Maria Gomes da Silva

Differentiation of human peripheral blood Vδ1+ T cells expressing the natural cytotoxicity receptor NKp30 for recognition of lymphoid leukemia cells

The success of cancer immunotherapy depends on productive tumor cell recognition by killer lymphocytes. γδ T cells are a population of innate-like lymphocytes endowed with strong, MHC-unrestricted cytotoxicity against tumor cells. This notwithstanding, we recently showed that a large proportion of human hematologic tumors is resistant to γδ peripheral blood lymphocytes (PBLs) activated with specific agonists to the highly prevalent Vγ9Vδ2 TCR. Although this probably constitutes an important limitation to current γδ T cell-mediated immunotherapy strategies, we describe here the differentiation of a novel subset of Vδ2(-) Vδ1(+) PBLs expressing natural cytotoxicity receptors (NCRs) that directly mediate killing of leukemia cell lines and chronic lymphocytic leukemia patient neoplastic cells. We show that Vδ1(+) T cells can be selectively induced to express NKp30, NKp44 and NKp46, through a process that requires functional phosphatidylinositol 3-kinase (PI-3K)/AKT signaling on stimulation with γ(c) cytokines and TCR agonists. The stable expression of NCRs is associated with high levels of granzyme B and enhanced cytotoxicity against lymphoid leukemia cells. Specific gain-of-function and loss-of-function experiments demonstrated that NKp30 makes the most important contribution to TCR-independent leukemia cell recognition. Thus, NKp30(+) Vδ1(+) T cells constitute a novel, inducible and specialized killer lymphocyte population with high potential for immunotherapy of human cancer.

Poor Concordance among Nine Immunohistochemistry Classifiers of Cell-of-Origin for Diffuse Large B-Cell Lymphoma: Implications for Therapeutic Strategies

PURPOSE The opportunity to improve therapeutic choices on the basis of molecular features of the tumor cells is on the horizon in diffuse large B-cell lymphoma (DLBCL). Agents such as bortezomib exhibit selective activity against the poor outcome activated B-cell type (ABC) DLBCL. In order for targeted therapies to succeed in this disease, robust strategies that segregate patients into molecular groups with high reliability are needed. Although molecular studies are considered gold standard, several immunohistochemistry (IHC) algorithms have been published that claim to be able to stratify patients according to their cell-of-origin and to be relevant for patient outcome. However, results are poorly reproducible by independent groups. EXPERIMENTAL DESIGN We investigated nine IHC algorithms for molecular classification in a dataset of DLBCL diagnostic biopsies, incorporating immunostaining for CD10, BCL6, BCL2, MUM1, FOXP1, GCET1, and LMO2. IHC profiles were assessed and agreed among three expert observers. A consensus matrix based on all scoring combinations and the number of subjects for each combination allowed us to assess reliability. The survival impact of individual markers and classifiers was evaluated using Kaplan-Meier curves and the log-rank test. RESULTS The concordance in patients classification across the different algorithms was low. Only 4% of the tumors have been classified as germinal center B-cell type (GCB) and 21% as ABC/non-GCB by all methods. None of the algorithms provided prognostic information in the R-CHOP (rituximab plus cyclophosphamide-adriamycin-vincristine-prednisone)-treated cohort. CONCLUSION Further work is required to standardize IHC algorithms for DLBCL cell-of-origin classification for these to be considered reliable alternatives to molecular-based methods to be used for clinical decisions.

