[68Ga]Ga/[177Lu]Lu-BL01, a Novel Theranostic Pair for Targeting C-X-C Chemokine Receptor 4.

C-X-C chemokine receptor type 4 (CXCR4) is overexpressed in hematological and solid malignancies. LY2510924 is a potent peptide antagonist of CXCR4. A derivative of LY2510924, BL01, was evaluated for theranostic applications targeting CXCR4. Methods: BL01 was synthesized by solid phase approach. A Lys(ivDde) residue was added at the C-terminus of LY2510924 (cyclo[Phe-Tyr-Lys(iPr)-d-Arg-2-Nal-Gly-d-Glu]-Lys(iPr)-NH2). A DOTA chelator was conjugated to the side chain of the deprotected exogenous Lys residue. The binding affinity of Ga/Lu-BL01 was determined by competitive radioligand binding assays. BL01 was radiolabeled with 68GaCl3 or 177LuCl3. Biodistribution studies were performed in mice bearing Daudi Burkitt's lymphoma tumor xenografts at selected time points. PET imaging studies were performed with [68Ga]Ga-BL01, with blocking experiments performed with preinjection of LY2510924. The stability of [68Ga]Ga/[177Lu]Lu-BL01 was assessed in mouse plasma. Results: Ga-BL01 and Lu-BL01 have nanomolar affinity for CXCR4. [68Ga]Ga-BL01 was obtained in 58 ± 5% decay-corrected radiochemical yields and >99% radiochemical purity with a molar activity of 40 ± 11 GBq/µmol, while [177Lu]Lu-BL01 was obtained in 65 ± 6% decay-corrected radiochemical yields and >99% radiochemical purity with a molar activity of 120 ± 21 GBq/µmol. [68Ga]Ga-BL01 and [177Lu]Lu-BL01 were excreted primarily through the renal pathway. Daudi xenografts were clearly delineated in PET images with good contrast. On the basis of biodistribution data, tumor uptake of [68Ga]Ga-BL01 was 10.2 ± 2.56% injected dose per gram (%ID/g) at 1 h postinjection (p.i.). Spleen (12.6 ± 2.36 %ID/g) and lungs (13.2 ± 2.98 %ID/g), organs that express CXCR4, had high uptake as well. Preinjection of LY2510924 reduced average uptake of [68Ga]Ga-BL01 in tumors by 88%, demonstrating target specificity. The uptake of [68Ga]Ga-BL01 in tumor increased to 15.3 ± 1.86 %ID/g at 2 h p.i., with improved contrast. [177Lu]Lu-BL01 has similar pharmacokinetics as [68Ga]Ga-BL01 at 1 h p.i. The highest uptake was observed in tumor (14.0 ± 1.11 %ID/g), followed by the lungs (13.0 ± 1.27 %ID/g) and spleen (11.6 ± 1.78 %ID/g). The tumor uptake increased to 16.2 ± 2.69 %ID/g at 4 h p.i., before declining slightly to 10.1 ± 1.41 %ID/g at 24 h p.i. Both compounds were stable in vivo, as no metabolites were observed at 5 min p.i. Conclusions: [68Ga]Ga-BL01 and [177Lu]Lu-BL01 are a promising theranostic pair for imaging and endoradiotherapy of CXCR4-expressing malignancies.

Hyperfractionated radiation therapy in Burkitt's lymphoma: a reconsideration aspect.

Burkitt's lymphoma (BL) is an aggressive non-Hodgkin's B-cell lymphoma with an extremely short doubling time that often presents in extra nodal sites or as an acute leukaemia. Nowadays, with the rapid response to chemotherapy and the diffuse nature of BL, there is no established role for radiation therapy (RT) even in localized disease. Regarding the relapsed/refractory BL, the treatment recommendations remain undefined. We present a 56-year-old woman, diagnosed with BL refractory to 6 cycles of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone), who had disease progression on R-DHAP (rituximab, dexamethasone, high dose cytarabine and cisplatin) with intrathecal methotrexate, then a partial response on RICE (rituximab, ifosfamide, carboplatin and etoposide). Patient received high dose chemotherapy and autologous haematopoietic stem cell transplantation. Then, she was treated with hyperfractionated involved-field RT regimen. Currently, the patient remains disease free for around 2 years after remission. We acknowledge that RT is not a standard treatment of BL, especially in patients who attain complete response (CR) after first-line multi-agent chemotherapy or even in those who have a CR after second-line chemotherapy pre-transplant. Yet, the use of a superfractionated regimen of consolidative radiation could be justified in the treatment of recurrent/refractory localized BL who do not achieve a CR even with second-line salvage chemotherapy. Radiation therapy in this context, given that it is a well-tolerated treatment, is a modality worthy of being re-considered in relapsed/refractory BL. Copyright © 2016 John Wiley & Sons, Ltd.

