A phase 3b study of sofosbuvir plus ribavirin in treatment-naive and treatment-experienced Korean patients chronically infected with genotype 2 hepatitis C virus.

In Korea, patients with chronic hepatitis C virus (HCV) infection are typically treated with pegylated interferon-alpha plus ribavirin, but interferons are contraindicated in many patients and are often poorly tolerated, particularly by the elderly and those with advanced liver disease. No interferon-free treatment regimens are approved in Korea. Sofosbuvir is an oral nucleotide analog inhibitor of the HCV nonstructural 5B RNA polymerase. It is approved in the USA, European Union and Japan for treating a number of HCV genotypes, including genotype 2. Genotype 2 has a seroprevalence of 38-46% in Korea. This single-arm, phase 3b study (NCT02021643) examined the efficacy and safety of sofosbuvir plus ribavirin (12-week duration) in chronic genotype 2 HCV-infected treatment-naive and treatment-experienced Korean patients with and without cirrhosis. The proportion of patients with sustained virologic response 12 weeks after treatment discontinuation (SVR12) was 97% (125/129), with 96% (101/105) of treatment-naive and 100% (24/24) of treatment-experienced patients achieving SVR12. Two patients experienced virologic failure (n = 1, on-treatment failure; n = 1, relapse). No patient discontinued study treatment due to an adverse event (AE). The most common treatment-emergent AEs were headache (18%, 23/129) and pruritus (15%, 19/129). Few patients had grade 3 AEs (5%, 6/129) or grade 3 laboratory abnormalities (12%, 15/129). No grade 4 AE was reported. These data suggest that 12 weeks of treatment with the all-oral, interferon-free regimen of sofosbuvir plus ribavirin is effective and well tolerated in Korean patients with chronic genotype 2 HCV infection.

Overview of studies on experimental radioimmunotherapy.

Experimental radioimmunotherapy (RIT) has significantly contributed to the development of clinical RIT. In this overview, the current status of experimental RIT is reviewed, including general principles of RIT and determinants of RIT effects. Areas of active research are reviewed, and the usefulness of multicell spheroids is compared to animal models. The radiobiology of RIT is discussed, and studies that have compared the relative efficacy of RIT with external beam radiation therapy are summarized. Approaches for increasing the therapeutic index of RIT are reviewed, including improvements in antibodies, labeling/chelation chemistry, selection of radionuclides, delivery, fractionated therapy, clearance of unbound radiolabeled monoclonal antibodies, protection of normal tissues, radiosensitization of tumors, utilization of colony-stimulating factors and bone marrow transplantation, and the use of novel targets for RIT and topoisomerase I inhibitors. RIT is a promising new therapy for a wide variety of malignancies that can best be optimized by continued research in the field of experimental RIT. Important areas of future research are discussed that may ultimately potentiate the efficacy and decrease the toxicity of RIT and help determine how to optimally combine RIT with other therapeutic modalities.

The effect of stem cell factor on irradiated human bone marrow.

This study evaluates the effect of recombinant human stem cell factor (SCF) on the in vitro response of human bone marrow progenitor cells to irradiation. Light density nonadherent mononuclear cells were isolated from human bone marrow and resuspended in either semisolid culture or liquid culture with or without 100 ng/ml SCF. After 24 h in culture, cells were irradiated and assessed for survival of erythroid burst-forming unit, granulocyte colony-forming unit(s), or granulocyte-macrophage colony-forming unit precursors in the presence of erythropoietin, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor, respectively. Incubation with SCF prior to irradiation (0-300 cGy) resulted in an increase in both absolute colony number and surviving fraction for erythroid burst-forming units, granulocyte colony-forming units, and granulocyte-macrophage colony-forming units as compared to cultures that did not contain SCF. The mean surviving fraction enhancement ratio after 100 cGy ranged from 1.2 to 3.7. An increased fraction of CD34+ progenitors in S-phase after exposure to SCF may explain in part the apparent radioprotective effect of SCF on human bone marrow progenitor cells.