Identification of a panel of ten cell surface protein antigens associated with immunotargeting of leukemias and lymphomas by peripheral blood γδ T cells

Background Vγ9Vδ2 T lymphocytes are regarded as promising mediators of cancer immunotherapy due to their capacity to eliminate multiple experimental tumors, particularly within those of hematopoietic origin. However, Vγ9Vδ2 T-cell based lymphoma clinical trials have suffered from the lack of biomarkers that can be used as prognostic of therapeutic success. Design and Methods We have conducted a comprehensive study of gene expression in acute lymphoblastic leukemias and non-Hodgkin’s lymphomas, aimed at identifying markers of susceptibility versus resistance to Vγ9Vδ2 T cell-mediated cytotoxicity. We employed cDNA microarrays and quantitative real-time PCR to screen 20 leukemia and lymphoma cell lines, and 23 primary hematopoietic tumor samples. These data were analyzed using state-of-the-art bioinformatics, and gene expression patterns were correlated with susceptibility to Vγ9Vδ2 T cell mediated cytolysis in vitro. Results We identified a panel of 10 genes encoding cell surface proteins that were statistically differentially expressed between “γδ-susceptible” and “γδ-resistant” hematopoietic tumors. Within this panel, 3 genes (ULBP1, TFR2 and IFITM1) were associated with increased susceptibility to Vγ9Vδ2 T-cell cytotoxicity, whereas the other 7 (CLEC2D, NRP2, SELL, PKD2, KCNK12, ITGA6 and SLAMF1) were enriched in resistant tumors. Furthermore, some of these candidates displayed a striking variance of expression among primary follicular lymphomas and T-cell acute lymphoblastic leukemias. Conclusions Our results suggest that hematopoietic tumors display a highly variable repertoire of surface proteins that can impact on Vγ9Vδ2 cell-mediated immunotargeting. The prognostic value of the proposed markers can now be evaluated in upcoming Vγ9Vδ2 T cell-based lymphoma/leukemia clinical trials.

Activity of the clinical-stage CK2-specific inhibitor CX-4945 against chronic lymphocytic leukemia

Activity of the clinical-stage CK2-specific inhibitor CX-4945 against chronic lymphocytic leukemia

Lenalidomide Maintenance Compared With Placebo in Responding Elderly Patients With Diffuse Large B-Cell Lymphoma Treated With First-Line Rituximab Plus Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone

Purpose The standard treatment of patients with diffuse large B-cell lymphoma (DLBCL) is rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Lenalidomide, an immunomodulatory agent, has shown activity in DLBCL. This randomized phase III trial compared lenalidomide as maintenance therapy with placebo in elderly patients with DLBCL who achieved a complete response (CR) or partial response (PR) to R-CHOP induction. Methods Patients with previously untreated DLBCL or other aggressive B-cell lymphoma were 60 to 80 years old, had CR or PR after six or eight cycles of R-CHOP, and were randomly assigned to lenalidomide maintenance 25 mg/d or placebo for 21 days of every 28-day cycle for 24 months. The primary end point was progression-free survival (PFS). Results A total of 650 patients were randomly assigned. At the time of the primary analysis (December 2015), with a median follow-up of 39 months from random assignment, median PFS was not reached for lenalidomide maintenance versus 58.9 months for placebo (hazard ratio, 0.708; 95% CI, 0.537 to 0.933; P = .01). The result was consistent among analyzed subgroups (eg, male v female, age-adjusted International Prognostic Index 0 or 1 v 2 or 3, age younger than 70 v ≥ 70 years), response (PR v CR) after R-CHOP, and positron emission tomography status at assignment (negative v positive). With longer median follow-up of 52 months (October 2016), overall survival was similar between arms (hazard ratio, 1.218; 95% CI, 0.861 to 1.721; P = .26). Most common grade 3 or 4 adverse events associated with lenalidomide versus placebo maintenance were neutropenia (56% v 22%) and cutaneous reactions (5% v 1%), respectively. Conclusion Lenalidomide maintenance for 24 months after obtaining a CR or PR to R-CHOP significantly prolonged PFS in elderly patients with DLBCL.

