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Severe Radiation Toxicity Associated with a Germline PTEN Mutation
E620
International Journal of Radiation Oncology Biology Physics
Materials/Methods: Genetically engineered mice with a mutant Kras and p53 deletion driven by a pancreatic Cre recombinase (KPC) were inbred to pure C57BL/6 to normalize background effects in normal tissue. Pancreatic tumors were diagnosed at 12-16 weeks of age by small animal ultrasonography. When tumors reached 5mm in any dimension, the KPC mouse was enrolled onto one of three cohorts: sham RT (NORT), EDR with vehicle alone (RT-VE), or EDR with FG-4592 for radioprotection (RT-FG). If indicated, KPC mice were gavaged daily with the EGLN inhibitor FG-4592 (40mg/kg) or vehicle control 6 hours prior to radiation. Mice were anesthetized supine in an XRAD225 small animal irradiator. A 15mm subxiphoid field was verified daily with cone beam CT prior to delivering a total dose of 75Gy/15 daily fractions (BED10Z112.5Gy) using AP/PA technique. Blinded necropsies were performed for each mouse to evaluate tumor size, cause of death, radiation-induced damage and presence of micro- and macrometastases. Survival time was measured from tumor diagnosis to death. Kaplan-Meier survival curves were generated for survival using log rank test to compare the groups. Differences in causes of death and metastatic disease were compared between groups using chi square or Fisher’s exact test where appropriate. Results: EDR with radioprotection by FG-4592 improved survival (median 43 days [39-47]; NZ8) compared to RT-VE (37 days [36-38]; NZ7) or NORT (9 days [7-11]; NZ23; p<.0001). Death due to radiation-related toxicity was significantly higher for RT-VE (33.3%) versus RT-FG (5.9%; p<.0001), and intestinal bleeding was the chief source of mortality. Presence of micrometastases at death was significantly higher for RT-VE (100%) than for RT-FG (58%; pZ0.03), suggesting an anti-metastatic effect of FG-4592. For deaths unrelated to treatment toxicity, death attributed to local, rather than metastatic disease, was higher in NORT arms vs RT arms (76% vs 60% and 69%; pZ0.03). Conclusion: In this preclinical study, EGLN inhibition reduced treatmentrelated GI toxicity sufficiently to enable EDR treatments, which improved local control, and reduced micrometastases. FG-4592 did not radioprotect tumors, and if anything, reduced metastatic spread, which contributed favorably towards survival. Our results suggest a new paradigm where selective GI radioprotection afforded by an EGLN inhibitor like FG-4592 could allow for potentially curative EDR treatments for LAPC. Author Disclosure: L.E. Colbert: None. T.N. Fujimoto: None. J.M. Molkentine: None. L. Baseler: None. Y. Huang: None. A. Deorukhkar: None. C.M. Taniguchi: None.
after only 54 Gy, due to unusually severe desquamation. She recovered completely about 1 month thereafter. Her subsequent brain metastases prompted a course of WBRT to 30 Gy. Notably, she experienced near total hair loss in the first week of WBRT, despite the scalp receiving <10 Gy (due to IMRT in accordance with CC-001 guidelines). She re-presented 4 months after completing WBRT with emesis, ataxia, and headache. MRI demonstrated increased intracranial edema and nodular enhancement. Subsequent PET favored an etiology of treatment-induced changes rather than disease progression, particularly since the intracranial tumors were smaller compared to pre-WBRT imaging. Her symptoms improved after steroids and supportive care over an 8-day admission. Imaging one week later demonstrated reduction of edema. Another MRI performed one month after discharge confirmed no evidence of tumor progression. At 2 months post-admission, her ataxia has continued to improved, however she has some persistent balance instability. Conclusion: To our knowledge, this is the first reported case of unexpected normal tissue toxicity following radiotherapy in the setting of a germline PTEN mutation. This observation is consistent with prior experimental studies in cell culture, which have predicted an association between germline PTEN loss-of-function mutations and hypersensitivity of normal tissues to radiotherapy. Caution with radiotherapy is recommended until this observation can be studied further in larger cohorts of Cowden syndrome patients. Author Disclosure: K. Tatebe: None. S.J. Chmura: Employee; Astellas. P.P. Connell: None.
