Cross-border referral for early breast cancer: an analysis of radiation fractionation patterns.

Because of increasing waiting times for adjuvant radiation in the province of Ontario, patients from one Canadian centre were referred to two centres in the United States. This situation provided an opportunity to compare radiation practices.We performed a retrospective review of radiation prescribed to patients following breast-conserving surgery for invasive breast cancer. Patients with positive margins, 4 or more positive lymph nodes, recurrent disease, or large tumours (>5 cm) were excluded. For comparison, we reviewed a random sample of similar patients treated at the Canadian centre during the same period. A total of 120 referred and 217 non-referred patients were eligible for comparison. The analysis included 98 pairs of patients (N = 196), fully matched on age, nodal status, T stage, grade, and estrogen receptor (er) status.Mean patient age was 60.7 years. The median total dose and number of fractions differed between centres [6040 cGy in 32 fractions (United States) vs. 4250 cGy in 16 fractions (Canadian), both p < 0.001). Boost was used more often in the United States (97% vs. 9%, p < 0.001). Variation in prescribing patterns was seen. In the United States, seven different schedules for whole-breast irradiation were used; at the Canadian centre, two schedules were prescribed. Predicted radiobiologic effects of these schedules were calculated to be similar.Differences in fractionation patterns were observed between and within U.S. and Canadian centres. Such variability is likely to affect patient convenience and resource utilization. Although patient selection, referring surgeon, and change in policies may account for some of the observed differences, further research is necessary to better understand the causes.

Limitations of reduced-field irradiated volume and technique in conventional radiation therapy of prostate cancer: implications for conformal 3-D treatment.

In order to define technical limitations of conventional external beam irradiation for clinically localized prostate cancer, we evaluated the impact of several reduced-field treatment factors, such as reduced-field (RF) irradiated volume, RF technique, photon energy of treatment, and dose on survival endpoints and local control in a retrospective series. Several survival endpoints, such as disease-specific survival, freedom from relapse survival, biochemical no-evidence of disease (bNED) survival, and local control were associated with several treatment variables using univariate and multivariate analyses in 329 patients. Reduced-field technique appeared to predict survival outcome, with patients treated by bilateral 120 degrees arcs faring less well than those treated by full 360 degrees rotational fields. The irradiated volume of the reduced-field was also significantly associated with survival outcome, with patients treated with smaller volumes faring less well. Local failure rates also appeared increased, although not statistically, in patients treated with smaller RF sizes. In an attempt to explain these detected deficiencies, dose-volume histograms for prostate coverage were created for a small sample of patients. The deficiencies related to small reduced-field volume appeared to be largely attributable to poor dosimetric coverage of the prostate. These results underscore the limitations of conventional external beam treatment for prostate carcinoma when conventional techniques are employed, particularly if small reduced fields are used, and further supports the development of improved treatment techniques, such as conformal irradiation, as alternatives.

NCCN Practice Guidelines for Prostate Cancer.

Systemic therapies for prostate cancer are likely to improve, and as they do, they will have enormous impact on the treatment of high-risk and locally advanced cancers. Further technical improvements in radiotherapy and alternative local modalities, such as cryoablation, are also likely, and will bring even more options for local control. It is certain these guidelines will continue to evolve.

Protein changes associated with ionizing radiation-induced apoptosis in human prostate epithelial tumor cells.

Ionizing radiation (IR) is an important component in the therapy of localized prostate cancer. Identification of protein alterations during IR-induced apoptosis prostate cancer cells is an important step toward understanding the new metabolic status of the dying cell. In the present study, we report changes in protein profile that define the execution phase of the apoptotic response in the in vitro model of tumorigenic radiation-transformed SV40-immortalized human prostate epithelial cells (267B1-XR), induced to undergo programmed cell death by IR. We employed an approach that involves use of analytical two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) coupled with Western blotting with specific antisera. Our results point out that apoptotic cells experience significant reduction in the levels of the intermediate filament proteins, keratins-18, 19, vimentin and the associated 14-3-3 adapter proteins. At the same time, molecular chaperones such as glucose-regulated protein 94, calreticulin, calnexin, and protein disulfide isomerase exhibit marked accumulation in these dying cells. The present data indicate that apoptosis-associated processes in prostate epithelial cells include solubilization of the rigid intermediate filament network by specific proteolysis as well as increased levels of endoplasmic reticulum (ER) proteins with chaperone functions.