Therapeutic options in relapsed or refractory peripheral T-cell lymphoma

Peripheral T-cell lymphoma (PTCL) represents a relatively rare group of heterogeneous non-Hodgkin lymphomas with a very poor prognosis. Current therapies, based on historical regimens for aggressive B-cell lymphomas, have resulted in insufficient patient outcomes. The majority of patients relapse rapidly, and current 5-year overall survival rates are only 10-30%. It is evident that new approaches to treat patients with PTCL are required. In recent years, prospective studies in PTCL have been initiated, mainly in patients with relapsed/refractory disease. In some of these, selected histologic subtypes have been evaluated in detail. As a consequence, numerous new therapies have been developed and shown activity in PTCL, including: agents targeting the immune system (e.g. brentuximab vedotin, alemtuzumab, lenalidomide); histone deacetylase inhibitors (romidepsin, belinostat); antifolates (pralatrexate); fusion proteins (denileukin diftitox); nucleoside analogs (pentostatin, gemcitabine); and other agents (e.g. alisertib, plitidepsin, bendamustine, bortezomib). A variety of interesting novel combinations is also emerging. It is hoped that these innovative approaches, coupled with a greater understanding of the clinicopathologic features, pathogenesis, molecular biology, and natural history of PTCL will advance the field and improve outcomes in this challenging group of diseases. This review summarizes the currently available clinical evidence on the various approaches to treating relapsed/refractory PTCL, including the role of stem cell transplantation, with an emphasis on potential new drug therapies.

Revisiting the immune microenvironment of diffuse large B-cell lymphoma using a tissue microarray and immunohistochemistry: robust semi-automated analysis reveals CD3 and FoxP3 as potential predictors of response to R-CHOP

Gene expression studies have identified the microenvironment as a prognostic player in diffuse large B-cell lymphoma. However, there is a lack of simple immune biomarkers that can be applied in the clinical setting and could be helpful in stratifying patients. Immunohistochemistry has been used for this purpose but the results are inconsistent. We decided to reinvestigate the immune microenvironment and its impact using immunohistochemistry, with two systems of image analysis, in a large set of patients with diffuse large B-cell lymphoma. Diagnostic tissue from 309 patients was arrayed onto tissue microarrays. Results from 161 chemoimmunotherapy-treated patients were used for outcome prediction. Positive cells, percentage stained area and numbers of pixels/area were quantified and results were compared with the purpose of inferring consistency between the two semi-automated systems. Measurement cutpoints were assessed using a recursive partitioning algorithm classifying results according to survival. Kaplan-Meier estimators and Fisher exact tests were evaluated to check for significant differences between measurement classes, and for dependence between pairs of measurements, respectively. Results were validated by multivariate analysis incorporating the International Prognostic Index. The concordance between the two systems of image analysis was surprisingly high, supporting their applicability for immunohistochemistry studies. Patients with a high density of CD3 and FoxP3 by both methods had a better outcome. Automated analysis should be the preferred method for immunohistochemistry studies. Following the use of two methods of semi-automated analysis we suggest that CD3 and FoxP3 play a role in predicting response to chemoimmunotherapy in diffuse large B-cell lymphoma.

Delta One T cells for immunotherapy of chronic lymphocytic leukemia: clinical-grade expansion/ differentiation and preclinical proof-of-concept

Purpose: The Vδ1+ subset of γδ T lymphocytes is a promising candidate for cancer immunotherapy, but the lack of suitable expansion/differentiation methods has precluded therapeutic application. We set out to develop and test (preclinically) a Vδ1+ T-cell–based protocol that is good manufacturing practice compatible and devoid of feeder cells for prompt clinical translation. Experimental design: We tested multiple combinations of clinical-grade agonist antibodies and cytokines for their capacity to expand and differentiate (more than 2–3 weeks) Vδ1+ T cells from the peripheral blood of healthy donors and patients with chronic lymphocytic leukemia (CLL). We characterized the phenotype and functional potential of the final cellular product, termed Delta One T (DOT) cells, in vitro and in vivo (xenograft models of CLL). Results: We describe a very robust two-step protocol for the selective expansion (up to 2,000-fold in large clinical-grade cell culture bags) and differentiation of cytotoxic Vδ1+ (DOT) cells. These expressed the natural cytotoxicity receptors, NKp30 and NKp44, which synergized with the T-cell receptor to mediate leukemia cell targeting in vitro. When transferred in vivo, DOT cells infiltrated tumors and peripheral organs, and persisted until the end of the analysis without showing signs of loss of function; indeed, DOT cells proliferated and produced abundant IFNγ and TNFα, but importantly no IL17, in vivo. Critically, DOT cells were capable of inhibiting tumor growth and preventing dissemination in xenograft models of CLL. Conclusions: We provide a clinical-grade method and the preclinical proof of principle for application of a new cellular product, DOT cells, in adoptive immunotherapy of CLL. Clin Cancer Res; 22(23); 5795–804. ©2016 AACR.

