Kristian Cibulskis

• Broad Institute of MIT and Harvard

Comprehensively mapping the genetic basis of human disease across diverse individuals is a long-standing goal for the field of human genetics 1–4 . The All of Us Research Program is a longitudinal cohort study aiming to enrol a diverse group of at least one million individuals across the USA to accelerate biomedical research and improve human healt...

The depletion of disruptive variation caused by purifying natural selection (constraint) has been widely used to investigate protein-coding genes underlying human disorders1-4, but attempts to assess constraint for non-protein-coding regions have proved more difficult. Here we aggregate, process and release a dataset of 76,156 human genomes from th...

Recessive diseases arise when both copies of a gene are impacted by a damaging genetic variant. When a patient carries two potentially causal variants in a gene, accurate diagnosis requires determining that these variants occur on different copies of the chromosome (that is, are in trans) rather than on the same copy (that is, in cis). However, cur...

In this Article, author Marquis P. Vawter was missing from the Genome Aggregation Database Consortium list. They are associated with the affiliation: ‘Department of Psychiatry & Human Behavior, University of California Irvine, Irvine, CA, USA’, and contributed to the generation of the primary data incorporated into the gnomAD resource. In addition,...

In this Article, author Marquis P. Vawter was missing from the Genome Aggregation Database Consortium list. They are associated with the affiliation: ‘Department of Psychiatry & Human Behavior, University of California Irvine, Irvine, CA, USA’, and contributed to the generation of the primary data incorporated into the gnomAD resource. The original...

A Correction to this paper has been published: https://doi.org/10.1038/s41591-020-01185-6.

Upstream open reading frames (uORFs) are tissue-specific cis -regulators of protein translation. Isolated reports have shown that variants that create or disrupt uORFs can cause disease. Here, in a systematic genome-wide study using 15,708 whole genome sequences, we show that variants that create new upstream start codons, and variants disrupting s...

Human genetic variants predicted to cause loss-of-function of protein-coding genes (pLoF variants) provide natural in vivo models of human gene inactivation and can be valuable indicators of gene function and the potential toxicity of therapeutic inhibitors targeting these genes1,2. Gain-of-kinase-function variants in LRRK2 are known to significant...

Genetic variants that inactivate protein-coding genes are a powerful source of information about the phenotypic consequences of gene disruption: genes that are crucial for the function of an organism will be depleted of such variants in natural populations, whereas non-essential genes will tolerate their accumulation. However, predicted loss-of-fun...

Structural variants (SVs) rearrange large segments of DNA1 and can have profound consequences in evolution and human disease2,3. As national biobanks, disease-association studies, and clinical genetic testing have grown increasingly reliant on genome sequencing, population references such as the Genome Aggregation Database (gnomAD)4 have become int...

Naturally occurring human genetic variants that are predicted to inactivate protein-coding genes provide an in vivo model of human gene inactivation that complements knockout studies in cells and model organisms. Here we report three key findings regarding the assessment of candidate drug targets using human loss-of-function variants. First, even e...

The acceleration of DNA sequencing in samples from patients and population studies has resulted in extensive catalogues of human genetic variation, but the interpretation of rare genetic variants remains problematic. A notable example of this challenge is the existence of disruptive variants in dosage-sensitive disease genes, even in apparently hea...

Mutect2 is a somatic variant caller that uses local assembly and realignment to detect SNVs and indels. Assembly implies whole haplotypes and read pairs, rather than single bases, as the atomic units of biological variation and sequencing evidence, improving variant calling. Beyond local assembly and alignment, Mutect2 is based on several probabili...

FireCloud, one of three NCI Cloud Pilots, is a collaborative genome analysis platform built on a cloud computing infrastructure. FireCloud aims to solve the many challenges presented by the increasingly large data sets and computing requirements employed in cancer research. However, cost uncertainty associated with cloud computing's pay-as-you-go m...

