Anti-erbB receptor strategy as a gene therapeutic intervention to improve radiotherapy in malignant human tumours.

PURPOSE: We explored and quantified the therapeutic potential of using dominant-negative EGFR transduction with replication-incompetent adenovirus (Ad-EGFR-CD533 or Ad-CD533) as a genetic approach for radiosensitization in different carcinoma and malignant glioma cell lines in vitro and in established tumour xenografts in vivo. MATERIAL AND METHODS: The cell lines MDA-MB-231, A-431, U-373 MG, U-87 MG and T47D were used. The ErbB expression profiles were quantified by Western blotting. MAPK immune complex assay measured MAPK activity with or without EGFR-CD533 expression after ionizing radiation. Radiosensitization was determined and quantified in vitro by colony-formation assays, in vivo by use of an ex vivo-in vitro colony-formation assay after intratumoral infusion of the adenoviral vectors expressing EGFR-CD533 or the control LacZ. RESULTS: Western blotting demonstrated widely varied expression levels of the ErbB receptors in the tested cell lines. Expression of EGFR-CD533 effectively blocked the radiation-induced activation of MAPK, leading to significant radiosensitization in vitro and in vivo. CONCLUSIONS: The radiation-induced ErbB activation can be effectively modulated by a gene therapeutic approach of over-expressing EGFR-CD533 leading to tumour cell radiosensitization after single and repeated radiation exposures both in vitro and in vivo.

The effect of setup uncertainty on normal tissue sparing with IMRT for head-and-neck cancer.

PURPOSE: Intensity-modulated radiotherapy (IMRT) is being evaluated in the management of head-and-neck cancers at several institutions, and a Radiation Therapy Oncology Group study of its utility in parotid sparing is under development. There is an inherent risk that the sharper dose gradients generated by IMRT amplify the potentially detrimental impact of setup uncertainty. The International Commission on Radiation Units and Measurements Report 62 (ICRU-62) defined planning organ-at-risk volume (PRV) to account for positional uncertainties for normal tissues. The purpose of this study is to quantify the dosimetric effect of employing PRV for the parotid gland and to evaluate the use of PRV on normal-tissue sparing in the setting of small clinical setup errors. METHODS AND MATERIALS: The optimized nine-beam IMRT plans for three head-and-neck cancer patients participating in an institutional review board approved parotid-sparing protocol were used as reference plans. A second optimized plan was generated for each patient by adding a PRV of 5 mm for the contralateral parotid gland. The effect of these additions on the quality of the plans was quantified, in terms of both target coverage and normal-tissue sparing. To test the value of PRV in a worst-case scenario, systematic translational setup uncertainties were simulated by shifting the treatment isocenter 5 mm superiorly, inferiorly, left, right, anteriorly, and posteriorly, without altering optimized beam profiles. At each shifted isocenter, dose distributions were recalculated, producing a total of six shifted plans without PRV and six shifted plans with PRV for each patient. The effect of setup uncertainty on parotid sparing and the value of PRV in compensating for the uncertainty were evaluated. RESULTS: The addition of the PRV and reoptimization did not significantly affect the dose to gross tumor volume, spinal cord, or brainstem. In contrast, without any shift, the PRV did increase parotid sparing and reduce coverage of the nodal region adjacent to the parotid gland. As expected, when the plans were shifted, the greatest increase in contralateral parotid irradiation was noted with shifts toward the contralateral parotid gland. With these shifts, the average volume of contralateral parotid receiving greater than 30 Gy was reduced from 22% to 4% when a PRV was used. This correlated with a reduction in the average normal-tissue complication probability (NTCP) from 22% to 7%. CONCLUSIONS: The use of PRV may limit the volume of normal tissue structures, such as the parotid gland, exceeding tolerance dose as a result of setup errors. Consequently, it will be important to incorporate the nomenclature of ICRU-62 into the design of future IMRT studies, if the clinical gains of increased normal-tissue sparing are to be realized.

Adenovirus-mediated overexpression of dominant negative epidermal growth factor receptor-CD533 as a gene therapeutic approach radiosensitizes human carcinoma and malignant glioma cells.

