Martin Moorhead

Diverse somatic mutation patterns and pathway alterations in human cancers.

The systematic characterization of somatic mutations in cancer genomes is essential for understanding the disease and for developing targeted therapeutics. Here we report the identification of 2,576 somatic mutations across approximately 1,800 megabases of DNA representing 1,507 coding genes from 441 tumours comprising breast, lung, ovarian and prostate cancer types and subtypes. We found that mutation rates and the sets of mutated genes varied substantially across tumour types and subtypes. Statistical analysis identified 77 significantly mutated genes including protein kinases, G-protein-coupled receptors such as GRM8, BAI3, AGTRL1 (also called APLNR) and LPHN3, and other druggable targets. Integrated analysis of somatic mutations and copy number alterations identified another 35 significantly altered genes including GNAS, indicating an expanded role for galpha subunits in multiple cancer types. Furthermore, our experimental analyses demonstrate the functional roles of mutant GNAO1 (a Galpha subunit) and mutant MAP2K4 (a member of the JNK signalling pathway) in oncogenesis. Our study provides an overview of the mutational spectra across major human cancers and identifies several potential therapeutic targets.

Population structure, differential bias and genomic control in a large-scale, case-control association study

The main problems in drawing causal inferences from epidemiological case-control studies are confounding by unmeasured extraneous factors, selection bias and differential misclassification of exposure. In genetics the first of these, in the form of population structure, has dominated recent debate. Population structure explained part of the significant +11.2% inflation of test statistics we observed in an analysis of 6,322 nonsynonymous SNPs in 816 cases of type 1 diabetes and 877 population-based controls from Great Britain. The remainder of the inflation resulted from differential bias in genotype scoring between case and control DNA samples, which originated from two laboratories, causing false-positive associations. To avoid excluding SNPs and losing valuable information, we extended the genomic control method by applying a variable downweighting to each SNP.

Deep-sequencing approach for minimal residual disease detection in acute lymphoblastic leukemia

The persistence of minimal residual disease (MRD) during therapy is the strongest adverse prognostic factor in acute lymphoblastic leukemia (ALL). We developed a high-throughput sequencing method that universally amplifies antigen-receptor gene segments and identifies all clonal gene rearrangements (ie, leukemia-specific sequences) at diagnosis, allowing monitoring of disease progression and clonal evolution during therapy. In the present study, the assay specifically detected 1 leukemic cell among greater than 1 million leukocytes in spike-in experiments. We compared this method with the gold-standard MRD assays multiparameter flow cytometry and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) using diagnostic and follow-up samples from 106 patients with ALL. Sequencing detected MRD in all 28 samples shown to be positive by flow cytometry and in 35 of the 36 shown to be positive by ASO-PCR and revealed MRD in 10 and 3 additional samples that were negative by flow cytometry and ASO-PCR, respectively. We conclude that this new method allows monitoring of treatment response in ALL and other lymphoid malignancies with great sensitivity and precision. The www.clinicaltrials.gov identifier number for the Total XV study is NCT00137111.

Prognostic value of deep sequencing method for minimal residual disease detection in multiple myeloma

We assessed the prognostic value of minimal residual disease (MRD) detection in multiple myeloma (MM) patients using a sequencing-based platform in bone marrow samples from 133 MM patients in at least very good partial response (VGPR) after front-line therapy. Deep sequencing was carried out in patients in whom a high-frequency myeloma clone was identified and MRD was assessed using the IGH-VDJH, IGH-DJH, and IGK assays. The results were contrasted with those of multiparametric flow cytometry (MFC) and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR). The applicability of deep sequencing was 91%. Concordance between sequencing and MFC and ASO-PCR was 83% and 85%, respectively. Patients who were MRD(-) by sequencing had a significantly longer time to tumor progression (TTP) (median 80 vs 31 months; P < .0001) and overall survival (median not reached vs 81 months; P = .02), compared with patients who were MRD(+). When stratifying patients by different levels of MRD, the respective TTP medians were: MRD ≥10(-3) 27 months, MRD 10(-3) to 10(-5) 48 months, and MRD <10(-5) 80 months (P = .003 to .0001). Ninety-two percent of VGPR patients were MRD(+). In complete response patients, the TTP remained significantly longer for MRD(-) compared with MRD(+) patients (131 vs 35 months; P = .0009).