Bcl-2 overexpression results in enhanced capacitative calcium entry and resistance to SKF-96365-induced apoptosis.

Although there is evidence that changes in cellular ionic concentrations are important early events in apoptosis, the regulation of ion fluxes across the plasma membrane during this process is poorly understood. We report here that Bcl-2 overexpression results in up-regulation of capacitative Ca2+ entry (CCE) and that SKF-96365, an inhibitor of CCE, is a potent inducer of apoptosis. Cells that overexpress Bcl-2 are resistant to SKF-96365-mediated apoptosis and to its inhibition of CCE. Enhanced CCE can be reversed with ouabain, suggesting that Bcl-2-associated plasma membrane hyperpolarization plays a role in up-regulating CCE and may partially explain the antiapoptotic effect of Bcl-2.

Local hyperthermia and SR 4233 enhance the antitumor effects of radioimmunotherapy in nude mice with human colonic adenocarcinoma xenografts.

Local hyperthermia and the hypoxic cytotoxin SR 4233 were administered to nude mice with 693 +/- 47 mm3 (mean +/- SE) s.c. HCT-8 human colonic adenocarcinoma xenografts in an attempt to enhance the antitumor effects of radioimmunotherapy. Biodistribution studies revealed preferential binding of NR-Lu-10, a murine monoclonal antibody, to the tumors compared with an isotype-matched control antibody, CCOO16-3.A single injection of 25 microCi 90Y-NR-Lu-10 significantly inhibited tumor growth (control versus 90Y-NR-Lu-10: P = 0.048). The administration of hyperthermia at 41.5 degrees C for 1 h immediately following the injection of 111In-labeled NR-Lu-10 up-regulated tumor-associated antigen expression and increased antibody uptake in the tumors by 73% (P = 0.001) without significantly affecting antibody uptake in normal tissues. However, the heat treatment did not produce a more homogeneous distribution of the antibodies in the tumors and did not significantly enhance the tumor growth delay produced by 90Y-NR-Lu-10 (P = 0.07). The administration of local hyperthermia at 43.0 degrees C for 1 h, on the other hand, had direct cytotoxic effects (P = 0.03) and enhanced the tumor growth delay produced by 90Y-NR-Lu-10 (P = 0.01). SR 4233 also enhanced the tumor growth delay produced by 90Y-NR-Lu-10 (P = 0.03). The greatest antitumor effects were observed when both hyperthermia at 43.0 degrees C and SR 4233 were administered in combination with 90Y-NR-Lu-10 (P = 0.002). No toxicity was produced by the local hyperthermia, and the only toxicities produced by 90Y-NR-Lu-10 and SR 4233 were neutropenia and weight loss.

Effects of stem cell factor on the growth and radiation survival of tumor cells.

Recombinant human stem cell factor (SCF) binds to the c-kit receptor on human bone marrow progenitor cells and enhances their survival following irradiation. Since the c-kit receptor has also been detected on malignant cells, experiments were performed to study the effect of SCF on the proliferation and radiation survival of a variety of both c-kit-positive and -negative human tumor cell lines using [3H]thymidine incorporation and colony formation assays. The addition of SCF to both c-kit-positive and -negative cell line cultures had no significant effect on the stimulation index (in [3H]thymidine assay). In contrast, colony formation by H69 (small cell lung cancer cell line), H128 (small cell lung cancer cell line), and HEL (erythroid leukemia cell line) cells was enhanced by SCF in a dose-dependent manner, but SCF did not promote the in vivo growth of H128 xenograft tumors in terms of graft rate, time from implantation to tumor detection, or tumor size. Furthermore, SCF did not significantly increase the surviving fraction of either c-kit-positive or -negative cell lines following radiation, and there were no statistically significant differences between D0 [defined by the slope of the terminal exponential region of the two-component (single-hit multitarget model) survival curve where slope = 1/D0], Dq (quasithreshold dose), n (extrapolation number), alpha, and beta values for any of the cell lines studied that were irradiated with and without SCF. Finally, nude mice with transplanted human LG425 cutaneous T-cell lymphoma (c-kit positive) were treated with 10 Gy with or without SCF (100 micrograms/kg i.p. 20 h before, 2 h before, and 4 h after irradiation). There were no significant differences in the median tumor quadrupling time between groups that received either no treatment or SCF alone, or between groups treated with 10 Gy and SCF or 10 Gy alone (P > 0.05). These results are encouraging and suggest that SCF does not stimulate tumor cell proliferation in vivo or enhance the survival of tumor cells following irradiation.

