Saskia Agm Cillessen

Molecular targeted therapies for diffuse large B-cell lymphoma based on apoptosis profiles

Diffuse large B‐cell lymphoma (DLBCL) is the most common type of adult non‐Hodgkin lymphoma and is treated with chemotherapy in combination with rituximab. Despite this aggressive therapy, the disease is fatal in 30–40% of patients. Inhibition of the apoptosis signalling pathways is strongly related to response to chemotherapy and eventual clinical outcome. In order to survive, lymphoma cells depend on disruption of the apoptosis pathway by mutations in apoptosis inducing genes or by continuous expression of anti‐apoptotic proteins. The development of molecules targeting these apoptosis inhibitors provides a very promising opportunity to specifically target tumour cells without toxicity to non‐malignant cells in DLBCL patients. Sensitivity for most of these antagonists can be predicted based on biological markers, suggesting the possibility of pre‐defining patients who will most likely benefit from these targeted therapies. Experimental therapies aimed at restoring the upstream apoptosis pathway or targeting apoptosis inhibitors are currently being tested in clinical trials and are expected to be effective particularly in chemotherapy‐refractory DLBCL, providing hope for patients who are refractory to current therapies. Copyright

Methylation-mediated repression of PRDM14 contributes to apoptosis evasion in HPV-positive cancers.

Promoter methylation of the transcription factor PRDM14 (PRDI-BF1 and RIZ domain containing 14) represents a highly frequent event in human papillomavirus (HPV)-induced cervical cancers and cancer precursor lesions. Here, we aimed to assess the functional consequences of PRDM14 promoter methylation in HPV-induced carcinogenesis. PRDM14 promoter methylation, expression and consequences of ectopic PRDM14 expression were studied in HPV16-positive cervical and oral cancer cell lines (SiHa, CaSki and 93VU147T), human embryonic kidney 293 (HEK293T) cells and primary human foreskin keratinocytes (HFK). PRDM14 mRNA expression was restricted to HEK293T and HFK cells, and could be upregulated in SiHa cells upon DNA methylation inhibition. Ectopic expression of PRDM14 in SiHa, CaSki and 93VU147T cells resulted in significantly more apoptotic cells, as measured by annexin V labelling, compared to HEK293T and HFK cells. MRNA profiling of 41 apoptosis regulators identified NOXA and PUMA as candidate target genes involved in PRDM14-mediated apoptosis induction. Full-length PRDM14 transactivated both NOXA and PUMA promoters. Transactivation was abolished upon deletion of the PRDM14 DNA binding domain. This suggests that NOXA and PUMA expression is directly regulated by PRDM14, which in case of NOXA was linked to a consensus PRDM14 binding motif in the promoter region. Taken together, these results suggest that PRDM14 acts as a regulator of NOXA and PUMA-mediated apoptosis induction, thereby providing evidence for a tumour suppressive role in HPV-induced carcinogenesis. The contribution of methylation-mediated gene silencing of PRDM14 to apoptosis evasion in HPV-positive cancer cells offers novel therapeutic options for HPV-induced cancers.

Exploring dendritic cell based vaccines targeting survivin for the treatment of head and neck cancer patients.

BackgroundNew treatment modalities are needed for the treatment of cancers of the head and neck region (HNSCC). Survivin is important for the survival and proliferation of tumor cells and may therefore provide a target for immunotherapy. Here we focused on the ex vivo presence and in vitro induction of survivin specific T cells.MethodsTetramer staining and ELIspot assays were used to document the presence of survivin specific T cells in patient derived material, and to monitor the presence and persistence of survivin specific T cells after repeated in vitro stimulation with autologous dendritic cells.ResultsEx vivo analysis showed the presence of survivin-specific T cells in the peripheral blood (by tetramer analysis) and in the draining lymph node (by ELIspot analysis) in a HNSCC and a locally advanced breast cancer patient respectively. However, we were unable to maintain isolated survivin specific T cells for prolonged periods of time. For the in vitro generation of survivin specific T cells, monocyte derived DC were electroporated with mRNA encoding full length survivin or a survivin mini-gene together with either IL21 or IL12 mRNA. Western blotting and immunohistochemical staining of dendritic cell cytospin preparations confirmed translation of the full length survivin protein. After repeated stimulation we observed an increase, followed by a decrease, of the number of survivin specific T cells. FACS sorted or limiting dilution cloned survivin specific T cells could not be maintained on feeder mix for prolonged periods of time. Protein expression analysis subsequently showed that activated, but not resting T cells contain survivin protein.ConclusionsHere we have shown that survivin specific T cells can be detected ex vivo in patient derived material. Furthermore, survivin specific T cells can be induced in vitro using autologous dendritic cells with enforced expression of survivin and cytokines. However, we were unable to maintain enriched or cloned survivin specific T cells for prolonged periods of time. Endogenous expression of survivin in activated T cells and subsequent fratricide killing might explain our in vitro observations. We therefore conclude that survivin, although it is a universal tumor antigen, might not be the ideal target for immunotherapeutic strategies for the treatment of cancer of the head and neck.