PURPOSE: Epidermal growth factor receptor (EGFR) and other members of the ErbB family of receptor tyrosine kinases (RTK) mediate autocrine growth regulation in a wide spectrum of human tumor cells. We have previously demonstrated that in stably transfected mammary carcinoma cells a dominant negative (DN) mutant of EGFR, EGFR-CD533 is a potent inhibitor of EGFR and its cytoprotective signaling after exposure to ionizing radiation. In the present study, we further investigate the capacity of a genetic approach, using replication-incompetent adenovirus (Ad)-mediated transfer of EGFR-CD533 (Ad-EGFR-CD533), to enhance the radiosensitivity in vitro of four cell lines representative of three major cancer phenotypes. METHODS AND MATERIALS: The cell lines MDA-MB-231 and T-47D mammary carcinoma, A-431 squamous carcinoma, and U-373 MG malignant glioma cells were used. The ErbB expression profiles and the EGFR tyrosine phosphorylation (Tyr-P) levels following irradiation were quantified by Western blotting. The relative radiosensitivities of tumor cells were assessed by standard colony formation assays after infection with control vector (Ad-LacZ) or Ad-EGFR-CD533. RESULTS: The expression profiles demonstrated varying levels of EGFR, ErbB2, ErbB3, and ErbB4 expression. The overexpression of EGFR-CD533 after infection with Ad-EGFR-CD533 completely inhibited the radiation-induced stimulation of EGFR Tyr-P relative to the immediate 2.4- to 3.1-fold increases in EGFR Tyr-P in control infected cells (Ad-LacZ). Ad-EGFR-CD533-infected cells demonstrated significant (p < 0.001) radiosensitization over a range of radiation doses (1-8 Gy), yielding dose-enhancement ratios (DER) between 1.4 and 1.7. This radiosensitization was maintained under conditions of repeated radiation exposures, using 3 x 2 Gy, yielding DERs of 1.6 and 1.7 for MDA-MB-231 and U-373 cells, respectively. CONCLUSIONS: Overexpression of EGFR-CD533 significantly sensitizes human carcinoma and glioma cells to single and repeated radiation exposures irrespective of their ErbB expression levels. Therefore, transduction of human tumor cells with EGFR-CD533 holds promise as a gene therapeutic approach for the radiosensitization of neoplastic cells that are growth-regulated by EGFR or other ErbB receptors.

Epidermal growth factor receptor as a genetic therapy target for carcinoma cell radiosensitization.

BACKGROUND: Exposure of human cancer cells to ionizing radiation activates the epidermal growth factor receptor (EGFR), which, in turn, mediates a cytoprotective response that reduces the cells' sensitivity to ionizing radiation. Overexpression of a dominant-negative EGFR mutant, EGFR-CD533, disrupts the cytoprotective response by preventing radiation-induced activation of the receptor and its downstream effectors. To investigate whether gene therapy with EGFR-CD533 has the potential to increase tumor cell radiosensitivity, we introduced an adenoviral vector containing EGFR-CD533 into xenograft tumors in nude mice and evaluated the tumor response to ionizing radiation. METHODS: Xenograft tumors established from the human mammary carcinoma cell line MDA-MB-231 were transduced via infusion with the adenoviral vector Ad-EGFR-CD533 or a control vector containing the beta-galactosidase gene, Ad-LacZ. The transduced tumors were then exposed to radiation in the therapeutic dose range, and radiation-induced EGFR activation was assessed by examining the tyrosine phosphorylation of immunoprecipitated EGFR. Radiosensitization was determined in vitro by colony-formation assays. All statistical tests were two-sided. RESULTS: The transduction efficiency of MDA-MB-231 tumors by Ad-LacZ was 44%. Expression of EGFR-CD533 in tumors reduced radiation-induced EGFR activation by 2.94-fold (95% confidence interval [CI] = 2.23 to 4.14). The radiosensitivity of Ad-EGFR-CD533-transduced tumors was statistically significantly higher (46%; P<.001) than that of Ad-LacZ-transduced tumors, yielding a dose-enhancement ratio of 1.85 (95% CI = 1.54 to 2.51). CONCLUSIONS: Transduction of MDA-MB-231 xenograft tumors with Ad-EGFR-CD533 conferred a dominant-negative EGFR phenotype and induced tumor radiosensitization. Therefore, disruption of EGFR function through overexpression of EGFR-CD533 may hold promise as a gene therapeutic approach to enhance the sensitivity of tumor cells to ionizing radiation.