High treatment efficacy by dual targeting of Burkitt's lymphoma xenografted mice with a (177)Lu-based CD22-specific radioimmunoconjugate and rituximab.

PURPOSE: Dual-targeted therapy has been shown to be a promising treatment option in recurrent and/or refractory B-cell non-Hodgkin's lymphoma (B-NHL). We generated radioimmunoconjugates (RICs) comprising either a novel humanized anti-CD22 monoclonal antibody, huRFB4, or rituximab, and the low-energy ß-emitter (177)Lu. Both RICs were evaluated as single agents in a human Burkitt's lymphoma xenograft mouse model. To increase the therapeutic efficacy of the anti-CD22 RIC, combination therapy with unlabelled anti-CD20 rituximab was explored. METHODS: The binding activity of CHX-A″-DTPA-conjugated antibodies to target cells was analysed by flow cytometry. To assess tumour targeting of (177)Lu-labelled antibodies, in vivo biodistribution experiments were performed. For radioimmunotherapy (RIT) studies, non-obese diabetic recombination activating gene-1 (NOD-Rag1 (null) ) interleukin-2 receptor common gamma chain (IL2rγ (null) ) null mice (NRG mice) were xenografted subcutaneously with Raji Burkitt's lymphoma cells. (177)Lu-conjugated antibodies were administered at a single dose of 9.5 MBq per mouse. For dual-targeted therapy, rituximab was injected at weekly intervals (0.5 - 1.0 mg). Tumour accumulation of RICs was monitored by planar scintigraphy. RESULTS: Conjugation of CHX-A"-DTPA resulted in highly stable RICs with excellent antigen-binding properties. Biodistribution experiments revealed higher tumour uptake of the (177)Lu-labelled anti-CD22 IgG than of (177)Lu-labelled rituximab. Treatment with (177)Lu-conjugated huRFB4 resulted in increased tumour growth inhibition and significantly longer survival than treatment with (177)Lu-conjugated rituximab. The therapeutic efficacy of the anti-CD22 RIC could be markedly enhanced by combination with unlabelled rituximab. CONCLUSION: These findings suggest that dual targeting with (177)Lu-based CD22-specific RIT in combination with rituximab is a promising new treatment option for refractory B-NHL.

Liposomal cytarabine in prophylaxis or curative treatment of central nervous system involvement in Burkitt leukemia/lymphoma.

In recent years, the outcome of Burkitt leukemia/lymphoma (BL) has improved significantly. Central nervous system (CNS) involvement continues to be a poor prognostic indicator. High doses of intravenous polychemotherapy, intrathecal chemotherapy, and cranio-spinal radiation therapy are used by numerous groups. Majority of patients are cured after this strategy. The next challenge is to decrease toxicities of treatment, including long-term toxicities secondary to cranio-spinal radiation therapy observed in these cured patients. Liposomal cytarabine could be a good alternative to cranio-spinal radiation therapy as already reported in acute lymphoblastic leukemia. We report here eleven patients treated in our center for BL, with liposomal cytarabine instead of cranio-spinal radiation therapy as prophylactic or curative treatment for CNS involvement. Treatment was safe with no short-term grade >3 adverse events. Moreover, no long-term side effects and no impact on outcome were observed. We conclude that LC could be a good option to decrease short/long-term side effects of cranio-spinal radiation therapy in BL and could be evaluated in a future clinical trial.

Curcumin improves the antitumor effect of X-ray irradiation by blocking the NF-κB pathway: an in-vitro study of lymphoma.

Curcumin, a phenolic compound from the rhizomes of Curcuma longa, inhibits the growth of a variety of malignant cell types including lymphoma cells. We investigated the role of curcumin in modulating the response of Burkitt's lymphoma cells to ionizing radiation (IR) in vitro and explored the mechanisms that mediated this effect. We treated three Burkitt's lymphoma cell lines with vehicle, curcumin, IR, and curcumin in combination with IR. Cell viability, apoptosis, and cell cycle distribution were determined to ascertain the radiosensitization effect of curcumin. Nuclear factor-kappa B (NF-κB) activation was assessed by nuclear translocation of p65. Apoptosis-related proteins were monitored by western blot assay and real-time RT-PCR. Pretreatment of curcumin sensitized lymphoma cells to IR-induced apoptosis and increased G2/M phase arrest in the cell cycle distribution. Accordingly, the antiapoptotic Bcl-xL protein, cell cycle modulating protein CDC2, and cyclin B1 were downregulated by the curcumin treatment. IR activated NF-κB as evidenced by an increased nuclear p65 translocation and cytoplasmic IκBα expression. However, pretreatment with curcumin significantly decreased the nuclear translocation of p65 and cytoplasmic IκBα degradation. Survivin and hexokinase II, downstream effectors of NF-κB that mediate the antiapoptotic effect of NF-κB, were suppressed by the pretreatment of curcumin. These observations suggest that the activated NF-κB pathway plays a prosurvival role in Burkitt's lymphoma in response to IR. Curcumin blocks this pathway and has therapeutic potential for improving the antitumor effects of radiotherapy.