Comprehensive multiplatform analysis of 80 uveal melanomas (UM) identifies four molecularly distinct, clinically relevant subtypes: two associated with poor-prognosis monosomy 3 (M3) and two with better-prognosis disomy 3 (D3). We show that BAP1 loss follows M3 occurrence and correlates with a global DNA methylation state that is distinct from D3-U...

To further our understanding of the somatic genetic basis of uveal melanoma, we sequenced the protein-coding regions of 52 primary tumors and 3 liver metastases together with paired normal DNA. Known recurrent mutations were identified in GNAQ, GNA11, BAP1, EIF1AX, and SF3B1. The role of mutated EIF1AX was tested using loss of function approaches i...

Genes with significant changes in translational efficiency following EIF1AX knockdown. P-value and cluster is indicated for each gene. (XLS)

Kaplan-Meier analysis of recurrent alterations in cohort. (A and B) Kaplan-Meier analysis showing primary uveal melanoma patients with evaluable OS data who had either monosomy 3 (n = 20) or disomy 3 (n = 23) tumors. OS reflective of death from metastatic uveal melanoma; the median survival was 1216 days in the monosomy 3 cohort and not reached in...

Clinical characteristics of 52 uveal melanomas. (XLS)

EIF1AX dependency across 216 cell lines from Project Achilles. (A) Histograms represent shRNA level scores (normalized log fold change) for 5 EIF1AX (top) and 5 RPS6 (bottom) shRNAs from 216 cell lines in Achilles v2.4.3. Lower values represent more depletion indicating more dependency. EIF1AX shRNAs used in this study are bolded. (B) Immunoblot an...

Validation of selected mutations by targeted resequencing. (XLS)

All somatic alterations identified in trio analysis. (XLS)

Trio mutation validation and clinical timeline. (A) IGV screenshot of SMARCA4 mutation from exome sequencing and targeted validation of UM45. (B) Trio 2 biopsy and treatment are indicated across time course. (PDF)

Supplementary materials and methods. (DOC)

Sequencing metrics of 52 uveal melanomas. (XLS)

All somatic alterations identified by whole exome sequencing in 52 pairs. (XLS)

Copy number profiles of 52 uveal melanomas. Coverage values from whole exome sequencing were converted into segmentation files and visualized using IGV. Samples are ordered by chromosome 3 status, or labeled Indeterminate (Ind.) due to noise across the genome. 1AX indicates samples with an EIF1AX mutation. (PDF)

EIF1AX sequence and protein expression across UM cell lines. (A) Exon 2 sequencing trace displays putative EIF1AXG6D mutation in 92.1 cell line. (B) Immunoblot analysis of EIF1AX protein levels. (PDF)

EIF1AX sequence and expression levels from polysome profiling RNAseq. (A) IGV screenshot of EIF1AX exon 2 start indicates exclusive mRNA expression of the G6D variant in the 92.1 cell line. (B) Heatmap displays EIF1AX expression levels in RPKM for total and polysome-associated mRNA in 92.1 cells expressing indicated shRNAs. (C) As in (B), but for O...

We performed genomic, epigenomic, transcriptomic, and proteomic characterizations of uterine carcinosarcomas (UCSs). Cohort samples had extensive copy-number alterations and highly recurrent somatic mutations. Frequent mutations were found in TP53, PTEN, PIK3CA, PPP2R1A, FBXW7, and KRAS, similar to endometrioid and serous uterine carcinomas. Transc...

Oesophageal cancers are prominent worldwide; however, there are few targeted therapies and survival rates for these cancers remain dismal. Here we performed a comprehensive molecular analysis of 164 carcinomas of the oesophagus derived from Western and Eastern populations. Beyond known histopathological and epidemiologic distinctions, molecular fea...

Resistance to the Brutonâ € s tyrosine kinase (BTK) inhibitor ibrutinib has been attributed solely to mutations in BTK and related pathway molecules. Using whole-exome and deep-Targeted sequencing, we dissect evolution of ibrutinib resistance in serial samples from five chronic lymphocytic leukaemia patients. In two patients, we detect BTK-C481S mu...