Effect of oncogene expression on telomerase activation and telomere length in human endothelial, fibroblast and prostate epithelial cells.

Although strong evidence is mounting that telomerase reactivation and the thereof resulting stabilization of telomeres is a major mechanism for human cells to overcome replicative senescence, a causal relationship linking telomerase activation conclusively to tumorigenesis remains to be established. Thus, the possibility exists that telomerase activation is passively co-selected as tumors develop. To elucidate the function of telomerase during tumorigenesis, we followed telomerase reactivation during immortalization of human primary cell types with in vitro transforming agents and determined the tumorigenic potential of these cells at various stages of transformation. The effects of SV40, v-Ki-ras, HPV-18 and HPV-16 E6/E7 oncoproteins on telomerase expression was examined in primary and immortalized human prostate epithelial (HPE), human prostate fibroblast (HPF), and umbilical vein endothelial cells (HUVEC). All of five SV40-transformed HPE and HPF lines were telomerase positive and had shorter telomeres than primary cells. The two HPV-18 immortalized HPE cell lines also expressed telomerase activity. In contrast, E6 or E7 alone could not produce immortalized HUVEC and did not reactivate telomerase. Life-span, however, was extended. The E6/E7 immortalized HUVEC had telomerase activity and short but stable telomeres. HPE, HPF or HUVEC cells which had been transformed by one oncoprotein alone were not tumorigenic although they had overcome cellular senescence and re-activated telomerase. However, if these cells were transformed by a second agent, either infection with v-Ki-ras or X-ray treatment, they were able to form tumors in nude mice. This suggests that tumorigenesis is a multistep process and that telomerase activation alone is not sufficient for malignant transformation in human cells.

Apoptosis-associated proteolysis of vimentin in human prostate epithelial tumor cells.

Vimentin intermediate filaments (IF) are responsible for regulation of cell attachment and subcellular organization. Using an in vitro model system of human prostate epithelial cells (267B1-XR), we demonstrate that a series of vimentin proteolytic fragments represent some of the differentially expressed proteins in 2D-gel profiles of the apoptotic cells undergoing ionizing radiation-induced cell death. A caspase-sensitive motif search suggests that the type III IF protein (vimentin) is subject to proteolysis to promote the execution phase of apoptosis, in a manner similar to the well-established type V (lamins) and type I (keratins 18, 19) IF proteins. Furthermore, vimentin and a few of its derived polypeptides, reported to be specific to the apoptotic process, correspond to ubiquinated proteins, thus pointing to the complex interrelationships of protein ubiquination in solubilizing the IF network during apoptosis.

Expression of cytokeratin-19 as a marker of neoplastic progression of human prostate epithelial cells.

BACKGROUND: Our earlier studies demonstrated neoplastic transformation of SV40-immortalized neonatal human prostate epithelial cells (267B1) by fractionated doses of ionizing radiation or by introduction of v-ki-ras oncogene. X-ray-treated 267B1 cells represent three different stages of neoplastic progression: nontumorigenic F3-SAC cells that acquired morphological changes and anchorage independence when treated with 2 x 2 Gy of X-rays; malignantly transformed 267B1-XR and 267B1-SXR cells that received 2-Gy doses to a total of 30 Gy. We also reported alterations in cell size, morphology, actin stress fibers, and levels of actin-binding proteins in these transformed human prostate cells. METHODS: We analyzed intermediate filament-nuclear matrix (IF-NM) protein expression in the various 267B1 cells as a consequence of neoplastic progression by two-dimensional gel electrophoresis and immunofluorescence. RESULTS: Our present study revealed that the 267B1 cells experienced progressive changes in their intermediate filament protein composition during the process of neoplastic conversion, achieved either by X-rays or by ras-oncogene. In particular, we observed a stepwise downregulation of cytokeratin-19 in these in vitro transformed 267B1 cells. CONCLUSIONS: Our data suggest that loss of expression of cytokeratin-19 accompanied the morphological alterations associated with in vitro neoplastic transformation of SV40-immortalized prostate epithelial cells.

