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Activating mutations in the Vav guanine nucleotide exchange factor 1 (VAV1) gene are reported in various subtypes of mature T-cell neoplasms (TCNs). However, oncogenic activities associated with VAV1 mutations in TCNs remain unclear. To define them, we established transgenic mice expressing VAV1 mutants cloned from human TCNs. Although we observed no tumors in these mice for up to a year, tumors did develop in comparably aged mice on a p53-null background (p53-/-VAV1-Tg), and p53-/-VAV1-Tg mice died with shorter latencies than did p53-null (p53-/-) mice. Notably, various TCNs with tendency of maturation developed in p53-/-VAV1-Tg mice, whereas p53-/- mice exhibited only immature TCNs. Mature TCNs in p53-/-VAV1-Tg mice mimicked a subtype of human peripheral T-cell lymphoma (PTCL-GATA3) and exhibited features of type 2 T helper (Th2) cells. Phenotypes seen following transplantation of either p53-/-VAV1 or p53-/- tumor cells into nude mice were comparable, indicating cell-autonomous tumor-initiating capacity. Whole-transcriptome analysis showed enrichment of multiple Myc-related pathways in TCNs from p53-/-VAV1-Tg mice relative to p53-/- or wild-type T cells. Remarkably, amplification of the Myc locus was found recurrently in TCNs of p53-/-VAV1-Tg mice. Finally, treatment of nude mice transplanted with p53-/-VAV1-Tg tumor cells with JQ1, a bromodomain inhibitor that targets the Myc pathway, prolonged survival of mice. We conclude that VAV1 mutations function in malignant transformation of T cells in vivo and that VAV1-mutant-expressing mice could provide an efficient tool for screening new therapeutic targets in TCNs harboring these mutations., (© 2020 by The American Society of Hematology.)

Follicular T-cell lymphoma (FTCL) is considered to originate from follicular helper T-cell (Tfh) cells. Angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphomas with the Tfh phenotype, derived from Tfh cells, often harbor RHOA G17V mutation. We investigated whether RHOA mutations affect the clinicopathological features of FTCL. We performed deep sequencing and Sanger sequencing for RHOA exon 2 in 16 cases of FTCL. Nine cases showed RHOA mutations, including eight with c.G50T, p.Gly17Val and one with c.G50A, p.Gly17Glu, c.A52G, p.Lys18Glu, c.T102C, p.Tyr34Tyr and c.G145T, p.Asp49Tyr. Compared to the RHOA mutation-negative group, the RHOA mutation-positive group had a higher tendency for B-immunoblasts (P = 0.06), the AITL component (P = 0.09), and higher positive rate for CD10 (P = 0.09) and BCL6 (P = 0.09), and a significantly higher positive rate for CXCL13 (P = 0.04). Although not statistically significant, the RHOA mutation-positive group showed higher values for almost all characteristic AITL features. There was no significant difference in overall survival between RHOA mutation-positive and -negative groups. The RHOA mutation may play an important role in clinicopathological characteristics and lymphomagenesis of FTCL. A more detailed investigation is needed to highlight the importance of RHOA mutations in FTCL., (© 2020 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Cell-free DNA (cfDNA) analysis to detect circulating tumor DNA has been focused on monitoring malignant lymphomas. However, clonal hematopoiesis of indeterminate potential (CHIP)-associated mutations can also be detected by cfDNA analysis. Our aim is to investigate the origin of mutations detected in cfDNA among B-cell lymphoma patients. MYD88/CD79B, DNMT3A, and TP53 were chosen as genes of interest, representing each of the following categories: lymphoma driver genes, CHIP-related genes, and genes shared between lymphoma and CHIP. Seventy-five B-cell lymphoma patients were included in this retrospective study. Serum cfDNAs at time of complete metabolic response (CMR) were sequenced for TP53 (N = 75) and DNMT3A (N = 49). MYD88 p.L265P and CD79B p.Y196C/H mutations were analyzed in diffuse large B-cell lymphoma (DLBCL) patients whose tumor samples were available (N = 29). Two and seven mutations in TP53 and DNMT3A, respectively, were detected in cfDNA at CMR. These mutations were detected in either bone marrow mononuclear cells (BMMC) or PBMC. Although four DNMT3A mutations were also detected in tumors, median variant allele frequencies in the tumors (<1.0%) were significantly lower than those in both BMMC (6.1%) and serum (5.2%) obtained before the therapy. Conversely, five MYD88 and three CD79B mutations detected in tumors were confirmed in cfDNA before therapy, but not in BMMC nor in cfDNA at CMR. Thus, all TP53 and DNMT3A mutations detected in cfDNA at remission seemed to originate from CHIP rather than from residual disease. Results of liquid biopsy should be carefully interpreted, especially in genes shared between lymphomas and CHIP., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare subtype of myeloid neoplasm. Clonal evolution in the development of BPDCN remains to be elucidated. In the present study, we examined clonal evolution in a case of BPDCN by analyzing the distribution of gene mutations in tumor cells and non-tumor blood cells. The p.D1129fs and p.K1005fs TET2 mutations, p.P95H SRSF2 mutation, and p.L287fs NPM1 mutation were identified in a skin tumor at diagnosis and peripheral blood mononuclear cells at relapse. Notably, the p.D1129fs TET2 and p.L287fs NPM1 mutations were observed only in tumor cells, while the p.K1005fs TET2 and p.P95H SRSF2 mutations were found in both tumor cells and non-tumor blood cells. Recent genetic studies have suggested that some blood cancers may originate from clonal hematopoiesis, harboring somatic mutations. In the present case, the data suggest that BPDCN originated from clonal hematopoiesis with the p.K1005fs TET2 and p.P95H SRSF2 mutations via acquisition of the additional p.D1129fs TET2 and p.L287fs NPM1 mutations.

