Clinical whole-genome sequencing from routine formalin-fixed, paraffin-embedded specimens: pilot study for the 100,000 Genomes Project.

PURPOSE: Fresh-frozen (FF) tissue is the optimal source of DNA for whole-genome sequencing (WGS) of cancer patients. However, it is not always available, limiting the widespread application of WGS in clinical practice. We explored the viability of using formalin-fixed, paraffin-embedded (FFPE) tissues, available routinely for cancer patients, as a source of DNA for clinical WGS. METHODS: We conducted a prospective study using DNAs from matched FF, FFPE, and peripheral blood germ-line specimens collected from 52 cancer patients (156 samples) following routine diagnostic protocols. We compared somatic variants detected in FFPE and matching FF samples. RESULTS: We found the single-nucleotide variant agreement reached 71% across the genome and somatic copy-number alterations (CNAs) detection from FFPE samples was suboptimal (0.44 median correlation with FF) due to nonuniform coverage. CNA detection was improved significantly with lower reverse crosslinking temperature in FFPE DNA extraction (80 °C or 65 °C depending on the methods). Our final data showed somatic variant detection from FFPE for clinical decision making is possible. We detected 98% of clinically actionable variants (including 30/31 CNAs). CONCLUSION: We present the first prospective WGS study of cancer patients using FFPE specimens collected in a routine clinical environment proving WGS can be applied in the clinic.

Clinical applicability and cost of a 46-gene panel for genomic analysis of solid tumours: Retrospective validation and prospective audit in the UK National Health Service.

BACKGROUND: Single gene tests to predict whether cancers respond to specific targeted therapies are performed increasingly often. Advances in sequencing technology, collectively referred to as next generation sequencing (NGS), mean the entire cancer genome or parts of it can now be sequenced at speed with increased depth and sensitivity. However, translation of NGS into routine cancer care has been slow. Healthcare stakeholders are unclear about the clinical utility of NGS and are concerned it could be an expensive addition to cancer diagnostics, rather than an affordable alternative to single gene testing. METHODS AND FINDINGS: We validated a 46-gene hotspot cancer panel assay allowing multiple gene testing from small diagnostic biopsies. From 1 January 2013 to 31 December 2013, solid tumour samples (including non-small-cell lung carcinoma [NSCLC], colorectal carcinoma, and melanoma) were sequenced in the context of the UK National Health Service from 351 consecutively submitted prospective cases for which treating clinicians thought the patient had potential to benefit from more extensive genetic analysis. Following histological assessment, tumour-rich regions of formalin-fixed paraffin-embedded (FFPE) sections underwent macrodissection, DNA extraction, NGS, and analysis using a pipeline centred on Torrent Suite software. With a median turnaround time of seven working days, an integrated clinical report was produced indicating the variants detected, including those with potential diagnostic, prognostic, therapeutic, or clinical trial entry implications. Accompanying phenotypic data were collected, and a detailed cost analysis of the panel compared with single gene testing was undertaken to assess affordability for routine patient care. Panel sequencing was successful for 97% (342/351) of tumour samples in the prospective cohort and showed 100% concordance with known mutations (detected using cobas assays). At least one mutation was identified in 87% (296/342) of tumours. A locally actionable mutation (i.e., available targeted treatment or clinical trial) was identified in 122/351 patients (35%). Forty patients received targeted treatment, in 22/40 (55%) cases solely due to use of the panel. Examination of published data on the potential efficacy of targeted therapies showed theoretically actionable mutations (i.e., mutations for which targeted treatment was potentially appropriate) in 66% (71/107) and 39% (41/105) of melanoma and NSCLC patients, respectively. At a cost of £339 (US$449) per patient, the panel was less expensive locally than performing more than two or three single gene tests. Study limitations include the use of FFPE samples, which do not always provide high-quality DNA, and the use of "real world" data: submission of cases for sequencing did not always follow clinical guidelines, meaning that when mutations were detected, patients were not always eligible for targeted treatments on clinical grounds. CONCLUSIONS: This study demonstrates that more extensive tumour sequencing can identify mutations that could improve clinical decision-making in routine cancer care, potentially improving patient outcomes, at an affordable level for healthcare providers.

Targeted Next-Generation Sequencing of Plasma DNA from Cancer Patients: Factors Influencing Consistency with Tumour DNA and Prospective Investigation of Its Utility for Diagnosis.

