Eric Zhao

Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs). Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL. We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations, and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations. Genes with roles in histone modification were frequent targets of somatic mutation. For example, 32% of DLBCL and 89% of FL cases had somatic mutations in MLL2, which encodes a histone methyltransferase, and 11.4% and 13.4% of DLBCL and FL cases, respectively, had mutations in MEF2B, a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones. Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis.

Lessons learned from the application of whole-genome analysis to the treatment of patients with advanced cancers

Given the success of targeted agents in specific populations it is expected that some degree of molecular biomarker testing will become standard of care for many, if not all, cancers. To facilitate this, cancer centers worldwide are experimenting with targeted “panel” sequencing of selected mutations. Recent advances in genomic technology enable the generation of genome-scale data sets for individual patients. Recognizing the risk, inherent in panel sequencing, of failing to detect meaningful somatic alterations, we sought to establish processes to integrate data from whole-genome analysis (WGA) into routine cancer care. Between June 2012 and August 2014, 100 adult patients with incurable cancers consented to participate in the Personalized OncoGenomics (POG) study. Fresh tumor and blood samples were obtained and used for whole-genome and RNA sequencing. Computational approaches were used to identify candidate driver mutations, genes, and pathways. Diagnostic and drug information were then sought based on these candidate “drivers.” Reports were generated and discussed weekly in a multidisciplinary team setting. Other multidisciplinary working groups were assembled to establish guidelines on the interpretation, communication, and integration of individual genomic findings into patient care. Of 78 patients for whom WGA was possible, results were considered actionable in 55 cases. In 23 of these 55 cases, the patients received treatments motivated by WGA. Our experience indicates that a multidisciplinary team of clinicians and scientists can implement a paradigm in which WGA is integrated into the care of late stage cancer patients to inform systemic therapy decisions.

Getting ready for the human phenome project: the 2012 forum of the human variome project

A forum of the Human Variome Project (HVP) was held as a satellite to the 2012 Annual Meeting of the American Society of Human Genetics in San Francisco, California. The theme of this meeting was “Getting Ready for the Human Phenome Project.” Understanding the genetic contribution to both rare single‐gene “Mendelian” disorders and more complex common diseases will require integration of research efforts among many fields and better defined phenotypes. The HVP is dedicated to bringing together researchers and research populations throughout the world to provide the resources to investigate the impact of genetic variation on disease. To this end, there needs to be a greater sharing of phenotype and genotype data. For this to occur, many databases that currently exist will need to become interoperable to allow for the combining of cohorts with similar phenotypes to increase statistical power for studies attempting to identify novel disease genes or causative genetic variants. Improved systems and tools that enhance the collection of phenotype data from clinicians are urgently needed. This meeting begins the HVPs effort toward this important goal.

Investigation of PD-L1 Biomarker Testing Methods for PD-1 Axis Inhibition in Non-squamous Non–small Cell Lung Cancer

Inhibitors of the programmed cell death 1 (PD-1) signaling axis have recently demonstrated efficacy and are rapidly being incorporated into the treatment of non–small cell lung cancers (NSCLCs). Despite clear benefits to certain patients, the association of these responses with a predictive biomarker remains uncertain. Several different biomarkers have been proposed, with differing results and conclusions. This study compares multiple methods of biomarker testing for treatment of NSCLCs with PD1-axis inhibitors. Tissue microarrays of matched primary and metastatic NSCLCs were used to compare four different PD-1 ligand (PD-L1) IHC techniques, as well as RNA ISH. Additional cases with whole genome and transcriptome data were assessed for molecular correlates of PD-L1 overexpression. Eighty cases were included in the IHC study. Multiple IHC methodologies showed a high rate of agreement (Kappa = 0.67). When calibrated to RNA expression, agreement improved significantly (Kappa = 0.90, p=0.0049). PD-L1 status of primary and metastatic tumors was discordant in 17 (22%) cases. This study suggests that different IHC methodologies for PD-L1 assessment provide slightly different results. There is significant discordance between the PD-L1 status of primary tumors and lymph node metastases. RNA ISH may be a useful adjunct to complement PD-L1 IHC testing.

