Targeting cytokine- and therapy-induced PIM1 activation in preclinical models of T-cell acute lymphoblastic leukemia and lymphoma.

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell acute lymphoblastic lymphoma (T-LBL) are aggressive hematological malignancies that are currently treated with high-dose chemotherapy. Over the last several years, the search toward novel and less-toxic therapeutic strategies for T-ALL/T-LBL patients has largely focused on the identification of cell-intrinsic properties of the tumor cell. However, non-cell-autonomous activation of specific oncogenic pathways might also offer opportunities that could be exploited at the therapeutic level. In line with this, we here show that endogenous interleukin 7 (IL7) can increase the expression of the oncogenic kinase proviral integration site for Moloney-murine leukemia 1 (PIM1) in CD127+ T-ALL/T-LBL, thereby rendering these tumor cells sensitive to in vivo PIM inhibition. In addition, using different CD127+ T-ALL/T-LBL xenograft models, we also reveal that residual tumor cells, which remain present after short-term in vivo chemotherapy, display consistent upregulation of PIM1 as compared with bulk nontreated tumor cells. Notably, this effect was transient as increased PIM1 levels were not observed in reestablished disease after abrogation of the initial chemotherapy. Furthermore, we uncover that this phenomenon is, at least in part, mediated by the ability of glucocorticoids to cause transcriptional upregulation of IL7RA in T-ALL/T-LBL patient-derived xenograft (PDX) cells, ultimately resulting in non-cell-autonomous PIM1 upregulation by endogenous IL7. Finally, we confirm in vivo that chemotherapy in combination with a pan-PIM inhibitor can improve leukemia survival in a PDX model of CD127+ T-ALL. Altogether, our work reveals that IL7 and glucocorticoids coordinately drive aberrant activation of PIM1 and suggests that IL7-responsive CD127+ T-ALL and T-LBL patients could benefit from PIM inhibition during induction chemotherapy.

Richter transformation driven by Epstein-Barr virus reactivation during therapy-related immunosuppression in chronic lymphocytic leukaemia.

The increased risk of Richter transformation (RT) in patients with chronic lymphocytic leukaemia (CLL) due to Epstein-Barr virus (EBV) reactivation during immunosuppressive therapy with fludarabine other targeted agents remains controversial. Among 31 RT cases classified as diffuse large B-cell lymphoma (DLBCL), seven (23%) showed EBV expression. In contrast to EBV- tumours, EBV+ DLBCLs derived predominantly from IGVH-hypermutated CLL, and they also showed CLL-unrelated IGVH sequences more frequently. Intriguingly, despite having different cellular origins, clonally related and unrelated EBV+ DLBCLs shared a previous history of immunosuppressive chemo-immunotherapy, a non-germinal centre DLBCL phenotype, EBV latency programme type II or III, and very short survival. These data suggested that EBV reactivation during therapy-related immunosuppression can transform either CLL cells or non-tumoural B lymphocytes into EBV+ DLBCL. To investigate this hypothesis, xenogeneic transplantation of blood cells from 31 patients with CLL and monoclonal B-cell lymphocytosis (MBL) was performed in Rag2-/- IL2γc-/- mice. Remarkably, the recipients' impaired immunosurveillance favoured the spontaneous outgrowth of EBV+ B-cell clones from 95% of CLL and 64% of MBL patients samples, but not from healthy donors. Eventually, these cells generated monoclonal tumours (mostly CLL-unrelated but also CLL-related), recapitulating the principal features of EBV+ DLBCL in patients. Accordingly, clonally related and unrelated EBV+ DLBCL xenografts showed indistinguishable cellular, virological and molecular features, and synergistically responded to combined inhibition of EBV replication with ganciclovir and B-cell receptor signalling with ibrutinib in vivo. Our study underscores the risk of RT driven by EBV in CLL patients receiving immunosuppressive therapies, and provides the scientific rationale for testing ganciclovir and ibrutinib in EBV+ DLBCL. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Identification of distinct subgroups of EBV-positive post-transplant diffuse large B-cell lymphoma.

