Feasibility and toxicity of concomitant radio/immunotherapy with MabThera (Rituximab®) for patients with non-Hodkin's Lymphoma: results of a prospective phase I/II study.

PURPOSE: Non-Hodgkin's lymphomas (NHL) have a high radio- and chemosensitivity. Although initially responsive, approximately 50% of low grade B-cell lymphomas relapse after 10-15 years. Besides chemo- and radiotherapy, rituximab, a mouse/human chimeric antibody targeting CD20 antigen on the surface of B-cell lymphoma cells, is another treatment approach. In vitro data showed potentiation of radiation-induced apoptosis by addition of rituximab. The purpose of this study was to evaluate the feasibility and toxicity of radiotherapy with concomitant application of rituximab in NHL patients. PATIENTS AND METHODS: A total of 21 patients with B-cell lymphoma (stage I: n = 11; II: n = 5; III: n = 1; IV: n = 4) were included in this study, treated with radiotherapy of 30-40 Gy and weekly application of rituximab (375 mg/m²). Nine patients had R-CHOP chemotherapy previously, 1 patient leuceran chemotherapy, and 2 patients an initial treatment with 6 cycles of rituximab. Mean time of follow-up was 41.7 months. RESULTS: No grade 4 toxicity or treatment-related death was observed. In 1 patient, rituximab application had to be stopped after 3 cycles due to radiation-induced side effects. No late toxicities were reported. All patients were in complete remission after treatment. Progression or relapse was observed in 6 patients (28%); the mean time to progression was 27 months. The mean overall survival (OS) was 53 months. CONCLUSION: Combined radio/immunotherapy is feasible and safe. Treatment was well tolerated, no late toxicities were observed, and treatment outcome is promising. Randomized trials are necessary to clarify the benefit of this treatment approach and its applicability.

Epithelial-to-mesenchymal transition and c-myc expression are the determinants of cetuximab-induced enhancement of squamous cell carcinoma radioresponse.

PURPOSE: Radiation therapy cures malignant tumors of the head and neck region more effectively when it is combined with application of the anti-EGFR monoclonal antibody cetuximab. Despite the successes achieved, we still do not know how to select patients who will respond to this combination of anti-EGFR monoclonal antibody and radiation. This study was conducted to elucidate possible mechanisms which cause the combined treatment with cetuximab and irradiation to fail in some cases of squamous cell carcinomas. METHODS AND MATERIALS: Mice bearing FaDu and A431 squamous cell carcinoma xenograft tumors were treated with cetuximab (total dose 3 mg, intraperitoneally), irradiation (10 Gy) or their combination at the same doses. Treatment was applied when tumors reached 8mm in size. To collect samples for further protein analysis (two-dimensional differential gel electrophoresis (2-D DIGE), mass spectrometry MALDI-TOF/TOF, Western blot analysis, and ELISA), mice from each group were sacrificed on the 8th day after the first injection of cetuximab. Other mice were subjected to tumor growth delay assay. RESULTS: In FaDu xenografts, treatment with cetuximab alone was nearly as effective as cetuximab combined with ionizing radiation, whereas A431 tumors responded to the combined treatment with significantly enhanced delay in tumor growth. Tumors extracted from the untreated FaDu and A431 xenografts were analysed for protein expression, and 34 proteins that were differently expressed in the two tumor types were identified. The majority of these proteins are closely related to intratumoral angiogenesis, cell adhesion, motility, differentiation, epithelial-to-mesenchymal transition (EMT), c-myc signaling and DNA repair. CONCLUSIONS: The failure of cetuximab to enhance radiation response in FaDu xenografts was associated with the initiation of the program of EMT and with c-myc up-regulation in the carcinoma cells. For this reason, c-myc and EMT-related proteins (E-cadherin, vimentin) may be considered as potential biomarkers to predict squamous cell carcinoma response after treatment with cetuximab in combination with radiation.

Intracellular signaling pathways regulating radioresistance of human prostate carcinoma cells.

