Reconstruction of rearranged T-cell receptor loci by whole genome and transcriptome sequencing gives insights into the initial steps of T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is an aggressive tumor with leukemic presentation of mature T-lymphocytes. Here, we aimed at characterizing the initial events in the molecular pathogenesis of T-PLL and particularly, at determining the point in T-cell differentiation when the hallmark oncogenic events, that is, inv(14)(q11q32)/t(14;14)(q11;q32) and t(X;14)(q28;q11) occur. To this end, we mined whole genome and transcriptome sequencing data of 17 and 11 T-PLL cases, respectively. Mapping of the 14q32.1 locus breakpoints identified only TCL1A, which was moreover significantly overexpressed in T-PLL as compared to benign CD4+ and CD8+ T-cells, as the only common oncogenic target of aberrations. In cases with t(14;14), the breakpoints mapped telomeric and in cases with inv(14) centromeric or in the 3'-untranslated region of TCL1A. Regarding the T-cell receptor alpha (TRA) locus-TCL1A breakpoint junctions, all 17 breakpoints involved recombination signal sequences and 15 junctions contained nontemplated (N-) nucleotides. All T-PLL cases studied carried in-frame TRA rearrangements on the intact allele, which skewed significantly toward usage of distal/central TRAV/TRAJ gene segments as compared to the illegitimate TRA rearrangements. Our findings suggest that the oncogenic TRA-TCL1A/MTCP1 rearrangements in T-PLL occur during opening of the TRA locus, that is, during the progression from CD4+ immature single positive to early double positive thymocyte stage, just before physiologic TCL1A expression is silenced. The cell carrying such an oncogenic event continues maturation and rearranges the second TRA allele to achieve a functional T-cell receptor. Thereafter, it switches off RAG and DNTT expression in line with the mature T-cell phenotype at presentation of T-PLL.

The new biology of PTCL-NOS and AITL: current status and future clinical impact.

Peripheral T-cell lymphomas (PTCL) comprise a heterogeneous group of aggressive lymphoproliferative disorders almost all of which are associated with poor clinical outcomes. Angioimmunoblastic T-cell lymphoma (AITL) and some peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) have similarities to normal CD4+ T-cell subsets in their gene expression profiles. A cell of origin model is, therefore, emerging and is likely to be refined in the future. Follicular helper (Tfh) T cells are now established as the cell of origin of AITL and about 20% of PTCL-NOS. Sequencing studies have identified recurrent genetic alterations in epigenetic modifiers, T-cell receptor signalling pathway intermediates or RHOA, most commonly a specific mutation leading to RHOA G17V. While PTCL-NOS remains a diagnosis of exclusion, advances in genomics have identified subgroups expressing transcription factors TBX 21 (Th1-like origin) and GATA3 (Th2-like origin). These findings suggest new biomarkers and new therapeutic avenues including the hypomethylating agent azacytidine, or inhibitors of proximal T-cell receptor (TCR) signalling and potentially certain monoclonal antibodies. The advances over the past few years, therefore, prompt stratified medicine approaches to test biologically based treatments and determine the clinical utility of the new disease classifications.

Follicular helper T-cells: expanding roles in T-cell lymphoma and targets for treatment.

Follicular helper T-cells (Tfh cells) are a subset of CD4(+) T-cells that are essential for normal production of high affinity antibodies. Tfh cells characteristically produce IL21 and IL4 and show high expression of surface markers CXCR5, ICOS, PDCD1 (PD-1) and the chemokine CXCL13. In this review we will focus on the emerging links between Tfh cells and subtypes of T-cell non-Hodgkin lymphoma: angioimmunoblastic T-cell lymphoma (AITL) and ~20% of peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) have surface marker features of Tfh cells and share a spectrum of genetic abnormalities. The recurrent genetic abnormalities associated with AITL include mutations in epigenetic modifiers such as TET2 and DNMT3A and the motility and adhesion gene, RHOA, is mutated in up to 70% of cases. ~20% of PTCL-NOS demonstrate RHOA mutations and have other characteristics suggesting an origin in Tfh cells. The recognition that specific genetic and surface markers are associated with malignant Tfh cells suggests that the next few years will bring major changes in diagnostic and treatment possibilities. For example, antibodies against IL21, PDCD1 and ICOS are already in clinical trials for autoimmune disease or other malignancies and antibodies against CXCL13 are in pre-clinical development.

Safety of bendamustine for the treatment of indolent non-Hodgkin lymphoma: a UK real-world experience.

