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

Robbe, Pauline; Popitsch, Niko; Knight, Samantha J. L.; Antoniou, Pavlos; Becq, Jennifer; He, Mingyue; Kanapin, Alexander; Samsonova, Anastasia; Vavoulis, Dimitrios V; Ross, Mark T.; Kingsbury, Zoya; Cabes, Maite; Ramos, S.; Page, Suzanne; Dreau, Hélène; Ridout, Kate; Jones, Louise; Tuff-Lacey, Alice; Henderson, Shirley; Mason, Joanne; Buffa, Francesca M.; Verrill, Clare; Maldonado-Pérez, David; Roxanis, Ioannis; Collantes, Elena; Browning, Lisa; Dhar, S.; Damato, Stephen; Davies, S. J.; Caulfield, Mark J.; Bentley, David; Taylor, Jenny C.; Turnbull, Clare; Schuh, Anna

doi:10.1038/gim.2017.241

Cited by 134 publications

(124 citation statements)

References 39 publications

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“…Our comparison of mutational rates in primary vs persistent/recurrent LSCC needs to be interpreted in this light. However, several recent studies have demonstrated high (ie, > 90%) concordance rates in detection of SNVs and CNVs in coding genomic regions between matched fresh‐frozen and FFPE tumor samples . In addition, comparison of amplicon‐based, targeted exome sequencing to TCGA dataset is a well‐established practice in the HNSCC literature .…”

Section: Discussionmentioning

confidence: 99%

“…However, several recent studies have demonstrated high (ie, > 90%) concordance rates in detection of SNVs and CNVs in coding genomic regions between matched freshfrozen and FFPE tumor samples. [28][29][30] In addition, comparison of amplicon-based, targeted exome sequencing to TCGA dataset is a well-established practice in the HNSCC literature. 11,13 Thus, we believe that these differences in DNA sequencing methodology do not significantly diminish the validity of our comparisons and conclusions.…”

Section: Discussionmentioning

confidence: 99%

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Mutational profiles of persistent/recurrent laryngeal squamous cell carcinoma

et al. 2018

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Background We sought to describe targeted DNA sequencing data of persistent/recurrent laryngeal squamous cell carcinoma (LSCC) and to compare gene‐specific alteration frequencies with that of primary, untreated LSCC specimens from The Cancer Genome Atlas (TCGA). Methods The tumors of 21 patients with persistent/recurrent LSCC were subjected to targeted DNA sequencing using the Ion AmpliSeq Comprehensive Cancer Panel. Gene‐specific alteration frequencies were compared (Chi‐Square test) to primary, untreated LSCC sequencing data from TCGA using the cBioPortal platform. Results Persistent/recurrent LSCC was characterized by a high rate of inactivating alterations in TP53 (38.1%) and CDKN2A (33%), amplification events of CCND1 (19.1%), and ERBB2 (14.3%), and NOTCH1 (19.1%) mutations. Comparison of primary vs persistent/recurrent LSCC revealed significant differences in alteration frequencies of eight critical genes: BAP1, CDKN2A, DCUN1D1, MSH2, MTOR, PIK3CA, TET2, and TP53. Conclusions Our results provide preliminary support for a distinct mutational profile of persistent/recurrent LSCC that requires validation in larger cohorts.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Mutational profiles of persistent/recurrent laryngeal squamous cell carcinoma

et al. 2018

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“…Despite optimization of DNA extraction methods, formalin-fixation induces DNA artefacts that increase the false positive mutation rate 22 . We therefore applied a stringent variant allele frequency (VAF) filter (see Methods) on all FFPE-derived data, and chose a genomic region unlikely to be affected by RS driver mutations (i.e.…”

Section: Mutation Burdenmentioning

confidence: 99%

“…Paraffin was removed from FFPE 10μm sections scraped from 5-10 slides using the Adaptive Focused Acoustics (AFA™) on the M220 Focused-ultrasonicator. DNA was thereafter purified with the truXTRAC FFPE DNA Kit according to the manufacturer's protocol (Covaris Inc., Woburn, MA, USA), but using optimized reverse cross-linking conditions as previously described 22 . Peripheral blood samples were subjected to a ficoll gradient centrifugation to isolate mononuclear cells.…”

