apy. This has potentially important implications for the development of future management strategies that specifically targethypoxia in this disease. Our study continue to accrue and mature, and will ultimately allow us to rigorously evaluate theindependent prognostic important of hypoxia in these patients, the patterns of recurrence in patients with oxic and hypoxictumors, and the influence of oxygen levels on the interaction between hormonal therapy and radiation.

174 Prognostic Significance of CK19 mRNA Expression Measured by Reverse-Transcription Polymerase ChainReaction (RT-PCR) in Peripheral Blood of Patients with Non-Small Cell Lung Cancers Treated by Chemo-Radiation

T. Chen, G. Jiang, X. Fu, L. Wang, H. Qian, K. Wu, S. Zhao

Radiation Oncology, Fudan University Cancer Hospital, Shanghai, China

Purpose/Objective: To investigate prognostic significance of micrometastasis in peripheral blood of patients with non-smallcell lung cancer (NSCLC) treated by chemo-radiation.

Materials/Methods: From Feb. 2002 to Mar. 2003 we measured CK19 mRNA, as the marker of epidermoid carcinoma cells,by nested RT-PCR in the peripheral blood, taken from 67 NSCLC patients twice, i.e., before and after 2 cycles of chemotherapyand irradiation. Among 67 patients, 60 were male and 7 female with median age of 62 years (40–75). The clinical characteristicswere as follows: adenocarcinoma 32, squamous cell carcinoma 32, and adenosquamous carcinomas 3; stage I-II 6, stage IIIa16 and stage IIIb 45. The chemotherapy regimen was cis-platinum based. Irradiation was carried out by conventionalfractionation with median dose of 62Gy/31fx, 6.2wks.

Results: The sensitivity of RT-PCR was one cancer cell per ten million PBMN to detect CK19 mRNA in peripheral blood. Thepositive rates of CK19 mRNA in peripheral blood were 66 percent and 33 percent, respectively, before and after the treatment.In the two detections, 14 cases showed persistent positive for CK19 mRNA; 15 persistent negative; 30 with the negative priorto treatment turned to the positive after treatment (negative-to-positive) and 8 with the positive before therapy changed to thenegative (positive-to-negative) after treatments.

Univariate analyses showed that the positive rates of CK19 mRNA in peripheral blood before treatments were closely incorrelation with both N-stage ( p�0.014) and cancer cell differentiation ( p � 0.051). However, it turned out to be more closelyin relation with histological types (adenocarcinoma vs. non-adenocarcinoma) (p � 0.019), weight loss ( p � 0.010), KPS status( p � 0.027) as well as N-stage ( p � 0.014) in the measurement after chemo-radiation. The negative of CK19 mRNA was infavor of squamous cell carcinoma, less weight loss, good KPS and early N stage. There was no survival difference betweenpatients with CK19 mRNA positive and negative measured before treatments. However, the status of CK19 mRNA detectedafter chemo-radiation could predict the prognoses. Patients with the negative of CK19 mRNA survived longer than those withthe positive with median survival time of 46 weeks and 23 weeks and 40-week survival rate of 57 percent and 21 percent,respectively, for the former and the later (p � 0.0002). There were also differences in survival among the NSCLC patients withpersistent positive, persistent negative, positive-to-negative and negative-to-positive of CK19 mRNA in their peripheral bloodduring the twice measurements with median survival of 23 weeks, �36 weeks, 40 weeks and 29 weeks, respectively, and40-week survival rate of 24 percent, 45 percent, 0 and 75 percent, respectively,(p � 0.0013).

Multivariate analyses showed that the positive of CK19 mRNA after chemo-radiation was an independent unfavorableprognostic factor (p � 0.017). 35-week metastasis rates for NSCLC patients with positive and negative of CK19 mRNAexpression in their peripheral blood were 88 percent and 43 percent, respectively (p � 0.0001). Moreover, there were significantdifferences in metastasis rates among NSCLC patients with persistent positive, persistent negative, positive-to-negative andnegative-to-positive during two measurements with 35-week metastasis rates of 84 percent, 29 percent, 49 percent and 100percent, respectively, ( p � 0.0002).