The relative role of ErbB1-4 receptor tyrosine kinases in radiation signal transduction responses of human carcinoma cells.

Activation of the epidermal growth receptor (ErbB1) occurs within minutes of a radiation exposure. Immediate downstream consequences of this activation are currently indistinguishable from those obtained with growth factors (GF), e.g. stimulation of the pro-proliferative mitogen-activated protein kinase (MAPK). To identify potential differences, the effects of GFs and radiation on other members of the ErbB family have been compared in mammary carcinoma cell lines differing in their ErbB expression profiles. Treatment of cells with EGF (ErbB1-specific) or heregulin (ErbB4-specific) resulted in a hierarchic transactivations of ErbB2 and ErbB3 dependent on GF binding specificity. In contrast, radiation indiscriminately activated all ErbB species with the activation profile reflecting that cell's ErbB expression profile. Downstream consequences of these ErbB interactions were examined with MAPK after specifically inhibiting ErbB1 (or 4) with tyrphostin AG1478 or ErbB2 with tyrphostin AG825. MAPK activation by GFs or radiation was completely inhibited by AG1478 indicating total dependance on ErbB1 (or 4) depending on which ErbB is expressed. Inhibiting ErbB2 caused an enhanced MAPK response simulating an amplified ErbB1 (or 4) response. Thus ErbB2 is a modulator of ErbB1 (or 4) function leading to different MAPK response profiles to GF or radiation exposure.

Radiosensitization of malignant glioma cells through overexpression of dominant-negative epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) plays an important role in neoplastic growth control of malignant gliomas. We have demonstrated that radiation activates EGFR Tyr-phosphorylation (EGFR Tyr-P) and the proliferation of surviving human carcinoma cells, a likely mechanism of accelerated cellular repopulation, a major cytoprotective response after radiation. We now investigate the importance of radiation-induced activation of EGFR on the radiosensitivity of the human malignant glioma cells U-87 MG and U-373 MG. The function of EGFR was inhibited through a genetic approach of transducing cells with an Adenovirus (Ad) vector containing dominant-negative (DN) EGFR-CD533 (Ad-EGFR-CD533) at efficiencies of 85-90%. The resulting cells are referred to as U-87-EGFR-CD533 and U-373-EGFR-CD533. After irradiation at 2 Gy, both of the cell lines exhibited a mean 3-fold increase in EGFR Tyr-P. The expression of EGFR-CD533 completely inhibited the radiation-induced activation of EGFR. In clonogenic survival assays after a single radiation exposure, the radiation dose for a survival of 37% (D37) for U-87-EGFR-CD533 cells was 1.4- to 1.5-fold lower, relative to cells transduced with AdLacZ or untransduced U-87 MG cells. This effect was amplified with repeated radiation exposures (3 x 2 Gy) yielding a D37 ratio of 1.8-2.0. In clonogenic survival studies with U-373 MG cells, the radiosensitizing effect of EGFR-CD533 was similar. Furthermore, in vivo studies with U-87 MG xenografts confirmed the effect of EGFR-CD533 on tumor radiosensitization (dose enhancement ratio, 1.8). We conclude that inhibition of EGFR function via Ad-mediated gene transfer of EGFR-CD533 results in significant radiosensitization. As underlying mechanism, we suggest the disruption of a major cytoprotective response involving EGFR and its downstream effectors, such as mitogen-activated protein kinase. The experiments demonstrate for the first time that radiosensitization of malignant glioma cells through disruption of EGFR function may be achieved by genetic therapy approaches.

Long-term local control and survival after concomitant boost accelerated radiotherapy for locally advanced cervix cancer.