Adenocarcinoma of the fallopian tube: results of a multi-institutional retrospective analysis of 72 patients.

PURPOSE/OBJECTIVE: To determine the prognostic factors for predicting outcome of patients with adenocarcinoma of the fallopian tube and to evaluate the impact of treatment modalities in managing this uncommon disease. MATERIALS AND METHODS: A retrospective analysis of the tumor registries from 6 major medical centers from January 1, 1960 up to March 31, 1995 yielded 72 patients with primary adenocarcinoma of the fallopian tube. The Dodson modification of the FIGO surgical staging as it applies to carcinoma of the fallopian tube was utilized. Endpoints for outcome included overall and disease-free survival. Univariate analysis of host, tumor, and treatment factors was performed to determine prognostic significance, and patterns of failure were reviewed. RESULTS: The median age of the study cohort was 61 years (range 30-79 years). Stage distribution was 24 (33%) Stage I; 20 (28%) Stage II; 24 (33%) Stage III; and 4 (6%) Stage IV. Adjuvant chemotherapy was administered to 54 (75%) patients, and postoperative radiotherapy was employed in 22 (31%). In the latter treatment group, 14 (64%) had whole pelvic external beam irradiation, 5 (23%) whole abdominal radiotherapy, 2 (9%) P-32 instillation, and 1 (4%) vaginal brachytherapy alone. Chemotherapy was used in 67% of Stage I and in 79% of Stages II/III/IV disease (not significant); radiotherapy was more commonly employed in Stage I than in Stages II/III/IV (46% vs. 23%, p = 0.05). The 5-, 8-, 15-year overall and disease-free survival for the study patients were 44.7%, 23.8%, 18.8% and 27.3%, 17%, 14%, respectively. Significant prognostic factors of overall survival included Stage I vs. II/III/IV (p = 0.04) and age < or = 60 years vs. > 60 years at diagnosis (p = 0.03). Only Stage I vs. II/III/IV (p = 0.05) was predictive of disease-free survival. Patterns of failure included 18% pelvic, 36% upper abdominal, and 19% distant. For all patients, upper abdominal failures were more frequently found in Stages II/III/IV (29%) than in Stage I (7%) (p = 0.03). Relapses solely outside of what would be included in standard whole abdominal radiotherapy portals occurred for only 15% of patients (6 of 40) with failures. Furthermore, patients having any recurrence, including the upper abdomen, were more likely (p = 0.001) to die (45%) than those without any type of relapse (18%). CONCLUSION: This retrospective, multi-institutional study demonstrated the importance of FIGO stage in predicting the overall and disease-free survival of patients with carcinoma of the fallopian tube. Future investigations should consider exploring whole abdominal irradiation as adjunctive therapy, particularly in Stage II and higher.

Treatment of female urethral carcinoma in medically inoperable patients using external beam irradiation and high dose rate intracavitary brachytherapy.

PURPOSE: We developed and present our experience with high dose rate brachytherapy for treatment of carcinoma of the urethra in medically inoperable women. MATERIALS AND METHODS: Since 1991, 4 women with localized urethral cancer, medically unable to undergo resection or interstitial implantation, were treated with external beam and high dose rate intracavitary implantation rather than external beam irradiation alone. The fractionated implants were delivered with a high dose rate remote afterloader using a shielded vaginal applicator and modified urethral catheter. The urethral catheter was inserted through the lumen of a 20F Foley tube to improve depth dose. Homogeneous dose distribution was achieved and customized to the individual patient. RESULTS: All high dose rate brachytherapy treatments were given at the clinic without use of sedation or anesthesia. Treatment was well tolerated, and all patients maintained voluntary urinary function and local control at 12 to 55 months after therapy. Chronic morbidity due to urethral, bladder, vaginal or rectal injury, including urethral stenosis, necrosis or fistula, was not noted. Isodose distributions were compared among this technique, interstitial implantation and external beam radiotherapy alone. CONCLUSIONS: Although we prefer interstitial implantation as the boost technique for women with urethral cancer, high dose rate brachytherapy is a reasonable option for medically inoperable patients. This outpatient treatment is well tolerated, preserves voluntary urinary function and enhances quality of life.

