Michael F. Berger

The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.

The systematic translation of cancer genomic data into knowledge of tumour biology and therapeutic possibilities remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacological annotation is available. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacological profiles for 24 anticancer drugs across 479 of the cell lines, this collection allowed identification of genetic, lineage, and gene-expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Together, our results indicate that large, annotated cell-line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of ‘personalized’ therapeutic regimens.

The Mutational Landscape of Head and Neck Squamous Cell Carcinoma

The mutational profile of head and neck cancer is complex and may pose challenges to the development of targeted therapies. Head and neck squamous cell carcinoma (HNSCC) is a common, morbid, and frequently lethal malignancy. To uncover its mutational spectrum, we analyzed whole-exome sequencing data from 74 tumor-normal pairs. The majority exhibited a mutational profile consistent with tobacco exposure; human papillomavirus was detectable by sequencing DNA from infected tumors. In addition to identifying previously known HNSCC genes (TP53, CDKN2A, PTEN, PIK3CA, and HRAS), our analysis revealed many genes not previously implicated in this malignancy. At least 30% of cases harbored mutations in genes that regulate squamous differentiation (for example, NOTCH1, IRF6, and TP63), implicating its dysregulation as a major driver of HNSCC carcinogenesis. More generally, the results indicate the ability of large-scale sequencing to reveal fundamental tumorigenic mechanisms.

The genomic complexity of primary human prostate cancer

Prostate cancer is the second most common cause of male cancer deaths in the United States. However, the full range of prostate cancer genomic alterations is incompletely characterized. Here we present the complete sequence of seven primary human prostate cancers and their paired normal counterparts. Several tumours contained complex chains of balanced (that is, ‘copy-neutral’) rearrangements that occurred within or adjacent to known cancer genes. Rearrangement breakpoints were enriched near open chromatin, androgen receptor and ERG DNA binding sites in the setting of the ETS gene fusion TMPRSS2–ERG, but inversely correlated with these regions in tumours lacking ETS fusions. This observation suggests a link between chromatin or transcriptional regulation and the genesis of genomic aberrations. Three tumours contained rearrangements that disrupted CADM2, and four harboured events disrupting either PTEN (unbalanced events), a prostate tumour suppressor, or MAGI2 (balanced events), a PTEN interacting protein not previously implicated in prostate tumorigenesis. Thus, genomic rearrangements may arise from transcriptional or chromatin aberrancies and engage prostate tumorigenic mechanisms.

Capturing intra-tumor genetic heterogeneity by de novo mutation profiling of circulating cell-free tumor DNA: a proof-of-principle

