Novel GPR34 and CCR6 mutation and distinct genetic profiles in MALT lymphomas of different sites.

Mucosa-associated lymphoid tissue (MALT) lymphoma originates from a background of diverse chronic inflammatory disorders at various anatomic sites. The genetics underlying its development, particularly in those associated with autoimmune disorders, is poorly characterized. By whole exome sequencing of 21 cases of MALT lymphomas of the salivary gland and thyroid, we have identified recurrent somatic mutations in 2 G-protein coupled receptors (GPR34 and CCR6) not previously reported in human malignancies, 3 genes (PIK3CD, TET2, TNFRSF14) not previously implicated in MALT lymphoma, and a further 2 genes (TBL1XR1, NOTCH1) recently described in MALT lymphoma. The majority of mutations in GPR34 and CCR6 were nonsense and frameshift changes clustered in the C-terminal cytoplasmic tail, and would result in truncated proteins that lack the phosphorylation motif important for β-arrestin-mediated receptor desensitization and internalization. Screening of these newly identified mutations, together with previously defined genetic changes, revealed distinct mutation profiles in MALT lymphoma of various sites, with those of salivary gland characterized by frequent TBL1XR1 and GPR34 mutations, thyroid by frequent TET2, TNFRSF14 and PIK3CD mutations, and ocular adnexa by frequent TNFAIP3 mutation. Interestingly, in MALT lymphoma of the salivary gland, there was a significant positive association between TBL1XR1 mutation and GPR34 mutation/translocation (P=0.0002). In those of ocular adnexa, TBL1XR1 mutation was mutually exclusive from TNFAIP3 mutation (P=0.049), but significantly associated with IGHV3-23 usage (P=0.03) and PIK3CD mutation (P=0.009). These findings unravel novel insights into the molecular mechanisms of MALT lymphoma and provide further evidence for potential oncogenic co-operation between receptor signaling and genetic changes.The research was supported by grants from Bloodwise (13006, 15002, 15019) UK, and Kay Kendal Leukaemia Fund (KKL582), UK. SM was initially supported by a PhD studentship from Medical Research Council, Department of Pathology, University of Cambridge and Addenbrooke’s Charitable Trust

Mutation screening using formalin-fixed paraffin-embedded tissues: a stratified approach according to DNA quality.

DNA samples from formalin-fixed paraffin-embedded tissues are highly degraded with variable quality, and this imposes a big challenge for targeted sequencing due to false positives, largely caused by PCR errors and cytosine deamination. To eliminate false positives, a common practice is to validate the detected variants by Sanger sequencing or perform targeted sequencing in duplicate. Technically, PCR errors could be removed by molecular barcoding of template DNA prior to amplification as in the HaloPlexHS design. Nonetheless, it is uncertain to what extent variants detected using this approach should be further validated. Here, we addressed this question by correlating variant reproducibility with DNA quality using HaloPlexHS target enrichment and Illumina HiSeq4000, together with an in-house validated variant calling algorithm. The overall sequencing coverage, as shown by analyses of 70 genes in 266 cases of large B-cell lymphoma, was excellent (98%) in DNA samples amenable for PCR of ≥400 bp, but suboptimal (92%) and poor (80%) in those amenable for PCR of 300 bp and 200 bp respectively. By mutation analysis in duplicate in 93 cases, we demonstrated that 20 alternative allele depth (AAD) was an optimal cut-off value for separating reproducible from non-reproducible variants in DNA samples amenable for PCR of ≥300 bp, with 97% sensitivity and 100% specificity. By cross validation with a previously established targeted sequencing protocol by Fluidigm-PCR and Illumina MiSeq, the HaloPlexHS protocol was shown to be highly sensitive and specific in mutation screening. To conclude, we proposed a stratified approach for mutation screening by HaloplexHS and Illumina HiSeq4000 according to DNA quality. DNA samples with good quality (≥400 bp) are amenable for mutation analysis with a single replicate, with only variants at 15-20 AAD requiring for further validation, while those with suboptimal quality (300 bp) are better analysed in duplicate with reproducible variants at >15 AAD regarded as true genetic changes

Distinct genetic changes reveal evolutionary history and heterogeneous molecular grade of DLBCL with MYC / BCL2 double-hit