ABSTRACT: Bendamustine is among the most effective chemotherapeutics for indolent B-cell non-Hodgkin lymphomas (iNHL), but trial reports of significant toxicity, including opportunistic infections and excess deaths, led to prescriber warnings. We conducted a multicenter observational study evaluating bendamustine toxicity in real-world practice. Patients receiving at least 1 dose of bendamustine with/without rituximab (R) for iNHL were included. Demographics, lymphoma and treatment details, and grade 3 to 5 adverse events (AEs) were analyzed and correlated. In total, 323 patients were enrolled from 9 National Health Service hospitals. Most patients (96%) received bendamustine-R, and 46%, R maintenance. Overall, 21.7% experienced serious AEs (SAE) related to treatment, including infections in 12%, with absolute risk highest during induction (63%), maintenance (20%), and follow-up (17%) and the relative risk highest during maintenance (54%), induction (34%), and follow-up (28%). Toxicity led to permanent treatment discontinuation for 13% of patients, and 2.8% died of bendamustine-related infections (n = 5), myelodysplastic syndrome (n = 3), and cardiac disease (n = 1). More SAEs per patient were reported in patients with mantle cell lymphoma, poor preinduction performance status (PS), poor premaintenance PS, and abnormal preinduction total globulins and in those receiving growth factors. Use of antimicrobial prophylaxis was variable, and 3 of 10 opportunistic infections occurred despite prophylaxis. In this real-world analysis, bendamustine-related deaths and treatment discontinuation were similar to those of trial populations of younger, fitter patients. Poor PS, mantle cell histology, and maintenance R were potential risk factors. Infections, including late onset events, were the most common treatment-related SAE and cause of death, warranting extended antimicrobial prophylaxis and infectious surveillance, especially for maintenance-treated patients.

Enhancement of CD154/IL4 proliferation by the T follicular helper (Tfh) cytokine, IL21 and increased numbers of circulating cells resembling Tfh cells in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) cells encounter T-cells and proliferate in response to T-cell signals in the lymph node microenvironment. In this report we determined interleukin 21 (IL21) function in CLL and showed that IL21 and interleukin 4 (IL4) act co-operatively to promote leukaemic cell proliferation without apoptosis or differentiation We further show that IL21 increased side population (SP) cells, which are associated with resistance to chemotherapy and increased self-renewal capacity in CLL. IL21 and IL4 are the major cytokines produced by the recently described CD4(+) T follicular helper (Tfh) cell subset. Determination of Tfh cells in peripheral blood showed that patients had significantly increased numbers as compared to normal subjects although no association was found between Tfh numbers and IGHV gene mutational status or clinical stage. Our data suggests that the Tfh cytokines, IL4 and IL21, contribute to driving leukaemic cell proliferation in the lymph node microenvironment, and may contribute to the specific production of cells resistant to conventional chemotherapy. We suggest that increased circulating Tfh cells is a component of T-cell dysregulation in CLL. Our findings have implications for the therapeutic use of IL21.

Systemic ALCL Treated in Routine Clinical Practice: Outcomes Following First-Line Chemotherapy from a Multicentre Cohort.

INTRODUCTION: Brentuximab vedotin (BV)-CHP is the new standard regimen for first-line treatment of systemic anaplastic large cell lymphoma (sALCL). We undertook a retrospective analysis of consecutive patients diagnosed with sALCL, treated in routine practice, to serve as a benchmark analysis for comparison BV-CHP efficacy in routine practice. METHODS: Patients aged 16 years or older with sALCL treated in seven UK and Australian centres and from 14 additional centres from the UK Haematological Malignancy Research Network database (n = 214). Treatment allocation was clinician choice and included best supportive care (BSC). Main outcomes were time to treatment failure (TTF) and overall survival (OS). Multivariable analysis for predictors of both TTF and OS was also undertaken. RESULTS: The median age 52 years (range 16-93), 18% ECOG ≥ 3 and 40% of cases were ALK positive. CHOP (cyclophosphamide, adriamycin, vincristine, prednisolone) was employed in 152 (71%) of patients and CHOEP (CHOP + etoposide) in 4% of patients. For CHOP-treated patients overall response rate (ORR) was 65% and complete response (CR) 47%. Only 9% of patients underwent autologous stem cell transplant (ASCT). With 57 months median follow-up, 4-year TTF and OS were 41.2% (95% CI 33.1-49.1) and 58.9% (95% CI 50.3-66.5) respectively. Multivariable analysis showed ALK+ status was independently associated with superior TTF (HR 0.36, 95% CI 0.21-0.63) but not OS (0.44, 95% CI 0.18-1.07). DISCUSSION: We present a retrospective analysis with mature follow-up of one of the largest multicentre populations of sALCL available, comparable to similar large retrospective studies. ALK status remains a strong predictor of outcomes. CONCLUSION: These data serve as a robust benchmark for BV-CHP as the new standard of care for sALCL. Similar real-world evidence with BV-CHP will be desirable to confirm the findings of ECHELON-2.

