Ken Doig

Massively‐parallel sequencing assists the diagnosis and guided treatment of cancers of unknown primary

The clinical management of patients with cancer of unknown primary (CUP) is hampered by the absence of a definitive site of origin. We explored the utility of massively‐parallel (next‐generation) sequencing for the diagnosis of a primary site of origin and for the identification of novel treatment options. DNA enrichment by hybridization capture of 701 genes of clinical and/or biological importance, followed by massively‐parallel sequencing, was performed on 16 CUP patients who had defied attempts to identify a likely site of origin. We obtained high quality data from both fresh‐frozen and formalin‐fixed, paraffin‐embedded samples, demonstrating accessibility to routine diagnostic material. DNA copy‐number obtained by massively‐parallel sequencing was comparable to that obtained using oligonucleotide microarrays or quantitatively hybridized fluorescently tagged oligonucleotides. Sequencing to an average depth of 458‐fold enabled detection of somatically acquired single nucleotide mutations, insertions, deletions and copy‐number changes, and measurement of allelic frequency. Common cancer‐causing mutations were found in all cancers. Mutation profiling revealed therapeutic gene targets and pathways in 12/16 cases, providing novel treatment options. The presence of driver mutations that are enriched in certain known tumour types, together with mutational signatures indicative of exposure to sunlight or smoking, added to clinical, pathological, and molecular indicators of likely tissue of origin. Massively‐parallel DNA sequencing can therefore provide comprehensive mutation, DNA copy‐number, and mutational signature data that are of significant clinical value for a majority of CUP patients, providing both cumulative evidence for the diagnosis of primary site and options for future treatment. Copyright

Reversion of BRCA1/2 Germline Mutations Detected in Circulating Tumor DNA From Patients With High-Grade Serous Ovarian Cancer

Purpose Germline BRCA1 or BRCA2 mutations in patients with high-grade serous ovarian cancer (HGSC) are associated with favorable responses to chemotherapy. However, secondary intragenic (reversion) mutations that restore protein function lead to clinically significant rates of acquired resistance. The goal of this study was to determine whether reversion mutations could be found in an unbiased manner in circulating cell-free DNA (cfDNA) to predict treatment response in HGSC. Patients and Methods Plasma and tumor samples were obtained from 30 patients with HGSC with either BRCA1 or BRCA2 germline mutation. Two cohorts were ascertained: patients with a malignancy before undergoing primary HGSC debulking surgery (n = 14) or patients at disease recurrence (n = 16). Paired tumor and plasma samples were available for most patients (24 of 30). Targeted amplicon, next-generation sequencing was performed using primers that flanked germline mutations, whose design did not rely on prior knowledge of reversion sequences. Results Five patients were identified with intragenic mutations predicted to restore BRCA1/2 open reading frames, including two patients with multiple independent reversion alleles. Reversion mutations were only detected in tumor samples from patients with recurrent disease (five of 16) and only in cfDNA from patients with a tumor-detected reversion (three of five). Findings from a rapid autopsy of a patient with multiple independent reversions indicated that reversion-allele frequency in metastatic sites is an important determinant of assay sensitivity. Abundance of tumor-derived DNA in total cell-free DNA, as measured by TP53 mutant allele frequency, also affected assay sensitivity. All patients with reversions detected in tumor-derived DNA were resistant to platin- or poly ADP ribose polymerase inhibitor-based chemotherapy. Conclusion Reversion mutations can be detected in an unbiased analysis of cfDNA, suggesting clinical utility for predicting chemotherapy response in recurrent HGSC.

Bioinformatics pipelines for targeted resequencing and whole-exome sequencing of human and mouse genomes: a virtual appliance approach for instant deployment.

Targeted resequencing by massively parallel sequencing has become an effective and affordable way to survey small to large portions of the genome for genetic variation. Despite the rapid development in open source software for analysis of such data, the practical implementation of these tools through construction of sequencing analysis pipelines still remains a challenging and laborious activity, and a major hurdle for many small research and clinical laboratories. We developed TREVA (Targeted REsequencing Virtual Appliance), making pre-built pipelines immediately available as a virtual appliance. Based on virtual machine technologies, TREVA is a solution for rapid and efficient deployment of complex bioinformatics pipelines to laboratories of all sizes, enabling reproducible results. The analyses that are supported in TREVA include: somatic and germline single-nucleotide and insertion/deletion variant calling, copy number analysis, and cohort-based analyses such as pathway and significantly mutated genes analyses. TREVA is flexible and easy to use, and can be customised by Linux-based extensions if required. TREVA can also be deployed on the cloud (cloud computing), enabling instant access without investment overheads for additional hardware. TREVA is available at http://bioinformatics.petermac.org/treva/.