Between 1989 and 1994, a prospective clinical trial tested the safety and efficacy of concomitant boost accelerated superfractionated (CBASF) radiotherapy for patients with locally advanced cervix cancer. CBASF radiotherapy included 45 Gy/25 fractions to the pelvis and a 14.4 Gy/9 fraction concomitant boost to the primary tumor, followed by brachytherapy for a total point A dose of 85 Gy to 90 Gy. The 22 patients of International Federation of Gynecology and Obstetrics stages IIIA-IVA who received CBASF radiotherapy now have a median follow-up time of more than 8 years. The 7-year actuarial rates of local control and overall survival are 81% and 36%, respectively. Serious late toxicity included bowel injury requiring colostomy in eight patients within 2.5 years after treatment, but no other severe toxicity was observed after longer follow-up intervals. The local control and survival rates achieved with CBASF radiotherapy were higher than those observed within a matched contemporaneous cohort of patients treated with standard radiotherapy alone at the same institution (p = 0.1 for local control, 0.09 for survival). The encouraging trend toward improved tumor control, tempered by the complication rate, suggests an opportunity to apply more sophisticated radiotherapy techniques that might sustain the favorable effects of dose intensification while mitigating the normal tissue toxicity.

A phase I study of RSR13, a radiation-enhancing hemoglobin modifier: tolerance of repeated intravenous doses and correlation of pharmacokinetics with pharmacodynamics.

PURPOSE: Preclinical studies indicate that RSR13 oxygenates and radiosensitizes hypoxic solid tumors by decreasing the oxygen (O(2))-binding affinity of hemoglobin (Hb). A Phase I open-label, multicenter dose and frequency escalation study was conducted to assess the safety, tolerance, pharmacokinetics, and pharmacodynamic effect of daily RSR13 administration to cancer patients receiving concurrent palliative radiotherapy (RT). METHODS AND MATERIALS: Eligibility criteria included the following: ECOG performance status < or =2; resting and exercise arterial oxygen saturation (SaO(2)) > or =90%; an indication for palliative RT, 20-40 Gy in 10-15 fractions. RSR13 was administered i.v. via central vein over 60 min immediately before RT. Patients received supplemental O(2) via nasal cannula at 4 L/min during RSR13 infusion and RT. Plasma, red blood cell (RBC), and urine RSR13 concentrations were assayed. The pharmacodynamic effect of RSR13 on Hb-O(2) binding affinity was quantified by multipoint tonometry and expressed as an increase in p50, defined as the partial pressure of O(2) that results in 50% SaO(2). The RSR13 dose in the first cohort was 75 mg/kg once a week for two doses; successive cohorts received higher, more frequent doses up to 100 mg/kg/day for 10 days during RT. RESULTS: Twenty patients were enrolled in the study. Repeated daily doses of RSR13 were generally well tolerated. Two adverse events of note occurred: (1) A patient with pre-existing restrictive lung disease had transient persistent hypoxemia after the sixth RSR13 dose; (2) a patient with a recurrent glioma receiving high-dose corticosteroids had edema after the seventh RSR13 dose, likely due to the daily high-volume fluid infusions. Both patients recovered to baseline status with conservative management. Maximum pharmacodynamic effect occurred at the end of RSR13 infusion and was proportional to the RBC RSR13 concentration. After an RSR13 dose of 100 mg/kg, the peak increase in p50 averaged 8.1 mm Hg, consistent with the targeted physiologic effect, and then diminished with a half-life of approximately 5 h. CONCLUSIONS: RSR13 was well tolerated in daily doses up to 100 mg/kg administered for 10 days during RT. The combined administration of RSR13 with 4 L/min supplemental O(2) yielded pharmacodynamic conditions in which hypoxic tumor radiosensitization can occur. Ongoing Phase II and Phase III studies are evaluating the combination of RT and RSR13 for selected indications, including primary brain tumors, brain metastases, and non-small-cell lung cancer.

Interstitial high-dose-rate brachytherapy boost: the feasibility and cosmetic outcome of a fractionated outpatient delivery scheme.