Modulation of c-Myc activity and apoptosis in vivo.

We have developed an animal tumor model system to study the effects of c-Myc activation on apoptosis induction in vivo. Tumors were generated in SCID mice from Rat-1 fibroblasts that constitutively express an inactive c-Myc-estrogen receptor fusion protein (T.D. Littlewood et al, Nucleic Acids Res., 23: 1686 -1690, 1995), which is activated in vivo by the administration of 4-hydroxytamoxifen in time release pellets. We demonstrate that activation of c-Myc results in a substantial increase in the number of apoptotic tumor cells and that this apoptosis is predominant in regions of tumor hypoxia. c-Myc-induced apoptosis of hypoxic cells is inhibited in tumors that overexpress the human Bcl-2 protein. Bcl-2, however, does not prevent p53 protein accumulation or the down-regulation of the cyclin-cdk inhibitor p27 protein following c-Myc activation by 4-hydroxytamoxifen. This result suggests that Bcl-2 does not affect c-Myc function directly but acts downstream of c-Myc to inhibit apoptosis. We propose that the ability of activated c-Myc to enhance cellular proliferation might contribute to the genesis of early neoplasms that are held in check by the alternate ability of c-Myc to induce apoptosis of cells that have outgrown their supply of oxygen or other factors associated with hypoxic regions of solid tumors. Secondary genetic lesions downstream of c-Myc that suppress the apoptotic potential of tumor cells, such as Bcl-2 overexpression, might play an important role in the malignant progression of these tumors because they would disrupt the balance between apoptosis and proliferation initiated by c-Myc deregulation.

Direct evidence that apoptosis enhances tumor responses to fractionated radiotherapy.

Currently, the contribution of cellular apoptotic sensitivity to tumor response after radiation therapy remains controversial. To address this issue, the survival of Rat-1 fibroblasts containing a 4-hydroxytamoxifen-regulated c-Myc allele, c-MycER (T. D. Littlewood et al., Nucleic Acids Res., 23: 1686-1690, 1995), after single and fractionated doses of radiation was investigated. This model system allows pharmacological regulation of apoptosis sensitivity in the same cells in vitro and as xenograft tumors derived from these cells in vivo (G. I. Evan et al., Cell, 69: 119-128, 1992; R. M. Alarcon et al., Cancer Res., 56: 4315-4319, 1996). Activating c-MycER in vitro resulted in marked sensitization of Rat-1 fibroblasts to the effects of both single-dose and fractionated irradiation as measured by the induction of apoptosis and clonogenic survival. Overexpression of the antiapoptosis protein Bcl-2 suppressed the induction of apoptosis and increased clonogenic survival in cells with activated c-Myc after single-dose and fractionated radiation. Systemic time-release implant delivery of 4-hydroxytamoxifen to severe combined immunodeficient mice bearing Rat-1-MycER tumors over the course of either single-dose (10 Gy) or fractionated (five fractions of 2 Gy) radiotherapy resulted in prolonged tumor growth delay relative to identical tumors from mice that received placebo implants. Furthermore, tumors derived from Rat-1-MycER cells that overexpressed Bcl-2 exhibited shorter tumor growth delays relative to similarly treated Rat-1-MycER tumors. The length of tumor growth delay after single-dose or fractionated radiotherapy strongly correlated with the extent of radiation-induced apoptosis in the xenograft tumors as measured by terminal deoxynucleotidyl transferase-mediated nick end labeling. These in vivo results provide direct evidence that increasing the sensitivity of tumor cells to die by apoptosis increases the efficacy of fractionated radiotherapy by reducing tumor cell clonogenic survival.

