Susan J. Knox

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-Hodgkins 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.

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 (γGCS), 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 24 h. Early activation and nuclear translocation of the nuclear factor kappa B subunit Rel A was observed at approximately 3 h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24 h 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 24 h 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 (ΔΨm) remained intact in most of the cells during the 72 h observation period, indicating that ΔΨm 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.

70-Gene Signature as an Aid to Treatment Decisions in Early-Stage Breast Cancer.

BACKGROUND The 70-gene signature test (MammaPrint) has been shown to improve prediction of clinical outcome in women with early-stage breast cancer. We sought to provide prospective evidence of the clinical utility of the addition of the 70-gene signature to standard clinical-pathological criteria in selecting patients for adjuvant chemotherapy. METHODS In this randomized, phase 3 study, we enrolled 6693 women with early-stage breast cancer and determined their genomic risk (using the 70-gene signature) and their clinical risk (using a modified version of Adjuvant! Online). Women at low clinical and genomic risk did not receive chemotherapy, whereas those at high clinical and genomic risk did receive such therapy. In patients with discordant risk results, either the genomic risk or the clinical risk was used to determine the use of chemotherapy. The primary goal was to assess whether, among patients with high-risk clinical features and a low-risk gene-expression profile who did not receive chemotherapy, the lower boundary of the 95% confidence interval for the rate of 5-year survival without distant metastasis would be 92% (i.e., the noninferiority boundary) or higher. RESULTS A total of 1550 patients (23.2%) were deemed to be at high clinical risk and low genomic risk. At 5 years, the rate of survival without distant metastasis in this group was 94.7% (95% confidence interval, 92.5 to 96.2) among those not receiving chemotherapy. The absolute difference in this survival rate between these patients and those who received chemotherapy was 1.5 percentage points, with the rate being lower without chemotherapy. Similar rates of survival without distant metastasis were reported in the subgroup of patients who had estrogen-receptor-positive, human epidermal growth factor receptor 2-negative, and either node-negative or node-positive disease. CONCLUSIONS Among women with early-stage breast cancer who were at high clinical risk and low genomic risk for recurrence, the receipt of no chemotherapy on the basis of the 70-gene signature led to a 5-year rate of survival without distant metastasis that was 1.5 percentage points lower than the rate with chemotherapy. Given these findings, approximately 46% of women with breast cancer who are at high clinical risk might not require chemotherapy. (Funded by the European Commission Sixth Framework Program and others; ClinicalTrials.gov number, NCT00433589; EudraCT number, 2005-002625-31.).

Tositumomab and Iodine-131 Tositumomab Produces Durable Complete Remissions in a Subset of Heavily Pretreated Patients With Low-Grade and Transformed Non-Hodgkin’s Lymphomas

PURPOSE This study is an integrated efficacy analysis of the five clinical trials of tositumomab and iodine-131 tositumomab in patients with relapsed or refractory low-grade, follicular, or transformed low-grade non-Hodgkins lymphoma (NHL) that resulted in the regulatory approval of the iodine-131 tositumomab by the US Food and Drug Administration. PATIENTS AND METHODS This integrated analysis included 250 patients. Patients received a single course of iodine-131 tositumomab. Responses were assessed by an independent panel of radiologists and oncologists. RESULTS Response rates in the five trials ranged from 47% to 68%; complete response rates ranged from 20% to 38%. With a median follow-up of 5.3 years, the 5-year progression-free survival was 17%. Eighty-one (32%) of 250 patients had a time to progression of > or = 1 year (termed durable response population). For the durable response population, 44% had not progressed at > or = 2.5 to > or = 9.5 years and had a median duration of response of 45.8 months. The median duration of complete response was not reached. The durable response population had many poor prognostic characteristics, including bone marrow involvement (41%), bulky disease > or = 5 cm (49%), and transformed histology (23%). Forty-three percent of the patients had been treated with more than four prior therapies and 36% had not responded to their most recent therapy. CONCLUSION The tositumomab and iodine-131 tositumomab therapeutic regimen produces high response rates in patients with relapsed or refractory low-grade, follicular, and transformed low-grade NHL, with a sizable subgroup of patients achieving long-term durable responses.

