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Novel strategies, such as chemosensitization with targeted agents, that build on the success of standard immunochemotherapy show promise for the treatment of non-Hodgkin lymphoma (NHL). Here, we report a phase 1b study investigating dose escalation of the BCL2 inhibitor, venetoclax, in combination with rituximab or obinutuzumab and cyclophosphamide, doxorubicin, vincristine, and prednisone (R-/G-CHOP) chemotherapy in B-cell NHL. Objectives included safety assessment and determination of a recommended phase 2 dose (RP2D). Fifty-six patients were enrolled, most with follicular lymphoma (43%) or diffuse large B-cell lymphoma (DLBCL; 32%). Dose-limiting toxicities were reported in 3/14 patients at the first venetoclax dose (200 mg/d), after which dosing was changed from daily to 10 days per cycle and escalated to 800 mg. A further reduction to 5 days per cycle occurred at the 800-mg dose level in the G-CHOP arm. Cytopenias were predominant among grade 3/4 events and reported at a higher rate than expected, particularly in the G-CHOP arm; however, safety was manageable. Overall response rates were 87.5% (R-CHOP and G-CHOP combinations); complete response (CR) rates were 79.2% and 78.1%, respectively. Most double-expressor (BCL2+and MYC+) DLBCL patients (87.5%; n = 7/8) achieved CR. Although the maximum tolerated dose was not reached, the RP2D for venetoclax with R-CHOP was established at 800 mg days 4 to 10 of cycle 1 and days 1 to 10 of cycles 2 to 8; higher doses were not explored, and this dosing schedule demonstrated an acceptable safety profile. This regimen is subsequently being evaluated in first-line DLBCL in the phase 2 portion of the study. This trial was registered at www.clinicaltrials.govas #NCT02055820.

Background:

We investigated whether the outcome prediction of patients with aggressive B-cell lymphoma can be improved by combining clinical, molecular genotype, and radiomics features. MYC, BCL2, and BCL6rearrangements were assessed using fluorescence in situ hybridization. Seventeen radiomics features were extracted from the baseline positron emission tomography–computed tomography of 323 patients, which included maximum standardized uptake value (SUVmax), SUVpeak, SUVmean, metabolic tumor volume (MTV), total lesion glycolysis, and 12 dissemination features pertaining to distance, differences in uptake and volume between lesions, respectively. Logistic regression with backward feature selection was used to predict progression after 2 years. The predictive value of (1) International Prognostic Index (IPI); (2) IPI plus MYC; (3) IPI, MYC, and MTV; (4) radiomics; and (5) MYCplus radiomics models were tested using the cross-validated area under the curve (CV-AUC) and positive predictive values (PPVs). IPI yielded a CV-AUC of 0.65 ± 0.07 with a PPV of 29.6%. The IPI plus MYCmodel yielded a CV-AUC of 0.68 ± 0.08. IPI, MYC, and MTV yielded a CV-AUC of 0.74 ± 0.08. The highest model performance of the radiomics model was observed for MTV combined with the maximum distance between the largest lesion and another lesion, the maximum difference in SUVpeakbetween 2 lesions, and the sum of distances between all lesions, yielding an improved CV-AUC of 0.77 ± 0.07. The same radiomics features were retained when adding MYC(CV-AUC, 0.77 ± 0.07). PPV was highest for the MYCplus radiomics model (50.0%) and increased by 20% compared with the IPI (29.6%). Adding radiomics features improved model performance and PPV and can, therefore, aid in identifying poor prognosis patients.

Background:Patients with MYCrearrangement positive large B cell lymphoma other than Burkitt lymphoma (MYC+ LBCL), have a dismal prognosis following standard first line therapy with R-CHOP. Retrospective studies report complete remission rates < 50% and 2-year overall survival (OS) of approximately 35%. Lenalidomide is an immunomodulatory drug and is able to down-regulate MYC and its target genes and proteins in B cells that harbor a MYCrearrangement. We report data of a prospective phase II study evaluating the efficacy of lenalidomide in combination with R-CHOP (R2CHOP) in newly diagnosed MYC+ LBCL patients.

CD37 is a tetraspanin molecule expressed on mature B-cells, but absent on normal stem cells and plasma cells. Due to this cellular distribution, which is highly similar to CD20, CD37 has gained attention as a target for B-cell lymphoma, especially for rituximab-resistant and relapsed patients.

