Single-Nucleotide Polymorphisms (SNPs) Associated With Radiation Proctitis From Genome-wide False Discovery Rate Analysis

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International Journal of Radiation Oncology  Biology  Physics

Author Disclosure: D. Blakaj: None. M. Garg: None. Z. Chen: None. R. Smith: None. M. Prystowsky: None. R. Burk: None. N. Schlecht: None. C. Guha: None. S. Kalnicki: None.

after therapy in order to determine possible markers for increased radiation sensitivity. Materials/Methods: Three hundred forty-nine patients treated with prostate brachytherapy between 1998 and 2010 provided saliva samples from which DNA was extracted for this research ethics board approved study. In the cohort of patients with at least 2 years of follow-up, 34 patients were identified as having high late urinary toxicity. These were patients with  RTOG grade 2 GU toxicity identified during follow-up, during chart review, or on the study enrollment questionnaires. 87 patients were identified as controls without high late urinary toxicities, and at least 2 years of follow-up. We analyzed 15 potential SNPs from 13 genes (MSH6, GSTA1, SOD2, NOS3, GSTP1, ATM, LIG4, XRCC1, XRCC3, RAD51, TP53, TGFB1, ERCC2) for correlation with increased late toxicity. Patient factors and dosimetric parameters were also studied in the analysis. Results: All 15 proposed SNPs demonstrated polymorphism within our population. We implemented a univariate analysis to examine the correlation between variant allele SNPs (versus wild type) and the presence of increased toxicity. This revealed a statistically significant relationship in 6 of the SNPs. Variants for SNPs rs1800470 (TGFB1), rs1801320 (RAD51), rs1805386 (LIG4), rs3213245 (XRCC1), rs4880 (SOD2), and rs861539 (XRCC3) were correlated with increased late urinary toxicity (all p < 0.05). Diabetes, coronary vascular disease, hormone therapy, hypertension, age at brachytherapy implantation and dosimetric parameters such as the prostate V150, V100, D90 and rectal V100 were also all significant for an association with late toxicity (p < 0.10). None of the variables were correlated with late toxicity in the multivariate analysis, which may have been limited by the small sample size. Conclusion: Our study demonstrates 6 SNPs in 6 genes (LIG4, TGFB1, RAD51, XRCC1, XRCC3 and SOD2) which are statistically significant for association with late urinary toxicity. This preliminary data may be utilized in future studies to identify patients at risk for complications after brachytherapy. Author Disclosure: N. Leong: None. M. Parliament: None. K. Martell: None. S. Ghosh: None. N. Pervez: None. J. Pedersen: None. D. Yee: None. A. Murtha: None. J. Amanie: None. N. Usmani: None.