Homologous Recombination Deficiency and Platinum-Based Therapy Outcomes in Advanced Breast Cancer

Purpose: Recent studies have identified mutation signatures of homologous recombination deficiency (HRD) in over 20% of breast cancers, as well as pancreatic, ovarian, and gastric cancers. There is an urgent need to understand the clinical implications of HRD signatures. Whereas BRCA1/2 mutations confer sensitivity to platinum-based chemotherapies, it is not yet clear whether mutation signatures can independently predict platinum response. Experimental Design: In this observational study, we sequenced tumor whole genomes (100× depth) and matched normals (60×) of 93 advanced-stage breast cancers (33 platinum-treated). We computed a published metric called HRDetect, independently trained to predict BRCA1/2 status, and assessed its capacity to predict outcomes on platinum-based chemotherapies. Clinical endpoints were overall survival (OS), total duration on platinum-based therapy (TDT), and radiographic evidence of clinical improvement (CI). Results: HRDetect predicted BRCA1/2 status with an area under the curve (AUC) of 0.94 and optimal threshold of 0.7. Elevated HRDetect was also significantly associated with CI on platinum-based therapy (AUC = 0.89; P = 0.006) with the same optimal threshold, even after adjusting for BRCA1/2 mutation status and treatment timing. HRDetect scores over 0.7 were associated with a 3-month extended median TDT (P = 0.0003) and 1.3-year extended median OS (P = 0.04). Conclusions: Our findings not only independently validate HRDetect, but also provide the first evidence of its association with platinum response in advanced breast cancer. We demonstrate that HRD mutation signatures may offer clinically relevant information independently of BRCA1/2 mutation status and hope this work will guide the development of clinical trials. Clin Cancer Res; 23(24); 7521–30. ©2017 AACR.

Molecular characterization of metastatic pancreatic neuroendocrine tumors (PNETs) using whole-genome and transcriptome sequencing

Pancreatic neuroendocrine tumors (PNETs) are a genomically and clinically heterogeneous group of pancreatic neoplasms often diagnosed with distant metastases. Recurrent somatic mutations, chromosomal aberrations, and gene expression signatures in PNETs have been described, but the clinical significance of these molecular changes is still poorly understood, and the clinical outcomes of PNET patients remain highly variable. To help identify the molecular factors that contribute to PNET progression and metastasis, and as part of an ongoing clinical trial at the BC Cancer Agency (clinicaltrials.gov ID: NCT02155621), the genomic and transcriptomic profiles of liver metastases from five patients (four PNETs and one neuroendocrine carcinoma) were analyzed. In four of the five cases, we identified biallelic loss of MEN1 and DAXX as well as recurrent regions with loss of heterozygosity. Several novel findings were observed, including focal amplification of MYCN concomitant with loss of APC and TP53 in one sample with wild-type MEN1 and DAXX. Transcriptome analyses revealed up-regulation of MYCN target genes in this sample, confirming a MYCN-driven gene expression signature. We also identified a germline NTHL1 fusion event in one sample that resulted in a striking C>T mutation signature profile not previously reported in PNETs. These varying molecular alterations suggest different cellular pathways may contribute to PNET progression, consistent with the heterogeneous clinical nature of this disease. Furthermore, genomic profiles appeared to correlate well with treatment response, lending support to the role of prospective genotyping efforts to guide therapy in PNETs.

Genomic profiling of pelvic genital type leiomyosarcoma in a woman with a germline CHEK2:c.1100delC mutation and a concomitant diagnosis of metastatic invasive ductal breast carcinoma

We describe a woman with the known pathogenic germline variant CHEK2:c.1100delC and synchronous diagnoses of both pelvic genital type leiomyosarcoma (LMS) and metastatic invasive ductal breast carcinoma. CHEK2 (checkpoint kinase 2) is a tumor-suppressor gene encoding a serine/threonine-protein kinase (CHEK2) involved in double-strand DNA break repair and cell cycle arrest. The CHEK2:c.1100delC variant is a moderate penetrance allele resulting in an approximately twofold increase in breast cancer risk. Whole-genome and whole-transcriptome sequencing were performed on the leiomyosarcoma and matched blood-derived DNA. Despite the presence of several genomic hits within the double-strand DNA damage pathway (CHEK2 germline variant and multiple RAD51B somatic structural variants), tumor profiling did not show an obvious DNA repair deficiency signature. However, even though the LMS displayed clear malignant features, its genomic profiling revealed several characteristics classically associated with leiomyomas including a translocation, t(12;14), with one breakpoint disrupting RAD51B and the other breakpoint upstream of HMGA2 with very high expression of HMGA2 and PLAG1. This is the first report of LMS genomic profiling in a patient with the germline CHEK2:c.1100delC variant and an additional diagnosis of metastatic invasive ductal breast carcinoma. We also describe a possible mechanistic relationship between leiomyoma and LMS based on genomic and transcriptome data. Our findings suggest that RAD51B translocation and HMGA2 overexpression may play an important role in LMS oncogenesis.

