Clonal germinal center B cells function as a niche for T-cell lymphoma.

Angioimmunoblastic T-cell lymphoma (AITL) is proposed to be initiated by age-related clonal hematopoiesis (ACH) with TET2 mutations, whereas the G17V RHOA mutation in immature cells with TET2 mutations promotes the development of T follicular helper (TFH)-like tumor cells. Here, we investigated the mechanism by which TET2-mutant immune cells enable AITL development using mouse models and human samples. Among the 2 mouse models, mice lacking Tet2 in all the blood cells (Mx-Cre × Tet2flox/flox × G17V RHOA transgenic mice) spontaneously developed AITL for approximately up to a year, while mice lacking Tet2 only in the T cells (Cd4-Cre × Tet2flox/flox × G17V RHOA transgenic mice) did not. Therefore, Tet2-deficient immune cells function as a niche for AITL development. Single-cell RNA-sequencing (scRNA-seq) of >50 000 cells from mouse and human AITL samples revealed significant expansion of aberrant B cells, exhibiting properties of activating light zone (LZ)-like and proliferative dark zone (DZ)-like germinal center B (GCB) cells. The GCB cells in AITL clonally evolved with recurrent mutations in genes related to core histones. In silico network analysis using scRNA-seq data identified Cd40-Cd40lg as a possible mediator of GCB and tumor cell cluster interactions. Treatment of AITL model mice with anti-Cd40lg inhibitory antibody prolonged survival. The genes expressed in aberrantly expanded GCB cells in murine tumors were also broadly expressed in the B-lineage cells of TET2-mutant human AITL. Therefore, ACH-derived GCB cells could undergo independent clonal evolution and support the tumorigenesis in AITL via the CD40-CD40LG axis.

[Genetic alterations in new category of Peripheral T-cell lymphoma, not otherwise specified].

Peripheral T-cell lymphomas (PTCLs) are mature T-cell and natural killer (NK) cell neoplasms with poor prognosis and no available standard treatment. The 2016 revision of the World Health Organization classification of hematological malignancies recognized a new group of nodal T-cell lymphoma with T follicular helper cell phenotype due to the presence of a group of PTCLs, not otherwise specified. This new subgroup of tumors showed clinical and molecular similarities with angioimmunoblastic T-cell lymphoma (AITL). AITL patients have mutations in the Vav guanine nucleotide exchange factor-1 (VAV1) gene. We established a transgenic mouse model harboring VAV1 gene mutation. These mice developed tumors that mimic human T-lymphoblastic lymphoma and the newly proposed PTCL-GATA3 subtype. To develop personalized treatment strategies by analyzing subgroup-specific mouse models, our study supports the establishment of PTCL subgroups based on genetic alterations or gene expression profiles.

Tet2 deficiency in immune cells exacerbates tumor progression by increasing angiogenesis in a lung cancer model.

Immune cells harboring somatic mutations reportedly infiltrate cancer tissues in patients with solid cancers and accompanying clonal hematopoiesis. Loss-of-function TET2 mutations are frequently observed in clonal hematopoiesis in solid cancers. Here, using a mouse lung cancer model, we evaluated the activity of Tet2-deficient immune cells in tumor tissues. Myeloid-specific Tet2 deficiency enhanced tumor growth in mice relative to that seen in controls. Single-cell sequencing analysis of immune cells infiltrating tumors showed relatively high expression of S100a8/S100a9 in Tet2-deficient myeloid subclusters. In turn, treatment with S100a8/S100a9 promoted Vegfa production by cancer cells, leading to a marked increase in the tumor vasculature in Tet2-deficient mice relative to controls. Finally, treatment of Tet2-deficient mice with an antibody against Emmprin, a known S100a8/S100a9 receptor, suppressed tumor growth. These data suggest that immune cells derived from TET2-mutated clonal hematopoiesis exacerbate lung cancer progression by promoting tumor angiogenesis and may provide a novel therapeutic target for lung cancer patients with TET2-mutated clonal hematopoiesis.

Molecular understanding of peripheral T-cell lymphomas, not otherwise specified (PTCL, NOS): A complex disease category.

Peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) includes various diseases. Attempts have been made to identify distinct properties of disease within the PTCL, NOS classification and evaluate their significance to prognosis. Comprehensive gene expression analysis and evaluation of genomic abnormalities have successfully identified specific diseases from heterogeneous PTCL, NOS cases. For example, cases with properties of T follicular helper cells have been identified and classified as an entity resembling angioimmunoblastic T-cell lymphoma (AITL), based on both immunohistochemistry and genomic features. Here, we focus on the molecular pathology of PTCL, NOS and discuss recent changes relevant to its classification.