PURPOSE: To evaluate the feasibility, potential toxicity, and cosmetic outcome of fractionated interstitial high dose rate (HDR) brachytherapy boost for the management of patients with breast cancer at increased risk for local recurrence. METHODS AND MATERIALS: From 1994 to 1996, 18 women with early stage breast cancer underwent conventionally fractionated whole breast radiotherapy (50-50.4 Gy) followed by interstitial HDR brachytherapy boost. All were considered to be at high risk for local failure. Seventeen had pathologically confirmed final surgical margins of less than 2 mm or focally positive. Brachytherapy catheter placement and treatment delivery were conducted on an outpatient basis. Preplanning was used to determine optimal catheter positions to enhance dose homogeneity of dose delivery. The total HDR boost dose was 15 Gy delivered in 6 fractions of 2.5 Gy over 3 days. Local control, survival, late toxicities (LENT-SOMA), and cosmetic outcome were recorded in follow-up. In addition, factors potentially influencing cosmesis were analyzed by logistic regression analysis. RESULTS: The minimum follow-up is 40 months with a median 50 months. Sixteen patients were alive without disease at last follow-up. There have been no in-breast failures observed. One patient died with brain metastases, and another died of unrelated causes without evidence of disease. Grade 1-2 late toxicities included 39% with hyperpigmentation, 56% with detectable fibrosis, 28% with occasional discomfort, and 11% with visible telangiectasias. Grade 3 toxicity was reported in one patient as persistent discomfort. Sixty-seven percent of patients were considered to have experienced good/excellent cosmetic outcomes. Factors with a direct relationship to adverse cosmetic outcome were extent of surgical defect (p = 0.00001), primary excision volume (p = 0.017), and total excision volume (p = 0.015). CONCLUSIONS: For high risk patients who may benefit from increased doses, interstitial HDR brachytherapy provides a convenient outpatient method for boosting the lumpectomy cavity following conventional whole breast irradiation without overdosing normal tissues. The fractionation scheme of 15 Gy in 6 fractions over 3 days is well tolerated. The volume of tissue removed from the breast at lumpectomy appears to dominate cosmetic outcome in this group of patients.

Extracorporeal irradiation of tumorous calvaria. Case report.

This patient with recurrent meningioma grossly involving the frontal bone underwent craniotomy and tumor resection. During the procedure a bone flap was irradiated extracorporeally at a very high dose (120 Gy) sufficient to sterilize residual tumor cells, and the bone was then successfully replaced orthotopically for reconstruction. The use of autologous irradiated bone in this setting offers advantages over cadaveric transplantation and prosthetic implants. Radiation might cause less disruption of the bone's architecture than other techniques of tumor cell eradication.

Signal transduction and cellular radiation responses.

Exposure of cells to ionizing radiation results in complex cellular responses resulting in cell death and altered proliferation states. The underlying cytotoxic, cytoprotective and cellular stress responses to radiation are mediated by existing signaling pathways, activation of which may be amplified by intrinsic cellular radical production systems. These signaling responses include the activation of plasma membrane receptors, the stimulation of cytoplasmic protein kinases, transcriptional activation, and altered cell cycle regulation. From the data presented, there is increasing evidence for the functional links between cellular signal transduction responses and DNA damage recognition and repair, cell survival, or cell death through apoptosis or reproductive mechanisms.

Enhanced radiosensitivity of malignant glioma cells after adenoviral p53 transduction.

OBJECT: The goal of this study was to determine whether adenoviral vector-mediated expression of human wildtype p53 can enhance the radiosensitivity of malignant glioma cells that express native wild-type p53. The p53 gene is thought to function abnormally in the majority of malignant gliomas, although it has been demonstrated to be mutated in only approximately 30%. This has led to studies in which adenoviral transduction with wild-type human p53 has been investigated in an attempt to slow tumor cell growth. Recent studies suggest that reconstitution of wild-type p53 can render cells more susceptible to radiation-mediated death, primarily by p53-mediated apoptosis. METHODS: Rat RT2 glioma cells were analyzed for native p53 status by reverse transcriptase-polymerase chain reaction and sequence analysis and for p53 expression by Western blot analysis. Clonogenic survival and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were used to characterize RT2 cell radiosensitivity and apoptosis, respectively, with and without prior transduction with p53-containing and control adenoviral vectors. Animal survival length was monitored after intracerebral implantation with transduced and nontransduced RT2 cells, with and without cranial radiation. The RT2 cells were demonstrated to express native rat wild-type p53 and to markedly overexpress human p53 following adenoviral p53 transduction. The combination of p53 transduction followed by radiation resulted in marked decreases in RT2 cell survival and increases in apoptosis at radiation doses from 2 to 6 Gy. Animals receiving cranial radiation after intracerebral implantation with RT2 cells previously transduced with p53 survived significantly longer than control animals (p<0.01). CONCLUSIONS: The ability to enhance the radiosensitivity of malignant glioma cells that express wild-type p53 by using adenoviral transduction to induce overexpression of p53 offers hope for this approach as a therapeutic strategy, not only in human gliomas that express mutant p53, but also in those that express wild-type p53.