Angioimmunoblastic T-cell lymphoma (AITL) is a subtype of nodal peripheral T-cell lymphoma (PTCL). Somatic RHOA mutations, most frequently found at the hotspot site c.50G > T, p.Gly17Val (G17V RHOA mutation) are a genetic hallmark of AITL. Detection of the G17V RHOA mutations assists prompt and appropriate diagnosis of AITL. However, an optimal detection method for the G17V RHOA mutation remains to be elucidated. We compared the sensitivity and concordance of next-generation sequencing (NGS), droplet digital PCR (ddPCR) and peptide nucleic acid-locked nucleic acid (PNA-LNA) clamp method for detecting the G17V RHOA mutation. G17V RHOA mutations were identified in 27 of 67 (40.3%) PTCL samples using NGS. ddPCR and PNA-LNA clamp method both detected G17V mutations in 4 samples in addition to those detected with NGS (31 of 67, 46.3%). Additionally, variant allele frequencies with ddPCR and those with NGS showed high concordance (P < .001). Three other RHOA mutations involving the p.Gly17 position (c.[49G > T;50G > T], p.Gly17Leu in PTCL198; c.[50G > T;51A > C], p.Gly17Val in PTCL216; and c.50G > A, p.Gly17Glu in PTCL223) were detected using NGS. These sequence changes could not appropriately be detected using the ddPCR assay and the PNA-LNA clamp method although both indicated that the samples might have mutations. In total, 34 out of 67 PTCL samples (50.7%) had RHOA mutations at the p.Gly17 position. In conclusion, our results suggested that a combination of ddPCR/PNA-LNA clamp methods and NGS are best method to assist the diagnosis of AITL by detecting RHOA mutations at the p.Gly17 position., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Angioimmunoblastic T-cell lymphoma (AITL) is an age-related malignant lymphoma, characterized by immune system-dysregulated symptoms. Recent sequencing studies have clarified the recurrent mutations in ras homology family member A (RHOA) and in genes encoding epigenetic regulators, tet methyl cytosine dioxygenase 2 (TET2), DNA methyl transferase 3 alpha (DNMT3A) and isocitrate dehydrogenase 2, mitochondrial (IDH2), as well as those related to the T-cell receptor signaling pathway in AITL. In this review, we focus on how this genetic information has changed the understanding of the developmental process of AITL and will in future lead to individualized therapies for AITL patients., (© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Recent progress in sequencing studies has suggested that somatic mutations can be used in clinical sequencing for predicting prognosis and selecting treatment options in myelodysplastic syndrome (MDS). A 48-year-old man was diagnosed with refractory cytopenia with multilineage dysplasia that is classified as a subtype of high-risk MDS based on both revised International Prognostic Scoring System and refined WHO classification based Prognostic Scoring System. He received a bone marrow transplant from an HLA-matched sibling donor at X＋87 months because of disease progression. Targeted sequencing of 69 genes in bone marrow cells at X＋82 months revealed mutations in BCOR and U2AF1 genes. Variant allele frequencies of these mutations were almost unchanged in the bone marrow examined from X＋9 months to X＋80 months, but they subsequently decreased. Neither of these mutations was detected in the bone marrow at X＋88 months, a month after transplantation. The mutations often found in secondary leukemia or high-risk MDS were not detected in our patient. These serial genetic conditions may correspond to the relatively stable disease course over a long time.

