The transformation of chronic lymphocytic leukemia (CLL) to high-grade B-cell lymphoma is known as Richter syndrome (RS), a rare event with dismal prognosis. In this study, we conducted whole-genome ...sequencing (WGS) of paired circulating CLL (PB-CLL) and RS biopsies (tissue-RS) from 17 patients recruited into a clinical trial (CHOP-O). We found that tissue-RS was enriched for mutations in poor-risk CLL drivers and genes in the DNA damage response (DDR) pathway. In addition, we identified genomic aberrations not previously implicated in RS, including the protein tyrosine phosphatase receptor (PTPRD) and tumor necrosis factor receptor–associated factor 3 (TRAF3). In the noncoding genome, we discovered activation-induced cytidine deaminase–related and unrelated kataegis in tissue-RS affecting regulatory regions of key immune-regulatory genes. These include BTG2, CXCR4, NFATC1, PAX5, NOTCH-1, SLC44A5, FCRL3, SELL, TNIP2, and TRIM13. Furthermore, differences between the global mutation signatures of pairs of PB-CLL and tissue-RS samples implicate DDR as the dominant mechanism driving transformation. Pathway-based clonal deconvolution analysis showed that genes in the MAPK and DDR pathways demonstrate high clonal-expansion probability. Direct comparison of nodal-CLL and tissue-RS pairs from an independent cohort confirmed differential expression of the same pathways by RNA expression profiling. Our integrated analysis of WGS and RNA expression data significantly extends previous targeted approaches, which were limited by the lack of germline samples, and it facilitates the identification of novel genomic correlates implicated in RS transformation, which could be targeted therapeutically. Our results inform the future selection of investigative agents for a UK clinical platform study. This trial was registered at www.clinicaltrials.gov as #NCT03899337.
•RS is a rare complication of CLL with dismal prognosis, as well as an area in which effective therapies are sorely needed.•WGS and RNA expression profiling of paired CLL and RS samples reveals clinically targetable genes and pathways in RS transformation.
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IJS, IMTLJ, KILJ, NLZOH, NUK, SAZU, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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.
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).
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase and a nuclease that restricts HIV-1 in noncycling cells. Germ-line mutations in SAMHD1 have been described in patients with ...Aicardi-Goutières syndrome (AGS), a congenital autoimmune disease. In a previous longitudinal whole genome sequencing study of chronic lymphocytic leukemia (CLL), we revealed a SAMHD1 mutation as a potential founding event. Here, we describe an AGS patient carrying a pathogenic germ-line SAMHD1 mutation who developed CLL at 24 years of age. Using clinical trial samples, we show that acquired SAMHD1 mutations are associated with high variant allele frequency and reduced SAMHD1 expression and occur in 11% of relapsed/refractory CLL patients. We provide evidence that SAMHD1 regulates cell proliferation and survival and engages in specific protein interactions in response to DNA damage. We propose that SAMHD1 may have a function in DNA repair and that the presence of SAMHD1 mutations in CLL promotes leukemia development.
•Acquired pathogenic mutations in SAMHD1 are found in up to 11% of relapsed/refractory patients with CLL.•SAMHD1 is mobilized to sites of DNA damage.
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IJS, IMTLJ, KILJ, NLZOH, NUK, SAZU, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Despite advances in chronic lymphocytic leukaemia (CLL) treatment, globally chemotherapy remains a central treatment modality, with chemotherapy trials representing an invaluable resource to explore ...disease-related/genetic features contributing to long-term outcomes. In 499 LRF CLL4 cases, a trial with >12 years follow-up, we employed targeted resequencing of 22 genes, identifying 623 mutations. After background mutation rate correction, 11/22 genes were recurrently mutated at frequencies between 3.6% (NFKBIE) and 24% (SF3B1). Mutations beyond Sanger resolution (<12% VAF) were observed in all genes, with KRAS mutations principally composed of these low VAF variants. Firstly, employing orthogonal approaches to confirm <12% VAF TP53 mutations, we assessed the clinical impact of TP53 clonal architecture. Whilst ≥ 12% VAF TP53mut cases were associated with reduced PFS and OS, we could not demonstrate a difference between <12% VAF TP53 mutations and either wild type or ≥12% VAF TP53mut cases. Secondly, we identified biallelic BIRC3 lesions (mutation and deletion) as an independent marker of inferior PFS and OS. Finally, we observed that mutated MAPK-ERK genes were independent markers of poor OS in multivariate survival analysis. In conclusion, our study supports using targeted resequencing of expanded gene panels to elucidate the prognostic impact of gene mutations.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Prognostication in patients with chronic lymphocytic leukemia (CLL) is challenging due to heterogeneity in clinical course. We hypothesize that constitutional genetic variation affects disease ...progression and could aid prognostication. Pooling data from seven studies incorporating 842 cases identifies two genomic locations associated with time from diagnosis to treatment, including 10q26.13 (rs736456, hazard ratio (HR) = 1.78, 95% confidence interval (CI) = 1.47-2.15; P = 2.71 × 10
) and 6p (rs3778076, HR = 1.99, 95% CI = 1.55-2.55; P = 5.08 × 10
), which are particularly powerful prognostic markers in patients with early stage CLL otherwise characterized by low-risk features. Expression quantitative trait loci analysis identifies putative functional genes implicated in modulating B-cell receptor or innate immune responses, key pathways in CLL pathogenesis. In this work we identify rs736456 and rs3778076 as prognostic in CLL, demonstrating that disease progression is determined by constitutional genetic variation as well as known somatic drivers.