Pretargeted versus directly targeted radioimmunotherapy combined with anti-CD20 antibody consolidation therapy of non-Hodgkin lymphoma.

UNLABELLED: We determined whether therapeutic responses using a bispecific antibody that pretargeted (90)Y-hapten-peptide radioimmunotherapy or a directly radiolabeled, humanized, (90)Y-anti-CD20 IgG (veltuzumab) could be improved by combining these treatments with unlabeled humanized antibodies against CD22 (epratuzumab), CD74 (milatuzumab), or veltuzumab. METHODS: Nude mice bearing established subcutaneous Ramos human Burkitt lymphoma were treated with antibodies alone or in combination with pretargeted radioimmunotherapy (PT-RAIT) or radioimmunotherapy, and tumor growth was monitored. Biodistribution studies examined the effect that predosing with unlabeled veltuzumab had on radioimmunotherapy and PT-RAIT targeting. RESULTS: None of the unconjugated antibodies was effective against established and rapidly growing xenografts, but PT-RAIT, at approximately 30% of its maximum tolerated dose, and radioimmunotherapy alone, at its maximum tolerated dose, were able to arrest growth and even entirely ablate tumors in some animals. Only combinations with veltuzumab improved therapeutic responses, most significantly when a veltuzumab regimen (weekly, 1.0 mg followed by 3 x 0.5 mg) was initiated 1 wk after PT-RAIT or (90)Y-veltuzumab. Biodistribution data indicated that when unlabeled veltuzumab (1.0 or 0.25 mg) was administered in advance of the radiolabeled veltuzumab or bispecific antibody injection, tumor uptake was significantly reduced ((111)In-veltuzumab, 47% and 25%, respectively; (111)In-hapten-peptide, 74% and 49%, respectively). Despite an approximately 50% decrease in radioactivity uptake in the tumor, antitumor responses were not diminished significantly for (90)Y-veltuzumab, and in the case of PT-RAIT responses were improved. However, higher amounts of predosed veltuzumab reduced the effects of PT-RAIT. CONCLUSION: These studies suggest that administering unlabeled anti-CD20 IgG therapy after the radioactivity dose provides the best efficacy and that the amount of unlabeled anti-CD20 IgG administered as a predose to anti-CD20-targeted radionuclide therapy should be minimized.

111In-LLP2A-DOTA Polyethylene Glycol-Targeting {alpha}4{beta}1 Integrin: Comparative Pharmacokinetics for Imaging and Therapy of Lymphoid Malignancies.

UNLABELLED: N-[[4-[[[(2-ethylphenyl)amino]carbonyl]amino]phenyl]acetyl]-N(epsilon)-6-[(2E)-1-oxo-3-(3-pyridinyl-2-propenyl)]-l-lysyl-l-2-aminohexanedioyl-(1-amino-1-cyclohexane)carboxamide (LLP2A) is a high-affinity, high-specificity peptidomimetic ligand (inhibitory concentration of 50% = 2 pM) that binds the activated alpha4beta1 integrin found on a variety of malignant lymphoid cell lines. To better determine whether this ligand holds promise for imaging and therapy in lymphoid malignancies, 6 LLP2A derivatives, as LLP2A-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (LLP2A-DOTA) and LLP2A-DOTA-polyethylene glycol (LLP2A-DOTA-PEG), were designed, synthesized, and radiolabeled with (111)In. Comparative pharmacokinetic studies in mice with Raji B-cell lymphoma xenografts were then complemented by small-animal PET of the lead molecular LLP2A format using (64)Cu-LLP2A-11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane ((64)Cu-LLP2A-CB-TE2A). METHODS: LLP2A-DOTA and LLP2A-CB-TE2A were prepared using solid-phase synthesis; LLP2A-DOTA-PEG(2,000), LLP2A-DOTA-PEG(5,000), LLP2A-DOTA-PEG(10,000), (LLP2A-DOTA)(2)PEG(10,000), and (LLP2A-DOTA)(4)PEG(10,000) were prepared by PEGylation. (111)In radiolabeling of DOTA and (64)Cu radiolabeling of CB-TE2A conjugates yielded 370-1,850 and 3,700-7,400 kBq/microg (10-50 and 100-200 microCi/microg), respectively. The pharmacokinetics of the six (111)In radioconjugates were studied in vivo using biodistribution data (4 and 24 h) and whole-body autoradiography (24 h) in mice with Raji tumor xenografts. (64)Cu-LLP2A-CB-TE2A was imaged (4 and 24 h) on a small-animal PET scanner in the same mouse model. RESULTS: The highest tumor uptake in pharmacokinetic studies was obtained with LLP2A-DOTA and (LLP2A-DOTA)(4)-PEG(10,000). For (111)In-LLP2A-DOTA (1 nM) at 4 and 24 h after injection, ratios of tumor to blood and tumor to nontumor (normal) organ (T/NT) were 8 to 35:1 for all organs or tissue except the spleen, marrow, and kidney, which were between 2:1 and 1:1. Tetravalent (LLP2A-DOTA)(4)-PEG(10,000) (1.1 nM) had tumor uptake similar to the univalent LLP2A-DOTA but higher liver, marrow, and kidney uptake. The excellent T/NT of LLP2A was also demonstrated by small-animal PET with (64)Cu-LLP2A-CB-TE2A at both 4 and 24 h after injection; obvious spleen targeting was apparent, but little kidney or liver activity was observed. CONCLUSION: Of the conjugates investigated, the univalent, non-PEGylated ligand (111)In-LLP2A-DOTA exhibited the best T/NT ratios and showed the greatest potential for imaging of alpha4beta1 in human lymphoma. Furthermore, this univalent non-PEGylated LLP2A format, as (64)Cu-LLP2A-CB-TE2A, demonstrated excellent tumor targeting by small-animal PET and warrants further investigation as an agent for the study of alpha4beta1 expression in human lymphoid malignancies.

