José Pablo Leone

Sensitive Detection of Mono- and Polyclonal ESR1 Mutations in Primary Tumors, Metastatic Lesions, and Cell-Free DNA of Breast Cancer Patients

Purpose: Given the clinical relevance of ESR1 mutations as potential drivers of resistance to endocrine therapy, this study used sensitive detection methods to determine the frequency of ESR1 mutations in primary and metastatic breast cancer, and in cell-free DNA (cfDNA). Experimental Design: Six ESR1 mutations (K303R, S463P, Y537C, Y537N, Y537S, D538G) were assessed by digital droplet PCR (ddPCR), with lower limits of detection of 0.05% to 0.16%, in primary tumors (n = 43), bone (n = 12) and brain metastases (n = 38), and cfDNA (n = 29). Correlations between ESR1 mutations in metastatic lesions and single (1 patient) or serial blood draws (4 patients) were assessed. Results: ESR1 mutations were detected for D538G (n = 13), Y537S (n = 3), and Y537C (n = 1), and not for K303R, S463P, or Y537N. Mutation rates were 7.0% (3/43 primary tumors), 9.1% (1/11 bone metastases), 12.5% (3/24 brain metastases), and 24.1% (7/29 cfDNA). Two patients showed polyclonal disease with more than one ESR1 mutation. Mutation allele frequencies were 0.07% to 0.2% in primary tumors, 1.4% in bone metastases, 34.3% to 44.9% in brain metastases, and 0.2% to 13.7% in cfDNA. In cases with both cfDNA and metastatic samples (n = 5), mutations were detected in both (n = 3) or in cfDNA only (n = 2). Treatment was associated with changes in ESR1 mutation detection and allele frequency. Conclusions: ESR1 mutations were detected at very low allele frequencies in some primary breast cancers, and at high allele frequency in metastases, suggesting that in some tumors rare ESR1-mutant clones are enriched by endocrine therapy. Further studies should address whether sensitive detection of ESR1 mutations in primary breast cancer and in serial blood draws may be predictive for development of resistant disease. Clin Cancer Res; 22(5); 1130–7. ©2015 AACR. See related commentary by Gu and Fuqua, p. 1034

Intrinsic Subtype Switching and Acquired ERBB2/HER2 Amplifications and Mutations in Breast Cancer Brain Metastases

Importance Patients with breast cancer (BrCa) brain metastases (BrM) have limited therapeutic options. A better understanding of molecular alterations acquired in BrM could identify clinically actionable metastatic dependencies. Objective To determine whether there are intrinsic subtype differences between primary tumors and matched BrM and to uncover BrM-acquired alterations that are clinically actionable. Design, Setting, and Participants In total, 20 cases of primary breast cancer tissue and resected BrM (10 estrogen receptor [ER]-negative and 10 ER-positive) from 2 academic institutions were included. Eligible cases in the discovery cohort harbored patient-matched primary breast cancer tissue and resected BrM. Given the rarity of patient-matched samples, no exclusion criteria were enacted. Two validation sequencing cohorts were used—a published data set of 17 patient-matched cases of BrM and a cohort of 7884 BrCa tumors enriched for metastatic samples. Main Outcomes and Measures Brain metastases expression changes in 127 genes within BrCa signatures, PAM50 assignments, and ERBB2/HER2 DNA-level gains. Results Overall, 17 of 20 BrM retained the PAM50 subtype of the primary BrCa. Despite this concordance, 17 of 20 BrM harbored expression changes (<2-fold or >2-fold) in clinically actionable genes including gains of FGFR4 (n = 6 [30%]), FLT1 (n = 4 [20%]), AURKA (n = 2 [10%]) and loss of ESR1 expression (n = 9 [45%]). The most recurrent expression gain was ERBB2/HER2, which showed a greater than 2-fold expression increase in 7 of 20 BrM (35%). Three of these 7 cases were ERBB2/HER2-negative out of 13 ERBB2/HER2-negative in the primary BrCa cohort and became immunohistochemical positive (3+) in the paired BrM with metastasis-specific amplification of the ERBB2/HER2 locus. In an independent data set, 2 of 9 (22.2%) ERBB2/HER2-negative BrCa switched to ERBB2/HER2-positive with 1 BrM acquiring ERBB2/HER2 amplification and the other showing metastatic enrichment of the activating V777L ERBB2/HER2 mutation. An expanded cohort revealed that ERBB2/HER2 amplification and/or mutation frequency was unchanged between local disease and metastases across all sites; however, a significant enrichment was appreciated for BrM (13% local vs 24% BrM; P < .001). Conclusions and Relevance Breast cancer BrM commonly acquire alterations in clinically actionable genes, with metastasis-acquired ERBB2/HER2 alterations in approximately 20% of ERBB2/HER2-negative cases. These observations have immediate clinical implications for patients with ERBB2/HER2–negative breast cancer and support comprehensive profiling of metastases to inform clinical care.