Determinants of the antitumor effect of radiolabeled monoclonal antibodies.

The murine B-cell lymphoma 38C13 model was used to study the radiobiological effect of 131I-monoclonal antibody (MAB) therapy compared with dose equivalent external beam irradiation. Continuous exponentially decreasing low dose rate (LDR) gamma-irradiation, and multiply fractionated (MF) X-irradiation were compared with dose equivalent 131I-MAB. The relative therapeutic efficacy of radioimmunotherapy, and the relative contribution of (a) low dose rate; (b) whole body irradiation; and (c) microdosimetry to the overall effect were determined. Groups of mice with or without B-cell lymphoma were treated with either (a) 131I-anti-idiotype MAB; (b) 131I-isotype-matched irrelevant control MAB; (c) 5-15 Gy 250 kV X-irradiation given as a single fraction; (d) 2.5-30 Gy 250 kV X-irradiation given in 10 fractions/2 weeks; or by (e) continuous exponentially decreasing gamma-irradiation via a 137Cs source, which simulated the effective t1/2 of the 131I-MAB. In tumor-free mice the LD50/30 was approximately 10 Gy for MF and LDR external irradiation, and 11-12 Gy for 131I-MAB. However, the effect of these modes of irradiation on tumor size differed significantly. The cumulative percentage of tumor reduction averaged over 12 days was 0.635 +/- 0.055%/Gy for MF, and 1.36 +/- 0.061%/Gy for LDR external irradiation (a relative efficacy factor of 1.63 for LDR irradiation; P = 0.01). Assuming homogeneous body distribution, the tumor reduction effect over 12 days for 131I-MAB was 2.064 +/- 0.133%/Gy for specific, and 1.742 +/- 0.1%/Gy for nonspecific isotype-matched irrelevant 131I-MAB (P = 0.02). When 131I-MAB was compared to LDR external irradiation, the relative efficacy factor was 1.99 (P less than 0.001). In summary, there was a dose rate effect on tumor response, which may in part explain the efficacy of radioimmunotherapy. The additional effect of 131I-MAB on tumor response was only partially explained by the cumulative concentration ratio of 131I-MAB tumor/131I-MAB whole body, which was on average 1.7. This relatively low concentration ratio was partly due to tumor-mediated dehalogenation. Thus, the overall tumor response was a function of the total dose, dose rate, and both the specific and nonspecific distribution of 131I-MAB.

Sofosbuvir plus ribavirin for the treatment of patients with chronic genotype 1 or 6 hepatitis C virus infection in Hong Kong.

BACKGROUND: In Hong Kong, most patients with hepatitis C virus (HCV) have either genotype 6a or 1b infection. AIM: To evaluate the efficacy and safety of sofosbuvir with ribavirin in treatment-naïve patients in Hong Kong with HCV genotype 1 or 6. METHODS: In an open-label study, patients were randomised to sofosbuvir 400 mg once daily plus ribavirin 1000-1200 divided twice daily for 12 (n = 10), 16 (n = 11) or 24 (n = 10) weeks. The primary endpoint was the percentage of patients with HCV RNA < LLOQ (lower limit of quantification, 25 IU/mL) 12 weeks after cessation of therapy (SVR12). RESULTS: All 31 patients (20 HCV genotype 1 and 11 genotype 6) had HCV RNA < LLOQ by Week 4 of treatment and at their last on-treatment visit. SVR12 rates were high in all treatment groups: 100% (10/10) for 12 weeks, 100% (11/11) for 16 weeks and 90% (9/10) for 24 weeks of therapy. The only patient who did not reach SVR12 had genotype 1 HCV and relapsed at post-treatment Week 4. Sofosbuvir with ribavirin was generally well tolerated. The most common adverse events were malaise (13%) and upper respiratory tract infection (13%), followed by anaemia (10%). No patients experienced serious adverse events. One patient discontinued treatment at Week 16 because of an adverse event. The event, upper respiratory tract infection, was not considered treatment related by the investigator. This subject achieved SVR12. CONCLUSIONS: The all-oral regimen sofosbuvir plus ribavirin is effective in treatment-naïve patients in Hong Kong with genotype 1 or 6 HCV. TRIAL REGISTRATION NUMBER: NCT02021643.

p53 mediates apoptosis induced by c-Myc activation in hypoxic or gamma irradiated fibroblasts.