Cosmetic outcome in patients receiving an interstitial implant as part of breast-conservation therapy.

PURPOSE: To study factors related to breast cosmetic outcome in patients treated with an interstitial implant as part of breast-conservation therapy. MATERIALS AND METHODS: One hundred fifty-six patients with stage I or II breast carcinoma who received 50 Gy of external-beam irradiation followed by a 20-Gy interstitial boost were examined. The dose homogeneity index (DHI) was calculated for each evaluable implant and was examined in light of other patient-, treatment-, and tumor-related variables previously demonstrated to affect cosmesis. RESULTS: Of the variables examined, both the DHI (P = .021) and the total excision volume (P = .019) were significantly related to cosmetic outcome (excellent vs less than excellent) in a univariate model. In the multivariate analysis, only the total excision volume remained significant (P = .032). The mean total excision volume +/- SD in patients with excellent cosmetic outcome (81.8 cm3 +/- 84.0) was significantly less than that in patients with less than excellent cosmetic outcome (120 cm3 +/- 84). The probability of excellent cosmetic outcome linearly increased with an increase in DHI. The mean DHI was 0.74 +/- 0.12 for the cases with excellent cosmetic outcome and 0.68 +/- 0.10 for those with less than excellent cosmetic outcome. CONCLUSION: To achieve optimal cosmesis, DHI should be maximized. The volume of tissue removed, however, remains the most significant determinant.

The influence of age and extensive intraductal component histology upon breast lumpectomy margin assessment as a predictor of residual tumor.

PURPOSE: Young age and extensive intraductal component (EIC) histology have been shown to be associated with increased local recurrence in women treated with breast conservation therapy. This study was conducted to determine if the status of the lumpectomy specimen margin consistently predicted for residual tumor burden risk irrespective of these variables. METHODS AND MATERIALS: As part of an institutional prospective approach for breast conservation therapy (BCT), 265 cases with AJCC Stage I/II carcinoma with an initial excision margin that was < or =2 mm or indeterminate were subjected to reexcision. The probability of residual tumor (+RE) was evaluated with respect to tumor size, histopathologic subtype (invasive ductal carcinoma, invasive ductal carcinoma with an EIC, and invasive lobular carcinoma), relative closeness of the measured margin, and the extent of margin positivity graded as focal, minimal, moderate, or extensive. The amount of residual tumor was graded as microscopic, small, medium, or large. All variables were analyzed for patient age < or =45 or >45 years. RESULTS: There was no significant difference in the incidence of a +RE according to age < or =45 versus >45 years when the margin was >0 < or =2 mm. Of the patients aged < or =45 years, the incidence of a +RE with a margin that was positive as compared to >0 < or =2 mm was 71% vs. 23%, respectively (p = 0.002). For women >45 years old, the difference in the incidence of +RE comparing margins that were positive or >0 < or =2 mm was not significant at 50% vs. 40%, respectively (p = 0.23). For all cases in aggregate, age < or =45 years was associated with a greater incidence of +RE as compared to patients aged >45 years with the discrepant incidence of a +RE by age strata most pronounced for focally positive margins (60% vs. 18%;p< or =0.05). In a logistic regression analysis, age (per year, as a continuous variable) and an EIC histology were significantly associated with the probability of a +RE (odds ratio [OR] = 0.80, p = 0.05 and OR = 1.9, p = 0.01, respectively). Tumor size was not significant (p = 0.23). In patients with an EIC histology, margin status is generally less predictive for differences in the incidence of a +RE. Further, the overall magnitude of difference in the incidence of a +RE related to age appears to be minimized when an EIC histology is present. In contrast, for cases classified as having non-EIC histology, there is a near-linear relationship for both age strata with respect to margin status and the incidence of a +RE. When histology is classified as non-EIC, age < or =45 years is consistently associated with a greater risk of residual tumor for all margin status categories. When the extent of margin positivity was graded as focal or minimal, residual tumor was semiquantitatively estimated as a medium/large amount in 33% versus 26% of cases aged < or =45 or >45 years, respectively (p = 0.62). CONCLUSION: For positive lumpectomy specimen margins, younger age is associated with an increased residual tumor risk. An EIC histology appears to be associated with an elevated risk of residual tumor irrespective of age and may undermine the predictive utility of margin status. Therefore, age and an EIC histology should be factored into risk assessments for residual tumor that rely upon margin assessment.