Targeted Therapies for Brain Metastases from Breast Cancer.

The discovery of various driver pathways and targeted small molecule agents/antibodies have revolutionized the management of metastatic breast cancer. Currently, the major targets of clinical utility in breast cancer include the human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF) receptor, mechanistic target of rapamycin (mTOR) pathway, and the cyclin-dependent kinase 4/6 (CDK-4/6) pathway. Brain metastasis, however, remains a thorn in the flesh, leading to morbidity, neuro-cognitive decline, and interruptions in the management of systemic disease. Approximately 20%–30% of patients with metastatic breast cancer develop brain metastases. Surgery, whole brain radiation therapy, and stereotactic radiosurgery are the traditional treatment options for patients with brain metastases. The therapeutic paradigm is changing due to better understanding of the blood brain barrier and the advent of tyrosine kinase inhibitors and monoclonal antibodies. Several of these agents are in clinical practice and several others are in early stage clinical trials. In this article, we will review the common targetable pathways in the management of breast cancer patients with brain metastases, and the current state of the clinical development of drugs against these pathways.

Prognostic factors and survival of patients with brain metastasis from breast cancer who underwent craniotomy

Brain metastasis (BM) in patients with breast cancer is a catastrophic event that results in poor prognosis. Identification of prognostic factors associated with breast cancer brain metastases (BCBM) could help to identify patients at risk. The aim of this study was to assess clinical characteristics, prognostic factors, and survival of patients with BCBM who had craniotomy and resection in a series of patients treated with modern multimodality therapy. We analyzed 42 patients with BCBM who underwent resection. Patients were diagnosed with breast cancer between April 1994 and May 2010. Cox proportional hazards regression was selected to describe factors associated with time to BM, survival from the date of first recurrence, and overall survival (OS). Median age was 51 years (range 24–74). Median follow‐up was 4.2 years (range 0.6–18.5). The proportion of the biological subtypes of breast cancer was ER+/HER2− 25%, ER+/HER2+ 15%, ER‐/HER2+ 30%, and ER‐/HER2− 30%. Median OS from the date of primary diagnosis was 5.74 years. Median survival after diagnosis of BM was 1.33 years. In multivariate Cox regression analyses, stage was the only factor associated with shorter time to the development of BM (P = 0.033), whereas age was the only factor associated with survival from the date of recurrence (P = 0.027) and with OS (P = 0.037). Stage at primary diagnosis correlated with shorter time to the development of BM, while age at diagnosis was associated with shorter survival in BCBM. None of the other clinical factors had influence on survival.

Novel approaches to target HER2-positive breast cancer: trastuzumab emtansine

The human epidermal growth factor receptor 2 (HER2) is overexpressed in 20% of breast carcinomas. Prior to the development of targeted therapies, HER2-positive breast cancer was associated with more aggressive disease and poor prognosis. Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that results from the combination of trastuzumab and DM1, a derivative of the antimicrotubule agent maytansine. This molecule has the ability to enhance cytotoxic drug delivery to specifically targeted cells that overexpress HER2, therefore, maximizing efficacy while sparing toxicity. In recent years, T-DM1 has shown to improve outcomes in metastatic HER2-positive breast cancer that is resistant to trastuzumab. In addition, T-DM1 is currently being tested in the neoadjuvant and adjuvant settings to identify patients who may benefit from this therapy. This review focuses on the mechanism of action, early and late-phase clinical trials, and ongoing studies of T-DM1 in HER2-positive breast cancer.

