Gisela Mir Arnau

Whole–genome characterization of chemoresistant ovarian cancer

Patients with high-grade serous ovarian cancer (HGSC) have experienced little improvement in overall survival, and standard treatment has not advanced beyond platinum-based combination chemotherapy, during the past 30 years. To understand the drivers of clinical phenotypes better, here we use whole-genome sequencing of tumour and germline DNA samples from 92 patients with primary refractory, resistant, sensitive and matched acquired resistant disease. We show that gene breakage commonly inactivates the tumour suppressors RB1, NF1, RAD51B and PTEN in HGSC, and contributes to acquired chemotherapy resistance. CCNE1 amplification was common in primary resistant and refractory disease. We observed several molecular events associated with acquired resistance, including multiple independent reversions of germline BRCA1 or BRCA2 mutations in individual patients, loss of BRCA1 promoter methylation, an alteration in molecular subtype, and recurrent promoter fusion associated with overexpression of the drug efflux pump MDR1.

UV-Associated Mutations Underlie the Etiology of MCV-Negative Merkel Cell Carcinomas

Merkel cell carcinoma (MCC) is an uncommon, but highly malignant, cutaneous tumor. Merkel cell polyoma virus (MCV) has been implicated in a majority of MCC tumors; however, viral-negative tumors have been reported to be more prevalent in some geographic regions subject to high sun exposure. While the impact of MCV and viral T-antigens on MCC development has been extensively investigated, little is known about the etiology of viral-negative tumors. We performed targeted capture and massively parallel DNA sequencing of 619 cancer genes to compare the gene mutations and copy number alterations in MCV-positive (n = 13) and -negative (n = 21) MCC tumors and cell lines. We found that MCV-positive tumors displayed very low mutation rates, but MCV-negative tumors exhibited a high mutation burden associated with a UV-induced DNA damage signature. All viral-negative tumors harbored mutations in RB1, TP53, and a high frequency of mutations in NOTCH1 and FAT1. Additional mutated or amplified cancer genes of potential clinical importance included PI3K (PIK3CA, AKT1, PIK3CG) and MAPK (HRAS, NF1) pathway members and the receptor tyrosine kinase FGFR2. Furthermore, looking ahead to potential therapeutic strategies encompassing immune checkpoint inhibitors such as anti-PD-L1, we also assessed the status of T-cell-infiltrating lymphocytes (TIL) and PD-L1 in MCC tumors. A subset of viral-negative tumors exhibited high TILs and PD-L1 expression, corresponding with the higher mutation load within these cancers. Taken together, this study provides new insights into the underlying biology of viral-negative MCC and paves the road for further investigation into new treatment opportunities.

Whole exome sequencing reveals activating JAK1 and STAT3 mutations in breast implant-associated anaplastic large cell lymphoma anaplastic large cell lymphoma

Systemic anaplastic large cell lymphoma (sALCL) is an aggressive T-cell non Hodgkin lymphoma which is divided into two categories based on the expression of the anaplastic lymphoma kinase (ALK) protein (i.e. ALK-positive ALCL and ALK-negative ALCL). ALK-negative sALCL typically presents with