BACKGROUND Plasma-derived cell-free tumor DNA (ctDNA) constitutes a potential surrogate for tumor DNA obtained from tissue biopsies. We posit that massively parallel sequencing (MPS) analysis of ctDNA may help define the repertoire of mutations in breast cancer and monitor tumor somatic alterations during the course of targeted therapy. PATIENT AND METHODS A 66-year-old patient presented with synchronous estrogen receptor-positive/HER2-negative, highly proliferative, grade 2, mixed invasive ductal-lobular carcinoma with bone and liver metastases at diagnosis. DNA extracted from archival tumor material, plasma and peripheral blood leukocytes was subjected to targeted MPS using a platform comprising 300 cancer genes known to harbor actionable mutations. Multiple plasma samples were collected during the fourth line of treatment with an AKT inhibitor. RESULTS Average read depths of 287x were obtained from the archival primary tumor, 139x from the liver metastasis and between 200x and 900x from ctDNA samples. Sixteen somatic non-synonymous mutations were detected in the liver metastasis, of which 9 (CDKN2A, AKT1, TP53, JAK3, TSC1, NF1, CDH1, MML3 and CTNNB1) were also detected in >5% of the alleles found in the primary tumor sample. Not all mutations identified in the metastasis were reliably identified in the primary tumor (e.g. FLT4). Analysis of ctDNA, nevertheless, captured all mutations present in the primary tumor and/or liver metastasis. In the longitudinal monitoring of the patient, the mutant allele fractions identified in ctDNA samples varied over time and mirrored the pharmacodynamic response to the targeted therapy as assessed by positron emission tomography-computed tomography. CONCLUSIONS This proof-of-principle study is one of the first to demonstrate that high-depth targeted MPS of plasma-derived ctDNA constitutes a potential tool for de novo mutation identification and monitoring of somatic genetic alterations during the course of targeted therapy, and may be employed to overcome the challenges posed by intra-tumor genetic heterogeneity. REGISTERED CLINICAL TRIAL: www.clinicaltrials.gov, NCT01090960.BACKGROUND Plasma-derived cell-free tumor DNA (ctDNA) constitutes a potential surrogate for tumor DNA obtained from tissue biopsies. We posit that massively parallel sequencing (MPS) analysis of ctDNA may help define the repertoire of mutations in breast cancer and monitor tumor somatic alterations during the course of targeted therapy. PATIENT AND METHODS A 66-year-old patient presented with synchronous estrogen receptor-positive/HER2-negative, highly proliferative, grade 2, mixed invasive ductal-lobular carcinoma with bone and liver metastases at diagnosis. DNA extracted from archival tumor material, plasma and peripheral blood leukocytes was subjected to targeted MPS using a platform comprising 300 cancer genes known to harbor actionable mutations. Multiple plasma samples were collected during the fourth line of treatment with an AKT inhibitor. RESULTS Average read depths of 287x were obtained from the archival primary tumor, 139x from the liver metastasis and between 200x and 900x from ctDNA samples. Sixteen somatic non-synonymous mutations were detected in the liver metastasis, of which 9 (CDKN2A, AKT1, TP53, JAK3, TSC1, NF1, CDH1, MML3 and CTNNB1) were also detected in >5% of the alleles found in the primary tumor sample. Not all mutations identified in the metastasis were reliably identified in the primary tumor (e.g. FLT4). Analysis of ctDNA, nevertheless, captured all mutations present in the primary tumor and/or liver metastasis. In the longitudinal monitoring of the patient, the mutant allele fractions identified in ctDNA samples varied over time and mirrored the pharmacodynamic response to the targeted therapy as assessed by positron emission tomography-computed tomography. CONCLUSIONS This proof-of-principle study is one of the first to demonstrate that high-depth targeted MPS of plasma-derived ctDNA constitutes a potential tool for de novo mutation identification and monitoring of somatic genetic alterations during the course of targeted therapy, and may be employed to overcome the challenges posed by intra-tumor genetic heterogeneity. REGISTERED CLINICAL TRIAL www.clinicaltrials.gov, NCT01090960.

Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing

As more clinically relevant cancer genes are identified, comprehensive diagnostic approaches are needed to match patients to therapies, raising the challenge of optimization and analytical validation of assays that interrogate millions of bases of cancer genomes altered by multiple mechanisms. Here we describe a test based on massively parallel DNA sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes from routine formalin-fixed and paraffin-embedded (FFPE) clinical specimens. We implemented a practical validation strategy with reference samples of pooled cell lines that model key determinants of accuracy, including mutant allele frequency, indel length and amplitude of copy change. Test sensitivity achieved was 95–99% across alteration types, with high specificity (positive predictive value >99%). We confirmed accuracy using 249 FFPE cancer specimens characterized by established assays. Application of the test to 2,221 clinical cases revealed clinically actionable alterations in 76% of tumors, three times the number of actionable alterations detected by current diagnostic tests.

Dissecting Therapeutic Resistance to RAF Inhibition in Melanoma by Tumor Genomic Profiling

A detailed understanding of the mechanisms by which tumors acquire resistance to targeted anticancer agents should speed the development of treatment strategies with lasting clinical efficacy. RAF inhibition in BRAF-mutant melanoma exemplifies the promise and challenge of many targeted drugs; although response rates are high, resistance invariably develops. Here, we articulate overarching principles of resistance to kinase inhibitors, as well as a translational approach to characterize resistance in the clinical setting through tumor mutation profiling. As a proof of principle, we performed targeted, massively parallel sequencing of 138 cancer genes in a tumor obtained from a patient with melanoma who developed resistance to PLX4032 after an initial dramatic response. The resulting profile identified an activating mutation at codon 121 in the downstream kinase MEK1 that was absent in the corresponding pretreatment tumor. The MEK1(C121S) mutation was shown to increase kinase activity and confer robust resistance to both RAF and MEK inhibition in vitro. Thus, MEK1(C121S) or functionally similar mutations are predicted to confer resistance to combined MEK/RAF inhibition. These results provide an instructive framework for assessing mechanisms of acquired resistance to kinase inhibition and illustrate the use of emerging technologies in a manner that may accelerate personalized cancer medicine.