The radioisotope contributes significantly to the activity of radioimmunotherapy

Purpose: A multicenter, randomized study was undertaken to estimate the single agent activity of Tositumomab and to determine the contribution of radioisotope-labeling with 131I to activity and toxicity by comparing treatment outcomes for Tositumomab and Iodine I 131 Tositumomab (BEXXAR) to an equivalent total dose of unlabeled Tositumomab. Experimental Design: Seventy-eight patients with refractory/relapsed non-Hodgkin’s lymphoma were randomized to either unlabeled Tositumomab or Iodine I 131 Tositumomab. Patients progressing after unlabeled Tositumomab could cross over to receive Iodine I 131 Tositumomab. The median follow-up at analysis was 42.6 months (range 1.9 to 71.5 months). Results: Responses in the Iodine I 131 Tositumomab versus unlabeled Tositumomab groups: overall response 55% versus 19% (P = 0.002); complete response 33% versus 8% (P = 0.012); median duration of overall response not reached versus 28.1 months (95% confidence interval: 7.6, not reached); median duration of complete response not reached in either arm; and median TTP 6.3 versus 5.5 months (P = 0.031), respectively. Of the patients who had a complete response after initial Iodine I 131 Tositumomab therapy, 71% (10 of 14) continued in complete response at 29.8 to 71.1 months. Two patients who achieved a complete response after unlabeled Tositumomab had ongoing responses at 48.1 to 56.9 months. Nineteen patients received Iodine I 131 Tositumomab crossover therapy. Responses after crossover versus prior response to unlabeled Tositumomab were as follows: complete response rates of 42% versus 0% (P = 0.008); overall response 68% versus 16% (P = 0.002); median durations of overall response 12.6 versus 7.6 months (P = 0.001); and median TTP 12.4 versus 5.5 months (P = 0.01), respectively. Hematologic toxicity was more severe and nonhematologic adverse events were more frequent after Iodine I 131 Tositumomab than after Tositumomab alone. Elevated thyrotropin occurred in 5% of patients. Seroconversion to human antimurine antibody after Iodine I 131 Tositumomab, unlabeled Tositumomab, and Iodine I 131 Tositumomab-crossover was 27%, 19%, and 0%, respectively. Conclusions: Unlabeled Tositumomab showed single agent activity, but in this direct comparison, all of the therapeutic outcome measures were significantly enhanced by the conjugation of 131I to Tositumomab.

The antiangiogenic agents SU5416 and SU6668 increase the antitumor effects of fractionated irradiation

Abstract Ning, S., Laird, D., Cherrington, J. M. and Knox, S. J. The Antiangiogenic Agents SU5416 and SU6668 Increase the Antitumor Effects of Fractionated Irradiation. Radiat. Res. 157, 45–51 (2002). Angiogenesis is critical for tumor development, growth and metastasis. The vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) and their tyrosine kinase receptors are major regulators of angiogenesis. Radiation induces the production of VEGF, FGF and PDGF in many tumor cells. We hypothesized that inhibition of the function of these growth factors could inhibit tumor angiogenesis and thereby enhance the efficacy of radiation therapy. To test this hypothesis, we used the small molecule inhibitors SU5416 (an inhibitor for Vegf receptor) and SU6668 (an inhibitor for Vegf, Fgf and Pdgf receptors) alone and in combination with fractionated irradiation to treat C3H mice bearing SCC VII carcinomas. The SCC VII tumors express Vegf, Fgf2 (also known as bFGF), Pdgf and their associated receptors. Animals were given either SU5416 or SU6668 daily before or after irradiation (2 Gy per fraction per day for 5 days). The results from these experiments demonstrate that administration of either SU5416 or SU6668 without radiation delayed tumor growth. Administration of SU5416 at a dose of 25 mg/kg per day (the maximum tolerated effective dose) inhibited tumor growth by 17.9% on day 7 (P < 0.05 compared to untreated control mice) and produced an average tumor growth delay time of 0.5–2.0 days. When combined with fractionated irradiation, administration of SU5416 increased the inhibition of tumor growth to 50–53% on day 7 and the tumor growth delay time to 5.7–6.5 days (P < 0.001 compared with SU5416 alone; P ≤ 0.05 compared with radiation alone). SU6668 alone inhibited tumor growth in a dose-dependent manner. Administration of SU6668 at a dose of 75 mg/kg per day (a suboptimal dose) inhibited tumor growth by 36% on day 7 and produced an average tumor growth delay time of 3.3 ± 1.4 days. The combination of SU6668 with fractionated radiation increased inhibition of tumor growth to 66–70% and the tumor growth delay time from 3.3 days to 11.9 days (P ≤ 0.001 compared with either radiation alone or SU6668 alone). Administration of these agents before or after irradiation produced similar results (P = 0.40 for SU5416; P = 0.98 for SU6668). SU5416 or SU6668 alone or in combination with radiation was very well tolerated with little or no toxicity. These results suggest that inhibition of Vegf, Fgf and Pdgf receptor function by SU5416 and SU6668 can enhance the efficacy of irradiation. The targeting of multiple tyrosine kinase receptors by SU6668 is more effective than inhibition of the Vegf receptor alone by SU5416 for the enhancement of tumor cell killing by fractionated irradiation.

Enhancement of murine intestinal stem cell survival after irradiation by keratinocyte growth factor.

Radiation-induced gastrointestinal toxicity is due in part to the killing of the clonogenic crypt cells and eventual depopulation of the villi. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family (FGF-7), has been shown to stimulate proliferation of cells along the murine digestive tract from the foregut to the colon. Using an in vivo microcolony assay, we found that 1.0 mg/kg KGF administered intravenously (i.v.) for 3 consecutive days (2 days before, 1 day before and 2 h after irradiation) increased the number of surviving crypts by a factor of 2.6, 2.7 and 2.4 in the duodenum, jejunum and ileum, respectively, after a single-dose whole-body irradiation (10-16 Gy) (P < 0.001). Treatment of mice with KGF i.v. significantly increased the D0 of the radiation survival curves by 0.37, 0.22 and 0.36 Gy, leading to dose modification factors of 1.28, 1.16 and 1.24 for duodenal, jejunal and ileal crypt cells, respectively. Similar results were obtained with KGF administered subcutaneously. Treatment with both KGF and stem cell factor (previously shown to enhance intestinal crypt survival after total-body irradiation) increased the number of surviving crypt cells after irradiation to levels similar to that in animals treated with KGF alone. Administration of KGF for 7 consecutive days (beginning 2 days prior to irradiation) increased the LD(50/10) from 5.50 Gy/day to 5.90 Gy/day (P = 0.05) for animals irradiated with five daily fractions to a local abdominal field. These results suggest that KGF may be of clinical value in reducing radiation toxicity to the intestine.