Male Breast CancerA Single-Institution Clinicopathologic and Immunohistochemical Study

OBJECTIVES A clinicopathologic study with an emphasis on tumor immunohistochemical profile is presented. METHODS Sixty-one cases of male invasive breast cancers were studied. Median age of the cohort was 65 years. RESULTS Ninety-seven percent were estrogen receptor positive+ and 10% human epidermal growth factor receptor 2 positive. The individual diagnostic marker positivity was 98% for GATA-binding protein 3, 95% for androgen receptor, 90% for progesterone receptor, 88% for deleted in pancreatic cancer 4, 75% for gross cystic disease fluid protein 15, 72% for cytokeratin 7, 55% for mammaglobin, and 15% for vimentin and Wilms tumor protein 1. Caudal type homeobox 2 protein, cytokeratin 20, Napsin A, paired box gene 8, prostate-specific antigen, thyroid transcription factor 1, and uroplakin II were negative in all cases. Survival analyses showed tumor stage, receptor status, and Nottingham prognostic index to be prognostic. The overall survival was 70%, but the breast cancer-specific survival was 92% (mean follow-up, 59 months); 33% developed second malignancy. The immunohistochemistry profile was similar to female breast cancers. CONCLUSIONS The second malignancies in this cohort affected overall survival and suggest the possibility of other germline mutations in addition to BRCA2 in male patients with breast cancer.Objectives: A clinicopathologic study with an emphasis on tumor immunohistochemical profile is presented. Methods: Sixty-one cases of male invasive breast cancers were studied. Median age of the cohort was 65 years. Results: Ninety-seven percent were estrogen receptor positive+ and 10% human epidermal growth factor receptor 2 positive. The individual diagnostic marker positivity was 98% for GATA-binding protein 3, 95% for androgen receptor, 90% for progesterone receptor, 88% for deleted in pancreatic cancer 4, 75% for gross cystic disease fluid protein 15, 72% for cytokeratin 7, 55% for mammaglobin, and 15% for vimentin and Wilms tumor protein 1. Caudal type homeobox 2 protein, cytokeratin 20, Napsin A, paired box gene 8, prostate-specific antigen, thyroid transcription factor 1, and uroplakin II were negative in all cases. Survival analyses showed tumor stage, receptor status, and Nottingham prognostic index to be prognostic. The overall survival was 70%, but the breast cancer–specific survival was 92% (mean follow-up, 59 months); 33% developed second malignancy. The immunohistochemistry profile was similar to female breast cancers. Conclusions: The second malignancies in this cohort affected overall survival and suggest the possibility of other germline mutations in addition to BRCA2 in male patients with breast cancer.

Expression of ERCC1 and TUBB3 in Locally Advanced Cervical Squamous Cell Cancer and its Correlation with Different Therapeutic Regimens

Background Several studies in solid tumors have shown that expression of excision repair cross-complementation group 1 (ERCC1) and class III β-tubulin (TUBB3) can predict response to chemoradiotherapy and might be prognostic factors. We assessed the role of ERCC1 and TUBB3 expressions as predictive and prognostic factors in locally advanced cervical squamous cell carcinoma (LACSCC) patients treated with different neoadjuvant regimens. Methods ERCC1 and TUBB3 were detected in 88 patients with LACSCC by immunohistochemical analysis. Sixty-two patients were included in 3 different prospective trials and grouped as follows: vinorelbine or docetaxel (group A, n = 44) and ifosfamide-vinorelbine-cisplatin (group B, n = 18). Both groups were compared with standard cisplatin chemoradiotherapy (group C, n = 26). Clinical data at baseline, disease-free survival (DFS) and overall survival (OS) were also collected. Univariate and multivariate Cox models were used to analyze the risk factors. Results Thirty-five patients (39.8%) and 18 (20.5%) had high ERCC1 and TUBB3 expression, respectively. Both proteins were overexpressed in tumors with unfavorable characteristics. High ERCC1 was associated with advanced FIGO stage (p = 0.034) and progressive disease (49% vs. 28%). Poor DFS (p = 0.021) and OS (p = 0.005) were observed in group C patients with high ERCC1 expression. Multivariate analysis showed that ERCC1 expression, FIGO stage and pretreatment hemoglobin level were significant prognostic factors (p = 0.002, p = 0.008 and p = 0.005, respectively). Conclusions ERCC1 expression could be a predictive and prognostic factor in LACSCC patients who receive cisplatin monotherapy. Conversely, TUBB3 had no impact on survival in patients treated with antimicrotubule agents.