18F-FDG or 3′-Deoxy-3′-18F-Fluorothymidine to Detect Transformation of Follicular Lymphoma

Considering the different treatment strategy for transformed follicular lymphoma (TF) as opposed to follicular lymphoma (FL), diagnosing transformation early in the disease course is important. There is evidence that 18F-FDG has utility as a biomarker of transformation. However, quantitative thresholds may require inclusion of homogeneous non-Hodgkin lymphoma subtypes to account for differences in tracer uptake per subtype. Moreover, because proliferation is a hallmark of transformation, 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) might be superior to 18F-FDG in this setting. To define the best tracer for detection of TF, we performed a prospective a head-to-head comparison of 18F-FDG and 18F-FLT in patients with FL and TF. Methods: 18F-FDG and 18F-FLT PET scans were obtained in 17 patients with FL and 9 patients with TF. We measured the highest maximum standardized uptake value (SUVmax), defined as the lymph node with the highest uptake per patient, and SUVrange, defined as the difference between the SUVmax of the lymph node with the highest and lowest uptake per patient. To reduce partial-volume effects, only lymph nodes larger than 3 cm3 (A50 isocontour) were analyzed. Scans were acquired 1 h after injection of 185 MBq of 18F-FDG or 18F-FLT. To determine the discriminative ability of SUVmax and SUVrange of both tracers for TF, receiver-operating-characteristic curve analysis was performed. Results: The highest SUVmax was significantly higher for TF than FL for both 18F-FDG and 18F-FLT (P < 0.001). SUVrange was significantly higher for TF than FL for 18F-FDG (P = 0.029) but not for 18F-FLT (P = 0.075). The ability of 18F-FDG to discriminate between FL and TF was superior to that of 18F-FLT for both the highest SUVmax (P = 0.039) and the SUVrange (P = 0.012). The cutoff value for the highest SUVmax of 18F-FDG aiming at 100% sensitivity with a maximum specificity was found to be 14.5 (corresponding specificity, 82%). For 18F-FLT, these values were 5.1 and 18%, respectively. When the same method was applied to SUVrange, the cutoff values were 5.8 for 18F-FDG (specificity, 71%) and 1.5 for 18F-FLT (specificity, 36%). Conclusion: Our data suggest that 18F-FDG PET is a better biomarker for TF than 18F-FLT PET. The proposed thresholds of highest SUVmax and SUVrange should be prospectively validated.

Treatment response in enteropathy associated T‐cell lymphoma; survival in a large multicenter cohort

Enteropathy‐associated T‐cell lymphoma (EATL) is a T‐cell Non‐Hodgkin Lymphoma which is highly associated with celiac disease. The prognosis of EATL has been considered poor and there are no standardized treatment protocols. Here, we evaluate treatment response and survival of EATL patients in a large multicenter cohort. A total of 61 patients diagnosed with EATL were analyzed. Various treatment regimens were applied in EATL during the past fifteen years including either monotherapy consisting of chemotherapy or resection, or combination therapy with both aforementioned regimens whether or not combined with stem‐cell transplantation (SCT). Overall, 50/61 patients (82%) died after a median of 7.4 months. One‐ and five‐year overall survival was 40 and 11%, respectively. Median follow‐up in the survivors was 26 months. Patients treated with the most aggressive treatment, that is, resection, chemotherapy and autologous SCT, showed the most favourable outcome with complete remission in all patients, the lowest relapse rate and one‐ and five‐year overall survival of 100 and 33%, respectively, although overall survival in this group was not significantly better as compared to patients treated with surgery and chemotherapy. This study indicates that combination treatment is superior compared to monotherapy. Whether or not consolidation therapy with autologous SCT may improve survival needs to be substantiated in a larger randomized international trial. Am. J. Hematol. 90:493–498, 2015.

ALK-negative anaplastic large cell lymphoma is sensitive to bortezomib through Noxa upregulation and release of Bax from Bcl-2

Clinical outcome in anaplastic large cell lymphoma (ALCL), especially in systemic anaplastic lymphoma kinase (ALK)-negative ALCL is frequently poor, despite intensive therapy regimens including CHOP (vincristine, doxorubicin, cyclophosphamide and prednisone), with or without etoposide. Although

A new and validated clinical prognostic model (EPI) for Enteropathy Associated T-cell Lymphoma

