Cooperative effects of adenoviral vector-mediated interleukin 12 gene therapy with radiotherapy in a preclinical model of metastatic prostate cancer.

We investigated the potential benefits of combining adenoviral vector mediated in situ interleukin-12 (AdmIL-12) gene therapy with radiation therapy (XRT) to enhance therapeutic efficacy. In a metastatic mouse prostate cancer cell line, 178-2 BMA, AdmIL-12+XRT demonstrated enhanced therapeutic activities in vitro as determined by clonogenic survival, apoptosis, and mIL-12 levels. At the molecular level, increased expression of tumor necrosis factor-alpha mRNA was specific for the combined therapy. In a subcutaneous 178-2 BMA in vivo model, the combination of AdmIL-12+XRT produced statistically significant tumor growth suppression compared to control vector Adbetagal, Adbetagal XRT, or AdmIL-12 as monotherapy. In addition, significant prolongation of survival was demonstrated for the combination of AdmIL-12+XRT. The combination of AdmIL-12+XRT significantly suppressed both spontaneous and pre-established lung metastases, and led to a prolonged elevation of serum IL-12 and significantly increased natural killer (NK) activities. Importantly, in vivo depletion of NK cells resulted in significant attenuation of the antimetastatic activities of AdmIL-12 alone or AdmIL-12+XRT. These combined effects suggest that AdIL-12 gene therapy together with radiotherapy may achieve maximal tumor control (both local and systemic) in selected prostate cancer patients via radio-gene therapy induced local cytotoxicity and local and systemic antitumor immunity.

Impact of margin on tumour and normal tissue dosimetry in prostate cancer patients treated with IMRT using an endorectal balloon for prostate immobilization.

In IMRT treatment, margin for planning target volume is determined by organ motion and set-up error. The margin width that achieves the desired dose escalation, while minimizing normal tissue exposure is dependent upon patient immobilization and/or organ localization techniques. In this study, we compare the impact of margin width on the dosimetry of tumour and normal tissues using an endorectal balloon filled with 100 cc of air. Plans were generated for ten patients using margin widths of 0, 3, 5, 8 and 10 mm. The prescription dose to prostate and seminal vesicles was 70 Gy in 35 fractions with 15% of bladder allowed to receive above 65 Gy, 15% of rectum above 68 Gy and 10% of femurs above 45 Gy. Margins above 5 mm produced significantly lower mean doses for both prostate and seminal vesicles without affecting TCP. For normal tissues, mean doses, percent volumes above prescription constraints and NTCP increased as a function of margin width, especially when this was 5 mm or above. We conclude that planning with tighter margins of < or =5 mm improves IMRT dosimetry for prostate and normal tissues and is only possible when target localization and/or immobilization devices are routinely used.

Enhanced therapeutic effect of multiple injections of HSV-TK + GCV gene therapy in combination with ionizing radiation in a mouse mammary tumor model.

