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The unlimited growth that occurs in tumors requires telomere maintenance. Yet, a portion of human tumors lack telomerase, and maintain telomeres using recombination-based mechanisms. Studies in other model organisms indicate that two different pathways of recombination-based mechanisms impact telomere maintenance and rely on the DNA repair proteins Rad50 and Rad51. In the Rad50-dependent pathway telomere recombination occurs within the telomere repeats. In contrast, recombination using the Rad51-dependent pathway occurs within repetitive sequences in the subtelomeres. Using a mouse B-cell lymphoma model lacking telomerase, Eμmyc+mTR-/-, and immortalized fibroblast cells lacking the RNA component of telomerase (mTR-/-) we have examined the impact of inhibiting Rad50 and Rad51a on telomere recombination. We find inhibiting Rad50 or Rad51a in Eμmyc+mTR-/- B-cell lymphomas, and in mTR-/- immortalized fibroblasts, has a synergistic effect on DNA damage sensitivity to mitomycin but not camptothecin. Inhibiting Rad50 in telomerase deficient cells also results in telomere shortening and in some tumors, reduced growth. In contrast, when Rad50 or Rad51a is inhibited in cells with telomerase, DNA damage sensitivity from mitomycin is not observed when compared to cells expressing a control shRNA. In addition inhibiting Rad50 in cells with telomerase does not significantly impact telomere length or recombination. Next we developed a comparative genomic hybridization (aCGH) approach that detects recombination events in the subtelomeres. Using these subtelomere arrays we find B-cell lymphomas lacking telomerase exhibit a significant increase in subtelomere recombination compared to primary cells. We also examined the impact of inhibiting Rad50 on subtelomere recombination events. Our findings using aCGH suggest that inhibiting Rad50 does not impact subtelomere recombination in Eμmyc+mTR-/- B-cell lymphomas. Overall, our findings suggest that inhibiting either Rad50 or Rad51a in mTR-/- cells has a synergistic impact on the sensitivity to DNA damaging agents in contrast to cells with mTR+/+. Currently we are testing the impact of inhibiting Rad51a on subtelomere recombination. In addition these results further support that Rad50 contributes to telomere recombination mechanisms in tumors lacking telomerase and will provide insight into the mechanism of subtelomere recombination in mammalian cells. Disclosures: No relevant conflicts of interest to declare.

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Preoperative breast radiation therapy is actively under investigation in clinical trials but practical experience with this technique and follow-up is limited. Our two single institutional trials evaluating preoperative partial breast irradiation are the largest US experience to date. In this study, we evaluated routine breast surveillance images following preoperative radiation in order to identify post-treatment mammographic imaging patterns unique to the preoperative approach. Materials/Methods: At one institution, women 55 years or older with clinically node negative, ER+ and/or PR+, HER2-, T1 invasive carcinomas or low-intermediate grade in situ disease <2cm were enrolled (nZ32). Intensity-modulated radiation therapy was used to deliver a single dose of 15, 18, or 21Gy to the tumor plus a 1.5cm margin. Lumpectomy was performed within 10 days of radiation. At the second institution, women with clinically node negative infiltrating ductal carcinomas measuring < 3 cm were enrolled. The pre-operative regimen consisted of 38.5 Gy in 3.85 Gy fractions delivered BID using NSABP B-39 expansions. Surgical resection followed at a median of 31 days (no sooner than 21 days) following completion of RT. For both cohorts, baseline mammography was reviewed and compared to all available posttreatment mammograms by an expert breast imager at each institution (nZ 31 at institution 1, n Z 27 at institution 2). Descriptive statistics were utilized to summarize findings. Results: In patients treated with larger single dose of preoperative radiation, median breast imaging follow-up is currently 26 months. In 17/31 (55%) of patients, exuberant and unusually well-demarcated fat necrosis beyond that expected following post-operative adjuvant radiation therapy was observed. For most patients (13/17; 76%), this change was evident by the first post-treatment mammogram (median 13 months) and persistent with additional follow-up. In contrast, patients treated with a fractionated course of preoperative radiation had a lower rate (2/27; 7%) of fat necrosis exceeding the typical post-BCT findings at a median follow-up of 38 months. However, 55% of patients were felt to have a wider area of scar tissue density at the lumpectomy cavity site. Conclusion: Postoperative MMG findings differ between two preoperative radiation therapy regimens. Patients treated with a large single dose of radiation, in contrast to fractionated radiation, demonstrate frequent and well-demarcated fat necrosis that appears to correlate with treated target volume. These imaging findings may be attributable to radiation-induced tissue ablation in the treated tissues, which differ according to treatment regimens. These findings have implications for surveillance in women treated with preoperative radiation.