On CK2 regulation of chronic lymphocytic leukemia cell viability

Specific inhibition of signaling elements essential for the viability of B-cell chronic lymphocytic leukemia (CLL) cells offers great promise for the design of more efficient therapies. The protein serine/threonine kinase CK2 is frequently upregulated in cancer, and it is overexpressed and hyperactivated in primary CLL cells from untreated patients. We have shown that inhibition of CK2 induces apoptosis of CLL cells, whereas it does not significantly impact normal lymphocytes, demonstrating the selectivity of the CK2 inhibitors toward leukemia cells. Notably, although co-culture with OP9 stromal cells and BCR stimulation both promote leukemia cell survival in vitro, they do not prevent apoptosis of CLL cells treated with CK2 inhibitors. PI3K signaling pathway was previously shown to be essential for CLL cell viability, an observation we confirmed in all patient samples analyzed. Further, we observed that CK2 blockade decreases PTEN phosphorylation, leading to PTEN activation, and that apoptosis of CLL cells upon CK2 inhibition is mediated by PKC inactivation. This suggests that activation of PI3K/PKC signaling pathway is involved in the pro-survival effects of CK2 in CLL cells. Sensitivity to CK2 inhibition does not correlate with expression of ZAP-70 or CD38, or with IGVH mutation status. However, it positively correlates with the percentage of CLL cells in the peripheral blood, β2 microglobulin levels, and Binet clinical stage. CK2 appears to play an important role in the biology of CLL and constitutes a promising target for the development of leukemia-specific therapies.

Three‐way translocation involves MLL, MLLT3, and a novel cell cycle control gene, FLJ10374, in the pathogenesis of acute myeloid leukemia with t(9;11;19)(p22;q23;p13.3)

The MLL gene, at 11q23, undergoes chromosomal translocation with a large number of partner genes in both acute lymphoblastic and acute myeloid leukemia (AML). We report a novel t(9;11;19)(p22;q23;p13.3) disrupting MLL in an infant AML patient. The 5′ end of MLL fused to chromosome 9 sequences on the der(11), whereas the 3′ end was translocated to chromosome 19. We developed long‐distance inverse–polymerase chain reaction assays to investigate the localization of the breakpoints on der(11) and der(19). We found that intron 5 of MLL was fused to intron 5 of MLLT3 at the der(11) genomic breakpoint, resulting in a novel in‐frame MLL exon 5–MLLT3 exon 6 fusion transcript. On the der(19), a novel gene annotated as FLJ10374 was disrupted by the breakpoint. Using reverse transcription–polymerase chain reaction analysis, we showed that FLJ10374 is ubiquitously expressed in human cells. Transfection of the FLJ10374 protein in different cell lines revealed that it localized exclusively to the nucleus. In serum‐starved NIH‐3T3 cells, the expression of FLJ10374 decreased the rate of the G1‐to‐S transition of the cell cycle, whereas the suppression of FLJ10374 through short interfering RNA increased cell proliferation. These results indicate that FLJ10374 negatively regulates cell cycle progression and proliferation. Thus, a single chromosomal rearrangement resulting in formation of the MLL–MLLT3 fusion gene and haplo‐insufficiency of FLJ10374 may have cooperated to promote leukemogenesis in AML with t(9;11;19).