Ian Bosdet

A physical map of the mouse genome

A physical map of a genome is an essential guide for navigation, allowing the location of any gene or other landmark in the chromosomal DNA. We have constructed a physical map of the mouse genome that contains 296 contigs of overlapping bacterial clones and 16,992 unique markers. The mouse contigs were aligned to the human genome sequence on the basis of 51,486 homology matches, thus enabling use of the conserved synteny (correspondence between chromosome blocks) of the two genomes to accelerate construction of the mouse map. The map provides a framework for assembly of whole-genome shotgun sequence data, and a tile path of clones for generation of the reference sequence. Definition of the human–mouse alignment at this level of resolution enables identification of a mouse clone that corresponds to almost any position in the human genome. The human sequence may be used to facilitate construction of other mammalian genome maps using the same strategy.

A physical map of the bovine genome

BackgroundCattle are important agriculturally and relevant as a model organism. Previously described genetic and radiation hybrid (RH) maps of the bovine genome have been used to identify genomic regions and genes affecting specific traits. Application of these maps to identify influential genetic polymorphisms will be enhanced by integration with each other and with bacterial artificial chromosome (BAC) libraries. The BAC libraries and clone maps are essential for the hybrid clone-by-clone/whole-genome shotgun sequencing approach taken by the bovine genome sequencing project.ResultsA bovine BAC map was constructed with HindIII restriction digest fragments of 290,797 BAC clones from animals of three different breeds. Comparative mapping of 422,522 BAC end sequences assisted with BAC map ordering and assembly. Genotypes and pedigree from two genetic maps and marker scores from three whole-genome RH panels were consolidated on a 17,254-marker composite map. Sequence similarity allowed integrating the BAC and composite maps with the bovine draft assembly (Btau3.1), establishing a comprehensive resource describing the bovine genome. Agreement between the marker and BAC maps and the draft assembly is high, although discrepancies exist. The composite and BAC maps are more similar than either is to the draft assembly.ConclusionFurther refinement of the maps and greater integration into the genome assembly process may contribute to a high quality assembly. The maps provide resources to associate phenotypic variation with underlying genomic variation, and are crucial resources for understanding the biology underpinning this important ruminant species so closely associated with humans.

A Clinically Validated Diagnostic Second-Generation Sequencing Assay for Detection of Hereditary BRCA1 and BRCA2 Mutations

Individuals who inherit mutations in BRCA1 or BRCA2 are predisposed to breast and ovarian cancers. However, identifying mutations in these large genes by conventional dideoxy sequencing in a clinical testing laboratory is both time consuming and costly, and similar challenges exist for other large genes, or sets of genes, with relevance in the clinical setting. Second-generation sequencing technologies have the potential to improve the efficiency and throughput of clinical diagnostic sequencing, once clinically validated methods become available. We have developed a method for detection of variants based on automated small-amplicon PCR followed by sample pooling and sequencing with a second-generation instrument. To demonstrate the suitability of this method for clinical diagnostic sequencing, we analyzed the coding exons and the intron-exon boundaries of BRCA1 and BRCA2 in 91 hereditary breast cancer patient samples. Our method generated high-quality sequence coverage across all targeted regions, with median coverage greater than 4000-fold for each sample in pools of 24. Sensitive and specific automated variant detection, without false-positive or false-negative results, was accomplished with a standard software pipeline using bwa for sequence alignment and samtools for variant detection. We experimentally derived a minimum threshold of 100-fold sequence depth for confident variant detection. The results demonstrate that this method is suitable for sensitive, automatable, high-throughput sequence variant detection in the clinical laboratory.

A BAC clone fingerprinting approach to the detection of human genome rearrangements

We present a method, called fingerprint profiling (FPP), that uses restriction digest fingerprints of bacterial artificial chromosome clones to detect and classify rearrangements in the human genome. The approach uses alignment of experimental fingerprint patterns to in silico digests of the sequence assembly and is capable of detecting micro-deletions (1-5 kb) and balanced rearrangements. Our method has compelling potential for use as a whole-genome method for the identification and characterization of human genome rearrangements.

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.

A population-based review of the feasibility of platinum-based combination chemotherapy after tyrosine kinase inhibition in EGFR mutation positive non-small cell lung cancer patients with advanced disease

INTRODUCTION The IPASS trial demonstrated superior progression free survival for Asian, light/never smoking, advanced, pulmonary adenocarcinoma patients treated with first-line gefitinib compared to carboplatin/paclitaxel, of which 59% of those tested were epidermal growth factor receptor (EGFR) mutation positive. In IPASS 39% of gefitinib treated patients went on to receive platin based polychemotherapy. We hypothesized that in a population-based setting fewer patients receive second-line platin based chemotherapy than those enrolled in a clinical trial. METHODS The Iressa Alliance program provided standardized EGFR mutation testing and appropriate access to gefitinib to all patients in British Columbia with advanced, non squamous non small cell lung cancer (NSCLC). We retrospectively analyzed clinical, pathologic data and outcomes for all patients tested in this program between March 2010 and June 2011. RESULTS A total of 548 patients were referred for testing and 22% of patients were mutation positive. Baseline characteristics of mutation negative and mutation positive; median age 67/65, male 41%/31%, Asian 15%/51%, never smoker 21%/58%, stage IV 80%/91%. Median overall survival was 12 months in mutation negative patients and not yet reached in mutation positive (p<0.0001). In mutation positive patients 5% of patients had a complete response, 46% partial response, 34% stable disease, 6% progressive disease. Twenty percent of patients continued on gefitinib after radiographic progression and clinical stability. Sixty-one gefitinib treated patients progressed at the time of analysis; 10% of patients received further gefitinib only, 38% platinum based doublet, 8% other chemotherapy and 44% no further treatment. Performance status most strongly predicted for delivery of second line chemotherapy. CONCLUSIONS This North American population based study shows similar efficacy of gefitinib in mutation positive patients compared to the IPASS trial. Contrary to our hypothesis, delivery of second line chemotherapy was feasible in a significant proportion of gefitinib treated patients.

Relationship of thyroid transcription factor 1 to EGFR status in non-small-cell lung cancer.

BACKGROUND Activating mutations of the epidermal growth factor receptor (EGFR) gene are known to drive a proportion of non-small-cell lung cancers. Identification of lung cancers harbouring such mutations can lead to effective treatment using one of the agents that targets and blocks egfr-mediated signalling. METHODS All specimens received at the BC Cancer Agency (Vancouver) for EGFR testing were prospectively identified and catalogued, together with clinical information and EGFR status, over a 14-month period. RESULTS Specimens from 586 patients were received for EGFR testing, and EGFR status was reported for 509 patients. No relationship between specimen type or site of origin and EGFR test failure rate was identified. Concurrent immunohistochemical (ihc) status for thyroid transcription factor 1 (ttf1) was available for 309 patients. The negative predictive value of ttf1-negative status by ihc was 94.2% for predicting negative EGFR status. CONCLUSIONS In patients with limited tissue available for testing, a surrogate for EGFR status would aid in timely management. Immunohistochemistry for ttf1 is readily available and correlates highly with EGFR status. In conjunction with genetic assays, ttf1 could be used to optimize an EGFR testing strategy.

Estimating deep molecular responses in chronic myelogenous leukemia: a Bayesian approach

The recent recommendations by the European Treatment Outcome Study (EUTOS) regarding the reporting of deep molecular responses are aimed at harmonizing laboratory practice.1