Rituximab sensitizes a Burkitt lymphoma cell line to cell killing by X-irradiation.

Clinical trials with rituximab in combination with chemotherapeutic regimens have shown promising results. Data on the effects of rituximab treatment in combination with irradiation are, however, limited and inconsistent. This study aims to investigate the effects of rituximab (R) on cell death induced by X-irradiation in Raji lymphoma cells and to evaluate its mechanisms. We found the cell growth inhibition by irradiation was enhanced by additional rituximab exposure both in cells precultured with rituximab followed by irradiation (R + irradiation) or in cells treated in the reverse sequence (irradiation + R). R + irradiation combination treatment induced more apoptotic cells than irradiation and irradiation + R treatment as early as 12 h after treatment. At 24 h, both combination treatments, R + irradiation and irradiation + R, showed apoptotic cells, which were significantly different from irradiation alone. G2/M cell cycle arrest was observed after irradiation alone and the combination treatment. The combination treatment revealed an elevation in reactive oxygen species (ROS) generation in a radiation dose-dependent manner. In addition, rituximab enhanced the cell growth inhibition and apoptotic cell death induced by the oxidative agent, H(2)O(2). We propose that rituximab mediates a significant in vitro radiosensitizing effect and induces cell cycle changes and apoptosis in Raji cells. ROS probably play an important role in these events.

t(8;14;18): a 3-way chromosome translocation in two patients with Burkitt's lymphoma/leukemia.

Burkitt's lymphoma (BL) is a heterogeneous group of highly aggressive mature B-cell malignancies. It is characterized by a high rate of turnover of malignant cells and deregulation of the c-myc gene. It is typically associated with a t(8;14) translocation. Dual translocation of t(8;14) (c-myc) and t(14;18) (bcl-2) was reported to be associated with extremely poor prognosis. This study reports a novel t(8;14;18) triple translocation in two patients with Burkitt's lymphoma.

Absence of prognostic impact of CD13 and/or CD33 antigen expression in adult acute lymphoblastic leukemia. Results of the GIMEMA ALL 0496 trial.

BACKGROUND AND OBJECTIVES: The prognostic value of myeloid antigen (MyAg) expression in adult acute lymphoblastic leukemia (ALL) is still controversial. The aim of this study was to correlate the expression of MyAg with clinical, hematologic and biological parameters, and to analyze the impact on response to treatment and prognosis in a large series of adult ALL uniformly characterized and treated. DESIGN AND METHODS: We analyzed the expression of the MyAg CD13 and/or CD33 in a cohort of 377 adult patients with de novo ALL enrolled and treated in the GIMEMA ALL 0496 protocol. RESULTS: MyAg expression was documented in 35% of the 377 adult ALL cases analyzed. MyAg were significantly more frequently associated with B-lineage ALL (38%) than with T-ALL (24%) (p=0.02). No difference was found with regard to clinical features at presentation; a difference was found only for white cell count (p=0.03), percentage of peripheral blasts (p=0.004) and platelet count (p=0.004). No difference was observed in the expression of MyAg between patients with normal or abnormal cytogenetics or between those with high-risk (BCR-ABL+, ALL1-AF4+, E2A-PBX1+) or low-risk B-lineage ALL. We failed to observe any difference between MyAg-positive and MyAg-negative cases in terms of achievement of complete remission, disease-free survival and overall survival at 5 years. INTERPRETATION AND CONCLUSIONS: Our data indicate that ALL MyAg expression in adults with ALL is not associated with adverse presenting clinical and biological features, and that response to treatment and prognosis is comparable in MyAg-positive and MyAg-negative ALL patients with regards to both complete remission rate and overall survival. We suppose that these result are due to more intensive treatment modalities adopted in the GIMEMA ALL 0496 protocol.