Radiation induces upregulation of cyclooxygenase-2 (COX-2) protein in PC-3 cells

PURPOSE To investigate the impact of gamma-irradiation on cyclooxygenase-2 (COX-2) expression and its enzymatic activity in PC-3 cells. Cell cycle redistribution, viability, and apoptosis were quantitated in control and irradiated cells with or without the COX-2 inhibitor NS-398. METHODS AND MATERIALS Western blot analysis was used to assess COX-2 protein expression. Prostaglandin (PGE(2)) was measured after addition of arachidonic acid (AA) using a Monoclonal Immunoassay Kit. Cell cycle and apoptosis were assessed using flow cytometry. RESULTS We observed a dose-dependent increase in COX-2 of 37.0%, 79.7%, and 97.5% following irradiation with 5, 10, and 15 Gy, respectively. The PGE(2) level of irradiated cells was higher than in controls (1512 +/- 157.5 vs. 973.7 +/- 54.2 rhog PGE(2)/mL; p < 0.005, n = 4) while cells irradiated in the presence of NS-398 had reduced PGE(2) levels (218.8 +/- 80.1 rhog PGE(2)/mL; p < 0.005; n = 4). We found no differences in cell cycle distribution or apoptosis between cells irradiated in the presence or absence of NS-398. CONCLUSIONS COX-2 protein is upregulated and enzymatically active after irradiation, resulting in elevated levels of PGE(2). This effect can be suppressed by NS-398, which has clinical implications for therapies combining COX-2 inhibitors with radiation therapy.

Expression and function of erythropoietin receptors in tumors: implications for the use of erythropoiesis-stimulating agents in cancer patients.

Safety concerns surrounding the use of recombinant human erythropoietin (Epo) to treat anemia in cancer patients were raised after 2 recent clinical studies reported a worse survival outcome in patients who received epoetin α or epoetin β compared with patients who received placebo. Although those findings contrasted with previous clinical studies, which demonstrated no difference in survival for cancer patients who received erythropoiesis‐stimulating agents (ESAs), some investigators have suggested a potential role for ESAs in promoting tumor growth through 1) stimulation of Epo receptors (EpoR) expressed in tumors, 2) stimulation and formation of tumor vessels, and/or 3) enhanced tumor oxygenation. The first and second hypotheses appeared to be supported by some EpoR expression and ESA in vitro studies. However, these conclusions have been challenged because of poor specificity of EpoR‐detection methodologies, conflicting data from different groups, and the lack of correlation between in vitro data and in vivo findings in animal tumor models. For this report, the authors reviewed the biology of EpoR in erythropoiesis and compared and contrasted the reported findings on the role of ESAs and EpoR in tumors. Cancer 2007.

Re-Treatment With I-131 Tositumomab in Patients With Non-Hodgkin's Lymphoma Who Had Previously Responded to I-131 Tositumomab

PURPOSE To study efficacy and safety of re-treatment with I-131 tositumomab in patients with low-grade, follicular, or transformed low-grade B-cell lymphoma who relapsed following a response to I-131 tositumomab. PATIENTS AND METHODS A prior response > or = 3 months to I-131 tositumomab was required. The single therapeutic dose following a dosimetric dose was adjusted to give the same total body dose (in Gy) as that used for the original dose, or was attenuated if the platelet count was less than 150,000 per mm(3) or if the prior treatment resulted in grade 4 cytopenias lasting longer than 7 days. RESULTS Of 32 patients enrolled, 28 completed the therapeutic dose. A median of four therapies were given before re-treatment. Eighteen (56%) of 32 patients had a complete or partial response (median duration, 15.2 months); eight (25%) had a complete response (median duration, 35 months). Five continue in response from 1.8 to 5.7 years, with a median follow-up of 35 months. The overall median response duration was not significantly different for the two treatments, with no clinical factors predicting response or its duration. Ten of 18 re-responders had longer responses with re-treatment, with five having responses > or = 1.5 years longer. Grade 3/4 neutropenia and thrombocytopenia occurred in 50% and 43% of patients, respectively, similar to initial treatment. Antimouse antibodies developed in 10% of patients, and 12% developed elevated serum thyroid-stimulating hormone. Six patients were diagnosed with second malignancies, including four patients who developed myelodysplastic syndrome (one who had not received the therapeutic dose) and one with acute myelogenous leukemia. CONCLUSION Re-treatment with I-131 tositumomab following a previous response can produce second responses that can be durable.