Melanoma genome sequencing reveals frequent PREX2 mutations

Melanoma is notable for its metastatic propensity, lethality in the advanced setting and association with ultraviolet exposure early in life. To obtain a comprehensive genomic view of melanoma in humans, we sequenced the genomes of 25 metastatic melanomas and matched germline DNA. A wide range of point mutation rates was observed: lowest in melanomas whose primaries arose on non-ultraviolet-exposed hairless skin of the extremities (3 and 14 per megabase (Mb) of genome), intermediate in those originating from hair-bearing skin of the trunk (5–55 per Mb), and highest in a patient with a documented history of chronic sun exposure (111 per Mb). Analysis of whole-genome sequence data identified PREX2 (phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2)—a PTEN-interacting protein and negative regulator of PTEN in breast cancer—as a significantly mutated gene with a mutation frequency of approximately 14% in an independent extension cohort of 107 human melanomas. PREX2 mutations are biologically relevant, as ectopic expression of mutant PREX2 accelerated tumour formation of immortalized human melanocytes in vivo. Thus, whole-genome sequencing of human melanoma tumours revealed genomic evidence of ultraviolet pathogenesis and discovered a new recurrently mutated gene in melanoma.

Genome Sequencing Identifies a Basis for Everolimus Sensitivity

Tumor genome sequencing reveals the molecular basis of a patient’s unexpected and dramatic response to a cancer drug. Cancer drugs often induce dramatic responses in a small minority of patients. We used whole-genome sequencing to investigate the genetic basis of a durable remission of metastatic bladder cancer in a patient treated with everolimus, a drug that inhibits the mTOR (mammalian target of rapamycin) signaling pathway. Among the somatic mutations was a loss-of-function mutation in TSC1 (tuberous sclerosis complex 1), a regulator of mTOR pathway activation. Targeted sequencing revealed TSC1 mutations in about 8% of 109 additional bladder cancers examined, and TSC1 mutation correlated with everolimus sensitivity. These results demonstrate the feasibility of using whole-genome sequencing in the clinical setting to identify previously occult biomarkers of drug sensitivity that can aid in the identification of patients most likely to respond to targeted anticancer drugs.

Variation in homeodomain DNA-binding revealed by high-resolution analysis of sequence preferences

Most homeodomains are unique within a genome, yet many are highly conserved across vast evolutionary distances, implying strong selection on their precise DNA-binding specificities. We determined the binding preferences of the majority (168) of mouse homeodomains to all possible 8-base sequences, revealing rich and complex patterns of sequence specificity and showing that there are at least 65 distinct homeodomain DNA-binding activities. We developed a computational system that successfully predicts binding sites for homeodomain proteins as distant from mouse as Drosophila and C. elegans, and we infer full 8-mer binding profiles for the majority of known animal homeodomains. Our results provide an unprecedented level of resolution in the analysis of this simple domain structure and suggest that variation in sequence recognition may be a factor in its functional diversity and evolutionary success.

ESR1 ligand-binding domain mutations in hormone-resistant breast cancer

Seventy percent of breast cancers express estrogen receptor (ER), and most of these are sensitive to ER inhibition. However, many such tumors for unknown reasons become refractory to inhibition of estrogen action in the metastatic setting. We conducted a comprehensive genetic analysis of two independent cohorts of metastatic ER-positive breast tumors and identified mutations in ESR1 affecting the ligand-binding domain (LBD) in 14 of 80 cases. These included highly recurrent mutations encoding p.Tyr537Ser, p.Tyr537Asn and p.Asp538Gly alterations. Molecular dynamics simulations suggest that the structures of the Tyr537Ser and Asp538Gly mutants involve hydrogen bonding of the mutant amino acids with Asp351, thus favoring the agonist conformation of the receptor. Consistent with this model, mutant receptors drive ER-dependent transcription and proliferation in the absence of hormone and reduce the efficacy of ER antagonists. These data implicate LBD-mutant forms of ER in mediating clinical resistance to hormonal therapy and suggest that more potent ER antagonists may be of substantial therapeutic benefit.