Deregulated c-Myc expression leads to a cellular state where proliferation and apoptosis are equally favored depending on the cellular microenvironment. Since the apoptotic sensitivity of many cells is influenced by the status of the p53 tumor suppressor gene, we investigated whether the induction of apoptosis by DNA damage or non-genotoxic stress are also influenced by the p53 status of cells with altered c-Myc activity. Rat-1 fibroblasts expressing a conditional c-Myc allele (c-MycER), were transfected to express an antisense RNA complimentary to p53 mRNA. Expression of antisense p53 RNA decreased p53 protein levels and delayed p53 accumulation following c-Myc activation. Under hypoxic or low serum conditions, cells expressing antisense p53 were substantially more resistant to c-Myc-induced apoptosis than were control cells. c-Myc activation also sensitized Rat-1 cells to radiation-induced apoptosis. Rat-1 cells expressing antisense p53 RNA were more resistant to apoptosis induced by the combined effects of c-Myc activation and gamma irradiation. In a similar manner, apoptosis induced by c-Myc in serum starved, hypoxic or gamma irradiated fibroblasts was also inhibited by Bcl-2. These data indicate that p53 is involved in c-Myc-mediated apoptosis under a variety of stresses which may influence tumor growth, evolution and response to therapy.

Over-expression of Bcl-2 protects against apoptosis induced by the bioreductive cytotoxic drug SR4233 (Tirapazamine).

The human B-cell lymphoma cell line PW undergoes radiation-induced programmed cell death (PCD). Bcl-2 transfected PW cells, that overexpressed Bcl-2, were significantly more radioresistant than parental or neomycin control transfected PW cells. The viability of Bcl-2 transfected cells was significantly greater than that of parental PW cells treated with the bioreductive cytotoxin SR4233 under aerobic conditions. Bcl-2 transfectants were also significantly more resistant to hypoxia-induced PCD. However, there was no significant difference in the viability of parental and Bcl-2 transfected cells exposed to SR4233 under hypoxic conditions (pO(2)<100 ppm). Incubation of parental PW cells with N-acetyl cysteine decreased the cytotoxicity of SR4233 under aerobic but not anaerobic conditions. Depletion of cellular glutathione with buthionine sulphoxamine killed nearly 100% of control PW cells, but none of the Bcl-2 transfectants under the same conditions. The TBARS assay for lipid peroxidation showed that Bcl-2 transfectants had a significantly lower level of lipid peroxidation than parental PW cells following a 24 hour constant exposure to SR4233 under aerobic conditions. These results suggest that Bcl-2 overexpression inhibits PCD induced by the bioreductive cytotoxin SR4233 under aerobic conditions as well as PCD induced by hypoxia, and that there are other pathways leading to PCD that are unaffected by Bcl-2 overexpression.

Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line.