Ionizing radiation stimulates existing signal transduction pathways involving the activation of epidermal growth factor receptor and ERBB-3, and changes of intracellular calcium in A431 human squamous carcinoma cells.

Previous studies demonstrated that ionizing radiation activates the epidermal growth factor receptor (EGFR), as measured by Tyr autophosphorylation, and induces transient increases in cytosolic free [Ca2+], [Ca2+]f. The mechanistic linkage between these events has been investigated in A431 squamous carcinoma cells with the EGFR Tyr kinase inhibitor, AG1478. EGFR autophosphorylation induced by radiation at doses of 0.5-5 Gy or EGF concentrations of 1-10 ng/ml is inhibited by >75% at 100 nM AG1478. Activation of EGFR enhances IP3 production as a result of phospholipase C (PLC) activation. At the doses used, radiation stimulates Tyr phosphorylation of both, PLCgamma and erbB-3, and also mediates the association between erbB-3 and PLCgamma not previously described. The increased erbB-3 Tyr phosphorylation is to a significant extent due to transactivation by EGFR as >70% of radiation- and EGF-induced erbB-3 Tyr phosphorylation is inhibited by AG 1478. The radiation-induced changes in [Ca2+]f are dependent upon EGFR, erbB-3 and PLCgamma activation since radiation stimulated IP3 formation and Ca2+ oscillations are inhibited by AG1478, the PLCgamma inhibitor U73122 or neutralizing antibody against an extracellular epitope of erbB-3. These results demonstrate that radiation induces qualitatively and quantitatively similar responses to EGF in stimulation of the plasma membrane-associated receptor Tyr kinases and immediate downstream effectors, such as PLCgamma and Ca2+.

Enhanced radiation late effects and cellular radiation sensitivity in an ATM heterozygous breast cancer patient.

We observed severe late effects in a patient treated with radiation therapy for breast cancer. Radiation survival studies of patient fibroblasts show an enhanced cellular radiation sensitivity (Do = 0.95 Gy). Genetic analysis reveals that the patient is heterozygous for a mutated ATM gene. Protein truncation test (PTT) and sequence analysis identified a truncation within the leucine zipper domain, corresponding to a fragment previously reported to exhibit dominant negative function. These findings demonstrate that ATM heterozygosity may be associated with enhanced clinical radiation sensitivity and suggest a clinical relevance to this truncation that results in a dominant negative-acting protein.

Dominant negative EGFR-CD533 and inhibition of MAPK modify JNK1 activation and enhance radiation toxicity of human mammary carcinoma cells.

Exposure of MDA-MB-231 human mammary carcinoma cells to an ionizing radiation dose of 2 Gy results in immediate activation and Tyr phosphorylation of the epidermal growth factor receptor (EGFR). Doxycycline induced expression of a dominant negative EGFR-CD533 mutant, lacking the COOH-terminal 533 amino acids, in MDA-TR15-EGFR-CD533 cells was used to characterize intracellular signaling responses following irradiation. Within 10 min, radiation exposure caused an immediate, transient activation of mitogen activated protein kinase (MAPK) which was completely blocked by expression of EGFR-CD533. The same radiation treatment also induced an immediate activation of the c-Jun-NH2-terminal kinase 1 (JNK1) pathway that was followed by an extended rise in kinase activity after 30 min. Expression of EGFR-CD533 did not block the immediate JNK1 response but completely inhibited the later activation. Treatment of MDA-TR15-EGFR-CD533 cells with the MEK1/2 inhibitor, PD98059, resulted in approximately 70% inhibition of radiation-induced MAPK activity, and potentiated the radiation-induced increase of immediate JNK1 activation twofold. Inhibition of Ras farnesylation with a concomitant inhibition of Ras function completely blocked radiation-induced MAPK and JNK1 activation. Modulation of EGFR and MAPK functions also altered overall cellular responses of growth and apoptosis. Induction of EGFR-CD533 or treatment with PD98059 caused a 3-5-fold increase in radiation toxicity in a novel repeated radiation exposure growth assay by interfering with cell proliferation and potentiating apoptosis. In summary, this data demonstrates that both MAPK and JNK1 activation in response to radiation occur through EGFR-dependent and -independent mechanisms, and are mediated by signaling through Ras. Furthermore, we have demonstrated that radiation-induced activation of EGFR results in downstream activation of MAPK which may affect the radiosensitivity of carcinoma cells.