Use of circulating tumour DNA (ctDNA) as a liquid biopsy has been proposed for potential identification and monitoring of solid tumours. We investigate a next-generation sequencing approach for mutation detection in ctDNA in two related studies using a targeted panel. The first study was retrospective, using blood samples taken from melanoma patients at diverse timepoints before or after treatment, aiming to evaluate correlation between mutations identified in biopsy and ctDNA, and to acquire a first impression of influencing factors. We found good concordance between ctDNA and tumour mutations of melanoma patients when blood samples were collected within one year of biopsy or before treatment. In contrast, when ctDNA was sequenced after targeted treatment in melanoma, mutations were no longer found in 9 out of 10 patients, suggesting the method might be useful for detecting treatment response. Building on these findings, we focused the second study on ctDNA obtained before biopsy in lung patients, i.e. when a tentative diagnosis of lung cancer had been made, but no treatment had started. The main objective of this prospective study was to evaluate use of ctDNA in diagnosis, investigating the concordance of biopsy and ctDNA-derived mutation detection. Here we also found positive correlation between diagnostic lung biopsy results and pre-biopsy ctDNA sequencing, providing support for using ctDNA as a cost-effective, non-invasive solution when the tumour is inaccessible or when biopsy poses significant risk to the patient.

Durable Response of Spinal Chordoma to Combined Inhibition of IGF-1R and EGFR.

Chordomas are rare primary malignant bone tumors arising from embryonal notochord remnants of the axial skeleton. Chordomas commonly recur following surgery and radiotherapy, and there is no effective systemic therapy. Previous studies implicated receptor tyrosine kinases, including epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGF-1R), in chordoma biology. We report an adult female patient who presented in 2003 with spinal chordoma, treated with surgery and radiotherapy. She underwent further surgery for recurrent chordoma in 2008, with subsequent progression in pelvic deposits. In June 2009, she was recruited onto the Phase I OSI-906-103 trial of EGFR inhibitor erlotinib with linsitinib, a novel inhibitor of IGF-1R/insulin receptor (INSR). Treatment with 100 mg QD erlotinib and 50 mg QD linsitinib was well-tolerated, and after 18 months a partial response was achieved by RECIST criteria. From 43 months, a protocol modification allowed intra-patient linsitinib dose escalation to 50 mg BID. The patient remained stable on trial treatment for a total of 5 years, discontinuing treatment in August 2014. She subsequently experienced further disease progression for which she underwent pelvic surgery in April 2015. Analysis of DNA extracted from 2008 (pre-trial) tissue showed that the tumor harbored wild-type EGFR, and a PIK3CA mutation was detected in plasma, but not tumor DNA. The 2015 (post-trial) tumor harbored a mutation of uncertain significance in ATM, with no detectable mutations in other components of a 50 gene panel, including EGFR, PIK3CA, and TP53. By immunohistochemistry, the tumor was positive for brachyury, the molecular hallmark of chordoma, and showed weak-moderate membrane and cytoplasmic EGFR. IGF-1R was detected in the plasma membrane and cytoplasm and was expressed more strongly in recurrent tumor than the primary. We also noted heterogeneous nuclear IGF-1R, which has been linked with sensitivity to IGF-1R inhibition. Similar variation in IGF-1R expression and subcellular localization was noted in 15 further cases of chordoma. In summary, this exceptionally durable response suggests that there may be merit in evaluating combined IGF-1R/INSR and EGFR inhibition in patients with chordomas that recur following failure of local treatment.

Vessel rejection secondary to human leucocyte antigen antibodies directed against the arterial conduit following pancreas transplantation from a separate donor.

Whole-organ pancreas transplantation is typically carried out using a Y-graft derived from the donor iliac vessels. We describe a case in which a 31-year-old male underwent a simultaneous pancreas-kidney transplant, but in which vessels from a different donor were used for the arterial anastomosis of the pancreas graft. Although initially there was good function, 18 months post-transplant the patient was admitted with diabetic ketoacidosis secondary to pancreas graft failure. Radiological investigations revealed complete occlusion of the vascular Y-graft, and laboratory investigations demonstrated donor-specific human leucocyte antigen (HLA) antibodies directed against HLA mismatches of the vessel donor. This case highlights the risks of using allogeneic vascular material for surgical anastomoses.

The experience of taking multiple drug therapies for persons with HIV infection: the Canadian perspective.

Advances in the understanding of HIV pathogenesis, clinical assessment with viral load testing, and the availability of potent combination antiretroviral therapy regimens have led to significant benefits for persons with HIV infection. Currently, the therapeutic strategy to achieve high-level suppression of viral replication involves many pills and doses each day. This regimen could adversely affect the quality of life of these clients. In reviewing the literature, it is evident that little is known about the experience of taking multiple drug therapies from the perspective of the individuals. For example, with the introduction of triple drug therapies has come constraints associated with the regimens, as well as drug interactions and short-term and long-term adverse effects. Little is known about the social and emotional impact related to successfully taking these or other therapies. Less is known regarding this experience from a Canadian perspective. This research study answered the following question: What is the experience of taking multiple drug therapies for persons with HIV infection? A phenomenological inquiry was used. A purposive sample of 19 participants was drawn from a large immunodeficiency clinic in a tertiary hospital. In-depth interviews were followed by a return interview to verify themes from the analysis. The findings revealed the complex process that these regimens demand and the major impact on clients' lives. The essence of the experience described by the participants was "learning to live with this process."