Multiple mutations at exon 2 of RHOA detected in plasma from patients with peripheral T-cell lymphoma.

The mutational landscape of peripheral T-cell lymphoma (PTCL) is being revealed through sequencing of lymph node samples, but there has been little work on the mutational load that is present in cell-free DNA (cfDNA) from plasma. We report targeted sequencing of cfDNA from PTCL patients to demonstrate c.50G>T (p.Gly17Val) in RHOA as previously described in angioimmunoblastic T-cell lymphoma (AITL) and a group of PTCL not otherwise specified (NOS) but also detect novel mutations at c.73A>G (p.Phe25Leu) and c.48A>T (p.Cys16*) of exon 2, which were confirmed by Sanger sequencing. In a group of AITL and PTCL-NOS analyzed by droplet digital polymerase chain reaction, 63% (12/19) showed c.50G>T (p.Gly17Val), 53% (10/19) c.73A>G (p.Phe25Leu), and 37% (7/19) c.48A>T (pCys16*). Sequencing of lymph node tissue in 3 out of 9 cases confirmed the presence of c.73A>G (p.Phe25Leu). Inspection of individual sequencing reads from individual patients showed that a single RHOA allele could contain >1 mutation, suggesting haplotypes of mutations at RHOA. Serial sampling showed changes to RHOA mutational frequency with treatment and the apparent occurrence of clones bearing specific haplotypes associated with relapse. Therefore, sequencing of RHOA from cfDNA has revealed new mutations and haplotypes. The clinical significance of these findings will need to be explored in clinical trials, but liquid biopsy might have potential for guiding treatment decisions in PTCL.

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry.

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin's lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

Structural and diffusion weighted MRI demonstrates responses to ibrutinib in a mouse model of follicular helper (Tfh) T-cell lymphoma.

Recent analyses of the genetics of peripheral T-cell lymphoma (PTCL) have shown that a large proportion of cases are derived from normal follicular helper (Tfh) T-cells. The sanroque mouse strain bears a mutation that increases Tfh cell number and heterozygous animals (Roquinsan/+) develop lymphomas similar to human Tfh lymphoma. Here we demonstrate the usefulness of Roquinsan/+ animals as a pre-clinical model of Tfh lymphoma. Long latency of development and incomplete penetrance in this strain suggests the lymphomas are genetically diverse. We carried out preliminary genetic characterisation by whole exome sequencing and detected tumor specific mutations in Hsp90ab1, Ccnb3 and RhoA. Interleukin-2-inducible kinase (ITK) is expressed in Tfh lymphoma and is a potential therapeutic agent. A preclinical study of ibrutinib, a small molecule inhibitor of mouse and human ITK, in established lymphoma was carried out and showed lymphoma regression in 8/12 (67%) mice. Using T2-weighted MRI to assess lymph node volume and diffusion weighted MRI scanning as a measure of function, we showed that treatment increased mean apparent diffusion coefficient (ADC) suggesting cell death, and that change in ADC following treatment correlated with change in lymphoma volume. We suggest that heterozygous sanroque mice are a useful model of Tfh cell derived lymphomas in an immunocompetent animal.

Comparison of interleukin-2-inducible kinase (ITK) inhibitors and potential for combination therapies for T-cell lymphoma.

Patients with peripheral T-cell lymphomas generally have poor clinical outcomes with conventional chemotherapy. Recent advances have demonstrated that a large subgroup of PTCL are derived from follicular helper (Tfh) T-cells. These cases show a characteristic pattern of gene expression, which includes high-level protein expression of interleukin-2-inducible kinase (ITK). ITK is a member of the TEC family of kinases and normally has essential functions in regulating T-cell receptor signalling and T-cell differentiation. Here we report a side-by-side comparison of four ITK inhibitors. We investigate effects on apoptosis, phosphorylation of signaling molecules, calcium flux and migration. In line with a specific mechanism of action ONO7790500 and BMS509744 did not inhibit MEK1/2 or AKT phosphorylation although other ITK inhibitors, ibrutinib and PF-06465469, did have this effect. Specific ITKi had modest effects on apoptosis alone but there was definite synergy with doxorubicin, pictilisib (PI3Ki) and idelalisib (PI3Kδi). ITKi repressed migration of Jurkat cells caused by CXCL12 and the CXCR4 antagonist, plerixafor enhanced this effect. Overall ITKi may have several mechanisms of action that will be therapeutically useful in PTCL including reduction in survival and perturbation of trafficking.