PURPOSE: Standard therapies for breast cancer lack tumor specificity and have significant risk for recurrence and toxicities. Herpes simplex virus-thymidine kinase (HSV-tk) gene therapy combined with radiation therapy (XRT) may be effective because of complementary mechanisms and distinct toxicity profiles. HSV-tk gene therapy followed by systemic administration of ganciclovir (GCV) enhances radiation-induced DNA damage by generating high local concentrations of phosphorylated nucleotide analogs that increase radiation-induced DNA breaks and interfere with DNA repair mechanisms. In addition, radiation-induced membrane damage enhances the "bystander effect" by facilitating transfer of nucleotide analogs to neighboring nontransduced cells and by promoting local and systemic immune responses. This study assesses the effect of single and multiple courses of HSV-tk gene therapy in combination with ionizing radiation in a mouse mammary cancer model. METHODS AND MATERIALS: Mouse mammary TM40D tumors transplanted s.c. in syngeneic immunocompetent BALB-c mice were treated with either adenoviral-mediated HSV-tk gene therapy or local radiation or the combination of gene and radiation therapy. A vector consisting of a replication-deficient (E1-deleted) adenovirus type 5 was injected intratumorally to administer the HSV-tk gene, and GCV was initiated 24 h later for a total of 6 days. Radiation was given as a single dose of 5 Gy 48 h after the HSV-tk injection. A metastatic model was developed by tail vein injection of TM40D cells on the same day that the s.c. tumors were established. Systemic antitumor effect was evaluated by counting the number of lung nodules after treating only the primary tumors with gene therapy, radiation, or the combination of gene and radiation therapy. To assess the therapeutic efficacy of multiple courses of this combinatorial approach, one, two, and three courses of HSV-tk + GCV gene therapy, in combination with radiation, were compared to HSV-tk or XRT alone and to sham-treated animals. (Treatments were repeated at 7-day intervals from the HSV-tk injection.) RESULTS: Both single-therapy modalities reduced tumor growth by 11% compared to controls, while the combined therapy resulted in a decrease of 29%. Median survival was 36 days in the combined therapy group, compared to 33 days in the monotherapy groups and 26 days in the control group. In the metastatic model, the number of lung nodules was reduced by 59.5% after HSV-tk gene therapy, whereas radiotherapy had no effect on metastatic growth. Combined therapy led to an additional 66.7% reduction in lung colonization. Compared to controls, local tumor growth was maximally suppressed by three courses of combined therapy (51.5%), followed by two courses of combined therapy (37.2%), and three sessions of XRT alone (35.6%). Median survival was also significantly prolonged to 58 days with the three courses of combined therapy, followed by two courses, to 45 days. All other treatment groups demonstrated median survival times between 26 and 35 days, while controls had a median survival of 24 days. CONCLUSIONS: These results indicate that multiple courses of HSV-tk therapy in combination with radiation improve the therapeutic efficacy of this approach and may provide therapeutic implications for the treatment of human breast cancer and other solid tumors.

Phase I/II trial evaluating combined radiotherapy and in situ gene therapy with or without hormonal therapy in the treatment of prostate cancer--a preliminary report.

PURPOSE: To report the preliminary results of a Phase I/II study combining radiotherapy and in situ gene therapy (adenovirus/herpes simplex virus thymidine kinase gene/valacyclovir) with or without hormonal therapy in the treatment of prostate cancer. METHODS AND MATERIALS: Arm A: low-risk patients (T1-T2a, Gleason score <7, pretreatment PSA <10) were treated with combined radio-gene therapy. A mean dose of 76 Gy was delivered to the prostate with intensity-modulated radiotherapy. Arm B: high-risk patients (T2b-T3, Gleason score >or=7, pretreatment PSA >or=10) were treated with combined radio-gene therapy and hormonal therapy. Hormonal therapy was comprised of a 4-month leuprolide injection and 2-week use of flutamide. Arm C: Stage D1 (positive pelvic lymph node) patients received the same regimen as Arm B, with the additional 45 Gy to the pelvic lymphatics. Treatment-related toxicity was assessed using Cancer Therapy Evaluation Program common toxicity score and Radiation Therapy Oncology Group (RTOG) toxicity score. RESULTS: Thirty patients (13 in Arm A, 14 in Arm B, and 3 in Arm C) completed the trial. Median follow-up was 5.5 months. Eleven patients (37%) developed flu-like symptoms (Cancer Therapy Evaluation Program Grade 1) of fatigue and chills/rigors after gene therapy injection but recovered within 24 h. Four patients (13%) and 2 patients (7%) developed Grade 1 and 2 fever, respectively. There was no patient with weight loss. One patient in Arm B developed Grade 3 elevation in liver enzyme, whereas 11 and 2 patients developed Grade 1 and 2 abnormal liver function tests. There was no Grade 2 or above hematologic toxicity. Three patients had transient rise in creatinine. There was no RTOG Grade 3 or above lower gastrointestinal toxicity. Toxicity levels were as follows: 4 patients (13%), Grade 2; 6 patients (20%), Grade 1; and 20 patients (67%), no toxicity. There was 1 patient with RTOG Grade 3 genitourinary toxicity, 12 patients (40%) with Grade 2, 8 patients (27%) with Grade 1, and 9 patients (30%) with no toxicity. No patient dropped out from the trial or had to withhold treatment because of severe toxicity. CONCLUSIONS: This is the first trial of its kind in the field of prostate cancer that aims to expand the therapeutic index of radiotherapy by combining in situ gene therapy. Initial experience has demonstrated the safety of this approach. There is no added toxicity to each therapy used alone. Long-term follow-up and larger cohort studies are warranted to evaluate long-term toxicity and efficacy.