Cancer 2015: a prospective, population-based cancer cohort-phase 1: feasibility of genomics-guided precision medicine in the clinic

“Cancer 2015” is a longitudinal and prospective cohort. It is a phased study whose aim was to pilot recruiting 1000 patients during phase 1 to establish the feasibility of providing a population-based genomics cohort. Newly diagnosed adult patients with solid cancers, with residual tumour material for molecular genomics testing, were recruited into the cohort for the collection of a dataset containing clinical, molecular pathology, health resource use and outcomes data. 1685 patients have been recruited over almost 3 years from five hospitals. Thirty-two percent are aged between 61–70 years old, with a median age of 63 years. Diagnostic tumour samples were obtained for 90% of these patients for multiple parallel sequencing. Patients identified with somatic mutations of potentially “actionable” variants represented almost 10% of those tumours sequenced, while 42% of the cohort had no mutations identified. These genomic data were annotated with information such as cancer site, stage, morphology, treatment and patient outcomes and health resource use and cost. This cohort has delivered its main objective of establishing an upscalable genomics cohort within a clinical setting and in phase 2 aims to develop a protocol for how genomics testing can be used in real-time clinical decision-making, providing evidence on the value of precision medicine to clinical practice.

Studying cancer genomics through next-generation DNA sequencing and bioinformatics.

Cancer is a complex disease driven by multiple mutations acquired over the lifetime of the cancer cells. These alterations, termed somatic mutations to distinguish them from inherited germline mutations, can include single-nucleotide substitutions, insertions, deletions, copy number alterations, and structural rearrangements. A patients cancer can contain a combination of these aberrations, and the ability to generate a comprehensive genetic profile should greatly improve patient diagnosis and treatment. Next-generation sequencing has become the tool of choice to uncover multiple cancer mutations from a single tumor source, and the falling costs of this rapid high-throughput technology are encouraging its transition from basic research into a clinical setting. However, the detection of mutations in sequencing data is still an evolving area and cancer genomic data requires some special considerations. This chapter discusses these aspects and gives an overview of current bioinformatics methods for the detection of somatic mutations in cancer sequencing data.

ASXL1 c.1934dup;p.Gly646Trpfs*12—a true somatic alteration requiring a new approach

The additional sex combs-like 1 (ASXL1) gene has a central role in the epigenetic regulation of chromatin remodelling and subsequent gene transcription via multiple mechanisms. These include the regulation of histone H2A deubiquitination as well as polycomb group repressor complex 2 mediated homeobox (HOX) gene transcription. ASXL1mutations are a recurrent finding in myeloid malignancies, where they are typically heterozygous in keeping with a haploinsufficiency effect. Mutated ASXL1 status has been associated with an inferior overall survival in acute myeloid leukaemia (AML), myelodysplastic syndromes (MDS), chronic myelomonocytic leukaemia (CMML), myelofibrosis, aplastic anaemia and age-related clonal haematopoiesis. The majority of ASXL1 exon 12 mutations are frameshift or nonsense and result in a C-terminal truncation of the resulting gene product. Missense mutations are also detected but these appear not to have an effect on clinical outcome and are of uncertain significance. The most commonly detected ASXL1 mutation is ASXL1 NM_015338.5:c.1934dup;p.Gly646Trpfs*12 (ASXL1 c.1934dupG), accounting for approximately half of somatic truncating mutations. This duplication of a single guanine occurs within an eight base-pair mononucleotide guanine repeat sequence (8G repeat) that extends from c.1927 to c.1934. Areas of repetitive sequence may be prone to accelerated mutagenesis due to replication slippage. This occurs when DNA polymerase pauses and dissociates from repeated areas of sequence allowing the terminal portion of the newly synthesised strand to anneal to a different yet still complimentary location on the template. Resumption of DNA replication completes the slippage event, which may result in duplications or deletions. This process, however, has also been described as a source of polymerase chain reaction (PCR) sequencing artefact. This fact, coupled with the detection by Sanger sequencing and mass spectrometry of ASXL1 c.1934dupG within the buccal DNA of individuals with myeloid malignancies and by Sanger sequencing in the granulocyte DNA of those without, has led some to assert that this variant is not a real somatic alteration. In addition, ASXL1 c.1934dupG has been reported at a frequency of between 0.001634% (Exome Aggregation Consortium) and 2.58% (Exome Sequencing Project) in the general population by whole-exome sequencing. Despite the fact that ASXL1 may be mutated in otherwise well individuals with agerelated clonal haematopoiesis, these detection frequencies may be overestimated due to artefact-related false-positive ASXL1 c.1934dupG detection. Various evidences in support of ASXL1 c.1934dupG being a true somatic alteration have been put forward. These include an inability to reproduce ASXL1 c.1934dupG detection consistently in samples known not to contain a myeloid malignancy (likely due to the use of high fidelity polymerases) and a failure to differentiate patients harbouring ASXL1 c.1934dupG and those with other truncating ASXL1 mutations by clinical outcome or gene expression profiling. However, these lines of evidence either rely on sequencing of the ASXL1 8G repeat or are circumstantial in nature. We aimed to evaluate the performance of various methodologies for the detection of ASXL1 c.1934dupG and to assess whether it is a true somatic alteration utilising a mutation-specific assay.