Expression of high affinity folate receptor in breast cancer brain metastasis

High affinity folate receptor (HFR) can be overexpressed in breast cancer and is associated with poor prognosis, however the expression in breast cancer brain metastases (BCBM) is unknown. The aim of this study was to analyze the rate of HFR expression in BCBM and its role in the prognosis of this high-risk cohort. We analyzed 19 brain metastasis (BM) and 13 primary tumors (PT) from a total of 25 patients. HFR status was assessed by immunohistochemistry. Median follow-up was 4.2 years (range 0.6-18.5). HFR was positive in 4/19 BM (21.1%) and in 1/13 PT (7.7%). Positive samples had low H-scores (range 1-50). 56% of patients had apocrine differentiation. OS was similar between patients with positive HFR (median OS 48 months) and negative HFR (median OS 69 months) (P = 0.25); and between patients with apocrine differentiation (median OS 63 months) and those without apocrine differentiation (median OS 69 months) (P = 0.49). To the best of our knowledge, this is the first analysis of HFR expression in BCBM. While previous studies associated the presence of HFR with worse prognosis; in our cohort HFR was positive in only 21.1% of BM with low levels of positivity. Neither HFR nor apocrine features had impact in OS.

Prognostic factors and survival according to tumour subtype in women presenting with breast cancer brain metastases at initial diagnosis

BACKGROUND The presence of brain metastases at the time of initial breast cancer diagnosis (BMIBCD) is uncommon. Hence, the prognostic assessment and management of these patients is very challenging. The aim of this study was to analyse the influence of tumour subtype compared with other prognostic factors in the survival of patients with BMIBCD. METHODS We evaluated women with BMIBCD, reported to Surveillance, Epidemiology and End Results program from 2010 to 2013. Patients with other primary malignancy were excluded. Univariate and multivariate analyses were performed to determine the effects of each variable on overall survival (OS). RESULTS We included 740 patients. Median OS for the whole population was 10 months, and 20.7% of patients were alive at 36 months. Tumour subtype distribution was: 46.6% hormone receptor (HR)+/HER2-, 17% HR+/HER2+, 14.1% HR-/HER2+ and 22.3% triple-negative. Univariate analysis showed that the presence of liver metastases, lung metastases and triple-negative patients (median OS 6 months) had worse prognosis. The HR+/HER2+ subtype had the longest OS with a median of 22 months. In multivariate analysis, older age (hazard ratio 1.8), lobular histology (hazard ratio 2.08), triple-negative subtype (hazard ratio 2.25), liver metastases (hazard ratio 1.6) and unmarried patients (hazard ratio 1.39) had significantly shorter OS. CONCLUSIONS Although the prognosis of patients with BMIBCD is generally poor, 20.7% were still alive 3 years after the diagnosis. There were substantial differences in OS according to tumour subtype. In addition to tumour subtype, other independent predictors of OS are age at diagnosis, marital status, histology and liver metastases.

Metastatic Adenoid Cystic Carcinoma of the Breast

A 68-year-old woman presented to dermatology clinic with a new scalp lesion. Patient had left breast adenoid cystic carcinoma in 2003 for which she underwent lumpectomy and adjuvant radiation therapy. Her tumor was negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). In 2011, she was incidentally found to have right upper lobe mass on chest imaging. PET-CT showed no other site of disease. Biopsy of lung mass showed metastatic adenoid cystic carcinoma and she underwent right upper lobectomy (Fig. 1). The scalp lesion was resected in July, 2014 (Fig. 2). Numerous ducts were present within the proliferation with cribriform appearance consistent with known history of adenoid cystic carcinoma; immunohistochemistry again was negative for