Supplementary Figures 1-6 and Supplementary Tables 1-4.

Mutation annotation file including cancer cell fraction estimates

Uveal melanoma (UM) is a rare type of melanoma that occurs in the iris, ciliary body, and choroid of the eye. Metastatic disease is typically found in the liver and has no effective therapeutic options. To improve our understanding of the genetic drivers of UM, we sequenced the exome of 61 primary tumors and 3 liver metastases, each with matched no...

Summary There is substantial heterogeneity among primary prostate cancers, evident in the spectrum of molecular abnormalities and its variable clinical course. As part of The Cancer Genome Atlas (TCGA), we present a comprehensive molecular analysis of 333 primary prostate carcinomas. Our results revealed a molecular taxonomy in which 74% of these t...

Which genetic alterations drive tumorigenesis and how they evolve over the course of disease and therapy are central questions in cancer biology. Here we identify 44 recurrently mutated genes and 11 recurrent somatic copy number variations through whole-exome sequencing of 538 chronic lymphocytic leukaemia (CLL) and matched germline DNA samples, 27...

Brain metastases are associated with a dismal prognosis. Whether brain metastases harbor distinct genetic alterations beyond those observed in primary tumors is unknown. We performed whole-exome sequencing of 86 matched brain metastases, primary tumors, and normal tissue. In all clonally related cancer samples, we observed branched evolution, where...

Detection of somatic mutations in human leukocyte antigen (HLA) genes using whole-exome sequencing (WES) is hampered by the high polymorphism of the HLA loci, which prevents alignment of sequencing reads to the human reference genome. We describe a computational pipeline that enables accurate inference of germline alleles of class I HLA-A, B and C...

Recent studies have revealed enhanced somatic mutation rate in HLA genes in several tumor types and has strongly implicated HLA dysfunction as a possible mediator of immune evasion. Mutation detection in this highly polymorphic and GC-rich locus, however, is complicated by suboptimal alignment to the canonical reference genome and lowered capture e...

s: AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC Background: Brain metastases are associated with a dismal prognosis. There is a limited understanding of the oncogenic alterations harbored by brain metastases and whether these are shared with their primary tumors. Our objectives were to (1) elucidate t...

We describe the landscape of genomic alterations in cutaneous melanomas through DNA, RNA, and protein-based analysis of 333 primary and/or metastatic melanomas from 331 patients. We establish a framework for genomic classification into one of four subtypes based on the pattern of the most prevalent significantly mutated genes: mutant BRAF, mutant R...

In Rspondin-based 3D cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. We report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely recapit...

The Cancer Genome Atlas profiled 279 head and neck squamous cell carcinomas (HNSCCs) to provide a comprehensive landscape of somatic genomic alterations. Here we show that human-papillomavirus-associated tumours are dominated by helical domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3, and amplification of the cell...

Pompe disease, an inherited deficiency of lysosomal acid alpha-glucosidase (GAA), is a metabolic myopathy with heterogeneous clinical presentations. Late-onset Pompe disease (LOPD) is a debilitating progressive muscle disorder that can occur anytime from early childhood to late adulthood. Enzyme replacement therapy (ERT) with recombinant human GAA...

We report somatic mutations of RNF43 in over 18% of colorectal adenocarcinomas and endometrial carcinomas. RNF43 encodes an E3 ubiquitin ligase that negatively regulates Wnt signaling. Truncating mutations of RNF43 are more prevalent in microsatellite-unstable tumors and show mutual exclusivity with inactivating APC mutations in colorectal adenocar...

Background: Cervical cancer is a major public health problem worldwide. We have recently identified novel significantly recurrent somatic mutations in HLA-B, ERBB2 and MAPK1 in cervical squamous cell carcinomas. However, the significance of somatic mutations and copy number alterations for clinical phenotype in cervical cancer is not well understoo...

