Luka Milas

The epidermal growth factor receptor mediates radioresistance

PURPOSE The epidermal growth factor (EGF) receptor is frequently overexpressed in malignant tumors, and its level is correlated with increased cellular resistance to ionizing radiation. However, no precedent studies have investigated whether expression of EGF receptor would by itself confer on cancer cells resistance to radiation. The current study is aimed to address this question. METHODS AND MATERIALS A full-length human EGF receptor expression vector was transfected into the OCA-I murine ovarian carcinoma cells for stable clones expressing various levels of EGF receptors. Apoptosis and cell clonogenic survival assays were used to evaluate the sensitivity of the resulting cell clones to ionizing radiation. RESULTS OCA-I cell clones expressing various levels of EGF receptor (OCA-I EGFR) were obtained. These clones showed an EGF receptor level-dependent increase in resistance to ionizing radiation, measured by apoptosis and cell clonogenic survival assays. Compared with the results for parental OCA-I and control vector-transfected OCA-I cells at the 10% cell survival level, the radioresistance was increased by a factor of 1.60 for EGFR-C5 (high level of EGF receptor expression), 1.37 for EGFR-C3 (intermediate level of EGF receptor expression), and 1.28 for EGFR-C1 (low level of EGF receptor expression). Treatment of the OCA-I EGF receptor transfectants with the anti-EGF receptor monoclonal antibody C225 downregulated the levels of EGF receptor, reduced the phosphorylation levels of EGF receptor downstream substrates (such as Akt and MAPK), and reversed the cellular radioresistance. CONCLUSION Our results demonstrate that overexpression of the EGF receptor conferred cellular resistance to ionizing radiation. The EGF receptor is thus a valid target for potential radiosensitization.

Apoptosis in irradiated murine tumors

Early radiation responses of transplantable murine ovarian (OCaI) and hepatocellular (HCaI) carcinomas were examined at 6, 24, 48, 96, and 144 h after single photon doses of 25, 35, or 45 Gy. Previous studies using tumor growth delay and tumor radiocurability assays had shown OCaI tumors to be relatively radiosensitive and HCaI tumors to be radioresistant. At 6 h, approximately 20% of nuclei in OCaI tumors showed aberrations characteristic of cell death by apoptosis. This contrasted to an incidence of 3% in HCaI tumors. Mitotic activity was eliminated in OCaI tumors but was only transiently suppressed in HCaI tumors. At 24-96 h, OCaI tumors continued to display apoptosis and progressive necrosis, whereas HCaI tumors responded by exhibiting marked pleomorphism. Factors other than mitotic activity may influence tumor radiosensitivity, and one of these may be susceptibility to induction of apoptosis (programmed cell death), because this was a prominent early radiation response by the radiosensitive OCaI tumors.

Heterogeneity in the development of apoptosis in irradiated murine tumours of different histologies.

Fifteen different murine tumours were evaluated with respect to the degree of apoptosis development that occurs in the tumour tissue in the first few hours following irradiation in vivo. Animals were killed at 3 or 6 h following irradiation with 0, 2.5, 10 or 25 Gy. Apoptosis was scored as percent aberrant nuclei by microscopic examination of histological sections made from the tumour specimens. Results showed that three of four mammary adenocarcinomas, one ovarian adenocarcinoma, and one lymphoma displayed at least 10% apoptotic cells after 25 Gy, whereas five sarcomas, three squamous cell carcinomas, and a hepatocarcinoma did not. The time courses and dose responses were similar in those tumours that responded. These data were compared with the known response of these same tumours when analysed using conventional assays. The tumours that did respond by significant apoptosis had longer specific growth delays and lower TCD50 (dose to cure 50% of animals) doses, thus suggesting that an acute apoptotic response following irradiation may be a feature of certain tumours that respond well to irradiation. Additionally, this analysis revealed heterogeneity in the apoptotic response both within an individual tumour specimen and among different tumour types. These observations of intra and intertumour heterogeneity are consistent with the idea that the propensity for apoptosis in tumours is genetically regulated.

Is the therapeutic index better with gemcitabine-based chemoradiation than with 5-fluorouracil-based chemoradiation in locally advanced pancreatic cancer?