A community-based model of rapid autopsy in end-stage cancer patients

To the Editor: Systematic genomic studies, including the Cancer Genome Atlas (TCGA)1 and the International Cancer Genome Consortium (ICGC)2, have provided an unprecedented catalog of driver mutations in human cancer. However, these studies use mainly primary, pre-treatment tumor material obtained at surgery with curative intent. There is an urgent need to identify and characterize resistance mechanisms to understand how cancers can evade even the best medical efforts and kill patients; therefore, access to end-stage disease is important. Solid cancers show considerable spatial3, temporal4,5 and genomic heterogeneity at diagnosis. Selective pressure and mutagenic impact of treatment6 drives intra-patient evolution of cancer cell populations4,7. Understanding acquired resistance requires access to paired preand post-treatment samples4,7; however, curative surgery is typically confined to patients with locoregional disease, and opportunities for tumor sampling in advanced disseminated disease are limited. Here, we describe Cancer Tissue Collection After Death (CASCADE), an autopsy program that overcomes logistical challenges to enable collection of samples at end stage for research in melanoma and breast, ovarian and prostate cancers. For the CASCADE study, we aimed to recruit cancer patients close to the end of life, including those outside the minority of patients who die in hospitals. To preserve tissue integrity, autopsies must commence within a few hours of death, requiring access to around-the-clock services. Intervention in the emotionally charged end-of-life environment must be managed in an ethical manner and to a high standard. Finally, we aimed for the study to be highly cost-effective. We believe our approach to meeting these challenges is applicable to researchers in other large urban centers. Here we summarize the main steps in CASCADE’s operating protocol and our experiences from the initial 3 years and 30 autopsies performed (Fig. 1). Information about institutional review board approvals (including a detailed patient informationand-consent form), the autopsy procedure and certain laboratory processes is given in Supplementary Methods and Supplementary Figure 1. Recruitment of participants was led by the clinicians. Such discussions require careful consideration, in timing and in language, and were initiated only if there was a perception that tissue donation would be acceptable to the patients and their families. Factors suggesting acceptability include the emotional stability of the participant and family members and their clarity about and acceptance of the terminal nature of the disease. On occasion, participants prompted discussion by asking about organ or body donation. Consent discussions typically involved oncologists and/or palliative care physicians employed at recruiting hospitals who had established a care relationship with the participant and their family during the patient’s cancer journey. Frequently, the study was introduced at one meeting and discussed over several subsequent clinic visits, allowing patients and their families time to consider participation. We view the involvement of family members in the consent process as essential to support the participant and facilitate decisionmaking. Involvement of family members also ensures that they are fully aware of the autopsy process and helps to clarify funeral arrangements for the study team. After obtaining consent, study investigators collated clinical information, including that related to past and current treatment and diagnostic procedures such as imaging, on an ongoing basis. Between September 2012 and August 2015, 40 patients were approached, and 37 (92.5%) expressed interest in participating. Of those 32 patients (80%) consented; the other 5 had rapid clinical deterioration precluding

Multiplexed transcriptome analysis to detect ALK, ROS1 and RET rearrangements in lung cancer

ALK, ROS1 and RET gene fusions are important predictive biomarkers for tyrosine kinase inhibitors in lung cancer. Currently, the gold standard method for gene fusion detection is Fluorescence In Situ Hybridization (FISH) and while highly sensitive and specific, it is also labour intensive, subjective in analysis, and unable to screen a large numbers of gene fusions. Recent developments in high-throughput transcriptome-based methods may provide a suitable alternative to FISH as they are compatible with multiplexing and diagnostic workflows. However, the concordance between these different methods compared with FISH has not been evaluated. In this study we compared the results from three transcriptome-based platforms (Nanostring Elements, Agena LungFusion panel and ThermoFisher NGS fusion panel) to those obtained from ALK, ROS1 and RET FISH on 51 clinical specimens. Overall agreement of results ranged from 86–96% depending on the platform used. While all platforms were highly sensitive, both the Agena panel and Thermo Fisher NGS fusion panel reported minor fusions that were not detectable by FISH. Our proof–of–principle study illustrates that transcriptome-based analyses are sensitive and robust methods for detecting actionable gene fusions in lung cancer and could provide a robust alternative to FISH testing in the diagnostic setting.

Circulating tumour DNA reflects treatment response and clonal evolution in chronic lymphocytic leukaemia

Several novel therapeutics are poised to change the natural history of chronic lymphocytic leukaemia (CLL) and the increasing use of these therapies has highlighted limitations of traditional disease monitoring methods. Here we demonstrate that circulating tumour DNA (ctDNA) is readily detectable in patients with CLL. Importantly, ctDNA does not simply mirror the genomic information contained within circulating malignant lymphocytes but instead parallels changes across different disease compartments following treatment with novel therapies. Serial ctDNA analysis allows clonal dynamics to be monitored over time and identifies the emergence of genomic changes associated with Richters syndrome (RS). In addition to conventional disease monitoring, ctDNA provides a unique opportunity for non-invasive serial analysis of CLL for molecular disease monitoring.

Breast ductal carcinoma in situ carry mutational driver events representative of invasive breast cancer

The spectrum of genomic alterations in ductal carcinoma in situ (DCIS) is relatively unexplored, but is likely to provide useful insights into its biology, its progression to invasive carcinoma and the risk of recurrence. DCIS (n=20) with a range of phenotypes was assessed by massively parallel sequencing for mutations and copy number alterations and variants validated by Sanger sequencing. PIK3CA mutations were identified in 11/20 (55%), TP53 mutations in 6/20 (30%), and GATA3 mutations in 9/20 (45%). Screening an additional 91 cases for GATA3 mutations identified a final frequency of 27% (30/111), with a high proportion of missense variants (8/30). TP53 mutations were exclusive to high grade DCIS and more frequent in PR-negative tumors compared with PR-positive tumors (P=0.037). TP53 mutant tumors also had a significantly higher fraction of the genome altered by copy number than wild-type tumors (P=0.005), including a significant positive association with amplification or gain of ERBB2 (P<0.05). The association between TP53 mutation and ERBB2 amplification was confirmed in a wider DCIS cohort using p53 immunohistochemistry as a surrogate marker for TP53 mutations (P=0.03). RUNX1 mutations and MAP2K4 copy number loss were novel findings in DCIS. Frequent copy number alterations included gains on 1q, 8q, 17q, and 20q and losses on 8p, 11q, 16q, and 17p. Patterns of genomic alterations observed in DCIS were similar to those previously reported for invasive breast cancers, with all DCIS having at least one bona fide breast cancer driver event. However, an increase in GATA3 mutations and fewer copy number changes were noted in DCIS compared with invasive carcinomas. The role of such alterations as prognostic and predictive biomarkers in DCIS is an avenue for further investigation.