Clinicopathological differences exist between CALR- and JAK2 -mutated myeloproliferative neoplasms despite a similar molecular landscape: data from targeted next-generation sequencing in the diagnostic laboratory

Mutations in CALR have recently been detected in JAK2-negative myeloproliferative neoplasms (MPNs) and are key pathological drivers in these diseases. CALR-mutated MPNs are shown to have numerous clinicopathological differences to JAK2-mutated MPNs. The basis of these differences is poorly understood. It is unknown whether these differences result directly from any differences in intracellular signalling abnormalities induced by JAK2/CALR mutations or whether they relate to other phenomena such as a differing spectrum of genetic lesions between the two groups. We aimed to review the clinicopathological and molecular features of CALR- and JAK2-mutated MPNs from samples referred for diagnostic testing using a custom-designed targeted next-generation sequencing (NGS) panel. Eighty-nine CALR-mutated cases were compared with 70 JAK2-mutated cases. CALR-mutated MPNs showed higher platelet counts and a female predominance as compared to JAK2-mutated MPNs in our cohort. We have also observed differences between CALR mutation subtypes in terms of disease phenotype, mutational frequency and allelic burden. Type 1 CALR mutations were found to be more common in myelofibrosis, associated with a higher frequency and number of additional mutations and a higher mutant allelic burden as compared to type 2 CALR mutations. Despite these biological differences, our molecular characterisation suggests that CALR- and JAK2-mutated MPNs are broadly similar in terms of the quantity, frequency and spectrum of co-occurring mutations and therefore observed biological differences are likely to not be heavily influenced by the nature and quantity of co-mutated genes.

Exploring the feasibility and utility of exome-scale tumour sequencing in a clinical setting: Feasibility of clinical exome sequencing

Technology has progressed from single gene panel to large‐scale genomic sequencing. This is raising expectations from clinicians and patients alike. The utility and performance of this technology in a clinical setting needs to be evaluated.

Sensitive NPM1 Mutation Quantitation in Acute Myeloid Leukemia Using Ultradeep Next-Generation Sequencing in the Diagnostic Laboratory

Context Detection of measurable residual disease after therapy is an important predictor of outcome in acute myeloid leukemia. Objective To investigate the feasibility of using next-generation sequencing (NGS) in the diagnostic laboratory to perform quantitative NPM1 mutation assessment using ultradeep (approximately 300 000×-500 000×) sequencing (NGS-q NPM1) as a method of assessing residual disease burden in patients with acute myeloid leukemia. Design A flexible NGS-based assay for the detection and quantitation of NPM1 mutations was developed by polymerase chain reaction amplification of target DNA sequences, sequencing on an Illumina (San Diego, California) MiSeq, and analyzing data with an in-house-designed bioinformatic pipeline. NGS-q NPM1 was compared with current NPM1 quantitation methods (real-time quantitative-polymerase chain reaction and multiparameter flow cytometry). Results The NGS-q NPM1 assay had a sensitivity of between 10-4 and 10-5 and showed high concordance and correlation with reference methodologies. Moreover, the NGS-q NPM1 assay was able to be integrated into the laboratorys existing, targeted amplicon-based sequencing workflow. Conclusions An NGS-based, quantitative NPM1-mutation assessment can be used to monitor patients with acute myeloid leukemia, and it has some practical advantages over existing modalities.