on behalf of 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.Genet Med advance online publication 1 February 2018
Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p.Arg200Trp mutation in the von Hippel-Lindau () gene. Since this discovery, additional mutations have been identified in patients with congenital erythrocytosis, in a homozygous or compound-heterozygous state. is a major tumor suppressor gene, mutations in which were first described in patients presenting with VHL disease, which is characterized by the development of highly vascularized tumors. Here, we identify a new cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. More importantly, we identify mutations in E1' in 7 families with erythrocytosis (1 homozygous case and 6 compound-heterozygous cases with a mutation in E1' in addition to a mutation in coding sequences) and in 1 large family with typical VHL disease but without any alteration in the other exons. In this study, we show that the mutations induced a dysregulation of splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In addition, we demonstrate a pathogenic role for synonymous mutations in exon 2 that altered splicing through E2-skipping in 5 families with erythrocytosis or VHL disease. In all the studied cases, the mutations differentially affected splicing, correlating with phenotype severity. This study demonstrates that cryptic exon retention and exon skipping are new alterations and reveals a novel complex splicing regulation of the gene. These findings open new avenues for diagnosis and research regarding the VHL-related hypoxia-signaling pathway.
The immunoglobulin heavy-chain variable region gene (IgHV) mutational status is considered the gold standard of prognostication in chronic lymphocytic leukemia (CLL) and is currently determined by Sanger sequencing that allows the analysis of the major clone. Using next-generation sequencing (NGS), we sequenced the IgHV gene from two independent cohorts: (A) 270 consecutive patient samples obtained at diagnosis and (B) 227 patients from the UK ARCTIC-AdMIRe clinical trials. Using complementary DNA from purified CD19+CD5+ cells, we demonstrate the presence of multiple rearrangements in independent experiments and showed that 24.4% of CLL patients express multiple productive clonally unrelated IgHV rearrangements. On the basis of IgHV-NGS subclonal profiles, we defined five different categories: patients with (a) multiple hypermutated (M) clones, (b) 1 M clone, (c) a mix of M-unmutated (UM) clones, (d) 1 UM clone and (e) multiple UM clones. In population A, IgHV-NGS classification stratified patients into five different subgroups with median treatment-free survival (TFS) of >280(a), 131(b), 94(c), 29(d), 15(e) months (P<0.0001) and a median OS of >397(a), 292(b), 196(c), 137(d) and 100(e) months (P<0.0001). In population B, the poor prognosis of multiple UM patients was confirmed with a median TFS of 2 months (P=0.0038). In conclusion, IgHV-NGS highlighted one quarter of CLL patients with multiple productive IgHV subclones and improves disease stratification and raises important questions concerning the pre-leukemic cellular origin of CLL.
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