Abstract 996

According to the classical HLA class II antigen presentation pathway, exogenous antigens are processed in the endosomal/lysosomal pathway and associate with HLA class II after exchange with the class II-associated invariant chain peptide (CLIP). For this reason, the relative amount of CLIP presented by HLA-DR (DR) molecules (CLIP/DR amount) can be considered as an indicator for HLA class II antigen loading. Previously, we showed that Invariant Chain (Ii) down-modulation in the Kasumi-1 and THP-1 AML cell lines led to marked declines in CLIP/DR amount [Van Luijn et al., Haematologica 2008; 93(s1), Abstract 0029]. In addition, the total amount of cell surface-expressed DR was reduced on Kasumi-1 blasts, in line with the need of Ii for the transport of newly synthesized HLA class II molecules into the endosomal/lysosomal pathway. Surprisingly, in THP-1 blasts, Ii down-modulation hardly affected DR expression at the cell surface. In the present study, we further explored the Ii-independent pathway of HLA class II antigen presentation in leukemic blasts. Not only in the THP-1, but also in another AML cell line, the KG-1, Ii down-modulation had no effect on DR expression levels, as determined by flow cytometry. Since DR expression does require peptide binding, Ii-independency in these AML blasts may be achieved by endogenous antigen loading in the endoplasmic reticulum (ER). To test this hypothesis, supply of endogenously derived peptides into the ER was blocked by viral proteins interfering with the function of the transporter associated with antigen processing (TAP). As expected, TAP inhibition in KG-1 blasts by the viral UL49.5 protein (which changes the conformation of TAP and mediates its degradation) resulted in a strong down-regulation (7.7-fold) of HLA class I. Strikingly, TAP inhibition also induced a clear DR−KG-1 blast population (52.3% of total; MFI=1.4) next to the original DR+KG-1 blast population (36.5% of total; MFI=288.9), demonstrating that DR expression is partly TAP-dependent in KG-1 blasts. Upon sorting of both populations, TAP−DR−blasts had a decreased expression of intracellular Ii as compared to TAP−DR+blasts (3.9-fold) and wild type blasts (4.7-fold). Additionally, confocal microscopy revealed that in TAP−DR+blasts, DR localised to the cell surface, indicating that Ii is able to rescue cell surface expression of DR. Indeed, Ii down-modulation in TAP−DR+blasts caused a 2.3-fold decline in DR expression. The observed differences in TAP, Ii and DR expression between these KG-1 variants were confirmed by Western blot analysis. Furthermore, blocking of proteasome function by the specific inhibitors MG-132 and Bortezomib also caused a marked decrease of DR expression on KG-1 blasts (MFI declined from 289.2 to respectively 76.4 and 114.5). Accompanying reduction in HLA class I levels ascertained specific proteasome inhibition. This confirmed that at least part of the antigens presented by DR on KG-1 blasts was derived from endogenous sources. Similar results were obtained with THP-1 blasts, as both TAP and proteasome inhibition clearly affected DR expression. In line with our observations that in Kasumi-1 blasts, DR expression is Ii-dependent, addition of the TAP and proteasome inhibitors to Kasumi-1 blasts did not affect cell surface expression of DR. In conclusion, our data reveal an alternative Ii-independent, but TAP- and proteasome-dependent cross-presentation pathway in different AML cell lines, which involves HLA class II loading of endogenous antigens in the ER. Therefore, this alternative pathway may serve as a potent immunomodulatory target in leukemic blasts to activate CD4+T cells specific for a broad range of leukemia-associated antigens.

Although acute myeloid leukemia (AML) can be cured with intensive treatment including myeloablative chemotherapy and haematopoietic stem cell transplantation, relapses occur in the majority of cases. A common feature of tumor cells is their ability to escape immune surveillance through adapted intrinsic mechanisms. Thus, it is a great challenge to develop optimal strategies that direct a specific cellular immune response against residual AML blasts in vivo. As CD4+T cells are needed to initiate a strong anti-leukemic CD8+T cell response, the mechanism through which HLA class-II restricted (leukemia-specific) antigens are presented on AML blasts could be an essential factor in immune surveillance. Previously, we showed that the self peptide Class-II Associated Invariant Chain Peptide (CLIP) important in HLA class-II antigen presentation appeared to be disadvantageous, as its expression on AML blasts predicted a shortened disease-free survival (Chamuleau et al. Canc. Res. 2004; 64(16):5546–50). We hypothesized that CLIP interferes with the presentation of specific tumor antigens on HLA class-II molecules, thereby preventing recognition of AML blasts by CD4+T cells. To investigate whether CLIP expression indeed has a functional effect on leukemia-specific T cell activation in patients, an AML cell line model with CLIP+and CLIP−leukemic blasts was set up. The Kasumi-1 and THP-1 AML cell lines were selected as both stained positive for extracellular HLA-DR (89%; MFI=31.3 and 91%; MFI=37.5 respectively) and CLIP expression (88%; MFI=37.2 and 91%; MFI=34.0 respectively) by flow cytometric analysis. These DR+CLIP+cell lines were specifically silenced for Invariant Chain (Ii) expression using RNA interference to down-modulate CLIP presentation on the cell surface. Indeed, Ii siRNA-treated cells not only showed a significant decrease of intracellular Ii expression (MFI decrease of 87.7% for Kasumi-1 and 82.7% for THP-1), but also a marked downregulation of relative CLIP amount per HLA-DR molecule (fold decline in CLIP/DR ratio of 1.4 for Kasumi-1 and 2.0 for THP-1). Wild type (DR+CLIP+) and modulated (DR+CLIP−) cells of Kasumi-1 or THP-1 origin acted as stimulators for alloreactive CD4+T cells in mixed leukocyte reactions using different stimulator to responder (S/R) ratios. Modulated DR+CLIP−Kasumi-1 and THP-1 cells induced a strong increase in alloreactive CD4+T cell proliferation as compared to DR+CLIP+wild type controls, both in an HLA-DR-specific and a S/R-dependent manner. At the highest S/R ratio, mean proliferation increases of 2.58-fold for Kasumi-1 (n=3) and 1.71-fold for THP-1 (n=2) were observed. These data support our hypothesis that the expression of CLIP on AML blasts plays an important role in immune surveillance, which might have impact on cellular immunotherapy with dendritic cell-based vaccines in AML.