Linfoma de Burkitt Primário de Mama: Relato de Caso / Primary Breast Burkitt Lymphoma: Case Report / Linfoma de Burkitt Primario de Mama: Relato de Caso

Introdução: O linfoma primário de mama (LPM) representa cerca de 0,5% das neoplasias mamárias, sendo considerado um tipo raro de tumor. Alguns dos tipos de LPM, por sua vez, possuem ligação intensa com o período gravídico e pós-parto em virtude do estímulo hormonal. O objetivo deste estudo é relatar um caso de LPM com apresentação rara de linfoma de Burkitt, considerando propostas terapêuticas eficazes para o seguimento. Relato do caso: Paciente do sexo feminino, 23 anos, portadora de tumoração periareolar em mama direita com aspecto de casca de laranja e crescimento rápido há um mês, queixas flogísticas no local da lesão, bom estado geral e sem outros sintomas associados. O quadro relatado iniciou-se sete meses após a expulsão fetal com feto morto. A paciente foi submetida à biópsia da lesão e encaminhada para seguimento ambulatorial sem sucesso, necessitando de retorno ao ambiente hospitalar por piora das condições clínicas e extensão da tumoração. Houve diagnóstico de linfoma de Burkitt, com tratamento multidisciplinar, sendo submetida a protocolo CODOX-M de quimioterapia, com óbito após 22 dias de acompanhamento hospitalar. Conclusão: Este relato demonstra uma situação rara em uma paciente jovem, ressaltando a importância de investigar as alterações mamárias, de maneira eficaz, para um diagnóstico precoce correto e um tratamento adequado, em todas as faixas etárias

Introduction: Primary breast lymphoma (PML) represents about 0.5% of breast cancers, being considered a rare type of tumor. Some of the types of PML, in turn, have an intense connection with the pregnancy and postpartum period due to hormonal stimulation. The aim of this study is to report a case of PML with a rare presentation of Burkitt's lymphoma, considering effective therapeutic proposals for follow-up. Case report: A 23-year-old female patient with a peri-areolar tumor in the right breast with orange peel aspect and rapid growth for one month, phlogistic complaints at the lesion site, good general condition and no other associated symptoms. The reported condition started seven months after fetal expulsion with a dead fetus. The patient underwent lesion biopsy and was referred to an outpatient follow-up with unsuccessful outcome, requiring return to the hospital due to worsening of clinical conditions and extension of the tumor. There was a diagnosis of Burkitt's lymphoma, with multidisciplinary treatment. She underwent the CODOX-M chemotherapy protocol, and died after 22 days of hospital follow-up. Conclusion: This report demonstrates a rare situation in a young patient, emphasizing the importance of effective investigation of breast changes so that correct early diagnosis and appropriate treatment can be made for all age groups

Introducción: El linfoma primario de mama (LMP) representa aproximadamente el 0,5% de los cánceres de mama, siendo considerado un tipo de tumor poco común. Algunos de los tipos de LPM, a su vez, tienen una conexión intensa con el embarazo y el posparto debido a la estimulación hormonal. El objetivo de este estudio es reportar un caso de LMP con rara presentación de linfoma de Burkitt, considerando propuestas terapéuticas efectivas para el seguimiento. Relato del caso: Paciente, 23 años, con un tumor periareolar en la mama derecha con aspecto de piel de naranja y rápido crecimiento durante un mes, quejas flogísticas en el sitio de la lesión, buen estado general y ningún otro. síntomas asociados. La condición reportada comenzó siete meses después de la expulsión fetal con un feto muerto. El paciente fue sometido a biopsia de la lesión y fue derivado sin éxito a seguimiento ambulatorio, requiriendo el retorno al entorno hospitalario por empeoramiento de la clínica y extensión del tumor. Hubo un diagnóstico de linfoma de Burkitt, con tratamiento multidisciplinario. Se sometió al protocolo de quimioterapia CODOX-M y murió a los 22 días de seguimiento hospitalario. Conclusión: Este informe demuestra una situación poco común en una paciente joven, enfatizando la importancia de investigar de manera efectiva los cambios en los senos para un diagnóstico temprano correcto y un tratamiento adecuado en todos los grupos de edad

Therapy with unlabeled and 131I-labeled pan-B-cell monoclonal antibodies in nude mice bearing Raji Burkitt's lymphoma xenografts.