3479 Severe Radiation Toxicity Associated with a Germline PTEN Mutation K. Tatebe, S.J. Chmura, and P.P. Connell; University of Chicago, Chicago, IL Purpose/Objective(s): Cowden syndrome is caused by germline loss-offunction mutations in PTEN, which result in the development of hamartomas and malignant tumors. The disease is inherited in an autosomal dominant fashion and has an approximate prevalence of at least 1 in 200,000. Experimental studies have demonstrated radiation sensitivity in cell lines after PTEN inhibition by gene targeting or RNAi. However, no report to our knowledge has identified hypersensitivity of a Cowden syndrome patient to radiotherapy. We describe a case of unexpected normal tissue toxicities in a patient with a confirmed germline PTEN mutation. Materials/Methods: The patient initially presented at age 39 with thyroid cancer, treated with thyroidectomy and I-131 ablation. She later developed bilateral breast cancer at age 44, treated with bilateral mastectomy and adjuvant radiation to the left chest wall and regional lymph nodes. Cowden syndrome was diagnosed during this time. She presented with brain metastases 15 months later, treated with resection of the two largest cerebellar masses, followed by whole brain radiotherapy (WBRT) with Memantine, on protocol, according to the hippocampal sparing arm of NRG CC-001. Results: Germline DNA sequencing demonstrated a heterozygous nonsense mutation in PTEN at K322, confirming a diagnosis of Cowden syndrome. Adjuvant breast treatment included 50 Gy to the left chest wall with a planned 10 Gy boost. This treatment required early discontinuation
3480 Downregulation of MicroRNA-203 Associated With Radioresistance in Esophageal Squamous Cell Carcinoma Cells K. Terashima,1 H. Hirata,2 H. Wakiyama,1 R. Uchi,3 T. Masuda,3 K. Sakamoto,1 M. Hirakawa,1 K. Mimori,3 and H. Honda2; 1Department of Radiology, Kyushu University Beppu Hospital, Beppu, Japan, 2Department of Clinical Radiology, Kyushu University, Fukuoka, Japan, 3Department of Surgery, Kyushu University Beppu Hospital, Beppu, Japan Purpose/Objective(s): Although radiotherapy is recognized as the standard treatment for esophageal squamous cell carcinoma (ESCC), radioresistance is one of the major obstacles in the treatment of ESCC. MicroRNAs (miRs) are known to affect the proliferation and the radiosensitivity on several human cancers by regulating the expressions of their target mRNAs. In this study, we aimed to identify miR involved in radioresistance in ESCC cells. Materials/Methods: Initially, MTT assay was performed to determine radiosensitivity of 10 ESCC cell lines irradiated with 8 Gy. Next, we compared miR expression between the most radiosensitive and the most radioresistant cell lines by miR array. Gene expression profiles of ESCC cells were also obtained from the public database in the Cancer Cell Line Encyclopedia (CCLE). Using CCLE data, we then performed gene set enrichment analysis (GSEA) to determine whether candidate miRs regulate the expressions of their targeted mRNAs. Finally, to investigate whether candidate miRs affect in vitro radiosensitivity in ESCC cells, cell lines were transfected with endogenous mature candidate miR mimic and radiosensitivity was evaluated by colony formation assay. Results: Among 10 ESCC cell lines, we found that TE1 cell line was the most radioresistant, whereas TE9 cell line was the most radiosensitive. Therefore miR array was performed using these two cell lines. Based on miR array data and the results of GSEA, we identified miR-203 as the downregulated miR in radioresistant cells compared to radiosensitive cells. In colony formation assay, overexpression of miR-203 inhibited cell proliferation in both radioresistant and radiosensitive cell lines. With regard to in vitro radiosensitivity, colony formation assay also revealed that overexpression of miR-203 impaired resistance to radiation in TE1 and second most radioresistant TE11 cells. Conclusion: Overexpression of miR-203 increased radiosensitivity of ESCC cell lines. Our findings indicate that miR-203 can be a target for overcoming the radioresistance of ESCC.