Despite that fact that 90% of cancer deaths are due to metastasis, relatively little is known about how metastases evolve from primary cancers. We subjected 101 trios consisting of primary tumor, brain metastasis, and normal reference tissue to whole exome sequencing (WES). To analyze the data, we developed novel computational tools to derive high...

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Comprehensive analysis of cancer genomes in clinical settings holds the promise to inform prognoses and guide the deployment of precise cancer treatments. A major barrier, however, is the inaccessibility of adequate metastatic tissue for accurate genomic analysis. The recognitio...

Recent genomic analyses of pathologically defined tumor types identify ''within-a-tissue'' disease sub-types. However, the extent to which genomic sig-natures are shared across tissues is still unclear. We performed an integrative analysis using five genome-wide platforms and one proteomic platform on 3,527 specimens from 12 cancer types, revealing...

Adenocarcinoma of the lung is the leading cause of cancer death worldwide. Here we report molecular profiling of 230 resected lung adenocarcinomas using messenger RNA, microRNA and DNA sequencing integrated with copy number, methylation and proteomic analyses. High rates of somatic mutation were seen (mean 8.9 mutations per megabase). Eighteen gene...

Background: Inhibition of the activated epidermal growth factor receptor (EGFR) with either enzymatic kinase inhibitors or anti-EGFR antibodies such as cetuximab, is an effective modality of treatment for multiple human cancers. Enzymatic EGFR inhibitors are effective for lung adenocarcinomas with somatic kinase domain EGFR mutations while, paradox...

Key Points Tumor neoantigens are a promising class of immunogens based on exquisite tumor specificity and the lack of central tolerance against them. Massively parallel DNA sequencing with class I prediction enables systematic identification of tumor neoepitopes (including from CLL).

Translating whole-exome sequencing (WES) for prospective clinical use may have an impact on the care of patients with cancer; however, multiple innovations are necessary for clinical implementation. These include rapid and robust WES of DNA derived from formalin-fixed, paraffin-embedded tumor tissue, analytical output similar to data from frozen sa...

Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we...

Urothelial carcinoma of the bladder is a common malignancy that causes approximately 150,000 deaths per year worldwide. So far, no molecularly targeted agents have been approved for treatment of the disease. As part of The Cancer Genome Atlas project, we report here an integrated analysis of 131 urothelial carcinomas to provide a comprehensive land...

Small cell lung carcinoma (SCLC) is a highly lethal, smoking-associated cancer with few known targetable genetic alterations. Using genome sequencing, we characterized the somatic evolution of a genetically engineered mouse model (GEMM) of SCLC initiated by loss of Trp53 and Rb1. We identified alterations in DNA copy number and complex genomic rear...

We performed massively parallel sequencing of paired tumor/normal samples from 203 multiple myeloma (MM) patients and identified significantly mutated genes and copy number alterations and discovered putative tumor suppressor genes by determining homozygous deletions and loss of heterozygosity. We observed frequent mutations in KRAS (particularly i...

Cervical cancer is responsible for 10-15% of cancer-related deaths in women worldwide. The aetiological role of infection with high-risk human papilloma viruses (HPVs) in cervical carcinomas is well established. Previous studies have also implicated somatic mutations in PIK3CA, PTEN, TP53, STK11 and KRAS as well as several copy-number alterations i...

Lung squamous cell carcinoma (SCC) is the second most prevalent type of lung cancer. Currently, no targeted therapeutics are approved for treatment of this cancer, largely because of a lack of systematic understanding of the molecular pathogenesis of the disease. To identify therapeutic targets and perform comparative analyses of lung SCC, we probe...

Treatment of BRAF-mutant melanoma with combined dabrafenib and trametinib, which target RAF and the downstream MAP–ERK kinase (MEK)1 and MEK2 kinases, respectively, improves progression-free survival and response rates compared with dabrafenib monotherapy. Mechanisms of clinical resistance to combined RAF/MEK inhibition are unknown. We performed wh...