Conclusions: Only after chemo-radiation could the measurement of CK19 mRNA in peripheral blood predict the prognoses ofNSCLC. Patients with the negative of CK19 mRNA survived longer and developed less distant metastases compared to thosewith the positive.

175 Mutagen Sensitivity Predicts Poor Overall and Disease Specific Survival in Patients With Stage III Non-Small Cell Lung Cancer Treated With Chemo/Radiotherapy

J. Y. Chang,1 X. Wu,2 R. Komaki,1 R. Sasaki,1 P. Allen,1 Z. Liao,1 C. W. Stevens,1 C. Lu,3 F. V. Fossella,3 J. D. Cox,1

M. R. Spitz2

1Radiation Oncology, MD Anderson Cancer Center, Houston, TX, 2Epidemiology, MD Anderson Cancer Center, Houston,TX, 3Medical Oncology, MD Anderson Cancer Center, Houston, TX

Purpose/Objective: In light of the dismal outcome of current treatment and the significant treatment related toxicities in lungcancer, we urgently need markers to individualize our patient’s management. The mutagen sensitivity assay was developed tomeasure indirectly an individual’s DNA repair capacity to assess susceptibility to tobacco carcinogenesis. Since DNA repaircapacity may also predict therapeutic effects and normal tissue repair after chemo/radiotherapy, we hypothesized that mutagensensitivity may likewise serve as a biomarker for clinical outcome in lung cancer. Patients with lung cancer may have geneticinstability unmasked by in vitro exposure of lymphocytes to a mutagen challenge such as bleomycin, a radiomimetic agent. Inthis study, we explored the role of bleomycin sensitivity in peripheral blood lymphocytes as a predictor of outcome in patientswith inoperable stage III non-small cell lung cancer treated with definitive chemo/radiotherapy.

Materials/Methods: 113 patients who were diagnosed with inoperable stage III non-small cell lung cancer at MD AndersonCancer Center between 1995–2001 were included in this analysis. All patients received definitive radiotherapy with or withoutchemotherapy. Pre-treatment peripheral blood lymphocytes were collected and cultured for three days. Bleomycin was addedfor 5 hours and cells were arrested in mitosis using colcemid. Bleomycin sensitivity was measured by counting the meannumber of chromatid breaks per cell in 50 metaphases. Patients with an average of more than 1.02 break/cell were consideredto exhibit the bleomycin sensitivity phenotype. The data were analyzed using Kaplan-Meier survival function with S-plus 6.1

S237Proceedings of the 46th Annual ASTRO Meeting

statistical software. The Log Rank test was used to compare different survivals. Spearman correlation test was used to analyzethe correlation between bleomycin sensitivity and complications.

Results: High bleomycin sensitivity (mean chromatid break/cell �1.02, designated as bleomycin sensitive, BS) predicted poordisease specific survival (DSS) and overall survival (OS). The 6 years DSS was 27 % in patients with BS compared with 46% in patients who didn’t exhibit the BS phenotype (p � 0.0094). The p value remained significant when adjusted (subgroupanalysis) for smoking status, age (dichotomized at 60 years), radiation doses (50–55 GY, 55–65 GY, �65 GY). The 6 yearsOS was 19 % for patients with BS and 29 % for patients without BS (p � 0.0193). There was a trend toward worse localregional control and worse disease free survival among patients with BS (p � 0.0972 and 0.1813 respectively). There was nodifference between two groups in distant metastasis free survival (p � 0.7695). To explore the predictive value of BS intreatment related complications, we analyzed the correlation between bleomycin sensitivity values with the toxicity grades ofcommon treatment related complications including esophagitis, pneumonitis, and lung fibrosis. However, there was nosignificant correlation between BS and the treatment related complications when the side effects were analyzed individually orcombined.