International Journal of Radiation Oncology Biology Physics
Materials/Methods: Genetically engineered mice with a mutant Kras and p53 deletion driven by a pancreatic Cre recombinase (KPC) were inbred to pure C57BL/6 to normalize background effects in normal tissue. Pancreatic tumors were diagnosed at 12-16 weeks of age by small animal ultrasonography. When tumors reached 5mm in any dimension, the KPC mouse was enrolled onto one of three cohorts: sham RT (NORT), EDR with vehicle alone (RT-VE), or EDR with FG-4592 for radioprotection (RT-FG). If indicated, KPC mice were gavaged daily with the EGLN inhibitor FG-4592 (40mg/kg) or vehicle control 6 hours prior to radiation. Mice were anesthetized supine in an XRAD225 small animal irradiator. A 15mm subxiphoid field was verified daily with cone beam CT prior to delivering a total dose of 75Gy/15 daily fractions (BED10Z112.5Gy) using AP/PA technique. Blinded necropsies were performed for each mouse to evaluate tumor size, cause of death, radiation-induced damage and presence of micro- and macrometastases. Survival time was measured from tumor diagnosis to death. Kaplan-Meier survival curves were generated for survival using log rank test to compare the groups. Differences in causes of death and metastatic disease were compared between groups using chi square or Fisher’s exact test where appropriate. Results: EDR with radioprotection by FG-4592 improved survival (median 43 days [39-47]; NZ8) compared to RT-VE (37 days [36-38]; NZ7) or NORT (9 days [7-11]; NZ23; p<.0001). Death due to radiation-related toxicity was significantly higher for RT-VE (33.3%) versus RT-FG (5.9%; p<.0001), and intestinal bleeding was the chief source of mortality. Presence of micrometastases at death was significantly higher for RT-VE (100%) than for RT-FG (58%; pZ0.03), suggesting an anti-metastatic effect of FG-4592. For deaths unrelated to treatment toxicity, death attributed to local, rather than metastatic disease, was higher in NORT arms vs RT arms (76% vs 60% and 69%; pZ0.03). Conclusion: In this preclinical study, EGLN inhibition reduced treatmentrelated GI toxicity sufficiently to enable EDR treatments, which improved local control, and reduced micrometastases. FG-4592 did not radioprotect tumors, and if anything, reduced metastatic spread, which contributed favorably towards survival. Our results suggest a new paradigm where selective GI radioprotection afforded by an EGLN inhibitor like FG-4592 could allow for potentially curative EDR treatments for LAPC. Author Disclosure: L.E. Colbert: None. T.N. Fujimoto: None. J.M. Molkentine: None. L. Baseler: None. Y. Huang: None. A. Deorukhkar: None. C.M. Taniguchi: None.
after only 54 Gy, due to unusually severe desquamation. She recovered completely about 1 month thereafter. Her subsequent brain metastases prompted a course of WBRT to 30 Gy. Notably, she experienced near total hair loss in the first week of WBRT, despite the scalp receiving <10 Gy (due to IMRT in accordance with CC-001 guidelines). She re-presented 4 months after completing WBRT with emesis, ataxia, and headache. MRI demonstrated increased intracranial edema and nodular enhancement. Subsequent PET favored an etiology of treatment-induced changes rather than disease progression, particularly since the intracranial tumors were smaller compared to pre-WBRT imaging. Her symptoms improved after steroids and supportive care over an 8-day admission. Imaging one week later demonstrated reduction of edema. Another MRI performed one month after discharge confirmed no evidence of tumor progression. At 2 months post-admission, her ataxia has continued to improved, however she has some persistent balance instability. Conclusion: To our knowledge, this is the first reported case of unexpected normal tissue toxicity following radiotherapy in the setting of a germline PTEN mutation. This observation is consistent with prior experimental studies in cell culture, which have predicted an association between germline PTEN loss-of-function mutations and hypersensitivity of normal tissues to radiotherapy. Caution with radiotherapy is recommended until this observation can be studied further in larger cohorts of Cowden syndrome patients. Author Disclosure: K. Tatebe: None. S.J. Chmura: Employee; Astellas. P.P. Connell: None.