Research grade Fresh Frozen (FF) DNA material is not yet routinely collected in clinical practice. Many hospitals, however, collect and store Formalin Fixed Paraffin Embedded (FFPE) tumor samples. ...Consequently, the sample size of whole genome cancer cohort studies could be increased tremendously by including FFPE samples, although the presence of artefacts might obfuscate the variant calling. To assess whether FFPE material can be used for cohort studies, we performed an in-depth comparison of somatic SNVs called on matching FF and FFPE Whole Genome Sequence (WGS) samples extracted from the same tumor.
Four variant callers (i.e. Strelka2, Mutect2, VarScan2 and Shimmer) were used to call somatic variants on matching FF and FFPE WGS samples from a metastatic prostate tumor. Using the variants identified by these callers, we developed a heuristic to maximize the overlap between the FF and its FFPE counterpart in terms of sensitivity and precision. The proposed variant calling approach was then validated on nine matched primary samples. Finally, we assessed what fraction of the discrepancy could be attributed to intra-tumor heterogeneity (ITH), by comparing the overlap in clonal and subclonal somatic variants.
We first compared variants between an FF and an FFPE sample from a metastatic prostate tumor, showing that on average 50% of the calls in the FF are recovered in the FFPE sample, with notable differences between callers. Combining the variants of the different callers using a simple heuristic, increases both the precision and the sensitivity of the variant calling. Validating the heuristic on nine additional matched FF-FFPE samples, resulted in an average F1-score of 0.58 and an outperformance of any of the individual callers. In addition, we could show that part of the discrepancy between the FF and the FFPE samples can be attributed to ITH.
This study illustrates that when using the correct variant calling strategy, the majority of clonal SNVs can be recovered in an FFPE sample with high precision and sensitivity. These results suggest that somatic variants derived from WGS of FFPE material can be used in cohort studies.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Background . Chronic lymphocytic leukaemia (CLL) is always preceded by preclinical clonal B-cell disorders such as monoclonal B-cell lymphocytosis (MBL). Pre-malignant clonal B-cells are identifiable ...in asymptomatic individuals, a minority of whom will progress to symptomatic CLL. While some people remain at pre-cancerous or asymptomatic early stage CLL, others experience clinically aggressive disease. There is currently no method to effectively stratify individuals at higher risk of developing symptomatic CLL. Despite recent advances in treatment options available, the disease remains incurable. Therefore, we need to expand our knowledge and improve current classification methods. Here, we propose a molecular classifier using whole genome sequencing (WGS). This classifier was obtained by performing genomic characterization in pre-malignancy / early stages of the disease and comparing the genomic subgroups with those of symptomatic CLL. Methods . Individuals with newly diagnosed monoclonal B-cell lymphocytosis and early asymptomatic chronic lymphocytic leukaemia were enrolled in the OXPLORED clinical trial (Oxford Pre-cancerous Lymphoproliferative Disorders: Analysis and Interception study). We performed WGS in 400 samples from 200 individuals (sorted CD19+ B-cells as tumour, and matched salivary DNA as germline) to detect somatic alterations. We derived immunoglobulin heavy chain variable (IGHV) mutational status, stereotype and an additional 186 genomic features as previously described (Robbe*, Ridout* et al. Nature Genetics 2022) including known and recently discovered candidate drivers of CLL, recurrent structural variants, mutational signatures, genomic complexity measures, mutational burden, and pathway alterations. All the data was compiled into a matrix to extract meaningful sets of features to cluster patients' genomes using non-negative matrix factorization (NMF). Genomic findings were compared to the largest CLL whole genome cohort of symptomatic patients requiring frontline treatment (n=443). Results . The IGHV mutational status was found unmutated in 31% of individuals and hypermutated in 69% (excluding missing data for 6 genomes). Next, we considered mutations and CNAs in 58 common CLL drivers and 34 regions with recurrent CNAs. Although numbers were lower than in frontline CLL, most individuals presented at least one driver (86%) or more (57%) (by comparison, the CLL frontline cohort presented at least one CLL driver in 98% of patients). Genomic complexity, defined as 4 or more CNAs was detected in 12%, and complex genome defined as the presence of both CN gains and CN losses in 22%, indicating that newly diagnosed monoclonal B-cell lymphocytosis and early asymptomatic CLL present a relatively high