Homologous Recombination DNA Repair Pathway Disruption and Retinoblastoma Protein Loss Are Associated with Exceptional Survival in High-Grade Serous Ovarian Cancer.

Purpose: Women with epithelial ovarian cancer generally have a poor prognosis; however, a subset of patients has an unexpected dramatic and durable response to treatment. We sought to identify clinical, pathological, and molecular determinants of exceptional survival in women with high-grade serous cancer (HGSC), a disease associated with the majority of ovarian cancer deaths. Experimental Design: We evaluated the histories of 2,283 ovarian cancer patients and, after applying stringent clinical and pathological selection criteria, identified 96 with HGSC that represented significant outliers in terms of treatment response and overall survival. Patient samples were characterized immunohistochemically and by genome sequencing. Results: Different patterns of clinical response were seen: long progression-free survival (Long-PFS), multiple objective responses to chemotherapy (Multiple Responder), and/or greater than 10-year overall survival (Long-Term Survivors). Pathogenic germline and somatic mutations in genes involved in homologous recombination (HR) repair were enriched in all three groups relative to a population-based series. However, 29% of 10-year survivors lacked an identifiable HR pathway alteration, and tumors from these patients had increased Ki-67 staining. CD8+ tumor-infiltrating lymphocytes were more commonly present in Long-Term Survivors. RB1 loss was associated with long progression-free and overall survival. HR deficiency and RB1 loss were correlated, and co-occurrence was significantly associated with prolonged survival. Conclusions: There was diversity in the clinical trajectory of exceptional survivors associated with multiple molecular determinants of exceptional outcome in HGSC patients. Concurrent HR deficiency and RB1 loss were associated with favorable outcomes, suggesting that co-occurrence of specific mutations might mediate durable responses in such patients. Clin Cancer Res; 24(3); 569–80. ©2017 AACR. See related commentary by Peng and Mills, p. 508

EIF1AX and NRAS Mutations Co-occur and Cooperate in Low-Grade Serous Ovarian Carcinomas

Low-grade serous ovarian carcinomas (LGSC) are associated with a poor response to chemotherapy and are molecularly characterized by RAS pathway activation. Using exome and whole genome sequencing, we identified recurrent mutations in the protein translational regulator EIF1AX and in NF1, USP9X, KRAS, BRAF, and NRAS RAS pathway mutations were mutually exclusive; however, we found significant co-occurrence of mutations in NRAS and EIF1AX Missense EIF1AX mutations were clustered at the N-terminus of the protein in a region associated with its role in ensuring translational initiation fidelity. Coexpression of mutant NRAS and EIF1AX proteins promoted proliferation and clonogenic survival in LGSC cells, providing the first example of co-occurring, growth-promoting mutational events in ovarian cancer. Cancer Res; 77(16); 4268-78. ©2017 AACR.

Circulating Tumor DNA Analysis and Functional Imaging Provide Complementary Approaches for Comprehensive Disease Monitoring in Metastatic Melanoma

PurposeCirculating tumor DNA (ctDNA) allows noninvasive disease monitoring across a range of malignancies. In metastatic melanoma, the extent to which ctDNA reflects changes in metabolic disease burden assessed by 18F-labeled fluorodeoxyglucose positron emission tomography (FDG-PET) is unknown. We assessed the role of ctDNA analysis in combination with FDG-PET to monitor tumor burden and genomic heterogeneity throughout treatment.Patients and MethodsWe performed a comprehensive analysis of serial ctDNA and FDG-PET in 52 patients who received systemic therapy for metastatic melanoma. Next-generation sequencing and digital polymerase chain reaction were used to analyze plasma samples from the cohort.ResultsctDNA levels were monitored across patients with mutant BRAF, NRAS, and BRAF/NRAS wild type disease. Mutant BRAF and NRAS ctDNA levels correlated closely with changes in metabolic disease burden throughout treatment. TERT promoter mutant ctDNA levels also paralleled changes in tumor burden, which provide ...