Abstract: Using a Burkitt lymphoma-like gene expression signature, we recently defined a high-risk molecular high-grade (MHG) group mainly within germinal centre B-cell like diffuse large B-cell lymphomas (GCB-DLBCL), which was enriched for MYC/BCL2 double-hit (MYC/BCL2-DH). The genetic basis underlying MHG-DLBCL and their aggressive clinical behaviour remain unknown. We investigated 697 cases of DLBCL, particularly those with MYC/BCL2-DH (n = 62) by targeted sequencing and gene expression profiling. We showed that DLBCL with MYC/BCL2-DH, and those with BCL2 translocation, harbour the characteristic mutation signatures that are associated with follicular lymphoma and its high-grade transformation. We identified frequent MYC hotspot mutations that affect the phosphorylation site (T58) and its adjacent amino acids, which are important for MYC protein degradation. These MYC mutations were seen in a subset of cases with MYC translocation, but predominantly in those of MHG. The mutations were more frequent in double-hit lymphomas with IG as the MYC translocation partner, and were associated with higher MYC protein expression and poor patient survival. DLBCL with MYC/BCL2-DH and those with BCL2 translocation alone are most likely derived from follicular lymphoma or its precursor lesion, and acquisition of MYC pathogenic mutations may augment MYC function, resulting in aggressive clinical behaviour

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Novel GPR34 and CCR6 mutation and distinct genetic profiles in MALT lymphomas of different sites

MALT lymphoma originates from a background of diverse chronic inflammatory disorders at various anatomic sites. The genetics underlying its development, particularly in those associated with autoimmune disorders, is poorly characterised. By whole exome sequencing of 21 cases of MALT lymphomas of the salivary gland and thyroid, we have identified recurrent somatic mutations in 2 G-protein coupled receptors (GPR34 and CCR6) not previously reported in human malignancies, 3 genes (PIK3CD, TET2, TNFRSF14) not previously implicated in MALT lymphoma, and a further 2 genes (TBL1XR1, NOTCH1) recently described in MALT lymphoma. The majority of mutations in GPR34 and CCR6 were nonsense and frameshift changes clustered in the C-terminal cytoplasmic tail, and would result in truncated proteins that lack the phosphorylation motif important for β-arrestin mediated receptor desensitization and internalisation. Screening of these newly identified mutations, together with previously identified genetic changes, identified distinct mutation profiles in MALT lymphoma of various sites, with those of salivary gland characterised by frequent TBL1XR1 and GPR34 mutations, thyroid by frequent TET2, TNFRSF14 and PIK3CD mutations, and ocular adnexa by frequent TNFAIP3 mutation. Interestingly, in MALT lymphoma of the salivary gland, there was a significant positive association between TBL1XR1 mutation and GPR34 mutation/translocation (P=0.0002). In those of ocular adnexa, TBL1XR1 mutation was mutually exclusive from TNFAIP3 mutation (P=0.049), but significantly associated with IGHV3-23 usage (P=0.03) and PIK3CD mutation (P=0.009). These findings unravel novel insights into the molecular mechanisms of MALT lymphoma and provide further evidence for potential oncogenic cooperation between receptor signalling and genetic changes.status: accepte

Mutation screening using formalin-fixed paraffin-embedded tissues: a stratified approach according to DNA quality

DNA samples from formalin-fixed paraffin-embedded tissues are highly degraded with variable quality, and this imposes a big challenge for targeted sequencing due to false positives, largely caused by PCR errors and cytosine deamination. To eliminate false positives, a common practice is to validate the detected variants by Sanger sequencing or perform targeted sequencing in duplicate. Technically, PCR errors could be removed by molecular barcoding of template DNA prior to amplification as in the HaloPlexHS design. Nonetheless, it is uncertain to what extent variants detected using this approach should be further validated. Here, we addressed this question by correlating variant reproducibility with DNA quality using HaloPlexHS target enrichment and Illumina HiSeq4000, together with an in-house validated variant calling algorithm. The overall sequencing coverage, as shown by analyses of 70 genes in 266 cases of large B-cell lymphoma, was excellent (98%) in DNA samples amenable for PCR of ≥400 bp, but suboptimal (92%) and poor (80%) in those amenable for PCR of 300 bp and 200 bp respectively. By mutation analysis in duplicate in 93 cases, we demonstrated that 20 alternative allele depth (AAD) was an optimal cut-off value for separating reproducible from non-reproducible variants in DNA samples amenable for PCR of ≥300 bp, with 97% sensitivity and 100% specificity. By cross validation with a previously established targeted sequencing protocol by Fluidigm-PCR and Illumina MiSeq, the HaloPlexHS protocol was shown to be highly sensitive and specific in mutation screening. To conclude, we proposed a stratified approach for mutation screening by HaloplexHS and Illumina HiSeq4000 according to DNA quality. DNA samples with good quality (≥400 bp) are amenable for mutation analysis with a single replicate, with only variants at 15–20 AAD requiring for further validation, while those with suboptimal quality (300 bp) are better analysed in duplicate with reproducible variants at &gt;15 AAD regarded as true genetic changes.</p

Delaying surgery for patients with a previous SARS-CoV-2 infection

Not availabl