3315 The Role of CD44-associated Stem Cells in the Recovery of Headand-Neck Squamous Cell Carcinoma Following Radiation G.D. Wilson, B.J. Thibodeau, S. Galoforo, B.L. Pruetz, B. Marples, J. Akervall, and J. Huang; Beaumont Health System, Royal Oak, MI Purpose/Objective(s): There is growing evidence that cancer stem cells (CSCs) may be an important factor in disease progression and resistance to current treatments. The rationale for this study was to investigate the possible role of CD44-associated CSCs in the recovery of head and neck cancer (HNSCC) xenografts following treatment with radiation. Materials/Methods: Flow cytometry was used to sort five untreated, low passage HNSCC cell lines (UT14, UT16A, UT24A, UT30, UT33) into CSC-enriched and CSC-depleted populations based upon expression of the putative cancer stem cell marker CD44. Gene expression microarray analysis was employed to develop a gene expression profile of the cancer stem cell population. In a separate study, UT14 cells were injected into the flanks of female nu/nu mice and allowed to grow to a size of 400-500mm3; they were then treated with either sham RT or 15 Gy in one fraction. Tumors were harvested at 4, 7, 12, and 21 days following radiation treatment and compared to untreated controls using immunohistochemistry (IHC) and gene expression microarray analysis. Two types of software were used to investigate the changes seen in the in vitro and in vivo results. Results: Microarray analysis of the in vitro data was used to develop a gene expression profile characteristic of CSCs that included 77 differentially expressed genes between the CSC-enriched and CSC-depleted populations. 15 Gy arrested tumors for the first 21 days after RT and growth resumed at a linear rate thereafter. Gene expression analysis showed that ten of the genes, identified in vitro, were also altered in the xenografts at a minimum of one timepoint following radiation; these included chemokine (C-X-C motif) ligand 11 (CXCL11) and metastasis associated in colon cancer 1 (MCC1). In comparison to the in vitro cell sorting data, the only timepoint that showed commonly altered categories of biological process as well as Ariadne sub-networks of genes regulating cell processes was at Day 7 following radiation treatment. This time point preceded a rapid rise in CD44 staining by IHC. The common biological processes included mitotic cell cycle, cell cycle, and mitosis and cell processes of G2/M transition and spindle assembly. Conclusions: This study suggests a possible role of cancer stem cells in the recurrence of head and neck squamous cell cancer following radiation treatment. Global gene expression patterns of CD44-positive CSCs demonstrate similarities to those of xenografts following radiation, particularly 7 days following treatment. Interestingly, Day 7 precedes the recovery of the xenograft from the sub-curative dose of radiation. From these studies we hope to gain greater insight into the importance of stem cells in HNSCC as well as the importance of targeting the stem cells to achieve more successful treatment options. Author Disclosure: G.D. Wilson: None. B.J. Thibodeau: None. S. Galoforo: None. B.L. Pruetz: None. B. Marples: None. J. Akervall: None. J. Huang: None.

3316 Single Nucleotide Polymorphisms (SNPs) Associated With Late Radiation Urinary Toxicity After Prostate Brachytherapy N. Leong,1 M. Parliament,1 K. Martell,2 S. Ghosh,1 N. Pervez,1 J. Pedersen,1 D. Yee,1 A. Murtha,1 J. Amanie,1 and N. Usmani1; 1Cross Cancer Institute, Edmonton, AB, Canada, 2University of Alberta, Edmonton, AB, Canada Purpose/Objective(s): Excessive toxicity from prostate brachytherapy treatment may be related to increased radiosensitivity from genetic polymorphisms. We aimed to identify particular single nucleotide polymorphisms (SNPs) that were associated with significant urinary toxicity

3317 Single-Nucleotide Polymorphisms (SNPs) Associated With Radiation Proctitis From Genome-wide False Discovery Rate Analysis M.B. Parliament,1 D. Broadhurst,1 S. Ghosh,1 G. Zhu,1 S. Kerns,2 B. Rosenstein,3 G. Khitrov,3 H. Ostrer,4 B. Warkentin,1 and D. Murray1; 1 University of Alberta, Edmonton, AB, Canada, 2Mt Sinai School of Medicine, New York, NY, 3Mt. Sinai School of Medicine, New York, NY, 4 Albert Einstein College of Medicine, Bronx, NY Purpose/Objective(s): Radiogenomics seeks to understand the relationship between genetic variants (usually SNPs) and the occurrence of radiation-related normal tissue complications. Candidate SNP association studies have been called into question due to the conflicting results obtained in validation studies and after a recent large-scale study failed to validate previously published associations. We report the initial findings from a genome-wide analysis of a cohort of prostate cancer patients who have previously undergone conformal RT or IMRT. Materials/Methods: One hundred fifty-five patients with adenocarcinoma of the prostate treated between 2000 and 2008 were recruited consecutively and consented to this IRB-approved study. Patients were treated with conventional fractionation (1.8 - 2 Gy/fx) or hypofractionation (2.72 Gy/fx) to doses of 68 - 83 Gy. Maximum occurrence of GU and GI toxicity >90 days from start of RT was graded retrospectively from patient records using the CTCAE version 3.0 scale. Genotyping was performed on patient genomic DNA and tests for association were performed. Results: The mean age of the cohort was 67 years (range, 45 - 82) and the vast majority of patients were of white ancestry. Diabetes was present in 17% while 32% had previous abdominopelvic surgery. Mean follow up