Recent sequencing studies demonstrated the MYD88 L265P mutation in more than 70% of primary central nervous system lymphomas (PCNSL), and the clinical significance of this mutation has been proposed as diagnostic and prognostic markers in PCNSL. In contrast, mutational analyses using cell-free DNAs have been reported in a variety of systemic lymphomas. To investigate how sensitively the MYD88 L265P mutation can be identified in cell-free DNA from PCNSL patients, we carried out droplet digital PCR (ddPCR) and targeted deep sequencing (TDS) in 14 consecutive PCNSL patients from whom paired tumor-derived DNA and cell-free DNA was available at diagnosis. The MYD88 L265P mutation was found in tumor-derived DNA from all 14 patients (14/14, 100%). In contrast, among 14 cell-free DNAs evaluated by ddPCR (14/14) and TDS (13/14), the MYD88 L265P mutation was detected in eight out of 14 (ddPCR) and in 0 out of 13 (TDS) samples, implying dependence on the detection method. After chemotherapy, the MYD88 L265P mutation in cell-free DNAs was traced in five patients; unexpectedly, the mutations disappeared after chemotherapy was given, and they remained undetectable in all patients. These observations suggest that ddPCR can sensitively detect the MYD88 L265P mutation in cell-free DNA and could be used as non-invasive diagnostics, but may not be applicable for monitoring minimal residual diseases in PCNSL., (© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Recent genetic analysis has identified frequent mutations in ten-eleven translocation 2 (TET2), DNA methyltransferase 3A (DNMT3A), isocitrate dehydrogenase 2 (IDH2) and ras homolog family member A (RHOA) in nodal T-cell lymphomas, including angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified. We examined the distribution of mutations in these subtypes of mature T-/natural killer cell neoplasms to determine their clonal architecture. Targeted sequencing was performed for 71 genes in tumor-derived DNA of 87 cases. The mutations were then analyzed in a programmed death-1 (PD1)-positive population enriched with tumor cells and CD20-positive B cells purified by laser microdissection from 19 cases. TET2 and DNMT3A mutations were identified in both the PD1+ cells and the CD20+ cells in 15/16 and 4/7 cases, respectively. All the RHOA and IDH2 mutations were confined to the PD1+ cells, indicating that some, including RHOA and IDH2 mutations, being specific events in tumor cells. Notably, we found that all NOTCH1 mutations were detected only in the CD20+ cells. In conclusion, we identified both B- as well as T-cell-specific mutations, and mutations common to both T and B cells. These findings indicate the expansion of a clone after multistep and multilineal acquisition of gene mutations., Competing Interests: The authors declare no conflict of interest.