Circulating Tfh1 (cTfh1) cell numbers and PD1 expression are elevated in low-grade B-cell non-Hodgkin's lymphoma and cTfh gene expression is perturbed in marginal zone lymphoma.

CD4+ T-cell subsets are found in the tumour microenvironment (TME) of low-grade B-cell non-Hodgkin's lymphomas such as marginal zone lymphoma (MZL) or follicular lymphoma (FL). Both numbers and architecture of activating follicular helper T-cells (Tfh) and suppressive Treg in the TME of FL are associated with clinical outcomes. There has been almost no previous work on CD4+ T-cells in MZL. It is now recognised that circulating CD4+CXCR5+ T-cells are the memory compartment of Tfh cells. We determined differences in number of circulating Tfh (cTfh) cells and cTfh subsets between normal subjects and patients with FL or MZL. Lymphoma patients showed increased numbers of cTfh1 and reduced cTfh17 cells due to decreased expression of the subset-defining marker CCR6 in patients. PD1, a surface marker associated with Tfh cells, showed increased expression on cTfh subsets in patients. Focusing on MZL we determined expression of 96 T-cell associated genes by microfluidic qRT-PCR. Analysis of differentially expressed genes showed significant differences between normal subjects and patients both for bulk cTfh (CCL4) and the cTfh1 subset (JAK3). While our findings require confirmation in larger studies we suggest that analysis of number and gene expression of circulating T-cells might be a source of clinically useful information as is the case for T-cells within lymphoma lymph nodes.

PD1hi cells associate with clusters of proliferating B-cells in marginal zone lymphoma.

BACKGROUND: Abnormally sustained immune reactions drive B-cell proliferation in some cases of marginal zone lymphoma but the CD4+ T-cell subsets, which are likely to contribute to the B-cell responses in the tumour microenvironment, are not well characterised and neither has the spatial distribution of the different subsets in involved lymph nodes been investigated. METHODS: Employing a workflow of multiplex semi-automated immunohistochemistry combined with image processing we investigated association between infiltrating T-cells and proliferating lymphoma B-cells. RESULTS: Both total numbers of activating follicular helper (Tfh) cells (defined by high expression of PD1) and suppressive regulatory (Treg) T-cells (defined by FOXP3+ expression) and the Tfh:Treg ratio, assessed over relatively large areas of tissue, varied among cases of marginal zone lymphoma. We determined spatial distribution and demonstrated that PD1hi cells showed significantly more clustering than did FOXP3+. To investigate the association of infiltrating T-cells with lymphoma B-cells we employed Pearson correlation and Morisita-Horn index, statistical measures of interaction. We demonstrated that PD1hi cells were associated with proliferating B-cells and confirmed this by nearest neighbour analysis. CONCLUSIONS: The unexpected architectural complexity of T-cell infiltration in marginal zone lymphoma, revealed in this study, further supports a key role for Tfh cells in driving proliferation of lymphoma B-cells. We demonstrate the feasibility of digital analysis of spatial architecture of T-cells within marginal zone lymphoma and future studies will be needed to determine the clinical importance of these observations.

Expression of PD-1 (CD279) and FoxP3 in diffuse large B-cell lymphoma.

The role of the microenvironment in high-grade lymphoma is not well defined. In this report, we employ immunohistochemistry to characterise programmed death-1 (PD-1/CD279) and FoxP3 expression in 70 cases of diffuse large B-cell lymphoma (DLBCL). PD-1 is a surface marker characteristic of follicular helper T-cells whilst FoxP3 is characteristic of Tregs. We demonstrate variable infiltration with CD4(+) T-cells (<10 to >50 % of all lymph node cells) and PD-1(hi) cells (0.1 to 1.5 % of all cells). CD4(+) T-cells can be distributed in clusters or more diffusely and PD-1(hi) cells, but not FoxP3(+) cells, are found in rosettes around lymphoma cells. Cases with high CD4(+) T-cell numbers tended to have higher numbers of both PD-1(hi) and FoxP3(+) cells. Cases with total CD4(+) T-cell, PD-1(hi) and FoxP3(+) numbers above the median associate with better clinical outcome. Overall, we demonstrate that infiltration by CD4(+) T-cells, including both FoxP3(+) and PD-1(hi) subsets, correlates with prognosis in DLBCL. In distinction to previous reported series, patients (91 %) were treated with rituximab-containing regimens, suggesting that the effects of CD4+ T-cell infiltration are maintained in the rituximab era. This work suggests that determinants of total CD4(+) T-cell infiltration, either molecular characteristics of the lymphoma or the patients' immune system, and not individual T-cell subsets, correlate with clinical outcome.