PURPOSE To retrospectively compare the toxicity and efficacy of concurrent gemcitabine-based chemoradiation with that of concurrent 5-fluorouracil (5-FU)-based chemoradiation in patients with unresectable pancreatic cancer. PATIENTS AND METHODS Between September 1996 and May 2000, 114 patients with localized unresectable adenocarcinoma of the pancreas were treated with concurrent chemoradiation. Locally advanced unresectable disease was defined as low-density tumor in contact with the superior mesenteric artery (SMA) or celiac artery, or occlusion of the superior mesenteric-portal venous confluence. Fifty-three patients were selected to receive gemcitabine in 7 weekly cycles (250-500 mg/m(2)) with concurrent radiotherapy (median dose 30 Gy, range 30-33 Gy in 10-11 fractions). The remaining 61 patients received continuous-infusion 5-FU (200-300 mg/m(2)) with concurrent radiotherapy (30 Gy in 10 fractions). Radiotherapy was delivered to the primary tumor and regional lymphatics. Patients receiving gemcitabine and those receiving 5-FU had a similar mean Karnofsky performance status (KPS, 89% vs. 86%), distribution of tumor grade (43% vs. 33% poorly differentiated), and percent weight loss (all p = NS). However, patients treated with gemcitabine had a significantly larger median maximum cross-sectional tumor area (TA, 8.8 cm(2) vs. 5.7 cm(2), p = 0.046) and were significantly younger (median age 60 vs. 68 years, p <0.001). Severe acute toxicity (ST) was defined as toxicity requiring a hospital stay of more than 5 days, mucosal ulceration with bleeding, more than 3 dose deletions of gemcitabine or discontinuation of 5-FU, or toxicity resulting in surgical intervention or death. Kaplan-Meier analysis was used to calculate the actuarial rate of local progression on imaging (LP), the rate of distant metastasis (DM), and the overall survival (OS) rate. The imaging was reviewed in resected patients. RESULTS Patients receiving gemcitabine developed significantly more ST during treatment (23% vs. 2%, p < 0.0001) than did those receiving 5-FU. Patients treated with gemcitabine had a similar 10-month LP rate (62% vs. 61%), 10-month DM rate (55% vs. 47%), 1-year OS rate (42% vs. 28%), and median OS duration (11 months vs. 9 months) to patients treated with 5 FU (all p = NS). Five patients who received gemcitabine and 1 patient who received 5-FU underwent margin-negative pancreaticoduodenectomy after chemoradiation. Three patients had a short segment (<or= 1 cm in length) of low-density tumor abutting the SMA, 1 had involvement of the common hepatic artery, and 1 had a short-segment occlusion of the superior mesenteric vein, amenable to venous resection and reconstruction. The other patient was thought to have inflammatory changes discontiguous with the tumor surrounding the SMA, which resolved after therapy. TA >10 cm(2) (p = 0.03) and poor differentiation (p = 0.07) were associated with a worse survival duration; however, other factors, such as KPS and weight loss >10% and age did not influence OS. CONCLUSION Despite the selection of healthier patients to receive gemcitabine, there was a significantly higher severe toxicity rate than with 5-FU. The median and 1-year survivals were not significantly different with the use of concurrent gemcitabine; however, the tumors treated were significantly larger. Additionally, a small number of patients with minimal arterial involvement whose disease met our radiographic definition of unresectable disease had margin-negative resections after treatment with gemcitabine-based chemoradiation. These possible benefits and the high rate of severe toxicity define a very narrow therapeutic index for concurrent gemcitabine-based chemoradiation given by this schedule of administration.

Kinetics of mitotic arrest and apoptosis in murine mammary and ovarian tumors treated with taxol

The kinetics of taxol-induced mitotic arrest and apoptosis in murine mammary carcinoma MCA-4 and ovarian carcinoma OCA-I tumors were determined to establish a possible causative relationship between mitotic arrest and apoptosis and to see whether these cellular effects of taxol would correlate with the extent of its antitumor efficacy. Mice bearing 8-mm tumors in a hind leg were given taxol i.v. at a dose of 10–80 mg/kg. Both tumors responded to taxol by significant growth delay or transient regression; in general, the response was greater as the dose of taxol was increased. For kinetics studies the mice were treated with 60 mg/kg taxol given once when tumors were 8 mm in size or twice, with the second dose being given 3 days after the first. At various times ranging from 1 to 96 h after treatment with taxol, tumors were histologically analyzed to quantify mitotic and apoptotic activity. After a single dose of taxol, mitotic arrest was visible at 1 h, and the mitotic index increased with time to reach peak values of 36% in MCA-4 tumors and 22% in OCA-I tumors at 9 h. The index then declined to a baseline of 1%–3% at 3 days for MCA-4 tumors and 1 day for OCA-I tumors. Apoptosis followed mitotic arrest, beginning at the time of peak mitotic arrest, increasing to the highest level of about 20% at 18–24 h after treatment and gradually declining to the normal level of 3%–6% after 3–4 days. Nuclear material progressively condensed in mitotically arrested cells, culminating in the frank appearance of multiple apoptotic bodies. The change in cell morphology plus the dynamics of apoptosis development imply that a large percentage of tumor cells arrested in mitosis by taxol die by apoptosis. Kinetic analysis undertaken after the second dose of taxol showed a considerably lower percentage of cells arrested in mitosis as compared with that seen after a single dose, and the induction of apoptosis by the second dose was minimal. However, the antitumor efficacy of the second dose of taxol was similar to or better than that of the first dose, implying that in addition to mitotic arrest and apoptosis, there exist other mechanisms by which taxol exerts its antitumor action.