Circulating tumour DNA and CT monitoring in patients with untreated diffuse large B-cell lymphoma: a correlative biomarker study

BACKGROUND Diffuse large-B-cell lymphoma is curable, but when treatment fails, outcome is poor. Although imaging can help to identify patients at risk of treatment failure, they are often imprecise, and radiation exposure is a potential health risk. We aimed to assess whether circulating tumour DNA encoding the clonal immunoglobulin gene sequence could be detected in the serum of patients with diffuse large-B-cell lymphoma and used to predict clinical disease recurrence after frontline treatment. METHODS We used next-generation DNA sequencing to retrospectively analyse cell-free circulating tumour DNA in patients assigned to one of three treatment protocols between May 8, 1993, and June 6, 2013. Eligible patients had diffuse large-B-cell lymphoma, no evidence of indolent lymphoma, and were previously untreated. We obtained serial serum samples and concurrent CT scans at specified times during most treatment cycles and up to 5 years of follow-up. VDJ gene segments of the rearranged immunoglobulin receptor genes were amplified and sequenced from pretreatment specimens and serum circulating tumour DNA encoding the VDJ rearrangements was quantitated. FINDINGS Tumour clonotypes were identified in pretreatment specimens from 126 patients who were followed up for a median of 11 years (IQR 6·8-14·2). Interim monitoring of circulating tumour DNA at the end of two treatment cycles in 108 patients showed a 5-year time to progression of 41·7% (95% CI 22·2-60·1) in patients with detectable circulating tumour DNA and 80·2% (69·6-87·3) in those without detectable circulating tumour DNA (p<0·0001). Detectable interim circulating tumour DNA had a positive predictive value of 62·5% (95% CI 40·6-81·2) and a negative predictive value of 79·8% (69·6-87·8). Surveillance monitoring of circulating tumour DNA was done in 107 patients who achieved complete remission. A Cox proportional hazards model showed that the hazard ratio for clinical disease progression was 228 (95% CI 51-1022) for patients who developed detectable circulating tumour DNA during surveillance compared with patients with undetectable circulating tumour DNA (p<0·0001). Surveillance circulating tumour DNA had a positive predictive value of 88·2% (95% CI 63·6-98·5) and a negative predictive value of 97·8% (92·2-99·7) and identified risk of recurrence at a median of 3·5 months (range 0-200) before evidence of clinical disease. INTERPRETATION Surveillance circulating tumour DNA identifies patients at risk of recurrence before clinical evidence of disease in most patients and results in a reduced disease burden at relapse. Interim circulating tumour DNA is a promising biomarker to identify patients at high risk of treatment failure. FUNDING National Cancer Institute and Adaptive Biotechnologies.

Minimal residual disease quantification using consensus primers and high-throughput IGH sequencing predicts post-transplant relapse in chronic lymphocytic leukemia

Quantification of minimal residual disease (MRD) following allogeneic hematopoietic cell transplantation (allo-HCT) predicts post-transplant relapse in patients with chronic lymphocytic leukemia (CLL). We utilized an MRD-quantification method that amplifies immunoglobulin heavy chain (IGH) loci using consensus V and J segment primers followed by high-throughput sequencing (HTS), enabling quantification with a detection limit of one CLL cell per million mononuclear cells. Using this IGH–HTS approach, we analyzed MRD patterns in over 400 samples from 40 CLL patients who underwent reduced-intensity allo-HCT. Nine patients relapsed within 12 months post-HCT. Of the 31 patients in remission at 12 months post-HCT, disease-free survival was 86% in patients with MRD <10−4 and 20% in those with MRD ⩾10−4 (relapse hazard ratio (HR) 9.0; 95% confidence interval (CI) 2.5–32; P<0.0001), with median follow-up of 36 months. Additionally, MRD predicted relapse at other time points, including 9, 18 and 24 months post-HCT. MRD doubling time <12 months with disease burden ⩾10−5 was associated with relapse within 12 months of MRD assessment in 50% of patients, and within 24 months in 90% of patients. This IGH–HTS method may facilitate routine MRD quantification in clinical trials.