The primary objective of this study was to determine the sequence of biochemical signaling events that occur after modulation of the cellular redox state in the B cell lymphoma line, PW, with emphasis on the role of mitochondrial signaling. L-Buthionine sulphoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), was used to modulate the cellular redox status. The sequence and role of mitochondrial events and downstream apoptotic signals and mediators was studied. After BSO treatment, there was an early decline in cellular glutathione (GSH), followed by an increase in reactive oxygen species (ROS) production, which induced a variety of apoptotic signals (detectable at different time points) in the absence of any external apoptotic stimuli. The sequence of biochemical events accompanying apoptosis included a 95% decrease in total GSH and a partial (25%) preservation of mitochondrial GSH, without a significant increase in ROS production at 24h. Early activation and nuclear translocation of the nuclear factor kappa B subunit Rel A was observed at approximately 3h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24h of BSO treatment. p53 protein expression was unchanged after redox modulation for up to 72 h, and p21waf1 independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a time-dependent manner, beginning at 24h after BSO incubation. Irreversible commitment to apoptosis occurred between 48 and 72 h after BSO treatment when mitochondrial GSH was depleted, and there was an increase in ROS production. Procaspase 3 protein levels showed a time-dependent reduction following incubation with BSO, notably after 48 h, that corresponded with increasing ROS levels. At 96 h, caspase 3 cleavage products were detectable. The pan-caspase inhibitor zVADfmk, partially blocked the induction of apoptosis at 48 h, and was ineffective after 72 h. PW cells could be rescued from apoptosis by removing them from BSO after up to 48, but not 72 h incubation with BSO. Mitochondrial transmembrane potential (DeltaPsi(m)) remained intact in most of the cells during the 72 h observation period, indicating that DeltaPsi(m) dissipation is not an early signal for the induction of redox dependent apoptosis in PW cells. These data suggest that a decrease in GSH alone can act as a potent early activator of apoptotic signaling. Increased ROS production following mitochondrial GSH depletion, represents a crucial event, which irreversibly commits PW cells to apoptosis.

Multicenter phase II study of iodine-131 tositumomab for chemotherapy-relapsed/refractory low-grade and transformed low-grade B-cell non-Hodgkin's lymphomas.

PURPOSE: This multicenter phase II study evaluated the efficacy, dosimetry methodology, and safety of iodine-131 tositumomab in patients with chemotherapy-relapsed/refractory low-grade or transformed low-grade non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: Patients received a dosimetric dose that consisted of 450 mg of anti-B1 antibody followed by 35 mg (5 mCi) of iodine-131 tositumomab. Serial total-body gamma counts were then obtained to calculate the patient-specific millicurie activity required to deliver the therapeutic dose. A therapeutic dose of 75 cGy total-body dose (attenuated to 65 cGy in patients with platelet counts of 101,000 to 149,000 cells/mm(3)) was given 7 to 14 days after the dosimetric dose. RESULTS: Forty-five of 47 patients were treated with a single dosimetric and therapeutic dose. Twenty-seven patients (57%) had a response. The response rate was similar in patients with low-grade (57%) or transformed low-grade (60%) NHL. The median duration of response was 9.9 months. Fifteen patients (32%) achieved a complete response (CR; 10 CRs and five clinical CRs), including five patients (50%) with transformed low-grade NHL. The median duration of CR was 19.9 months, and six patients have an ongoing CR. Treatment was well tolerated, with the principal toxicity being hematologic. The most common nonhematologic toxicities that were considered to be possibly related to the treatment included mild to moderate fatigue (32%), nausea (30%), fever (26%), vomiting (15%), infection (13%), pruritus (13%), and rash (13%). Additionally, one patient developed human-antimouse antibodies. CONCLUSION: Iodine-131 tositumomab produced a high overall response rate, and approximately one third of patients had a CR despite having chemotherapy-relapsed or refractory low-grade or transformed low-grade NHL.

Pivotal study of iodine I 131 tositumomab for chemotherapy-refractory low-grade or transformed low-grade B-cell non-Hodgkin's lymphomas.