Post-transplantation lymphoproliferative disorder is an aggressive complication of transplantation, most frequently of diffuse large B-cell lymphoma morphology and associated with Epstein-Barr virus (EBV) infection/reactivation. In this study the microenvironment of EBV+ (n=23) and EBV- (n=9) post-transplant non-germinal center B-cell diffuse large B-cell lymphoma was characterized. Of EBV+ cases somatic hypermutation analysis, gene expression profiling, and extensive phenotyping were performed. Our results demonstrated variable cytotoxic T-cell infiltration and significantly increased CD163+ M2 macrophage infiltration in EBV+ compared with EBV- post-transplant diffuse large B-cell lymphoma. On the basis of IgM staining and hypermutation analysis, two EBV+ post-transplant diffuse large B-cell lymphoma subgroups were identified: IgM+ tumors lacking somatic hypermutations and IgM- tumors harboring somatic hypermutations. IgM- tumors arose late following transplantation (median interval: 16 months), mainly in kidney recipients. IgM+ tumors on the other hand arose early (median interval: 3 months, P-value=0.0032), almost exclusively following stem cell transplantation and were associated with worse outcome (median survival 1 month for IgM+ versus 41 months for IgM- tumors, log-rank/Wilcoxon P-value 0.07/0.04). Notably, IgM+ tumors were characterized by plasma cell features (monotypic kappa/lambda expression, high MUM1 expression, and partial CD138 expression) and a high proliferation index. Consistent with the plasma cell phenotype, unfolded protein response signaling was upregulated. In contrast, IgM- EBV+ post-transplant diffuse large B-cell lymphoma did not express kappa, lambda, IgD, or CD138 and expressed limited MUM1. In these tumors T-cell signaling was enhanced associated with increased T-cell infiltration compared with IgM+ cases. Overall, our results allow further molecular classification of EBV+ post-transplant diffuse large B-cell lymphoma and provide a rationale for the use of subtype-specific-targeted therapies (eg, bortezomib in IgM+ tumors). Our findings also provide a biological basis for the clinical differences between post-transplant lymphoproliferative disorder following solid organ and stem cell transplantation, which are regarded as different disorders.

The accuracy of positron emission tomography in the detection of posttransplant lymphoproliferative disorder.

We investigated sensitivity, specificity, positive predictive value, negative predictive value and accuracy of 18F-fluorodeoxyglucose-positron emission tomography in 170 cases with suspected or biopsy-proven posttransplant lymphoproliferative disorder. All solid organ and hematopoietic stem cell transplant recipients who underwent an 18F-fluorodeoxyglucose-positron emission tomography scan between 2003 and 2010 in our center for the indication posttransplant lymphoproliferative disorder, were retrospectively reviewed and results were compared with tissue biopsy whenever possible. One hundred and seventy positron emission tomography scans in 150 patients were eligible for evaluation. In 45 cases, the patient had a biopsy-confirmed posttransplant lymphoproliferative disorder before positron emission tomography scanning and positron emission tomography was performed for staging purposes. In the remaining 125 cases, positron emission tomography was performed to differentiate between posttransplant lymphoproliferative disorder and other diseases. 18F-fluorodeoxyglucose-uptake was quantitatively expressed by calculation of maximum and mean standardized uptake value in the most intense lesion or, in the absence of attenuation corrected positron emission tomography scans, by comparing uptake in target lesion to liver and mediastinal uptake. We found an overall sensitivity of 89%, specificity of 89%, positive predictive value of 91% and negative predictive value of 87% for posttransplant lymphoproliferative disorder detection by 18F-fluorodeoxyglucose-positron emission tomography. In a subanalysis of the 125 scans performed for differentiating posttransplant lymphoproliferative disorder from other diseases, sensitivity, specificity, positive predictive value and negative predictive value were 90%, 89%, 85% and 93%, respectively. 18F-fluorodeoxyglucose-uptake in posttransplant lymphoproliferative disorder was generally high with a median mean and maximum standardized uptake value of 9.0 (range 2.0-18.6) and 17.4 (range 2.6-26.4). Posttransplant lymphoproliferative disorder often had an atypical presentation on positron emission tomography with high incidence of extranodal involvement. In conclusion, from these data, we can conclude that 18F-fluorodeoxyglucose-positron emission tomography is highly sensitive for detecting posttransplant lymphoproliferative disorder and has an excellent ability to differentiate posttransplant lymphoproliferative disorder from non-malignant diseases.

The spleen as a sanctuary site for residual leukemic cells following ABT-199 monotherapy in ETP-ALL.