Most patients with BRAFV600-mutant metastatic melanoma develop resistance to selective RAF kinase inhibitors. The spectrum of clinical genetic resistance mechanisms to RAF inhibitors and options for salvage therapy are incompletely understood. We performed whole-exome sequencing on formalin-fixed, paraffin-embedded tumors from 45 patients with BRAF...

The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib thwarts B cell receptor (BCR) signaling via irreversible inhibition of BTK, and induces durable remissions in relapsed/refractory CLL with a progression-free survival rate of 75% after 26 months of therapy (Byrd JC et al., NEJM 2013). However, a small fraction of patients treated with this targ...

The diagnosed incidence of small intestine neuroendocrine tumors (SI-NETs) is increasing, and the underlying genomic mechanisms have not yet been defined. Using exome- and genome-sequence analysis of SI-NETs, we identified recurrent somatic mutations and deletions in CDKN1B, the cyclin-dependent kinase inhibitor gene, which encodes p27. We observed...

Small cell lung carcinoma (SCLC) is a highly lethal tumor for which few targetable genetic alterations have been identified. SCLC is tightly associated with an extended history of tobacco use, and recent DNA sequencing studies have revealed highly mutated SCLC cancer genomes. Interestingly, two studies analyzing the human SCLC genome report distinc...

Clonal evolution is a key feature of cancer progression and relapse. We present a computational technique for analyzing whole exome sequence data to measure the fraction of cancer cells harboring each somatic mutation and resolve subclonal cell populations. We applied our methods to 149 chronic lymphocytic leukemia (CLL) samples to classify driver...

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The ability to harness the power of next generation sequencing for characterizing the cancer genome would be extremely valuable to the cancer research community and ultimately improve diagnosis and individualized therapy. We present a targeted approach using massively par...

Genetic changes underlying clear cell renal cell carcinoma (ccRCC) include alterations in genes controlling cellular oxygen sensing (for example, VHL) and the maintenance of chromatin states (for example, PBRM1). We surveyed more than 400 tumours using different genomic platforms and identified 19 significantly mutated genes. The PI(3)K/AKT pathway...

Major international projects are underway that are aimed at creating a comprehensive catalogue of all the genes responsible for the initiation and progression of cancer. These studies involve the sequencing of matched tumour-normal samples followed by mathematical analysis to identify those genes in which mutations occur more frequently than expect...

We performed an integrated genomic, transcriptomic and proteomic characterization of 373 endometrial carcinomas using array- and sequencing-based technologies. Uterine serous tumours and ∼25% of high-grade endometrioid tumours had extensive copy number alterations, few DNA methylation changes, low oestrogen receptor/progesterone receptor levels, an...

The analysis of exonic DNA from prostate cancers has identified recurrently mutated genes, but the spectrum of genome-wide alterations has not been profiled extensively in this disease. We sequenced the genomes of 57 prostate tumors and matched normal tissues to characterize somatic alterations and to study how they accumulate during oncogenesis an...

Characterizing the genomic evolution of cancer is critical to understanding disease progression and identifying potential therapeutic targets. By examining the clonal hierarchy of genomic lesions in common tumors, it would be possible to reconstruct the path of oncogenic events that drive carcinogenesis. Reliable assessment of such paths from high-...

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Cervical cancer is a major public health problem worldwide. The etiological role of human papilloma virus (HPV) infections in cervical cancer is well established. However, HPV infection is insufficient to account for the development of cervical cancers because...

The incidence of esophageal adenocarcinoma (EAC) has risen 600% over the last 30 years. With a 5-year survival rate of ∼15%, the identification of new therapeutic targets for EAC is greatly important. We analyze the mutation spectra from whole-exome sequencing of 149 EAC tumor-normal pairs, 15 of which have also been subjected to whole-genome seque...