Conclusions: BS correlated with poor overall survival and disease specific survival in these patients with stage III non-smallcell lung carcinoma treated with chemo/radiotherapy. There was also a trend indicating poor local regional control and poordisease free survival with BS. There was no significant correlation between BS and treatment related complications. BS mayfunction as a biomarker for poor clinical outcome for this group of patients. These data suggested the hypothesis that BS mayindicate a genetic instability of cancer that contributes to high tumor grade, high genetic heterogeneity, and possible treatmentresistance.

176 Cytokine Profiling to Predict Radiation-Induced Lung Injury

J. P. Hart,1 Z. N. Rabbani,2 S. V. Pizzo,1 Z. Vujaskovic,2 M. S. Anscher2

1Pathology, Duke University Medical Center, Durham, NC, 2Radiation Oncology, Duke University Medical Center,Durham, NC

Purpose/Objective: To determine whether pre- and post-radiation plasma cytokine profiles among patients receiving thoracicirradiation for lung cancer are predictive for the development radiation-induced lung injury.

Materials/Methods: Plasma samples were collected from patients undergoing thoracic irradiation for lung cancer according toan IRB approved protocol. Following completion of radiation therapy patients were monitored for the development ofsymptomatic radiation-induced lung injury according to a previously described clinical scoring system (Fu et al. Int J RadiatOncol Biol Phys 2001;4:899–908). Pre- and post-treatment plasma samples from patients with or without clinical symptomsof lung injury were analyzed using a multianalyte human cytokine assay (BioRad, Hercules, CA). Subjects in this study wereselected from patients included in a previous prospective analysis of the relationship between TGF-� and the incidence ofradiation-induced lung injury. From this pool, all patients with plasma samples available in the archive obtained prior to the startof treatment and at the end of treatment were eligible. We identified 11 patients with lung injury and all were included. Fromthe group without lung injury, 17 were selected at random using a random number generator. The cytokines analyzed includedIL-I�, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p70), IL-13, IL-17, GM-CSF, G-CSF, INF-�, TNF-�, MCP-1, andMIP-1�. Plasma concentrations of these 17 cytokines were then compared among patient populations with or without lunginjury. Control plasma samples were utilized from healthy patients without cancer (n � 8). T-test was performed to determinestatistical significance (p � 0.05).

Results: Analysis of pretreatment samples demonstrated increased levels of IL-7 and MIP-1� among patients that did notdevelop lung injury following radiation therapy (see table below). Both control patients and patients that developed lung injuryfollowing irradiation had lower plasma levels of these two cytokines. Analysis of post-treatment samples demonstrateddecreased levels of IL-6 in patients without radiation-induced lung injury as compared to both controls and patients with lunginjury (see table below(table 1). (Data are represented as mean � SEM. Pre-T, Pretreatment; Post-T, Post-treatment; p valuesrepresent comparisons of patients with or without lung injury within pre- or post-treatment groups.)

Conclusions: These preliminary observations suggest that plasma cytokine profiling may prove useful for determining the riskof developing radiation-induced lung injury. Indeed, patients with increased levels of IL-7 and MIP-1� were less likely todevelop pneumonitis following irradiation. Further, these findings suggest that pretreatment interactions between tumors and thehost immune system may affect tissue radiosensitivity. Previous studies have demonstrated that increased levels of TGF-�correlate with an increased risk of developing radiation-induced lung injury. Interestingly, IL-7 down regulates TGF-�production and studies in mice lacking the IL-7 receptor suggest that IL-7 protects intestinal stem cells from radiation-inducedapoptosis. IL-7 also increases expression of MIP-1� suggesting a possible link between the increased levels of these twocytokines observed in our patients which do not develop pneumonitis. Additionally, an analogue of MIP-1�, a chemokinehighly related to MIP-1�, protects the small intestine from radiation injury. Patients that did not develop pneumonitis also haddecreased levels of IL-6 following radiation therapy. Further studies are required to determine the predictive value of cytokinelevels in larger populations of patients receiving radiation therapy. These studies will also include subsets of patients withvarious tumor types to investigate tumor specific modulation of the host immune system and the subsequent effects on tissueradiosensitivity.

S238 I. J. Radiation Oncology ● Biology ● Physics Volume 60, Number 1, Supplement, 2004