Section: Chop-or Cohortmentioning

confidence: 99%

Differential genomic and transcriptomic events associated with high-grade transformation of Chronic Lymphocytic Leukemia

Klintman

Stamatopoulos

Ridout

et al. 2019

Preprint

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The transformation of chronic lymphocytic leukemia (CLL) to high-grade diffuse large B-cell lymphoma (DLBCL), also called Richter's Syndrome (RS), is a rare cancer with dismal prognosis.Drug discovery for RS is hampered by the lack of suitable experimental models, and effective therapies remain elusive rendering RS an area of high unmet clinical need. We performed whole genome sequencing (WGS) to interrogate paired CLL and RS samples from 17 patients enrolled in a prospective multicenter Phase 2 clinical trial (CHOP-OR) and we found that subclones affected by mutations in MAPK and PI3K pathways show a high expansion probability during transformation. We also demonstrate for the first time that non-coding mutation clusters in a PAX5 enhancer, situated 330kb upstream from the transcription initiation site, correlate with transformation. Finally, we confirm our findings by employing targeted DNA sequencing (TGS) andRNA expression profiling on an extended cohort of 38 patients. Statement of significanceThrough integrated analysis of WGS, TGS and RNA expression data, we identified drivers of transformation not previously implicated in RS, which can be targeted therapeutically and tested in the clinic. Our results have informed the design of a new clinical platform study, which is now open to recruitment in the UK.

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“…At 2-4x coverage, WGS provides thousands of read pairs per 100kb segment, a substantive amount enough to enable sensitive CNA detection. This provides an improved resolution compared to SNP microarrays in which there are approximately 60 probes per 100kb segment.Identifying copy number variation with high coverage WGS data have been studied extensively in basic and clinical research settings[29][30][31] . For example, in a recent systematic benchmark, Trost et al reported good performance of using >20x WGS data for identifying small-scale CNVs (1 -100kb)32 .…”

mentioning

confidence: 99%

Whole genome analysis identifies the association of TP53 genomic deletions with lower survival in Stage III colorectal cancer

Xia

Hummelen

Kubit

et al. 2019

Preprint

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DNA copy number aberrations (CNA) were frequently observed in colorectal cancers (CRC).There is an urgent call for CNA-based biomarkers in clinics, in particular for Stage III CRC, if combined with imaging or pathologic evidence, promise more precise care at the timing. We conducted this Stage III specific biomarker discovery with a cohort of 134 CRCs, and with a newly developed high-efficiency CNA profiling protocol. Specifically, we developed the profiling protocol for tumor-normal matched tissue samples based on low-coverage clinical wholegenome sequencing (WGS). We demonstrated the protocol's accuracy and robustness by a systematic benchmark with microarray, high-coverage whole-exome and -genome approaches, where the low-coverage WGS-derived CNA segments were highly accordant (PCC>0.95) with those derived from microarray, and they were substantially less variable if compared to exome-derived segments. A lasso-based model and multivariate cox regression analysis identified a chromosome 17p loss, containing the TP53 tumor suppressor gene, that was significantly associated with reduced survival (P=0.0139, HR=1.688, 95% CI = [1.112-2.562]), which was validated by an independent cohort of 187 Stage III CRCs. In summary, the new low-coverage WGS protocol has high sensitivity, high resolution and low cost and the identified 17p-loss is an effective poor prognosis marker for Stage III patients.

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

Cited by 134 publications

References 39 publications

Mutational profiles of persistent/recurrent laryngeal squamous cell carcinoma

Mutational profiles of persistent/recurrent laryngeal squamous cell carcinoma

Differential genomic and transcriptomic events associated with high-grade transformation of Chronic Lymphocytic Leukemia

Whole genome analysis identifies the association of TP53 genomic deletions with lower survival in Stage III colorectal cancer

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