PURPOSE: To evaluate the efficacy and safety of tositumomab and iodine I 131 tositumomab (Bexxar; Corixa Corp, Seattle, WA, and GlaxoSmithKline, Philadelphia, PA) in patients with chemotherapy-refractory low-grade or transformed low-grade non-Hodgkin's lymphoma (NHL) and to compare its efficacy to the patients' last qualifying chemotherapy (LQC) regimens. PATIENTS AND METHODS: Sixty patients who had been treated with at least two protocol-specified qualifying chemotherapy regimens and had not responded or progressed within 6 months after their LQC were treated with a single course of iodine I 131 tositumomab. RESULTS: Patients had received a median of four prior chemotherapy regimens. A partial or complete response (CR) was observed in 39 patients (65%) after iodine I 131 tositumomab, compared with 17 patients (28%) after their LQC (P <.001). The median duration of response (MDR) was 6.5 months after iodine I 131 tositumomab, compared with 3.4 months after the LQC (P <.001). Two patients (3%) had a CR after their LQC, compared with 12 (20%) after iodine I 131 tositumomab (P <.001). The MDR for CR was 6.1 months after the LQC and had not been reached with follow-up of more than 47 months after iodine I 131 tositumomab. An independent review panel verified that 32 (74%) of the 43 patients with nonequivalent durations of response (> 30 days difference) had a longer duration of response after iodine I 131 tositumomab (P <.001). Only one patient was hospitalized for neutropenic fever. Five patients (8%) developed human antimurine antibodies, and one (2%) developed an elevated TSH level after treatment. Myelodysplasia was diagnosed in four patients in follow-up. CONCLUSION: A single course of iodine I 131 tositumomab was significantly more efficacious than the LQC received by extensively pretreated patients with chemotherapy-refractory, low-grade, or transformed low-grade NHL and had an acceptable safety profile.

Phase II trial of yttrium-90-DOTA-biotin pretargeted by NR-LU-10 antibody/streptavidin in patients with metastatic colon cancer.

A Phase II study of yttrium-90-tetra-azacyclododecanetetra-acetic acid-biotin (90Y-DOTA-biotin) pretargeted by NR-LU-10 antibody/streptavidin (SA) was performed. The primary objectives of the study were to evaluate the efficacy and safety of this therapy in patients with metastatic colon cancer. Twenty-five patients were treated with a single dose of 110 mCi/m2 (mean administered dose, 106.5 +/- 10.3 mCi/m2) of 90Y-DOTA-biotin. There were three components of the therapy. Patients first received NR-LU-10/SA on day 1. A clearing agent (biotin-galactose-human serum albumin) was administered approximately 48 h after the NR-LU-10/SA to remove residual circulating unbound NR-LU-10/SA. Lastly, 24 h after administration of clearing agent, patients received biotin-DOTA-labeled with 110 mCi/m2 90Y. All three components of the therapy were administered i.v. Both hematological and nonhematological toxicities were observed. Diarrhea was the most frequent grade 4 nonhematological toxicity (16%; with 16% grade 3 diarrhea). Hematological toxicity was less severe with 8% grade 3 and 8% grade 4 neutropenia and 8% grade 3 and 16% grade 4 thrombocytopenia. The overall response rate was 8%. Two partial responders had freedom from progression of 16 weeks. Four patients (16%) had stable disease with freedom from progression of 10-20 weeks. Despite the relatively disappointing results of this study in terms of therapeutic efficacy and toxicity, proof of principle was obtained for the pretargeting approach. In addition, valuable new information was obtained about normal tissue tolerance to low-dose-rate irradiation that will help to provide useful guidelines for future study designs.

Yttrium-90-labeled anti-CD20 monoclonal antibody therapy of recurrent B-cell lymphoma.

A Phase I/II dose escalation study of 90Y-murine anti-CD20 monoclonal antibody (mAb) in patients with recurrent B-cell lymphoma was performed. The primary objectives of the study were: (a) to determine the effect of the preinfusion of unlabeled anti-CD20 mAb on the biodistribution of 111In-anti-CD20 mAb; (b) to determine the maximal tolerated dose of 90Y-anti-CD20 mAb that does not require bone marrow transplantation; and (c) to evaluate the safety and antitumor effect of 90Y-anti-CD20 mAb in patients with recurrent B-cell lymphoma. Eighteen patients with relapsed low- or intermediate-grade non-Hodgkin's lymphoma were treated. Biodistribution studies with 111In-anti-CD20 mAb were performed prior to therapy. Groups of three or four patients were treated at dose levels of approximately 13.5, 20, 30, 40, and 50 mCi 90Y-anti-CD20 mAb. Three patients were retreated at the 40-mCi dose level. The use of unlabeled antibody affected the biodistribution favorably. Nonhematological toxicity was minimal. The only significant toxicity was myelosuppression. The overall response rate following a single dose of 90Y-anti-CD20 mAb therapy was 72%, with six complete responses and seven partial responses and freedom from progression of 3-29+ months following treatment. Radioimmunotherapy with