VAV1 mutations contribute to development of T-cell neoplasms in mice.

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.

Molecular pathogenesis of progression to myeloid leukemia from TET-insufficient status.

Loss-of-function mutations in ten-eleven translocation-2 (TET2) are recurrent events in acute myeloid leukemia (AML) as well as in preleukemic hematopoietic stem cells (HSCs) of age-related clonal hematopoiesis. TET3 mutations are infrequent in AML, but the level of TET3 expression in HSCs has been found to decline with age. We examined the impact of gradual decrease of TET function in AML development by generating mice with Tet deficiency at various degrees. Tet2f/f and Tet3f/f mice were crossed with mice expressing Mx1-Cre to generate Tet2f/wtTet3f/fMx-Cre+ (T2ΔT3), Tet2f/fTet3f/wtMx-Cre+ (ΔT2T3), and Tet2f/fTet3f/fMx-Cre+ (ΔT2ΔT3) mice. All ΔT2ΔT3 mice died of aggressive AML at a median survival of 10.7 weeks. By comparison, T2ΔT3 and ΔT2T3 mice developed AML at longer latencies, with a median survival of ∼27 weeks. Remarkably, all 9 T2ΔT3 and 8 ΔT2T3 mice with AML showed inactivation of the remaining nontargeted Tet2 or Tet3 allele, respectively, owing to exonic loss in either gene or stop-gain mutations in Tet3. Recurrent mutations other than Tet3 were not noted in any mice by whole-exome sequencing. Spontaneous inactivation of residual Tet2 or Tet3 alleles is a recurrent genetic event during the development of AML with Tet insufficiency.

Dasatinib Is an Effective Treatment for Angioimmunoblastic T-cell Lymphoma.

Recurrent hotspot (p.Gly17Val) mutations in RHOA encoding a small GTPase, together with loss-of-function mutations in TET2 encoding an epigenetic regulator, are genetic hallmarks of angioimmunoblastic T-cell lymphoma (AITL). Mice expressing the p.Gly17Val RHOA mutant on a Tet2-null background succumbed to AITL-like T-cell lymphomas due to deregulated T-cell receptor (TCR) signaling. Using these mice to investigate therapeutics for AITL, we found that dasatinib, a multikinase inhibitor prolonged their survival through inhibition of hyperactivated TCR signaling. A phase I clinical trial study of dasatinib monotherapy in 5 patients with relapsed/refractory AITL was performed. Dasatinib was started at a dose of 100 mg/body once a day and continued until days 10-78 (median day 58). All the evaluable patients achieved partial responses. Our findings suggest that AITL is highly dependent on TCR signaling and that dasatinib could be a promising candidate drug for AITL treatment. SIGNIFICANCE: Deregulated T-cell receptor signaling is a critical molecular event in angioimmunoblastic T-cell lymphoma and can be targeted with dasatinib.

Droplet digital polymerase chain reaction assay and peptide nucleic acid-locked nucleic acid clamp method for RHOA mutation detection in angioimmunoblastic T-cell lymphoma.

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.

Review of the biologic and clinical significance of genetic mutations in angioimmunoblastic T-cell lymphoma.

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.

Quantification of bone marrow plasma cell infiltration in multiple myeloma: usefulness of bone marrow aspirate clot with CD138 immunohistochemistry.

Accurate quantification of plasma cells (PCs) in bone marrow (BM) is critical for diagnosis and assessment of treatment response in patients with multiple myeloma (MM). We compared the % of BM PC quantified by 250 cell differential count on May-Giemsa-stained BM smears, by counting 500 - 2500 cells in 2 - 5 representative microscopy fields in CD138-immunostained BM clot and biopsy sections, and CD38/CD45/CD138 gated BM PCs on flow cytometry (FCM) in 150 sets of BM samples from 120 patients. Percentages of PC were significantly correlated between BM biopsy and clot, and between smear and FCM (r = 0.96, 0.93, respectively). However, quantification by smear and FCM significantly underestimated the PC compared to biopsy or clot, and the degree of underestimation increased with blood dilution. FCM consistently showed lower % of PC compared to aspirate smears. Fifty-nine of 103 patients with M-protein level < 3000 mg/dL in serum or 500 mg/24 h in urine and diagnosed with monoclonal gammopathy of undetermined significance (MGUS) based on smear alone were reclassified as smoldering MM when reassessed using CD138-stained biopsy/clot sections. Among the 72 patients with sMM diagnosed by BM biopsy and/clot, three patients (4.2%) had extensive BM infiltration of PC (≥ 60%) and required treatment. Our data clearly showed the necessity of CD138 immunostaining of BM biopsy/clot specimens for correct diagnosis of MM and related disorders. Copyright © 2016 John Wiley & Sons, Ltd.