Mantle irradiation alone for pathologic stage I and II Hodgkin's disease: long-term follow-up and patterns of failure.

PURPOSE: We performed a retrospective study to determine the long-term outcome, patterns of failure, and prognostic factors for patients with pathologic Stage I or II Hodgkin's disease (HD) who were treated with mantle irradiation alone. METHODS AND MATERIALS: The medical records of 145 patients with pathologic Stage I or II supradiaphragmatic Hodgkin's disease treated with mantle irradiation alone between June 1967 and June 1991 were reviewed. Patterns of failure, overall survival (OS) rate, and progression-free survival (PFS) rate were determined. Univariate and multivariate analyses were performed to identify adverse prognostic factors for OS and PFS. The number of adverse prognostic factors per patient was counted, and a prognostic score was assigned to each patient. The log-rank test was used to compare the OS or PFS rates among patients with prognostic scores 0, 1, and 2. RESULTS: The median patient age was 27 years (range 10-66), with almost even male to female distribution. Every patient had splenectomy and negative laparotomy (LAP). Fifty-one patients had Stage I disease (IA-49, IB-2) and 94 Stage II (IIA-89, IIB-5). The histologic subtypes were nodular sclerosing in 110, mixed cellularity in 28, lymphocyte predominance in 5, lymphocyte depleted in 1, and unclassified in 1. Twelve patients with Stage II disease had >/= 3 sites of nodal involvement. Fifty-four patients had a prognostic score of 0, 70 of 1, and 21 of 2. The median follow-up time for the 109 surviving patients was 146 months (range 25-381). The 10- and 20-year actuarial OS rates for the whole group were 87.6% and 65.3%, respectively. The corresponding actuarial PFS rates were 75.3% and 74.2%, respectively. Thirty-six patients (9 Stage I, 27 Stage II) had relapses in a total of 41 sites. Failures by histology were 29 patients with nodular sclerosing, 6 with mixed cellularity, and 1 with lymphocyte predominance. Failures by sites were: trans-diaphragmatic, 22 (para-aortic nodes, 15; as the only site of progression in 12; visceral, 7; as the only site of progression in 5); within radiation field, 8; marginal miss, 8 (as the only site of failure in 2); and unknown, 3. The majority of the failures occurred within 5 years of diagnosis. Long-term side effects of radiation included cardiac complications in 30 patients, with 10- and 20-year actuarial cardiac complication rates of 12.6% and 35.1%, respectively; secondary solid tumors in 14, with 10- and 20-year actuarial rates of 2.3% and 25.7%, respectively; leukemia in 4; non- Hodgkin's lymphoma in 4, with the 10- and 20-year actuarial rates for leukemia and non-Hodgkin's lymphoma of 4.0% and 13.9%; and hypothyroidism in 38. Four adverse prognostic factors were identified for PFS: age > or = 40 years, > or = 3 sites of involvement, male sex, and constitutional symptoms. The prognostic score correlated with patients' outcome as indicated by PFS and OS rates. Patients with a prognostic score of 0 did significantly better than those with a score of 1 or 2. CONCLUSION: In this select group of patients with pathologic Stage I and II Hodgkin's disease treated with mantle irradiation alone, the OS and PFS rates at 10 and 20 years were comparable to those reported in the literature. The major pattern of disease progression was relapse below the diaphragm, therefore close surveillance of the abdomen is warranted. The prognostic score used in our series may predict the patient's outcome, and might be worth testing in a prospective trial. In our series, patients with a prognostic score of 0 had excellent long-term survival, indicating adequate treatment with mantle irradiation alone. Late complications of the treatment pose a significant threat for the patient's survival with long-term follow-up.