B-cell lymphoma 2 (BCL-2) has recently emerged as a therapeutic target for early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL), a high-risk subtype of human T-cell ALL. The major clinical challenge with targeted therapeutics, such as the BCL-2 inhibitor ABT-199, is the development of acquired resistance. We assessed the in vivo response of luciferase-positive LOUCY cells to ABT-199 monotherapy and observed specific residual disease in the splenic microenvironment. Of note, these results were confirmed by using a primary ETP-ALL patient-derived xenograft. Splenomegaly has previously been associated with poor prognosis in diverse types of leukemia. However, the exact mechanism by which the splenic microenvironment alters responses to specific targeted therapies remains largely unexplored. We show that residual LOUCY cells isolated from the spleen microenvironment displayed reduced BCL-2 dependence, which was accompanied by decreased BCL-2 expression levels. Notably, this phenotype of reduced BCL-2 dependence could be recapitulated by using human splenic fibroblast coculture experiments and was confirmed in an in vitro chronic ABT-199 resistance model of LOUCY. Finally, single-cell RNA-sequencing was used to show that ABT-199 triggers transcriptional changes in T-cell differentiation genes in leukemic cells obtained from the spleen microenvironment. Of note, increased expression of CD1a and sCD3 was also observed in ABT199-resistant LOUCY clones, further reinforcing the idea that a more differentiated leukemic population might display decreased sensitivity toward BCL-2 inhibition. Overall, our data reveal the spleen as a site of residual disease for ABT-199 treatment in ETP-ALL and provide evidence for plasticity in T-cell differentiation as a mechanism of therapy resistance.

Cyclin D2 overexpression drives B1a-derived MCL-like lymphoma in mice.

Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma with poor long-term overall survival. Currently, MCL research and development of potential cures is hampered by the lack of good in vivo models. MCL is characterized by recurrent translocations of CCND1 or CCND2, resulting in overexpression of the cell cycle regulators cyclin D1 or D2, respectively. Here, we show, for the first time, that hematopoiesis-specific activation of cyclin D2 is sufficient to drive murine MCL-like lymphoma development. Furthermore, we demonstrate that cyclin D2 overexpression can synergize with loss of p53 to form aggressive and transplantable MCL-like lymphomas. Strikingly, cyclin D2-driven lymphomas display transcriptional, immunophenotypic, and functional similarities with B1a B cells. These MCL-like lymphomas have B1a-specific B cell receptors (BCRs), show elevated BCR and NF-κB pathway activation, and display increased MALT1 protease activity. Finally, we provide preclinical evidence that inhibition of MALT1 protease activity, which is essential for the development of early life-derived B1a cells, can be an effective therapeutic strategy to treat MCL.

Targeting steroid resistance in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is characterized by a variable response to steroids during induction and/or consolidation therapy. Notably, recent work suggested that these differences in glucocorticoid sensitivity might, at least in part, be mediated by hyperactivation of specific oncogenic pathways such as RAS/MEK/ERK, PI3K/AKT and IL7R/JAK/STAT. In this review, we elaborate on putative associations between aberrant signaling, therapy resistance, incidence of relapse and clinical outcome in human T-ALL. Furthermore, we emphasize that this potential association with clinical parameters might also be mediated by the tumor microenvironment as a result of increased sensitivity of leukemic T-cells towards cytokine induced signaling pathway activation. With this in mind, we provide an overview of small molecule inhibitors that might have clinical potential for the treatment of human T-ALL in the near future as a result of their ability to overcome steroid resistance thereby potentially increasing survival rates in this aggressive hematological neoplasm.

Correction to: Secondary B-cell lymphoma associated with the Epstein-Barr virus in chronic lymphocytic leukemia patients.

[This corrects the article DOI: 10.1007/s12308-016-0273-8.].

Recent insights in the pathogenesis of post-transplantation lymphoproliferative disorders.

Post-transplant lymphoproliferative disorder (PTLD) is an aggressive complication of solid organ and hematopoietic stem cell transplantation that arises in up to 20% of transplant recipients. Infection or reactivation of the Epstein-Barr virus (EBV), a ubiquitous human herpesvirus, in combination with chronic immunosuppression are considered as the main predisposing factors, however insight in PTLD biology is fragmentary. The study of PTLD is complicated by its morphological heterogeneity and the lack of prospective trials, which also impede treatment optimization. Furthermore, the broad spectrum of underlying disorders and the graft type represent important confounding factors. PTLD encompasses different malignant subtypes that resemble histologically similar lymphomas in the general population. Post-transplant diffuse large B-cell lymphoma (PT-DLBCL), Burkitt lymphoma (PT-BL) and plasmablastic lymphoma (PT-PBL) occur most frequently. However, in many studies various EBV(+) and EBV(-) PTLD subtypes are pooled, complicating the interpretation of the results. In this review, studies of the gene expression pattern, the microenvironment and the genetic profile of PT-DLBCL, PT-BL and PT-PBL are summarized to better understand the mechanisms underlying post-transplantation lymphomagenesis. Based on the available findings we propose stratification of PTLD according to the histological subtype and the EBV status to facilitate the interpretation of future studies and the establishment of clinical trials.