Optimization of planar high-dose-rate implants.

PURPOSE: Brachytherapy has long been used to deliver localized radiation to the breast and other cancer sites. For interstitial implants, proper source positioning is critical in obtaining satisfactory dose distributions. The present work examines techniques for optimizing source guide placement in high-dose-rate (HDR) biplanar implants, and examines the effects of suboptimal catheter placement. METHODS AND MATERIALS: Control of individual dwell times in HDR implants allows a high degree of dose uniformity in planes parallel to the implant planes. Biplanar HDR implants can be considered optimized when the dose at the implant center is equal to the dose at the symmetric target boundaries. It is shown that this optimal dose uniformity is achieved when the interplanar separation is related to the target thickness T through the direct proportionality, s = T/square root2. To quantify the significance of source positioning, the average dose and a related quantity, equivalent uniform dose (EUD), were calculated inside the treatment volume for two conditions of suboptimal catheter geometry. In one case, the interplanar spacing was varied from 1 cm up to the target thickness T, while a second study examined the effects of off-center placement of the implant planes. RESULTS: Both the average dose and EUD were minimized when the interplanar spacing satisfied the relationship s = T/square root2. EUD, however, was significantly smaller than the average dose, indicating a reduced relative cell killing in the high dose regions near the dwell points. It was also noted that in contrast to the average dose, the EUD is a relatively weak function of catheter misplacement, suggesting that the biological consequences of suboptimal implant geometry may be less significant than is indicated by the increase in average dose. CONCLUSION: A concise formula can be used to determine the interplanar separation needed for optimal dose uniformity in Manchester-type implants. Deviations from optimal source geometry result in an increase in the average dose inside the treatment volume, but the weaker dependence of the EUD suggests that the surviving fraction of cells may not be not strongly affected by suboptimal source geometry.

Extent of margin positivity as a predictor for local recurrence after breast conserving irradiation.

The extent of positivity of the final excision margin in relationship to other relevant factors was evaluated as a predictor for local recurrence after breast conservation therapy (BCT). As part of an institutional practice policy for BCT in 509 stage I/II breast carcinomas, 105 cases had a final excision margin, which was positive. The median age for this cohort was 58 years and the median follow-up was 86 months. All positive margin patients received whole breast irradiation to 50 Gy-50.4 Gy followed by a boost to the tumor bed for an additional 20 Gy. The extent of positivity (EOP) of the excision margin was graded according to a four-point scale: focal, minimal, moderate, extensive. Cases were then analyzed for local failure according to EOP grade, histology, age, tumor size, total excision volume, re-excision, tamoxifen therapy, and chemotherapy. A focal or minimal EOP grade was found in 70% of specimens while an additional 26% were moderate or extensive. The incidence of invasive carcinoma with prominently associated DCIS was significantly greater in cases with an EOP grade of moderate/extensive. There were nine ipsilateral breast recurrences, eight of which could be evaluated for EOP grade. All recurrences were in or near the previous biopsy cavity. A Kaplan-Meier plot of freedom from local failure showed a significant (P = 0.008) difference between cases grouped by EOP grade of focal/minimal as compared to moderate/extensive. A Cox proportional hazards regression model found that the only variable significantly related at the P < or = 0.05 level to local failure was an EOP grade of moderate/extensive. For breast excision specimens with a positive final margin, an EOP grade of moderate/extensive is a predictor for local recurrence after BCT, which may be independent of other variables such as age or histology.