Enhanced therapeutic effect of HSV-tk+GCV gene therapy and ionizing radiation for prostate cancer.

Standard therapies for prostate cancer including radiation, prostatectomy, and hormone ablation have significant toxicities and recurrence risk. HSV-tk gene therapy may be effective in combination with radiation therapy due to complementary mechanisms and distinct toxicity profiles. Mouse prostate tumors transplanted subcutaneously were treated by either gene therapy involving intratumoral injection of AdV-tk followed by systemic ganciclovir or local radiation therapy or the combination of gene and radiation therapy. Both single-therapy modalities showed a 38% decrease in tumor growth compared to controls. The combined treatment resulted in a decrease of 61%. In addition the combined-therapy group had a mean survival of 22 days versus 16.6 days for single therapy and 13.8 days for nontreated controls. To analyze systemic anti-tumor activity, lung metastases were generated by tail vein injection of RM-1 prostate cancer cells on the same day that they were injected subcutaneously. The primary tumors were treated as before with AdV-tk followed by ganciclovir, radiation, or the combination. The number of lung nodules was reduced by 37% following treatment with AdV-tk, whereas radiotherapy alone had no effect on metastatic growth. The combination led to an additional 50% reduction in lung colonization. Primary tumors that received the combination therapy had a marked increase in CD4 T cell infiltrate. This is the first report showing a dramatic systemic effect following the local combination treatment of radiation and AdV-tk. A clinical study using this strategy has been initiated and patient accrual is ongoing.

ASTRO research fellowship: the role of BCL-2 and glutathione in an antioxidant pathway to prevent radiation-induced apoptosis.

PURPOSE: The expression of the bcl-2 proto-oncogene has been associated with resistance to radiation-induced apoptosis. There is evidence that the bcl-2 protein acts in an antioxidant pathway to block the effects of reactive oxygen species that mediate apoptosis possibly by increasing the levels of intracellular glutathione. Our hypothesis is that pretreatment of radiation-sensitive cells, known to lack bcl-2 expression, with antioxidants will reduce radiation-induced apoptosis. For this purpose, the apoptotic response to radiation and the intracellular levels of GSH were tested before and after pretreatment with antioxidants in two murine lymphoma cell lines, a radiation-resistant, bcl-2- expressing (LY-ar) line and a radiation-sensitive, non-bcl-2-expressing (LY-as) line. METHODS AND MATERIALS: LY-ar and LY-as cells were irradiated at 0,1,2,3, and 4 hours before collection. The intracellular levels of reduced (GSH) and oxidized (GSSG) glutathione were determined by the use of the fluorescent dye o-phthalaldehyde. LY-as cells were treated with GSH ethyl-ester for 1 and 2 hours after irradiation. Apoptotic response was measured by the DNA fragmentation assay. The radiation dose was 2.5 Gy. RESULTS: After irradiation, the apoptotic rate of LY-ar and LY-as cells was 10-20% and 50-70% respectively. LY-ar cells had higher intracellular GSH and GSSG levels compared to LY-as cells by 69.9% and 91.9% respectively and the GSH/GSSG ratio in LY-ar and LY-as cells was 15.09 and 17.09 respectively. GSH levels did not change during the first 2 hours after irradiation; however, there was a 49% and 84% reduction at 3 and 4 hours after irradiation, respectively, times at which the LY-as cells have already fragmented their DNA. Treatment of LY-as cells with GSH ethyl-ester at a concentration of 7 mM for 1 and 2 hours resulted in 70% and 231% increases in the intracellular GSH levels respectively. Treatment of LY-as cells with GSH ethyl-ester for 1 and 2 hours also conferred a 25-50% decrease in their apoptotic response after irradiation. CONCLUSIONS: GSH and GSSG levels are elevated in radiation-resistant, bcl-2-expressing murine lymphoma cells compared to radiation-sensitive, non-bcl-2-expressing cells. GSH levels decline only in radiation-sensitive cells after irradiation but this appears to occur at the time of apoptotic cell death. Exogenous thiols increase intracellular GSH levels and repress radiation-induced apoptosis. In conclusion, intracellular thiols appear to be involved in protecting cells from apoptotic cell death. Further investigation should be directed in identifying substances which by lowering intracellular thiols may result in sensitization to radiation.