Detection of bone marrow involvement in newly diagnosed post-transplant lymphoproliferative disorder: (18)F-fluorodeoxyglucose positron emission tomography/computed tomography versus bone marrow biopsy.

Detecting bone marrow involvement (BMI) in lymphoma is important as it adversely affects stage. Bone marrow biopsy (BMB) remains the standard to detect BMI but is prone to sampling error. We retrospectively investigated whether (18)F-fluorodeoxyglucose positron emission tomography with computed tomography ((18)F-FDG-PET/CT) could identify BMI in patients with post-transplant lymphoproliferative disorder (PTLD) with sufficient accuracy in comparison with staging BMB. Twenty-five patients diagnosed with PTLD who underwent (18)F-FDG-PET/CT and BMB within one month were evaluated. Based on our criteria, six patients (24%) were considered positive for BMI on (18)F-FDG-PET/CT compared to one by BMB. Although we cannot completely exclude false positive results on (18)F-FDG-PET/CT, our data indicate a significantly higher sensitivity of (18)F-FDG-PET/CT compared to BMB (100% vs 17%) but similar specificity. These data confirm the high diagnostic performance of (18)F-FDG-PET/CT for detecting BMI, but prospective studies are needed to determine whether (18)F-FDG-PET/CT could indeed replace staging BMB in PTLD.

Genetic landscape of T- and NK-cell post-transplant lymphoproliferative disorders.

Post-transplant lymphoproliferative disorders of T- or NK-cell origin (T/NK-PTLD) are rare entities and their genetic basis is unclear. We performed targeted sequencing of 465 cancer-related genes and high-resolution copy number analysis in 17 T-PTLD and 2 NK-PTLD cases. Overall, 377 variants were detected, with an average of 20 variants per case. Mutations of epigenetic modifier genes (TET2, KMT2C, KMT2D, DNMT3A, ARID1B, ARID2, KDM6B, n=11). and inactivation of TP53 by mutation and/or deletion(n=6) were the most frequent alterations, seen across disease subtypes, followed by mutations of JAK/STAT pathway genes (n=5). Novel variants, including mutations in TBX3 (n=3), MED12 (n=3) and MTOR (n=1), were observed as well. High-level microsatellite instability was seen in 1 of 14 (7%) cases, which had a heterozygous PMS2 mutation. Complex copy number changes were detected in 8 of 16 (50%) cases and disease subtype-specific aberrations were also identified. In contrast to B-cell PTLDs, the molecular and genomic alterations observed in T/NK-PTLD appear similar to those reported for peripheral T-cell lymphomas occurring in immunocompetent hosts, which may suggest common genetic mechanisms of lymphoma development.

Secondary B-cell lymphoma associated with the Epstein-Barr virus in chronic lymphocytic leukemia patients.

Up to 10 % of chronic lymphocytic leukemia (CLL) patients present with aggressive secondary B-cell lymphoma (most frequently diffuse large B-cell lymphoma, DLBCL) which may be clonally related to the CLL (i.e., Richter transformation, RT, 80 % of the cases) or de novo (20 % of the cases). Several genetic lesions associated with RT have already been identified, but the potential role of the Epstein-Barr virus (EBV) has been largely overlooked. In this study, we describe six CLL patients who developed a secondary EBV-positive (EBV+) B-cell lymphoma (five DLBCL, one Hodgkin lymphoma) and compare their clinicopathological characteristics to ten CLL patients with EBV-negative (EBV-) secondary B-cell lymphomas (all DLBCL). All 16 patients had a history of iatrogenic immunosuppression or chemotherapy. Eighty percent had received fludarabine as part of the CLL treatment. Most secondary lymphomas were clonally related to the previous CLL (3/4 EBV+, 7/7 EBV- cases tested). Notably EBV+ RT was associated with a trend for older age at onset (median 72 vs. 63 years, p value >0.05), longer interval between CLL and RT diagnosis (median 4.2 vs. 2.9 years, p value >0.05), and shorter overall survival (median 4 vs. 10 months, p value >0.05). These differences were not significant, probably due to small sample size. Immunohistochemical profiling suggested more frequent overexpression of TP53 and MYC in EBV- compared to EBV+ secondary lymphoma. Based on this small retrospective single center series, we hypothesize that EBV+ RT may constitute a separate subgroup of RT. Larger series are required to validate this suggestion.