Whole genome and whole transcriptome genomic profiling of a metastatic eccrine porocarcinoma

Eccrine porocarcinomas (EPs) are rare malignant tumours of the intraepidermic sweat gland duct and most often arise from benign eccrine poromas. Some recurrent somatic genomic events have been identified in these malignancies, but very little is known about the complexity of their molecular pathophysiology. We describe the whole genome and whole transcriptome genomic profiling of a metastatic EP in a 66-year-old male patient with a previous history of localized porocarcinoma of the scalp. Whole genome and whole transcriptome genomic profiling was performed on the metastatic EP. Whole genome sequencing was performed on blood-derived DNA in order to allow a comparison between germline and somatic events. We found somatic copy losses of several tumour suppressor genes including APC, PTEN and CDKN2A, CDKN2B and CDKN1A. We identified a somatic hemizygous CDKN2A pathogenic splice site variant. De novo transcriptome assembly revealed abnormal splicing of CDKN2A p14ARF and p16INK4a. Elevated expression of oncogenes EGFR and NOTCH1 was noted and no somatic mutations were found in these genes. Wnt pathway somatic alterations were also observed. In conclusion, our results suggest that the molecular pathophysiology of malignant EP features high complexity and subtle interactions of multiple key genes. Cell cycle dysregulation and CDKN2A loss of function was found to be a new potential driver in EP tumourigenesis. Moreover, the combination of somatic copy number variants and abnormal gene expression perhaps partly related to epigenetic mechanisms, all likely contribute to the development of this rare malignancy in our patient.

CancerMine: A literature-mined resource for drivers, oncogenes and tumor suppressors in cancer

Understanding a mutation in cancer requires knowledge of the different roles that genes play in cancer as drivers, oncogenes and tumor suppressors. We present CancerMine, a high-quality text-mined knowledgebase that catalogues over 856 genes as drivers, 2,421 as oncogenes and 2,037 as tumor suppressors in 426 cancer types. We compile 3,485 genes that are not in the IntOGen resource of drivers and complement the Cancer Gene Census with 3,136 new genes identified as oncogenes and tumor suppressors. CancerMine provides a method for gene-centric clustering of cancer types illustrating genetic similarities between cancer types of different organs and was validated against data from the Cancer Genome Atlas (TCGA) project. Finally with 178 novel cancer gene mentions in publications each month, this resource will be updated monthly, pre-empting the need to manually curate the ever-increasing number of novel cancer associated genes. CancerMine is viewable through a web portal (http://bionlp.bcgsc.ca/cancermine/) and available for download (https://github.com/jakelever/cancermine).

Whole-Genome Sequencing in Cancer

Genome sequencing of cancer has fundamentally advanced our understanding of the underlying biology of this disease, and more recently has provided approaches to characterize and monitor tumors in the clinic, guiding and evaluating treatment. Although cancer research is relying more on whole-genome characterization, the clinical application of genomics is largely limited to targeted sequencing approaches, tailored to capture specific clinically relevant biomarkers. However, as sequencing costs reduce, and the tools to effectively analyze complex and large-scale data improve, the ability to effectively characterize whole genomes at scale in a clinically relevant time frame is now being piloted. This ability effectively blurs the line between clinical cancer research and the clinical management of the disease. This leads to a new paradigm in cancer management in which real-time analysis of an individuals disease can have a rapid and lasting impact on our understanding of how clinical practices need to change to exploit novel therapeutic rationales. In this article, we will discuss how whole-genome sequencing (WGS), often combined with transcriptome analysis, has been used to understand cancer and how this approach is uniquely positioned to provide a comprehensive view of an evolving disease in response to therapy.