Volume 84  Number 3S  Supplement 2012 was 5.6 years (range, 2-10). Before QA filtering, there are 905,486 SNPs. Total genotyping rate was 99.6%. After removing SNPs with >10% missing genotypes or low allelic frequency (minor allele frequency <1%), there were 765,233 SNPs. There were 19 cases of grade 2+ proctitis and 136 cases of grade 0-1 proctitis. Unadjusted and adjusted case-control association tests were conducted across all 765,233 SNPs. For the adjusted association study different correction measures were obtained. P-values obtained from the False Discovery Rate (FDR) method were reported for the compensation for multiple parallel comparisons. Setting the FDR rate to 5% resulted in a critical p-value of 210 7. Thirteen SNPs had a pvalue < 210 7 thus supporting their inclusion in future validation experiments. Interestingly, these variants were all intronic or non-genic and map to the same region of chromosome 7. The strongest association with severe proctitis was found for rs10279669. Conclusions: Genome-wide analysis of radiation proctitis in a case-control discovery cohort of prostate cancer patients has revealed SNPs never previously associated with radiation-related normal tissue complications. False Discovery Rate analysis, and the fact that there are multiple hits from the same region, suggests that the SNPs identified may indeed be true positives. Author Disclosure: M.B. Parliament: None. D. Broadhurst: None. S. Ghosh: None. G. Zhu: None. S. Kerns: None. B. Rosenstein: None. G. Khitrov: None. H. Ostrer: None. B. Warkentin: None. D. Murray: None.

3318 Cell-free DNA as a Biomarker of Residual Disease Following Radiation Therapy for Non-small Cell Lung Cancer S.V. Bratman,1 N.C. Eclov,1 L.A. Modlin,1 J. Neal,1 B.W. Loo,1 G. Wu,2 K. Richardson,2 A.M. Newman,1 A. Alizadeh,1 and M. Diehn1; 1Stanford University, Stanford, CA, 2Transgenomic, Inc., Omaha, NE Purpose/Objective(s): Radiation therapy (RT) is an important component of curative therapy for stage I-III non-small-cell lung cancer (NSCLC). An urgent need exists to develop improved methods for identifying which patients are cured following RT and which patients may need closer monitoring or additional therapy. In this study, we evaluate the feasibility of analyzing tumor-specific cell-free DNA (cfDNA) derived from peripheral blood plasma as a means for monitoring residual disease following RT for NSCLC. Materials/Methods: We prospectively enrolled patients undergoing RT for NSCLC in a translational study that allows banking of peripheral blood samples. Blood from healthy controls was used to determine optimum processing procedures. We purified cfDNA from pre-RT plasma and measured the concentration of total cfDNA using quantitative PCR. The presence of tumor-specific cfDNA was determined for patients with tumors known to harbor mutations in EGFR, KRAS, or ALK. Results: The concentration and fragment size of cfDNA was more stable in plasma than in serum over a range of processing times and storage temperatures. In plasma, most cfDNA fragments were 80-200 bp long. There was a wide range of total cfDNA concentrations in pre-RT plasma (1.8 - 43.2 ng DNA per mL plasma; n Z 45). There were no statistically significant differences in cfDNA concentration between the NSCLC patients and a group of 15 healthy (p Z 0.25). In addition, there were no statistically significant differences in cfDNA concentrations when patients were grouped by stage (p Z 0.75) or treatment modality (i.e., chemoRT vs. stereotactic ablative RT) (p Z 0.38). ICECOLD PCR assays for EGFR and KRAS mutations detected the presence of tumor-specific cfDNA in pre-treatment plasma from 2 of 15 patients (15%). In both positive cases, the tumor-specific cfDNA became undetectable following treatment. For another patient with ALK-rearranged lung adenocarcinoma, we mapped the genomic breakpoint within tumor DNA to intron 20 of EML4 and intron 19 of ALK. A PCR amplicon specific for the EML4-ALK rearranged sequence was detected in pre-treatment plasma, and the relative concentration subsequently decreased with treatment. Conclusions: The concentration of total cfDNA in pre-treatment plasma of NSCLC patients undergoing RT is insufficient for determining the