High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (MIP) microarrays

BackgroundA major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE). DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. As FFPE protocols may vary widely between labs and samples may be stored for decades at room temperature, an ideal FFPE CN technology should work on diverse sample sets. Molecular Inversion Probe (MIP) technology has been applied successfully to obtain high quality CN and genotype data from cell line and frozen tumor DNA. Since the MIP probes require only a small (~40 bp) target binding site, we reasoned they may be well suited to assess degraded FFPE DNA. We assessed CN with a MIP panel of 50,000 markers in 93 FFPE tumor samples from 7 diverse collections. For 38 FFPE samples from three collections we were also able to asses CN in matched fresh frozen tumor tissue.ResultsUsing an input of 37 ng genomic DNA, we generated high quality CN data with MIP technology in 88% of FFPE samples from seven diverse collections. When matched fresh frozen tissue was available, the performance of FFPE DNA was comparable to that of DNA obtained from matched frozen tumor (genotype concordance averaged 99.9%), with only a modest loss in performance in FFPE.ConclusionMIP technology can be used to generate high quality CN and genotype data in FFPE as well as fresh frozen samples.

Analysis of molecular inversion probe performance for allele copy number determination

We have developed a new protocol for using molecular inversion probes to accurately and specifically measure allele copy number. The new protocol provides for significant improvements, including the reduction of input DNA (from 2 μg) by more than 25-fold (to 75 ng total genomic DNA), higher overall precision resulting in one order of magnitude lower false positive rate, and greater dynamic range with accurate absolute copy number up to 60 copies.

Antigen presentation profiling reveals recognition of lymphoma immunoglobulin neoantigens

Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. However, the personalized identification and validation of neoantigens remains a major challenge. Here we discover neoantigens in human mantle-cell lymphomas by using an integrated genomic and proteomic strategy that interrogates tumour antigen peptides presented by major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically characterize MHC ligands from 17 patients. Remarkably, all discovered neoantigenic peptides were exclusively derived from the lymphoma immunoglobulin heavy- or light-chain variable regions. Although we identified MHC presentation of private polymorphic germline alleles, no mutated peptides were recovered from non-immunoglobulin somatically mutated genes. Somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC class II. We isolated circulating CD4+ T cells specific for immunoglobulin-derived neoantigens and found these cells could mediate killing of autologous lymphoma cells. These results demonstrate that an integrative approach combining MHC isolation, peptide identification, and exome sequencing is an effective platform to uncover tumour neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.

Optimal genotype determination in highly multiplexed SNP data

High-throughput genotyping technologies that enable large association studies are already available. Tools for genotype determination starting from raw signal intensities need to be automated, robust, and flexible to provide optimal genotype determination given the specific requirements of a study. The key metrics describing the performance of a custom genotyping study are assay conversion, call rate, and genotype accuracy. These three metrics can be traded off against each other. Using the highly multiplexed Molecular Inversion Probe technology as an example, we describe a methodology for identifying the optimal trade-off. The methodology comprises: a robust clustering algorithm and assessment of a large number of data filter sets. The clustering algorithm allows for automatic genotype determination. Many different sets of filters are then applied to the clustered data, and performance metrics resulting from each filter set are calculated. These performance metrics relate to the power of a study and provide a framework to choose the most suitable filter set to the particular study.