Purpose: Enteropathy-associated T-cell lymphoma (EATL) is a rare intestinal non–Hodgkin lymphoma with a poor, though variable prognosis. The International Prognostic Index (IPI) and the prognostic index for peripheral T-cell lymphoma (PIT) have limited predictive value for outcome of EATL. The purpose of this study was to develop and validate a prognostic model for EATL, which can identify high-risk patients who need more aggressive therapy. Experimental Design: This retrospective multicenter study was based on 92 patients and included 45 patients diagnosed with EATL between 1999 and 2009 from the Netherlands and 47 patients from England and Scotland, diagnosed with EATL between 1994 and 1998. A new EATL prognostic index (EPI) was constructed using the RPART (recursive partitioning and regression trees) procedure. Validation was performed applying the bootstrap method. Results: Three risk groups were distinguished (P < 0.0001): a high-risk group, characterized by the presence of B-symptoms [median overall survival (OS) of 2 months]; an intermediate-risk group, comprising patients without B-symptoms and an IPI score ≥ 2 (7 months); and a low-risk group, representing patients without B-symptoms and an IPI score of 0 to 1 (34 months). Internal validation showed stability of statistical significance and prognostic discrimination. In contrast with the IPI and PIT, the EPI better classified patients in risk groups according to their clinical outcome. Conclusions: Our new, validated, prognostic model EPI accurately predicts survival outcome in EATL and may be used for patient selection for new therapeutic strategies and evaluation of clinical trials. Clin Cancer Res; 21(13); 3013–9. ©2015 AACR.

Abstract C69: Schedule-dependent synergy between the histone deacetylase inhibitor belinostat and the dihydrofolate reductase inhibitor pralatrexate in T-and B-cell lymphoma cells in vitro

Pralatrexate (Folotyn; FOL) and belinostat (Beleodaq; BEL) have recently been registered for the treatment of patients with peripheral T-cell lymphoma (PTCL) and have promising activity in other lymphoma. FOL is a folate analog and a potent dihydrofolate reductase (DHFR) inhibitor, designed to accumulate in cancer cells via the reduced folate carrier (RFC) and retained via efficient polyglutamylation. DHFR inhibition leads to an imbalance of deoxynucleotides (depletion of dTTP and an increase in dUTP) resulting in DNA strand breaks and inhibition of DNA synthesis. BEL is a hydroxamic acid-based pan-histone deacetylase (HDAC) inhibitor that inhibits all of the zinc-dependent HDAC enzymes, with high affinity for the Class I, II and IV isozymes. HDAC inhibition results in an alteration in the degree of histone and non-histone protein acetylation, which in turn affects transcription of genes essential in cellular proliferation, cell cycle and DNA repair. In this study we investigated whether folate transporters other than RFC, i.e. folate receptor α (FRα) and the proton-coupled folate transporter (PCFT) could contribute to the efficacy of FOL. Moreover, we explored whether in combination experiments BEL had the ability to potentiate the cytotoxicity of FOL. A panel of lymphoma cell lines was used for the combination studies: the B-cell SU-DHL-4, SU-DHL-5, HT, Jeko-1 and T-cell Karpas-299 and Hut-78. RFC-mediated uptake efficiency of FOL (in competition with [3H]-methotrexate), showed a 6-fold better RFC substrate affinity for FOL, and 2-fold better than levo-leucovorin (l-LV). FOL had a very poor substrate binding affinity for FRα (>100-fold lower than folic acid and > 10 lower than l-LV) and a low affinity for PCFT (>10-fold lower than folic acid and l-LV in [3H]-LV uptake competition experiments). Sensitivity (IC50 concentrations after 72 hr exposure) to FOL varied from 2.8 (Hut-78), 5.5 (SU-DHL4 and 5), 7.4 (HT) to 20 nM (Karpas-299 and Jeko-1) and for BEL from 100 (SU-DLH-4 and 5, Jeko-1 and Hut-78) to 200 nM (Karpas-299 and HT). The interaction between BEL and FOL was studied using the median-drug effect analysis. At a fixed ratio between the drugs based on the IC50 concentration the average combination index (CI) for all cell lines showed additivity (CI: ±1.0). In two selected cell lines (SU-DHL-4 and HT) sequential exposure (24 hr pretreatment with either BEL or FOL, followed by 48 hr to FOL + BEL), did not improve interaction (CI: 0.9-1.4). As an alternative approach a non-fixed ratio was used by exposing SU-DHL-4 and HT cells to IC25 concentrations of either BEL or FOL in combination with the other drug. Exposure to IC25 of FOL did not decrease the IC50 for BEL (CI around 1.2), but exposure to IC25 of BEL markedly increased FOL sensitivity (low CIs from 0.40-0.66). Mechanistic studies focused on induction of apoptosis, showed cleavage of caspase 8 and 9 in HT and SU-DHL-4 cells for both drugs at their IC50s, being similar in the combination setting. Moreover, at these concentrations, the drugs were shown to confer an S-phase arrest. In conclusion, the combination of FOL and BEL showed additivity in various lymphoma cell lines, with a schedule-dependent synergism. Based on these data, proficient inhibition of HDAC activity by BEL holds promise in sensitization of tumor cells to FOL. Citation Format: Godefridus J. Peters, Frank H. Van Gemert, I. Kathmann, Saskia A. Cillessen, Gerrit Jansen, Guru Reddy. Schedule-dependent synergy between the histone deacetylase inhibitor belinostat and the dihydrofolate reductase inhibitor pralatrexate in T-and B-cell lymphoma cells in vitro. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C69.