Protein expression changes associated with radiation-induced neoplastic progression of human prostate epithelial cells.

Carcinogenic progression in most epithelial systems is a multistep process and presents as numerous (un)stable intermediate stages prior to the development of a fully malignant phenotype. Recently, we reported the neoplastic transformation of an SV40 immortalized, neonatal human prostate epithelial cell line (267B1) by multiple exposures to X-rays [1, 2]. The parental 267B1 cells acquired anchorage-independence and exhibited morphological transformation following exposure to two consecutive doses of 2 Gy. Exposure of either the parental 267B1 cells or the anchorage-independent derivatives (F3-SAC) to a total dose of 30 Gy of X-rays yielded tumorigenic transformants (267B1-XR and 267B1-SXR, respectively). All of these radiation-treated derivatives (F3-SAC, 267B1-XR, and 267B1-SXR) were characterized by reduced cell size and poorly organized actin stress fibers [2, 3]. The present study examines the protein expression changes associated with cytoskeletal alterations during the different steps of neoplastic progression induced by X-rays in the in vitro human prostate cell system. This analysis was achieved by using the high resolving power of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) in the 267B1, F3-SAC, 267B1-XR, and 267B1-SXR cells. We report changes in the expression of gelsolin in the partially transformed, anchorage-independent, nontumorigenic (F3-SAC) cells and a progressive loss of expression of tropomyosin isoforms (TM-1 and TM-3), and myosin light chain-2 (MLC-2) in the tumorigenic (267B1-XR; 267B1-SXR) cells, respectively. In contrast, our results demonstrate that the levels of the small GTP-binding protein Rho-A, an active participant in the actin stress fiber organization, are not altered during neoplastic progression of these 267B1 cells. Thus the changes in synthesis of gelsolin, tropomyosins, and MLC-2 provide a rationale for the alterations in the actin stress fiber formation and reduction in cell size during the exposure of prostate epithelial cells to multiple doses of X-rays.

Malignant transformation of human prostate epithelial cells by N-nitroso-N-methylurea.

We report the malignant transformation of adult human prostate epithelial cells after multiple exposures to the chemical carcinogen N-nitroso-N-methylurea. Such transformants showed morphological alterations and anchorage-independent growth in soft agar and induced carcinomas when transplanted into nude mice. No p53 or ras mutations were observed. Stepwise chromosomal changes in the progression to tumorigenicity were observed. Loss of the p arms of chromosome 8 (p10>pter) and chromosome 10 (p10>pter) and gain of the q arm of chromosome 8 (q10>qter) were only observed in the tumor outgrows. These findings provide the first evidence of malignant transformation of human prostate epithelial cells exposed to a chemical carcinogen.

p53-Independent tumorigenic progression of human prostate cells.

We have previously described the development of radiation transformed human fetal prostate epithelial cells, 267B1. Using this in vitro model system, we investigated the molecular mechanisms of prostate carcinogenic progression by comparing nontumorigenic (267B1/B) and tumorigenic (267B1/D) cells. We examined the G1- to S-phase transition in synchronized cells to determine if the progression of 267B1 cells from nontumorigenic to tumorigenic was the consequence of a perturbation in the G1- to S-phase transition involving p53, pRb, p21, or p16. Nontumorigenic 267B1/B cells showed a time-dependent increase in the expression of p53 and a corresponding increase in p21 following exposure to ionizing radiation (6 Gy). The levels of pRb and p16 protein were virtually unchanged. In contrast, tumorigenic 267B1/D cells exhibited a p53-independent induction of p21 protein with a parallel increase in p16 protein in response to ionizing radiation, but no change in pRb was observed. These results suggest that the progression of 267B1 cells from nontumorigenic to tumorigenic involves p53-independent processes.

Management of locally advanced prostate cancer.