Clinical trials of radioimmunotherapy (RIT) of lymphoma have produced frequent tumor regressions and remissions, but it has been difficult to determine to what extent these tumor responses have been due to antibody-specific targeted radiation, nontargeted radiation, and/or cytotoxicity mediated by the carrier monoclonal antibody (MoAb). In this report, RIT was studied in athymic nude mice bearing s.c. Raji human Burkitt's lymphoma xenografts using two different pan-B-cell MoAbs, MB-1 (anti-CD37) and anti-B1 (anti-CD20), which differ in isotype (and thus the potential for interaction with host effector mechanisms) and isotype-matched control antibodies either in the unlabeled state or labeled with 131I. When a single i.p. injection of 300 microCi 131I-labeled MB-1 (IgG1) was compared to treatment with unlabeled MB-1 or 300 microCi 131I-labeled MYS control IgG1 MoAb, an antibody-specific targeted radiation effect of RIT was seen. 131I-labeled MB-1 produced a 44 +/- 19% (SEM) reduction in tumor size at 3 weeks posttreatment, while unlabeled MB-1 or 300 microCi 131I-labeled MYS control IgG1 antibody treatment resulted in continued tumor growth over this period of time. In vitro studies demonstrated that MB-1 was incapable of mediating antibody-dependent cellular cytotoxicity using Raji tumor cell targets and human peripheral blood mononuclear cells. Similar to the MB-1 studies, treatment with 300 microCi 131I-labeled anti-B1 produced a 64% reduction in mean tumor size, while 300 microCi of control antibody resulted in a 58% increase in tumor size over the same 3-week period. In contrast to MB-1, however, unlabeled anti-B1 (an IgG2a MoAb which in vitro studies showed to be capable of antibody-dependent cellular cytotoxicity) also had a substantial antitumor effect. Indeed, 300 microCi 131I-labeled anti-B1 and unlabeled anti-B1 treatment (using an equivalent amount of total protein in the treatment dose) produced a similar specific reduction in tumor size. Increasing the radionuclide dose of anti-B1 to 450 microCi in another experiment did not produce a significant difference in tumor regression compared to a 300-microCi dose. These results suggest that the antitumor effects of 131I-labeled anti-B1 treatment were dominated by antibody-mediated cytotoxicity mechanisms, such that an antibody-specific targeted radiation effect could not be distinguished. In contrast, antibody-specific targeting of radiation was the dominant mechanism of tumor killing with 131I-labeled MB-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Resistance to radiation-induced apoptosis in Burkitt's lymphoma cells is associated with defective ceramide signaling.

Increased sensitivity to ionizing radiation has been shown to be due to defects in double-strand break repair and mutations in the proteins that detect DNA damage. However, it is now recognized that the cellular radiation response is complex and that radioresistance/radiosensitivity may also be regulated at different levels in the radiation signal transduction pathway. Here, we describe a direct relationship between resistance to radiation-induced apoptosis and defective ceramide signaling. Radiation sensitivity in human tumor cells correlated with the immediate accumulation of the second messenger ceramide. In the BL30A Burkitt's lymphoma line, ceramide increased 4-fold by 10 min postirradiation (10 Gy), and in the moderately sensitive HL-60 leukemia cells, ceramide accumulated 2.5-fold above basal levels. In contrast, in all radioresistant tumor cells examined, including several Burkitt's lymphoma lines (BL30K, BL29, and BL36) and the MO59K glioma cell line, ceramide did not accumulate postirradiation. The ability to abrogate ceramide production by pretreatment with the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, conferred resistance to radiation-induced apoptosis in the sensitive BL30A cells. An isogenic subline of BL30A, BL30K, was resistant to both C8-ceramide (20 microM) and ionizing radiation-induced apoptosis. Bypassing the block in radiation-induced ceramide production by the addition of exogenous ceramide was not sufficient to induce apoptosis; this suggests the existence of a second ceramide-associated signaling defect in these radioresistant cells that confers resistance to ceramide-induced apoptosis. Thus, these results provide compelling evidence that ceramide is an essential mediator of radiation-induced apoptosis and that defective ceramide signaling confers an apoptosis-resistant phenotype in tumor cells.

Relationships between G1 arrest and stability of the p53 and p21Cip1/Waf1 proteins following gamma-irradiation of human lymphoma cells.

We investigated temporal relationships between ionizing radiation-induced G1 arrest and induction of the p53-regulated genes GADD45, CIP1/WAF1, and MDM2 in a series of Burkitt's lymphoma and lymphoblastoid cell lines that differed in p53 gene status. Emphasis was placed on characterization of the EW36 cell line, which despite expressing wild-type p53 genes, is defective in G1 arrest following gamma-irradiation (P. M. O'Connor et al., Cancer Res., 53: 4776-4780, 1993). Induction of CIP1/WAF1, GADD45, and to a lesser extent MDM2 mRNA was observed in all wild-type p53 lines that arrested in G1. Cell lines that contained only mutant p53 genes or were heterozygous for p53 mutations failed to induce appreciable levels of these p53-regulated transcripts and did not arrest in G1. G1 arrest in the wild-type p53 cell line WMN was more prolonged than elevation of CIP1/WAF1, GADD45, or MDM2 transcripts, suggesting that G1 arrest duration must be dependent upon stability of these newly synthesized proteins. In agreement, we found that p21Cip1/Waf1, a potent inhibitor of G1-S phase cyclin-dependent kinases, was maintained at elevated levels throughout the period that WMN cells remained arrested in G1. EW36 cells exhibited normal induction of CIP1/WAF1, GADD45, and MDM2 mRNA following gamma-irradiation, suggesting that the defect in G1 arrest must reside downstream of p53 transactivation. Investigations into the stability of p53 and p21Cip1/Waf1 revealed that EW36 cells failed to maintain elevated levels of these proteins following irradiation. p53 levels decreased within 4 h of irradiation, and p21Cip1/Waf1 levels decreased shortly after the normal decline of CIP1/WAF1 mRNA levels. Degradation of p21Cip1/Waf1 coincided with the escape of EW36 cells from G1 arrest. Our studies suggest that p21Cip1/Waf1 stability may determine G1 arrest duration and that premature degradation of this protein could provide an alternative route to subversion of the G1 checkpoint in cancer cells.