Adult T-cell leukemia/lymphoma (ATLL) is a distinct form of peripheral T-cell lymphoma with poor prognosis, which is caused by the human T-lymphotropic virus type 1 (HTLV-1). In contrast to the unequivocal importance of HTLV-1 infection in the pathogenesis of ATLL, the role of acquired mutations in HTLV-1 infected T cells has not been fully elucidated, with a handful of genes known to be recurrently mutated. In this study, we identified unique RHOA mutations in ATLL through whole genome sequencing of an index case, followed by deep sequencing of 203 ATLL samples. RHOA mutations showed distinct distribution and function from those found in other cancers. Involving 15% (30/203) of ATLL cases, RHOA mutations were widely distributed across the entire coding sequence but almost invariably located at the guanosine triphosphate (GTP)-binding pocket, with Cys16Arg being most frequently observed. Unexpectedly, depending on mutation types and positions, these RHOA mutants showed different or even opposite functional consequences in terms of GTP/guanosine diphosphate (GDP)-binding kinetics, regulation of actin fibers, and transcriptional activation. The Gly17Val mutant did not bind GTP/GDP and act as a dominant negative molecule, whereas other mutants (Cys16Arg and Ala161Pro) showed fast GTP/GDP cycling with enhanced transcriptional activation. These findings suggest that both loss- and gain-of-RHOA functions could be involved in ATLL leukemogenesis. In summary, our study not only provides a novel insight into the molecular pathogenesis of ATLL but also highlights a unique role of variegation of heterologous RHOA mutations in human cancers., (© 2016 by The American Society of Hematology.)

Recent advances in sequencing technologies led us to the discovery of mutations in epigenetic and metabolic regulators in peripheral T-cell lymphoma (PTCL). Frequencies of these mutations were found to be especially high in PTCL with features of follicular helper T cells (TFH). These mutations are also identifiable in myeloid malignancies as well as PTCL, suggesting that abnormalities in epigenetic and metabolic pathways are likely to be the fundamental mechanisms underlying various types of hematologic malignancies. The downstream pathways of these mutations have been extensively analyzed in myeloid malignancies, but have yet to be elucidated in PTCL. We clarified a downstream pathway of TET2 mutations in PTCL by analyzing TET2 knockdown mice.

Cohesin is a multimeric protein complex that is involved in the cohesion of sister chromatids, post-replicative DNA repair and transcriptional regulation. Here we report recurrent mutations and deletions involving multiple components of the cohesin complex, including STAG2, RAD21, SMC1A and SMC3, in different myeloid neoplasms. These mutations and deletions were mostly mutually exclusive and occurred in 12.1% (19/157) of acute myeloid leukemia, 8.0% (18/224) of myelodysplastic syndromes, 10.2% (9/88) of chronic myelomonocytic leukemia, 6.3% (4/64) of chronic myelogenous leukemia and 1.3% (1/77) of classical myeloproliferative neoplasms. Cohesin-mutated leukemic cells showed reduced amounts of chromatin-bound cohesin components, suggesting a substantial loss of cohesin binding sites on chromatin. The growth of leukemic cell lines harboring a mutation in RAD21 (Kasumi-1 cells) or having severely reduced expression of RAD21 and STAG2 (MOLM-13 cells) was suppressed by forced expression of wild-type RAD21 and wild-type RAD21 and STAG2, respectively. These findings suggest a role for compromised cohesin functions in myeloid leukemogenesis.

Myelodysplastic syndromes and related disorders (myelodysplasia) are a heterogeneous group of myeloid neoplasms showing deregulated blood cell production with evidence of myeloid dysplasia and a predisposition to acute myeloid leukaemia, whose pathogenesis is only incompletely understood. Here we report whole-exome sequencing of 29 myelodysplasia specimens, which unexpectedly revealed novel pathway mutations involving multiple components of the RNA splicing machinery, including U2AF35, ZRSR2, SRSF2 and SF3B1. In a large series analysis, these splicing pathway mutations were frequent (∼45 to ∼85%) in, and highly specific to, myeloid neoplasms showing features of myelodysplasia. Conspicuously, most of the mutations, which occurred in a mutually exclusive manner, affected genes involved in the 3'-splice site recognition during pre-mRNA processing, inducing abnormal RNA splicing and compromised haematopoiesis. Our results provide the first evidence indicating that genetic alterations of the major splicing components could be involved in human pathogenesis, also implicating a novel therapeutic possibility for myelodysplasia.