3479 Severe Radiation Toxicity Associated with a Germline PTEN Mutation K. Tatebe, S.J. Chmura, and P.P. Connell; University of Chicago, Chicago, IL Purpose/Objective(s): Cowden syndrome is caused by germline loss-offunction mutations in PTEN, which result in the development of hamartomas and malignant tumors. The disease is inherited in an autosomal dominant fashion and has an approximate prevalence of at least 1 in 200,000. Experimental studies have demonstrated radiation sensitivity in cell lines after PTEN inhibition by gene targeting or RNAi. However, no report to our knowledge has identified hypersensitivity of a Cowden syndrome patient to radiotherapy. We describe a case of unexpected normal tissue toxicities in a patient with a confirmed germline PTEN mutation. Materials/Methods: The patient initially presented at age 39 with thyroid cancer, treated with thyroidectomy and I-131 ablation. She later developed bilateral breast cancer at age 44, treated with bilateral mastectomy and adjuvant radiation to the left chest wall and regional lymph nodes. Cowden syndrome was diagnosed during this time. She presented with brain metastases 15 months later, treated with resection of the two largest cerebellar masses, followed by whole brain radiotherapy (WBRT) with Memantine, on protocol, according to the hippocampal sparing arm of NRG CC-001. Results: Germline DNA sequencing demonstrated a heterozygous nonsense mutation in PTEN at K322, confirming a diagnosis of Cowden syndrome. Adjuvant breast treatment included 50 Gy to the left chest wall with a planned 10 Gy boost. This treatment required early discontinuation
3480 Downregulation of MicroRNA-203 Associated With Radioresistance in Esophageal Squamous Cell Carcinoma Cells K. Terashima,1 H. Hirata,2 H. Wakiyama,1 R. Uchi,3 T. Masuda,3 K. Sakamoto,1 M. Hirakawa,1 K. Mimori,3 and H. Honda2; 1Department of Radiology, Kyushu University Beppu Hospital, Beppu, Japan, 2Department of Clinical Radiology, Kyushu University, Fukuoka, Japan, 3Department of Surgery, Kyushu University Beppu Hospital, Beppu, Japan Purpose/Objective(s): Although radiotherapy is recognized as the standard treatment for esophageal squamous cell carcinoma (ESCC), radioresistance is one of the major obstacles in the treatment of ESCC. MicroRNAs (miRs) are known to affect the proliferation and the radiosensitivity on several human cancers by regulating the expressions of their target mRNAs. In this study, we aimed to identify miR involved in radioresistance in ESCC cells. Materials/Methods: Initially, MTT assay was performed to determine radiosensitivity of 10 ESCC cell lines irradiated with 8 Gy. Next, we compared miR expression between the most radiosensitive and the most radioresistant cell lines by miR array. Gene expression profiles of ESCC cells were also obtained from the public database in the Cancer Cell Line Encyclopedia (CCLE). Using CCLE data, we then performed gene set enrichment analysis (GSEA) to determine whether candidate miRs regulate the expressions of their targeted mRNAs. Finally, to investigate whether candidate miRs affect in vitro radiosensitivity in ESCC cells, cell lines were transfected with endogenous mature candidate miR mimic and radiosensitivity was evaluated by colony formation assay. Results: Among 10 ESCC cell lines, we found that TE1 cell line was the most radioresistant, whereas TE9 cell line was the most radiosensitive. Therefore miR array was performed using these two cell lines. Based on miR array data and the results of GSEA, we identified miR-203 as the downregulated miR in radioresistant cells compared to radiosensitive cells. In colony formation assay, overexpression of miR-203 inhibited cell proliferation in both radioresistant and radiosensitive cell lines. With regard to in vitro radiosensitivity, colony formation assay also revealed that overexpression of miR-203 impaired resistance to radiation in TE1 and second most radioresistant TE11 cells. Conclusion: Overexpression of miR-203 increased radiosensitivity of ESCC cell lines. Our findings indicate that miR-203 can be a target for overcoming the radioresistance of ESCC.