Stage I Hodgkin disease: radiation therapy and chemotherapy at the University of Texas M. D. Anderson Cancer Center, 1996-1997.

PURPOSE: To summarize 30 years of experience in treatment of and prognosis for stage I Hodgkin disease. MATERIALS AND METHODS: The authors reviewed retrospectively the cases of 196 patients seen and followed up at one institution from 1967 to 1997. All patients were treated with radiation therapy, and 46 also received combination chemotherapy as part of their initial treatment. Radiation therapy techniques included involved or regional-field irradiation in 83 patients, extended field irradiation (mantle or inverted Y) in 74, and subtotal nodal irradiation in 39 (median radiation doses for subclinical and clinical disease were 30 and 40 Gy, respectively, at 1.5-2.0 Gy per fraction). Of 46 patients treated with combination and radiation therapy, 26 received subtotal nodal irradiation; in the remaining 20, chemotherapy was combined with more limited-field radiation therapy. Follow-up ranged from 3 to 356 months (median, 144 months). RESULTS: The actuarial overall survival, disease-specific survival, and freedom from progression at 10 and 20 years were 82% and 66%, 94% and 91%, and 77% and 70%, respectively. In multivariate analysis, age adversely influenced overall survival, and female sex favorably affected freedom from progression. Mixed cellularity histology and mantle field technique adversely influenced disease-specific survival. Laparotomy significantly influenced disease-specific survival but not overall survival. CONCLUSION: Radiation therapy results in an excellent outcome in patients with favorable stage I Hodgkin disease. Pathologic staging is no longer necessary.

Long-term outcome of treatment for Ann Arbor stage 1 Hodgkin's disease: patterns of failure, late toxicity and second malignancies.

PURPOSE: Radiation therapy results in excellent short-term survival in patients with early-stage Hodgkin's disease. However, the optimal therapeutic scheme that achieves the highest disease-free survival with the minimum long-term toxicity is yet to be determined. An analysis of the patterns of failure and late complications after radiation therapy was conducted to address this question. METHODS AND MATERIALS: A retrospective study was conducted of 145 patients with Stage I Hodgkin's disease treated at M. D. Anderson Cancer Center from 1967 through 1987. Follow-up extended from a minimum of 30 to 339 months, with a median period of observation of 16.5 years. All the patients were treated with radiation therapy and, and 16 received combination MOPP-based chemotherapy as part of their initial treatment. The radiotherapy technique, was involved/regional in 71 (49%), extended in 62 (43%), and subtotal nodal irradiation in 12 patients. The median total dose was 40 Gy. RESULTS: The actuarial freedom from progression at 10 and 20 years was 76% and 69%, respectively. Forty of 145 patients relapsed (27.6%). The site of primary disease was cervical adenopathy in 30 (75%), axillary in 7 (17.5%), mediastinal in 2 patients and subdiaphragmatic in one patient. Twenty-two patients were treated with involved/regional technique (55%), 17 with extended (42.5%), and 1 with subtotal nodal irradiation technique. There were three in field and four marginal recurrences. Six relapses occurred in non-irradiated nodal regions at the same side of the diaphragm and 17 in non-irradiated transdiaphragmatic lymph nodes (57.5%). Nine patients (22.5%) relapsed with visceral disease. Nineteen patients (47.5%) relapsed within the first 2 years, 15 (37.5%) 3 to 10 years after diagnosis and the remaining 6 (15%) after 10 years. Eleven of 40 patients died of disease after the first or subsequent relapses (27.5%). Three of six patients with late relapses had progression in viscera but only two died with disease. Thirty-eight of 145 patients developed late toxicity from the treatment (26.2%). Twenty-three patients experienced ischemic heart disease (15.9%), only 13 of whom received mediastinal irradiation (9%). Fifteen patients developed secondary malignant solid tumors (10.3%). Nine of those (6.2%) occurred within the irradiation field (two were also treated with chemotherapy). Two additional patients, one of whom received chemotherapy as part of the initial treatment, died of acute myelogenous leukemia. Non-Hodgkin's lymphoma and lung cancer were the most common second malignancies. CONCLUSIONS: Limited field radiotherapy results in a significant number of relapses in non-irradiated, especially transdiaphragmatic lymph nodes. Subtotal nodal irradiation can prevent some relapses and therefore improve freedom from progression. Careful design of the treatment fields may decrease the risk of morbidity and mortality from coronary artery disease and second malignancies in early-stage Hodgkin's disease. Careful long-term surveillance may permit early detection and management of late relapses and treatment complications.