Radiation therapy plays an important role in the management of prostate carcinoma. However, the problem of radioresistance and molecular mechanisms by which prostate carcinoma cells overcome cytotoxic effects of radiation therapy remains to be elucidated. In order to investigate possible intracellular mechanisms underlying the prostate carcinoma recurrences after radiotherapy, we have established three radiation-resistant prostate cancer cell lines, LNCaP-IRR, PC3-IRR, and Du145-IRR derived from the parental LNCaP, PC3, and Du145 prostate cancer cells by repetitive exposure to ionizing radiation. LNCaP-IRR, PC3-IRR, and Du145-IRR cells (prostate carcinoma cells recurred after radiation exposure (IRR cells)) showed higher radioresistance and cell motility than parental cell lines. IRR cells exhibited higher levels of androgen and epidermal growth factor (EGF) receptors and activation of their downstream pathways, such as Ras-mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K)-Akt and Jak-STAT. In order to define additional mechanisms involved in the radioresistance development, we determined differences in the proteome profile of parental and IRR cells using 2-D DIGE followed by computational image analysis and MS. Twenty-seven proteins were found to be modulated in all three radioresistant cell lines compared to parental cells. Identified proteins revealed capacity to interact with EGF and androgen receptors related signal transduction pathways and were involved in the regulation of intracellular routs providing cell survival, increased motility, mutagenesis, and DNA repair. Our data suggest that radioresistance development is accompanied by multiple mechanisms, including activation of cell receptors and related downstream signal transduction pathways. Identified proteins regulated in the radioresistant prostate carcinoma cells can significantly intensify activation of intracellular signaling that govern cell survival, growth, proliferation, invasion, motility, and DNA repair. In addition, such analyses may be utilized in predicting cellular response to radiotherapy.

Rituximab enhances radiation-triggered apoptosis in non-Hodgkin's lymphoma cells via caspase-dependent and - independent mechanisms.

Rituximab (RTX), a chimeric human anti-CD20 monoclonal antibody, is currently employed in the treatment of malignant non-Hodgkin's lymphoma (NHL) either alone or in combination with other cytotoxic approaches. The present study examines the effects of ionizing radiation in combination with RTX on proliferation and apoptosis development in B-lymphoma RL and Raji cells. RTX was used at a concentration of 10 microg/mL 24 hours prior to irradiation at a single dose of 9 Gy. CD20 expression, cell viability, apoptosis, mitochondrial membrane potential and apoptosis-related proteins were evaluated in the treated B cells. The constitutive level of CD20 expression in RL and Raji lymphoma cells did not play an essential role in RTX-induced cell growth delay. Both lymphoma cells showed similar inhibition of cell proliferation without apoptosis development in response to RTX treatment. Exposure to ionizing radiation induced cell growth delay and apoptosis in RL cells, whereas Raji cells showed moderate radio-resistance and activation of cell growth at 24 hours after irradiation, which was accompanied by increased radiation-triggered CD20 expression. The simultaneous exposure of lymphoma cells to ionizing radiation and RTX abrogated radioresistance of Raji cells and significantly enhanced cell growth delay and apoptosis in RL cells. X-linked inhibitor of apoptosis protein (XIAP) and the inducible form of heat shock protein 70 (Hsp70) were positively modulated by RTX in combination with ionizing radiation in order to induce apoptosis. Furthermore, it was demonstrated that mitochondrial membrane potential dissipation is not an essential component to induce apoptosis-inducing factor (AIF) maturation and apoptosis. Our results show that RTX-triggered enhancement of radiation-induced apoptosis and cell growth delay is achieved by modulation of proteins involved in programmed cell death.

Pretreatment with rituximab enhances radiosensitivity of non-Hodgkin's lymphoma cells.

The present study examines the effects of ionizing radiation in combination with rituximab (RTX), a chimeric human anti-CD20 monoclonal antibody, on proliferation, cell cycle distribution and apoptosis in B-lymphoma RL and Raji cells. Exposure to ionizing radiation (9 Gy) induced cell growth delay and apoptosis in RL cells, whereas Raji cells showed moderate radio-resistance. The simultaneous exposure of lymphoma cells to ionizing radiation and RTX (10 microg/mL) markedly enhanced apoptosis and cell growth delay in RL and Raji cells. Cooperative antiproliferative and apoptotic effects of RTX and radiation were achieved through the inhibition of c-myc and bcl-XL expression. Furthermore, RTX-modulated expression of cell cycle regulating proteins, such as p53, p21/WAF1, p27/KIP1, contributed to the development of radiation-induced cell killing and growth arrest. Each NHL cell line that underwent apoptosis induced by combination treatment revealed enhanced caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage as compared to only irradiated cells. These findings show that rituximab synergistically enhances radiation-induced apoptosis and cell growth delay through the expression of proteins involved in the programmed cell death and cell cycle regulation pathways.