified using Varscan2 and GISTIC2.0. Pathway and network analysis of mutations/CNAs was performed using Ingenuity Pathway Analysis and HOTNET. The genomic landscape of MaBCs was compared with that of FBCs of the same subtype analyzed as part of The Cancer Genome Atlas project. Results: All MaBCs were ER-positive and HER2-negative. Using the St. Gallen’s criteria, 37.5% and 62.5% were classified as luminal A-like or luminal B-like, respectively. The genes most frequently mutated in MaBCs were PIK3CA, GATA3, FLG and PLEC, with PIK3CA being the only significantly mutated gene as defined by MutSigCV (q=0.003). CNA analysis revealed recurrent gains of 1q and 8q and loss of 16q. GISTIC2.0 identified significantly recurrent high-level amplifications in 1q25.3, 8p11 (FGFR1, ZNF703), 8q24.3 (DEPTOR), 17q23 (PPM1D) and 15q26 (IGF1R) and deletions in 11q22 (ATM) and 21q22.12 (RUNX1). HOTNET analysis of the genes mutated and/or targeted by gene amplifications in MaBCs revealed significantly altered subnetworks involving genes related to DNA repair, PI3K and FGF signaling pathways. The most frequently mutated genes in luminal A-like MaBCs were PIK3CA and MLL3, while those of luminal B-like MaBCs were PIK3CA and GATA3. MLL3 and GATA3 mutations were only found in luminal A-like MaBCs (p=0.039) and luminal B-like MaBCs (p=0.048), respectively. Although the mutational landscapes of MaBCs and luminal FBCs were qualitatively similar, PIK3CA and TP53 were less frequently mutated in MaBCs (p Conclusions: MaBCs are preferentially of luminal subtype and are characterized by recurrent mutations in PIK3CA, GATA3, FLG and PLEC. Genetic alterations in MaBCs often target DNA repair and FGF signaling pathways. The known drivers of luminal FBCs appear to be less frequently altered in MaBCs. Given these important differences between MaBCs and FBCs, caution should be exercised in the extrapolation of biologic and clinical implications from studies in FBCs to the management of MaBCs. Citation Format: Salvatore Piscuoglio, Melissa Murray, Charlotte KY Ng, Elena Guerini Rocco, Luciano G Martelotto, Francois-Clement Bidard, Carey A Eberle, Nicola Fusco, Rita A Sakr, Leticia De Mattos-Arruda, Raymond Lim, Timour Baslan, James Hicks, Tari A King, Edi Brogi, Larry Norton, Britta Weigelt, Clifford A Hudis, Jorge S Reis-Filho. The genomic landscape of male breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S6-06.

Abstract 91: The mutational landscape of mucinous carcinomas of the breast

Background: Mucinous breast carcinoma (MBC) is a special histologic type of breast cancer, comprising approximately 2% of all invasive breast carcinomas. Morphologically, MBCs are characterized by clusters of tumor cells floating in large amounts of extracellular mucin. MBCs are preferentially of low histologic grade and estrogen receptor (ER)-positive and HER2-negative. MBCs have been shown to lack the hallmark 1q gains and 16q losses found in low-grade ER-positive invasive ductal carcinomas of no special type (IDC-NSTs). Here we sought to characterize the mutational landscape and copy number alterations (CNA) of MBCs by whole exome sequencing analysis. Material and Methods: Frozen sections from 25 pure MBCs (n = 13 mucinous A (paucicellular), n = 12 mucinous B (hypercellular)) were subjected to microdissection to ensure a tumor cell content >85%. In addition, five cases of mixed mucinous carcinomas composed of mucinous and IDC-NST components were included in this study. In these cases, the MBC and IDC-NST components were microdissected separately. DNA samples extracted from microdissected tumors and matched normal counterparts were subjected to whole exome sequencing on an Illumina HiSeq2000. Somatic single nucleotide variants (SNVs) were identified using MuTect and somatic insertions and deletions (indels) were identified using Strelka and Varscan2. Somatic CNAs were defined using FACETS. Mutational frequencies in MBCs were compared to those of luminal IDC-NSTs from The Cancer Genome Atlas. Results: A median of 26 (range 5-71) non-synonymous SNVs and indels were identified per MBC. GATA3, mutated in 28% of cases, was the only significantly mutated gene as defined by MutSigCV (q Conclusions: Our data suggest that the repertoires of somatic mutations and gene CNAs of MBCs are distinct from those of luminal IDC-NSTs, and that MBCs are an entity distinct from low-grade ER-positive IDC-NSTs not only at the morphologic but also at the genetic level. Citation Format: Salvatore Piscuoglio, Charlotte KY Ng, Felipe C. Geyer, Carey A. Eberle, Elena Guerini-Rocco, Caterina Marchio, Anne Vincent-Salomon, Jorge S. Reis-Filho, Britta Weigelt. The mutational landscape of mucinous carcinomas of the breast. [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 91.