Preclinical evaluation of a diabody-based (177)Lu-radioimmunoconjugate for CD22-directed radioimmunotherapy in a non-Hodgkin lymphoma mouse model.

Radioimmunotherapy is considered as treatment option in recurrent and/or refractory B-cell non-Hodgkin lymphoma (B-NHL). To overcome the dose limiting bone marrow toxicity of IgG-based radioimmunoconjugates (RICs), we modified a humanized diabody with 5-, 10-, or 20-kDa polyethylene glycol (PEG) for CD22-targeted radioimmunotherapy using the low-energy ß-emitter lutetium-177 ((177)Lu). A favorable pharmacokinetic profile was observed for the 10-kDa-PEG-diabody in nude mice being xenografted with subcutaneous human Burkitt lymphoma. Even at high doses of 16 MBq this diabody RIC was well tolerated by NOD Rag1(null) IL2rγ(null) (NRG) mice and did not reveal signs of organ long-term toxicity 80 days post injection. Combination therapy of the diabody RIC with unconjugated anti-CD20 Rituximab demonstrated therapeutic efficacy in established disseminated mantle cell lymphoma xenograft models. When compared with the combination of the IgG formatted (177)Lu anti-CD22 antibody and Rituximab, dual targeted therapy with the diabody RIC achieved an improved reduction of disease burden in the first nine days following treatment. The data indicate that the PEGylated anti-CD22 diabody may have potential for extending the repertoire of radiopharmaceuticals for the treatment of patients with B-NHL.

Antiviral agent cidofovir decreases Epstein-Barr virus (EBV) oncoproteins and enhances the radiosensitivity in EBV-related malignancies.

The Epstein-Barr virus (EBV) is involved in the carcinogenesis of several human cancers such as nasopharyngeal carcinoma (NPC) and Burkitt lymphoma (BL). Given the consistent role of EBV in transformation and maintenance of malignant phenotype, antiviral strategies provide an attractive approach to target EBV-expressing cells. In that aim, we have tested the Cidofovir, which is an acyclic nucleoside phosphonate analog known to exert an antiproliferative activity in some human virus-related tumors. Here, we show that Cidofovir induces a downregulation of the EBV oncoprotein LMP1 associated with a decrease of the antiapoptotic Bcl-2 and an increase of the proapoptotic Bax protein in Raji (BL) and C15 (NPC) cells. Using BL cell line BL2 B95-8 (BL2 infected with the B95.8 strain of EBV), we addressed the relation between EBV genome expression and modulation of viral oncoproteins by Cidofovir and/or ionizing radiation (IR). Cidofovir was able to significantly reduce LMP1 and EBNA2 mRNA and protein expression. This effect was associated with inhibition of proliferation, stimulation of apoptosis, and decrease of Bcl-2 expression in BL2 B95.8 cells. In addition, Cidofovir enhanced the radiation-induced apoptosis and the radiosensitivity through the proteolytic cleavage of death effectors caspase-9 and -3, which was specifically induced by combined treatment in EBV-positive cells compared to their negative counterparts. Furthermore, the combined treatment in nude mice led to a complete tumor remission without increasing toxicity in two human EBV-related cancer xenografts (Raji and C15). These results provide the basis for a novel anticancer strategy to enhance the therapeutic ratio of IR in EBV-related cancers.

Combined modality therapy for adults with small noncleaved cell lymphoma (Burkitt's and non-Burkitt's types).