degree of genomic complexity similar to what was observed in frontline CLL. The most common driver was del13q (60%), including as a sole driver in a third of these genomes. Other recurrent alterations were IGLL5 mutations (20%), trisomy 12 (9%), and mutations in MYD88 (8%), CREBBP (5%), NOTCH1 (5%), and SPEN (5%). Noncoding elements were also found mutated, including 30 promoters (including BIRC3 in 7%), five UTRs and eight enhancers, including BCL6 and PAX5 enhancers in 18% and 3.4% of individuals, respectively. When comparing this cohort with CLL genomes, we found that alterations in the main cancer pathways were depleted in OXPLORED, including known drivers such as ATM, NOTCH1, SF3B1. Finally, moving away from single-alteration grouping, we clustered the 643 genomes (443 CLL + 200 OXPLORED) using 186 genomic features by applying NMF. Importantly, we found distinct genomic profiles in the early disease cohort, including a subgroup like symptomatic CLL at frontline and another distinct subgroup, hinting towards identifying a genomic subgroup at higher risk of progressing to CLL. Conclusion: The comprehensive characterisation of genomic events that determine progression to malignancy are essential to understand the mechanisms of disease progression. Our study opens new horizons towards identifying individuals at higher risk and might pinpoint to individuals who need early intervention with curative intent. Therefore, we propose to analyse newly generated whole-genome sequencing (WGS) data to establish genomic subgroups in early CLL / pre-malignancy stages.
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IJS, IMTLJ, KILJ, NLZOH, NUK, SAZU, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Unmutated (UM) immunoglobulin heavy chain variable region (IgHV) status or IgHV3-21 gene usage is associated with poor prognosis in chronic lymphocytic leukemia (CLL) patients. Interestingly, ...IgHV3-21 is often co-expressed with light chain IgLV3-21, which is potentially able to trigger cell-autonomous BCR-mediated signaling. However, this light chain has never been characterized independently of the heavy chain IgHV3-21.
We performed total RNA sequencing in 32 patients and investigated IgLV3-21 prognostic impact in terms of treatment-free survival (TFS) and overall survival (OS) in 3 other independent cohorts for a total of 813 patients. IgLV3-21 presence was tested by real-time PCR and confirmed by Sanger sequencing.
Using total RNA sequencing to characterize 32 patients with high-risk CLL, we found a high frequency (28%) of IgLV3-21 rearrangements. Gene set enrichment analysis revealed that these patients express higher levels of genes responsible for ribosome biogenesis and translation initiation (
< 0.0001) as well as MYC target genes (
= 0.0003). Patients with IgLV3-21 rearrangements displayed a significantly shorter TFS and OS (
< 0.05), particularly those with IgHV mutation. In each of the three independent validation cohorts, we showed that IgLV3-21 rearrangements-similar to UM IgHV status-conferred poor prognosis compared with mutated IgHV (
< 0.0001). Importantly, we confirmed by multivariate analysis that this was independent of IgHV mutational status or subset #2 stereotyped receptor (
< 0.0001).
We have demonstrated for the first time that a light chain can affect CLL prognosis and that IgLV3-21 light chain usage defines a new subgroup of CLL patients with poor prognosis.
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Although TP53, NOTCH1, and SF3B1 mutations may impair prognosis of patients with chronic lymphocytic leukemia (CLL) receiving frontline therapy, the impact of these mutations or any other, alone or ...in combination, remains unclear at relapse. The genome of 114 relapsed/refractory patients included in prospective trials was screened using targeted next-generation sequencing of the TP53, SF3B1, ATM, NOTCH1, XPO1, SAMHD1, MED12, BIRC3, and MYD88 genes. We performed clustering according to both number and combinations of recurrent gene mutations. The number of genes affected by mutation was ≥2, 1, and 0 in 43 (38%), 49 (43%), and 22 (19%) respectively. Recurrent combinations of ≥2 mutations of TP53, SF3B1, and ATM were found in 22 (19%) patients. This multiple-hit profile was associated with a median progression-free survival of 12 months compared with 22.5 months in the remaining patients (P = .003). Concurrent gene mutations are frequent in patients with relapsed/refractory CLL and are associated with worse outcome.
•Targeted NGS of relapsed/refractory CLL reveals a high incidence of concurrent mutations that mostly affect the TP53, ATM, and SF3B1 genes.•Concurrent mutations of the TP53, ATM, and/or SF3B1 genes confer short survival in patients with relapsed/refractory CLL.
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IJS, IMTLJ, KILJ, NLZOH, NUK, SAZU, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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