Long-term outcome of treatment for Ann Arbor Stage I Hodgkin's disease: prognostic factors for survival and freedom from progression.

PURPOSE: The earliest stages of Hodgkin's disease are associated with excellent short-term survival with radiation therapy. This has led to controversies regarding pretreatment evaluation, the extent of irradiation, the role of chemotherapy, and the relative importance of prognostic factors. Long-term results were sought to address these controversies. METHODS AND MATERIALS: A retrospective study was conducted of patients with Stage I Hodgkin's disease treated at the M. D. Anderson Cancer Center from 1967 through 1987. The median age at presentation of 145 patients was 31 years, and the male-to-female ratio was 1.8. Pretreatment evaluation included lymphangiography and bone marrow aspiration and biopsy in all patients. Laparotomy was performed in 101 of the 145 patients (70%). There were 133 patients with supradiaphragmatic presentations; 12 patients had infradiaphragmatic adenopathy. Only five patients had B symptoms (3.5%). Histologic subtypes of the disease included lymphocyte predominance 17.9%, nodular sclerosis 40.7%, mixed cellularity 40.7%, and one unclassified Hodgkin's disease with primary splenic involvement. All patients were treated with radiotherapy, and 16 (11%) also received combination chemotherapy as part of their initial treatment. Radiotherapy techniques included involved/regional field in 49%, extended field in 42.7% (mantle or inverted Y), and subtotal nodal irradiation in 8.3%. Follow-up extended from a minimum of 30-339 months, with a median period of observation of 16.5 years. RESULTS: The median survival was 13.7 years. The 10- and 20-year survival rates were 83% and 66%, respectively. The only factor important for decreased survival was age >40 years at diagnosis (p < 0.0001). Out of 43 deaths, 11 were the result of Hodgkin's disease and the remaining 32 resulted from intercurrent disease, including treatment-related causes. Median freedom from progression was 10.5 years, and the 10- and 20-year freedom from progression were 76% and 69%, respectively. Out of 39 relapses, 5 (13%) occurred beyond 10 years. Women had higher freedom from progression (p = 0.0534) than men. Age, histology, bulk of disease, site of involvement including the mediastinal presentations, and the addition of chemotherapy did not influence the freedom of progression. Although very few patients (12 of 145) received subtotal nodal irradiation, the freedom from progression at 10 years was 91.7% for this group versus 64.7% for the group of patients who were treated with more limited techniques. CONCLUSION: Treatment with radiation therapy for patients with Stage I Hodgkin's disease leads to an excellent outcome, but patients require long-term surveillance as late relapses are not rare. Age is the only factor that affects survival, and gender marginally affects freedom from progression. Subtotal nodal irradiation may improve freedom from progression; further investigation of this treatment is justified.