Poster Viewing Abstracts S713 presence or burden of disease. Assays performed on cfDNA targeting specific mutations known to be present in each patient’s tumor may provide a personalized biomarker for disease burden following RT for NSCLC. Author Disclosure: S.V. Bratman: None. N.C. Eclov: None. L.A. Modlin: None. J. Neal: None. B.W. Loo: None. G. Wu: A. Employee; Transgenomic. K. Stock; Transgenomic. L. Stock Options; Transgenomic. K. Richardson: A. Employee; Transgenomic. L. Stock Options; Transgenomic. A.M. Newman: None. A. Alizadeh: None. M. Diehn: None.

3319 Determination of Predictive Markers of Chemoradiation Sensitivity and Resistance of Rectal Tumors B. Zaki,1 S.F. Bakhoum,2 A. Suriawinata,1 S. Ibrahim,1 and A.R. Eastman2; 1Dartmouth-Hitchcock Medical Center, Lebanon, NH, 2 Dartmouth-Hitchcock Medical School, Hanover, NH Purpose/Objective(s): Pathological response to neoadjuvant chemoradiation for rectal adenocarcinoma may have an impact on local relapse, metastases and overall survival. The results may also implicate the surgical approach including the ability to do a sphincter sparing procedure. The Mre11/Rad50/Nbs1 (MRN) complex is a regulator of cell cycle checkpoints and DNA repair. Defects in MRN can lead to defective S-phase arrest when cells are damaged. In this retrospective analysis, we examine the expression of MRN in rectal tumor specimens and its potential effect on radiosensitivity of cancer cells. Materials/Methods: One hundred eighty patients with clinical stage III rectal cancer were treated with neoadjuvant chemoradiation followed by surgical excision. The tumors were studied for pathological response using the Tumor Regression Grade (TRG). TRG1 indicated complete pathological response, while TRG5 indicated no apparent response to chemoradiation therapy. Twenty tumors representing the lower (TRG1) and upper (TRG3/4) deciles were identified. Their biopsies obtained prior to therapy were immunostained for p53, Ki-67, geminin, Mre11, carbonic anhydrase IX, thymidylate synthase and thymidine phosphorylase. The immunohistochemistry results were scored in a semi-quantitative fashion based on the intensity of the staining and the number of stained cells. Results: Metastases were noted in 5/10 patients in the TRG3/4 group while none were seen in the TRG1 group. 4/10 of the TRG3/4 group overexpressed Mre11 while 4/9 of the TRG1 underexpressed the same marker. 4/5 metastatic cases also overexpressed Mre11 in the original biopsy. None of the other markers was expressed differently between the 2 groups. Conclusions: Mre11 overexpression in rectal carcinoma may predict resistance to neoadjuvant chemoradiation and possible metastases. More biopsies are being tested. Author Disclosure: B. Zaki: None. S.F. Bakhoum: None. A. Suriawinata: None. S. Ibrahim: None. A.R. Eastman: None.

3320 Hyperspectral Imaging for Early Prediction of Chronic Microvascular Injury in Irradiated Skin M.S. Chin,1 B.B. Freniere,1 L. Lancerotto,2 J.H. Saleeby,1 Y. Lo,1 R.A. Ignotz,1 D.P. Orgill,2 J.F. Lalikos,1 and T.J. Fitzgerald1; 1University of Massachusetts Medical School, Worcester, MA, 2Brigham and Women’s Hospital, Boston, MA Purpose/Objective(s): Ionizing radiation is known to have significant effects on cutaneous tissues, including deleterious effects on dermal vasculature. Ischemia is postulated as a mechanism of chronic radiationinduced injury. Currently, there are no reliable biomarkers for early prediction of chronic vascular damage. Recently, hyperspectral imaging (HSI), a type of wide-field diffuse reflectance spectroscopy, has emerged as a tool to simultaneously investigate the oxy- and deoxy-hemoglobin (oxyHb and deoxy-Hb) content of cutaneous tissues. In previous studies, we have found deoxy-Hb levels decline significantly in the first week post-