A "whole-blood" technique for the quantitation of canine "T-lymphocyte" progenitors using a semisolid culture system.

A whole blood technique is described for the growth of concanavalin A (Con A) stimulated canine lymphocyte colonies in semisolid medium. By eliminating the routine Ficoll-Paque (F-P) gradient lymphocyte isolation, this method avoids potential problems of growth modulation due to elimination of non-lymphoid accessory cells and the influences on colony formation associated with the selective effects of F-P on lymphocyte subpopulations. Thus, the technique more closely approximates the in vivo milieu. The whole-blood method also produces higher cloning efficiencies than methods using gradient isolation of lymphocytes. Studies over a wide range of blood concentration produced a linear response of in vitro colony formation although extrapolation of the cell-dose colony-response curve did not intersect zero. Mitogen titration data indicates that a relatively large dose of Con A is required for whole blood colony formation compared to the standard F-P method. The colonies ultrastructurally were composed of lymphoblastic and lymphocytic elements which were negative for non-specific esterase activity. Characterization of cells retrieved from the colonies using rosetting techniques indicates a high percentage of the colony cells relative to canine peripheral blood cells form rosettes with human erythrocytes.

The selective growth of human T lymphocyte colonies from whole blood in a semisolid culture system.

Human T lymphocyte colonies may be selectively grown from whole blood in a single phase semisolid culture system following stimulation with PHA-P, Con-A, or PPD. This technique eliminates the requirement for gradient-enriched lymphocyte fractions, and provides a sensitive system for the study of T lymphocyte progenitors that more closely approximates the in vivo milieu. Whole blood colonies were composed of lymphoblasts and mature lymphocytes. Individual colony cells, identified as T lymphocytes, lacked lipase and specific esterase activity, formed E rosettes, did not phagocytize latex beads, and were largely ANAE positive. Whole blood was plated at a final concentration of 3%. Optimal mitogen/antigen concentrations were 125 microgram Con-A, 80 microgram PHA-P and 50 microgram PPD/ml culture media. Peak colony growth occurred between days 7 and 8. Colony formation increased as a power function over a wide range of cell concentrations (5 x 10(3)-5 x 10(4) lymphocytes plated). Maximal whole blood colony formation occurred when 5 x 10(4)-10(5) lymphocytes were plated. There was a significant increase in the cloning efficiency using whole blood as compared to gradient-separated cells. This method has wide application for the study of radiation effects, lymphocyte alterations in various disease states, antigen recognition, and the induction and amplification of T cell function.

Clinical radioimmunotherapy.

Radioimmunotherapy (RIT) is a promising new therapy for the treatment of a variety of malignancies. General principles of RIT are discussed, including important considerations in the selection of monoclonal antibodies (MAb) and radionuclides for RIT. Results of clinical trials using RIT for the treatment of lymphoma, leukemia, and solid tumors are summarized. The results from many of these trials are promising, especially for the treatment of lymphohematopoietic malignancies, in which a variety of MAb, radionuclides, and study designs have resulted in high response rates with a number of durable responses. Encouraging results have also been obtained using RIT to treat some solid tumors, primarily in patients with relatively low tumor burdens. RIT is generally well tolerated, with the primary toxicity being transient reversible myelosuppression in most nonmyeloablative studies. Nonhematologic toxicity, especially at nonmyeloablative doses, has been minimal in most studies. Approaches for increasing the therapeutic index of RIT are reviewed, which may further potentiate the efficacy and decrease the toxicity of RIT.