Clinicopathologic comparison of plasmablastic lymphoma in HIV-positive, immunocompetent, and posttransplant patients: single-center series of 25 cases and meta-analysis of 277 reported cases.

Plasmablastic lymphoma (PBL) is a rare B-cell non-Hodgkin lymphoma often associated with Epstein-Barr virus (EBV) infection. To gain insight in this aggressive lymphoma subtype, the clinicopathologic characteristics of 25 unpublished single-center PBLs (2 in acquired immunodeficiency syndrome patients, 11 in immunocompetent individuals [IC-PBL], 12 in transplant recipients [PT-PBL]) and of 277 reported PBLs were summarized. In the reported series, PBL patients were predominantly male (77%) with a median age at diagnosis of 46 years (range, 1.2 to 87 y). The majority of the biopsies (66%) was EBV positive. Extranodal presentation was most frequent (88%, of which 35% were oral, 18% gastrointestinal, 12% cutaneous). PBL was diagnosed in acquired immunodeficiency syndrome patients (50%), immunocompetent individuals (35%), and transplant recipients (14%). These subgroups differed in age at diagnosis (median: 41, 64, 47 y, respectively), primary localization (oral, oral, cutaneous, respectively), EBV positivity (75%, 50%, 67%, respectively), CD45 expression (31%, 33%, 70%, respectively), and C-MYC aberrations (78%, 44%, 38%, respectively). Ann Arbor stage I, EBV positivity, CD45 expression, and lack of C-MYC aberrations were associated with better outcome (P<0.05). Our series of IC-PBL and PT-PBL cases revealed differential expression of CD10 (0% vs. 42%, respectively), CD56 (22% vs. 42%, respectively), TP53 (67% vs. 8%, respectively), and BCL2 (88% vs. 25%, respectively). Gene expression analysis of 5 of our PT-PBLs revealed upregulation of DNMT3B, PTP4A3, and CD320 in EBV-positive PT-PBL and suggested a role for cancer/testis antigens. The results of this retrospective study suggest different pathogenic mechanisms of PBL in different immunologic settings and a potentially important impact of EBV and CD45 on prognosis.

Clinicopathological characteristics of posttransplant lymphoproliferative disorders of T-cell origin: single-center series of nine cases and meta-analysis of 147 reported cases.

T-cell or natural killer (NK)-cell posttransplant lymphoproliferative disorder (T-PTLD) is a rare but severe complication after transplant. Here we present the clinicopathological features of a single-center series of nine cases. Additionally, we summarize the clinicopathological findings of 147 cases of T/NK-cell PTLD reported in the literature in an attempt to define subtype-specific characteristics. T/NK-cell PTLD occurs in patients of all ages, usually extranodally, and most frequently after kidney transplant. Organ specific incidence, however, is highest following heart transplant. Approximately one-third of T-cell PTLDs are Epstein-Barr virus (EBV)-related, with peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) being the most prevalent EBV-associated T-cell PTLD. A male predominance is observed, which is most striking in the EBV(+) group, particularly in PTCL, NOS. With a median posttransplant interval of 72 months, T-cell PTLDs are among the late-occurring PTLDs. Of the most common T-cell PTLDs, anaplastic large cell lymphoma (ALCL) has the best prognosis, whereas PTCL, NOS and hepatosplenic T-cell lymphoma (HSTCL) have the worst prognosis. EBV(+) cases seem to have a longer survival than EBV(-) cases, suggesting a different pathogenetic mechanism.

Single-center analysis of biopsy-confirmed posttransplant lymphoproliferative disorder: incidence, clinicopathological characteristics and prognostic factors.

Hematopoietic stem cell and solid organ transplant recipients diagnosed with biopsy-confirmed posttransplant lymphoproliferative disorder (PTLD) at our institution from 1989 to 2010 were identified. Patient-, transplant- and disease-related characteristics, prognostic factors and outcome were collected and analyzed. One hundred and forty biopsy-proven cases of PTLD were included. Overall incidence in the transplant population was 2.12%, with heart transplant recipients carrying the highest risk. Most PTLDs were monomorphic (82%), with diffuse large B-cell lymphoma being the most frequent subtype. The majority of cases (70.7%) occurred > 1 year posttransplant, and 66% were Epstein-Barr virus positive. Following initial therapy the overall response rate was 68.5%. Three-year relapse-free and overall survivals were 59% and 49%, respectively. At last follow-up, 44% of the patients were alive. Multivariable analysis identified several classical lymphoma-specific poor prognostic factors for the different outcome measures. The value of the International Prognostic Index was confirmed in our analysis.