C225 antiepidermal growth factor receptor antibody enhances tumor radiocurability

PURPOSE Overexpression of epidermal growth factor receptor (EGFR) has been correlated with tumor resistance to radiation. Blockade of EGFR with C225 anti-EGFR antibody was previously shown to synergistically enhance radiation-induced tumor growth delay. The purpose of this study was to assess whether C225 can increase tumor cure by radiation. METHODS AND MATERIALS Nude mice bearing 8-mm-diameter A431 tumor xenografts in the hind leg were treated with C225 antibody, graded single doses of local tumor irradiation, or both. C225 was given i.p. at a dose of 1 mg/mouse 6 h before irradiation or 6 h before and 3 plus 6 days after irradiation. Tumor cure was the treatment endpoint assessed by the TCD(50) assay 120 days after treatment. The onset of recurrences of tumors not cured was also determined. RESULTS C225 antibody increased the antitumor effects of radiation by reducing TCD(50) values and delaying tumor recurrences. Tumor radiocurability was enhanced by a factor of 1.18 by a single dose and by a factor of 1.92 by three doses of C225. Likewise, the appearance of tumor recurrences was delayed by a factor of 1.37 by a single dose and by a factor of 2.13 by three doses of C225. CONCLUSION The data presented here demonstrate that C225 can increase tumor radiocurability and delay the appearance of recurrences of tumors not cured by radiation treatment.

Development of Apoptosis in Irradiated Murine Tumors as a Function of Time and Dose

In a previous paper (Radiat. Res. 127, 308-316, 1991), we reported that a moderately radiosensitive, transplantable murine ovarian carcinoma (OCaI) displayed apoptosis after irradiation whereas a radioresistant hepatocellular carcinoma (HCaI) did not. These initial observations have been followed up in this detailed analysis of the development of apoptosis in these two tumors as a function of time and dose. Histological sections of OCaI and HCaI carcinomas were scored at various times between 0.5 and 24 h after single doses of 2.5 or 25 Gy gamma radiation for the incidence of apoptosis. The percentage of nuclei undergoing apoptosis in untreated tumors was 5% in OCaI and 0.6% in HCaI. The peak in the number of apoptotic bodies occurred in the OCaI tumors 3-5 h after either dose. After 2.5 Gy, the peak incidence was about 20% and after 25 Gy it was about 30%. Irrespective of dose, HCaI tumors had an incidence of apoptosis of less than 3%. Based on the results of this time course, 4 h after irradiation was chosen for the determination of the dose response, over doses ranging from 2.5 to 25 Gy. The dose response for the OCaI tumors reached a plateau at 25-30% apoptotic nuclei after doses of about 7.5 Gy and above. Autoradiographic analysis of histological sections from mice injected with [3H]thymidine showed that some apoptotic bodies in the OCaI tumors arose from cycling cells. These results confirm that the apoptotic mode of cell death may represent an important response in some irradiated tumors.

Single Nucleotide Polymorphism at rs1982073:T869C of the TGFβ1 Gene Is Associated With the Risk of Radiation Pneumonitis in Patients With Non–Small-Cell Lung Cancer Treated With Definitive Radiotherapy