Between June 1979 and June 1984 18 adult patients with small noncleaved cell lymphoma (SNCL) (diffuse undifferentiated lymphoma, Burkitt's and non-Burkitt's types of the Rappaport classification) were treated with high-dose cyclophosphamide, doxorubicin, vincristine, prednisone, midcycle high-dose methotrexate, and intrathecal methotrexate. Early in the course of treatment, hyperfractionated radiotherapy (125 cGy, every two days, for 1,500 to 2,250 centigray [cGy]) was administered to unresected masses greater than 10 cm in their greatest dimension. Chemotherapy was administered every 21 days for six to ten cycles. Treatment was generally well tolerated; however, one patient died of probable tumor lysis syndrome. With a median follow-up of 1.2 years, actuarial survival was 66.8% and relapse-free survival (RFS) was 71.3% for the entire group. All treatment failures and deaths occurred in patients with stage D disease. RFS projected at 2 years was 100% for stages A and AR and 60.6% for stage B, C, and D (P = .13 Gehan). Two-year RFS for patients with stage A, AR, B, or C disease was 100 v 41% for those with stage D disease. Patients with adverse prognostic features (n = 7)--unresected bulk measuring greater than 10 cm, pretreatment serum lactate dehydrogenase (LDH) 500 IU/L (normal, 200) or involvement of CNS or bone marrow--had a projected RFS of 28.6% compared with 100% for those patients without these features (P = .002 Gehan). Too few patients received induction radiotherapy to assert its role in therapy. By using aggressive multiagent therapy, cure can be expected in a high percentage of adults with SNCL. In the subset with adverse prognostic features, more effective therapy is necessary.

Pretreatment with interferon-alpha radiosensitizes Daudi cells modulating gene expression and biomarkers.

BACKGROUND: Interferon (IFN) potentiates cytotoxicity by X-ray irradiation. To elucidate the mechanism of this potentiation, the biological markers related to DNA damage and cell survival were studied. MATERIALS AND METHODS: IFN-alpha-sensitive Daudi and its resistant cells were used. Survival after treatment was assessed by clonogenic assays. DNA breaks were studied by pulse-field gel electrophoresis (PFGE). Production of reactive oxygen metabolites was measured using flow cytometry. Messenger RNA and protein were examined by RT-PCR and immunoblot, respectively. RESULTS: IFN-alpha treatment for 24 h before irradiation potentiated the sensitivity of Daudi cells to X-rays. This combination induced 50 kb DNA fragmentation and activated caspase-3 in Daudi cells. Pretreatment with IFN-alpha inhibited the production of reactive oxygen species by irradiation. IFN-alpha pretreatment down-regulated most of the double-strand break (DSB) repair-related mRNAs, but did not affect the repair of DSBs studied by PFGE. The induction and phosphorylation of p21(Cip1/WAF1) (p21) was prominently suppressed in cells pretreated with IFN-a. CONCLUSION: Pretreatment with IFN-alpha potentiates the cytotoxic effects of X-rays. Inhibition of X-ray-induced p21 may cause the augmented sensitivity by IFN-alpha pretreatment.

Radioimmunotherapy for model B cell malignancies using 90Y-labeled anti-CD19 and anti-CD20 monoclonal antibodies.

In recent years, radioimmunotherapy (RIT) with beta(-) particle emitting radionuclides targeting the CD20 antigen on B cells in the treatment of non-Hodgkin's lymphoma has provided the most compelling human clinical data for the success of RIT. CD19, like CD20, is an antigen expressed on the surface of cells of the B lineage, and CD19 may provide an alternative target for radioimmunotherapy of B cell neoplasms. CD19 has been largely overlooked as a target for conventional 131I RIT, because the antigen rapidly internalizes upon binding of antibody, resulting in catabolism and significant release of 131I. Such modulation may be an advantage to RIT with radiometals such as 90Y, 177Lu, 213Bi and 225Ac. Herein, we have compared beta(-) particle RIT with antibodies targeting either CD19 or CD20. The anti-CD19 and anti-CD20 antibodies, B4 or C2B8, respectively, were appended with the SCN-CHX-A''-DTPA bifunctional chelating agent and labeled with 90Y. In the tumor model used, there were three times as many CD20 target sites on lymphoma cells as compared to CD19 sites (62000 vs 20000 binding sites, respectively). We compared the efficacy of the 90Y-labeled antibodies to reduce lymphoma in a nude mouse xenograft solid tumor model, after measurable lymphoma appeared. Reduction in tumor size began at day 3 in all three 90Y-treated groups, but tumor began to recur in many animals 9 days after the treatments. There was one cure in each specific treatment group. In contrast, the tumor in the two control groups showed no regression. There was a significant prolongation of median survival time from xenograft (P < 0.0001) in all the 90Y-labeled antibody construct-treated groups (32 days for 0.15 mCi 90Y-B4; 26 days for 0.20 mCi 90Y-C2B8, and 23 days for 0.15 mCi 90Y-C2B8) in comparison to the two control groups (11 days for 0.02 mg of C2B8 and 9 days for untreated growth controls). Specificity of the radioimmunotherapy was also shown. In conclusion, 90Y-labeled anti-CD19 antibody has efficacy comparable to 90Y-labeled anti-CD20 antibody in the treatment of mice bearing human lymphoma xenografts. These data suggest that CD19-targeted RIT merits further study.