Abnormal heavy/light chain ratio after treatment is associated with shorter survival in patients with IgA myeloma.

Immunoglobulin (Ig) heavy/light chain (HLC) assays enable the separate quantification of the different light chain types of each Ig class. We retrospectively analyzed the correlation of heavy/light chain ratio (HLCR) with clinical status and its impact on outcome in 120 patients with multiple myeloma (MM). Abnormal HLCR was seen more frequently in patients with poorer myeloma response, and it appeared to be more sensitive for detecting clonality in IgA myeloma compared to IgG myeloma after treatment. Among the 85 patients who achieved ≥VGPR, the patients remained HLCR abnormal were showed significantly shorter overall survival (OS) compared to those achieving a normal HLCR (not reached vs 55.5 months, P = 0.032). This correlation was seen in IgA myeloma patients (not reached vs 30.1 months, P = 0.014), but not in IgG myeloma patients when patients were analyzed separately. Univariate and multivariate analysis of factors that may affect survival identified abnormal HLCR at the best response as the only independent risk factor (hazard ratio, 4.7; 95% confidence interval, 1.4 - 15.26; P = 0.012) for shorter OS in this subset of patients. This study highlighted the HLC assay as a prognostic predictor in patients with IgA myeloma.

Prognostic impact of immunophenotypic complete response in patients with multiple myeloma achieving better than complete response.

To investigate the impact of immunophenotypic complete response [iCR, ≤10(-4) multiple myeloma (MM) cells defined by multicolor flow cytometry (MFC)] on survival in patients with MM, we retrospectively analyzed 78 patients that obtained conventional CR at our hospital. Survivals were landmarked at achievement of CR. The rate of stringent CR (sCR) among patients with CR was 88%, and iCR for CR and sCR patients were 44% and 49%, respectively. Achievement of iCR was associated with significantly longer disease-free survival (DFS) not only in CR patients (p = 0.009) but also in sCR patients (p = 0.002), while sCR attainment per se did not have statistically significant impact on DFS (p = 0.06) or overall survival (OS) (p = 0.587). Univariate and multivariate analyses indicated that attainment of iCR was independently associated with longer 2-year DFS in addition to creatinine (≤2.0 mg/dL) and maintenance therapy. This study highlights the importance of pursuing iCR even in patients with sCR.

Changes in survival rate of multiple myeloma after the introduction of bortezomib: a single institutional experience over 20 years.

The data of factors on the changes in survival before and after the introduction of bortezomib in unselected multiple myeloma (MM) patients is scarce in Asian population. We analyzed the clinical features and treatment outcomes of 270 consecutive MM patients admitted to our hospital between January 1995 and August 2014. The patients were divided into two groups, 1995­2005 (n=106) and 2006­2014 (n=164), based on bortezomib availability. There were no differences in baseline characteristics between the groups, except age and percentage of patients receiving autologous stem cell transplantation (auto-SCT). The proportion of patients obtaining ≥very good partial response (VGPR) was higher in the recent cohort, which was translated as better overall survival in both younger and older patients (36.1 vs. 79.8 months, P=0.024, and 40.0 vs. 110.7 months, respectively). Patients receiving bortezomib early after diagnosis showed significantly better survival. However, there was no difference in survival between patients obtaining ≥VGPR in the two groups. On multivariate analysis, age ≥75 and lactate dehydrogenase (LDH) >normal were associated with shorter survival, while early bortezomib use and auto-SCT were associated with longer survival. On Cox regression analysis, International Staging System (ISS) stage III, LDH, and treatment response

Clinical features and treatment outcome of very elderly patients over 80 years old with multiple myeloma: comparison with patients in different age groups in the era of novel agents.

We retrospectively analyzed the outcomes of 175 consecutive patients admitted to our hospital between April 2004 and June 2014, and identified 42 (24%), 80 (46%), and 53 (30%) patients ≥ 80, 66-79, and ≤ 65 years old, respectively. The median progression-free survival (PFS) and overall survival (OS) of the ≥ 80, 66-79, and ≤ 65 years old groups were 19.1, 26.3, and 54.3 months, and 31.9, 54.8, and 83.8 months, respectively. Patients ≥ 80 but not ≤ 79 years old with ECOG performance score (PS) ≥ 3 and/or Charlson comorbidity index (CCI) ≥ 5 showed significantly shorter survival. ECOG PS and CCI predicted the treatment outcome of patients ≥ 80 but did not predict ≤ 79 years old.