PURPOSE In search of reliable biologic markers to predict the risk of normal tissue damage by radio(chemo)therapy before treatment, we investigated the association between single nucleotide polymorphisms (SNPs) in the transforming growth factor 1 (TGFbeta1) gene and risk of radiation pneumonitis (RP) in patients with non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS Using 164 available genomic DNA samples from patients with NSCLC treated with definitive radio(chemo)therapy, we genotyped three SNPs of the TGFbeta1 gene (rs1800469:C-509T, rs1800471:G915C, and rs1982073:T869C) by polymerase chain reaction restriction fragment length polymorphism method. We used Kaplan-Meier cumulative probability to assess the risk of grade > or = 3 RP and Cox proportional hazards analyses to evaluate the effect of TGFbeta1 genotypes on such risk. RESULTS There were 90 men and 74 women in the study, with median age of 63 years. Radiation doses ranging from 60 to 70 Gy (median = 63 Gy) in 30 to 58 fractions were given to 158 patients (96.3%) and platinum-based chemotherapy to 147 (89.6%). Grade > or = 2 and grade > or = 3 RP were observed in 74 (45.1%) and 36 patients (22.0%), respectively. Multivariate analysis found CT/CC genotypes of TGFbeta1 rs1982073:T869C to be associated with a statistically significantly lower risk of RP grades > or = 2 (hazard ratio [HR] = 0.489; 95% CI, 0.227 to 0.861; P = .013) and grades > or = 3 (HR = 0.390; 95% CI, 0.197 to .774; P = 0.007), respectively, compared with the TT genotype, after adjustment for Karnofsky performance status, smoking status, pulmonary function, and dosimetric parameters. CONCLUSION Our results showed that CT/CC genotypes of TGFbeta1 rs1982073:T869C gene were associated with lower risk of RP in patients with NSCLC treated with definitive radio(chemo)therapy and thus may serve as a reliable predictor of RP.

Astro research fellowship: Apoptosis as a predictor of tumor response to radiation in Stage IB cervical carcinoma

PURPOSE Levels of apoptosis predict for tumor responsiveness to radiation in various animal systems. To investigate the potential role of apoptosis as a predictor of response in human tumors, a retrospective review was undertaken of patients with adenocarcinoma of the cervix whose primary lesion at presentation measured at least 4 cm and who underwent definitive radiation therapy. A previous report had indicated that roughly half this group of patients should have a long-term relapse free survival. METHODS AND MATERIALS Pretreatment biopsy specimens of 44 patients with Stage IB adenocarcinoma of the cervix, whose primary lesion at presentation measured at least 4 cm in greatest dimension, were scored for apoptosis by two independent investigators without knowledge of the treatment outcome, and the results were averaged. Actuarial methods were used to assess overall survival, disease-free survival, determinate survival, and local control as a function of the baseline level of apoptosis. Patients ranged in age from 21 to 87 years and were treated with definitive radiotherapy between 1964 and 1989. Follow-up for the surviving patients ranged from 1 to 278 months, with a mean of 101 months. RESULTS Patients whose tumors had a baseline level of apoptosis above the median value (2%) had a better overall survival than those with lower levels of apoptosis (p = 0.056). A similar trend for disease-free survival (p = 0.32) and determinate survival (p = 0.27) did not reach statistical significance, perhaps because of the small number of patients. Because only 6 of the 44 patients (13%) had a local tumor failure, it was not possible to establish a correlation between the pretreatment level of apoptosis and the local tumor control by radiation. CONCLUSION The baseline level of apoptosis predicted for survival in patients with Stage IB cervical adenocarcinoma. Further investigation of the measurement of apoptosis as a potential predictive assay is warranted in other human tumor systems.

Apoptosis in murine tumors treated with chemotherapy agents.

There is increasing attention directed to the hypothesis that apoptosis plays a role in the response to cancer treatment including chemotherapy. However, the evidence to support this hypothesis has come almost entirely from experiments conducted in cultured cell systems. To extend this hypothesis to the therapeutic setting it is necessary to address this critical question in tumors treated in vivo. We have therefore evaluated the extent of apoptosis induced in murine tumors treated in vivo with cancer chemotherapy agents. Seven different murine tumors, comprising a mammary adenocarcinoma (MCa-4), an ovarian adenocarcinoma (OCa-1), a lymphoma (LY-TH), three sarcomas (FSA, NFSA and SA-NH) and a squamous cell carcinoma (SSC-7), were examined 8 and 24 h after treatment with cisplatin or cyclophosphamide (CY). Apoptosis was scored by morphometric analysis of histological sections of the tumors. The results showed that MCa-4, OCa-1 and LY-TH had a significant apoptotic response to both cisplatin and CY, and the other tumors had essentially no apoptotic response. In addition, two of these tumors, MCa-4 and OCa-1, underwent apoptosis in response to adriamycin, 5-fluorouracil, Ara-C, etoposide, camptothecin and fludarabine. These observations demonstrate that apoptosis may be a feature of tumor response to chemotherapy in vivo, and illustrate the heterogeneity of apoptotic response amongst different tumor types and to different cytotoxic agents.