The staging and treatment of prostate cancer are complex, particularly in patients with clinical disease that has advanced locally beyond the confines of the gland. Management choices are made more difficult by a paucity of quality randomized and controlled studies. Staging has traditionally relied on digital rectal examination, which is now being augmented by improved noninvasive radiologic studies. Radiation is the most common form of treatment today, and newer techniques are being examined and compared to external-beam therapy. Surgical intervention as monotherapy has failed to show a survival advantage. Current approaches treatment appear to be evolving toward combination therapies, potentially incorporating hormonal manipulation. Patients with locally advanced disease should be encouraged to enter prospective clinical trials.

Radiation-induced neoplastic transformation of human prostate epithelial cells.

We report the malignant transformation of human prostate epithelial cells (267B1) after multiple exposures to ionizing radiation. Carcinogenic progression of cells from immortal growth to anchorage-independent growth in soft agar to tumorigenicity in athymic mice resulted after a cumulative X-ray dose of 30 Gy. The tumors were characterized histologically as poorly differentiated adenocarcinomas, expressed prostate-specific antigen, and stained positive for keratin. No p53 or ras mutations were observed. Numerous chromosomal defects were noted on karyotypes after radiation exposure. However, chromosome 3 and 8 translocations were observed predominantly in the tumor outgrowths. These findings provide the first evidence of malignant transformation of human prostate epithelial cells exposed to ionizing radiation.

Cytoskeletal changes during radiation-induced neoplastic transformation of human prostate epithelial cells.

We recently reported tumorigenic transformation of SV40-immortalized neonatal human prostate epithelial cells (267B1) by exposure to fractionated doses of X-rays. Altered morphology and anchorage independence were observed following two successive fractions of 2 Gy each (F3-SAC). Additional 2 Gy treatments to these non-tumorigenic cells to a total dose of 30 Gy resulted in radiation-transformed tumorigenic colonies (267B1-SXR). Malignant transformation of parental 267B1 cells was also achieved by consecutive 2 Gy exposures to a total dose of 30 Gy (267B1-XR). This study discusses the cytoskeletal changes in the F3-SAC, 267B1-XR and 267B1-SXR derivatives of these human prostate epithelial cells. Confocal and conventional fluorescence microscopy of filamentous actin showed numerous, well organized, evenly distributed stress fibers in the parental cells prior to irradiation, while the anchorage-independent cells and several tumorigenic derivatives exhibited poor stress fiber organization after radiation exposure. This disorganization of actin microfilaments in the radiation-transformed cells was also accompanied by changes in the expression of selective tropomyosin isoforms as judged by two-dimensional gel electrophoresis. These changes in actin organization and tropomyosin expression appear to be coincidental with morphological transformation and acquisition of tumorigenicity in the 267B1 cells following radiation exposure.

Radiation-induced neoplastic transformation of human cells.

Ionizing radiation can induce cancers in humans and animals and can cause in vitro neoplastic transformation of various rodent cell systems. However, numerous attempts to achieve neoplastic transformation of human cells by radiation have generally proven unsuccessful. Neoplastic transformation of immortalized human epidermal keratinocytes by X-ray irradiation has recently been reported. The carcinogenic effect of radiation on cultured human cells will be briefly reviewed. The current state-of-the-art in radiation-induced transformation of human cells in culture is presented. This will provide insight into the molecular and cellular mechanisms in the conversion of normal cells to a neoplastic state of growth.

Neoplastic transformation of a human prostate epithelial cell line by the v-Ki-ras oncogene.

Investigations of mechanisms of human prostate carcinogenesis are limited by the unavailability of a suitable in vitro model system. We have demonstrated that an immortal, but nontumorigenic, human epithelial cell line (267B1) established from fetal prostate tissue can be malignantly transformed by a biological carcinogen, and can serve as a useful model for investigations of the progression steps of carcinogenesis. Activated Ki-ras was introduced into 267B1 cells by infection with the Kirsten murine sarcoma virus. Morphological alterations and anchorage-independent growth were observed; when cells were injected into nude mice, poorly differentiated adenocarcinomas developed. These findings represent the first evidence of malignant transformation of human prostate epithelial cells in culture, and support a role for Ki-ras activation in a multistep process for prostate neoplastic transformation.