- Email: [email protected]
Mutational Analysis of Ionizing Radiation Induced Neoplasms
S72 1
Clinique et maternite´ Sainte-Elisabeth, Namur, Belgium, 2NARILIS, Namur, Belgium, 3UCL-CHU Dinant-Godinne, Yvoir, Belgium, 4University of Namur, Namur, Belgium, 5CH Mouscron, Mouscron, Belgium Purpose/Objective(s): Due to a risk of 18% to 45% of occult metastases among the clinically free of nodes head and neck squamous cell carcinoma (HNSCC) patients, prophylactic neck irradiation is often mandatory. Based on international guidelines for the selection of the neck node levels, this prophylactic treatment still leads to unnecessary large irradiation of normal tissues because bilateral drainage is the rule in only 30% to 50% of individuals, and 15% to 30% of the tumors drain in unpredicted nodal basins. Sentinel lymph node (SLN) single-photon emission computerized tomography (SPECT/CT) lymphoscintigraphy makes it possible to individually predict cervical subregions requiring prophylactic irradiation in cN0 patients. This ongoing prospective phase 2 study analyzes its oncological safety. Materials/Methods: Twenty-one patients with newly diagnosed cN0 SCC of the oral cavity, oropharynx, larynx, or hypopharynx were included. All patients were imaged with SPECT/CT after 99mTc nanocolloid injection around the tumor. The neck levels containing up to 4 hottest nodes were identified and selected for prophylactic irradiation (CTVn-LS) by volumetric modulated arc therapy. A comparative virtual planning was performed with volumes selected according to international guidelines (CTVn-IG). Results: Migration was observed in all of the 21 patients (1 with gamma probe only) with an average of 2.7 sentinel nodes detected per patient. CTVn-LS was totally encompassed by CTVn-IG in all patients but 2 with an unpredicted drainage in retropharyngeal levels. On average, CTVn-LS and related planning target volume (PTV) were 2 times smaller than IG ones. This led to significant dose decrease in identified organs at risk as well as remaining volume at risk. With a median follow-up of 14 months, no regional relapse was observed, while 2 patients had a local one (9%). Currently, 3 patients have died (1 patient from geriatric degradation and 2 experienced fatal local relapse). Conclusion: SPECT/CT lymphoscintigraphy of SLN allows individualization of prophylactic node CTV in cN0 HNSCC patients eligible for definitive radiation therapy. Both CTV and PTV are significantly reduced, which results in a significant dose decrease in all identified organs at risk. At a median follow-up of 14 months, no regional relapse was observed, but further follow-up and recruitment are necessary to ensure the oncological safety. Author Disclosure: E. Longton: None. G. Lawson: None. J. Installe´: None. B. Bihin: None. M. Laloux: None. T. Vander Borght: None. I. Mathieu: None. K. Thevissen: None. J. Daisne: None.
162 Mutational Analysis of Ionizing Radiation Induced Neoplasms J.L. Nakamura,1 A. Sherborne,2 P. Davidson,3 K. Yu,2 and A. Nakamura3; 1 University of California, San Francisco, San Francisco, CA, 2University of California San Francisco, San Francisco, CA, 3University of Alberta, Edmonton, AB, Canada Purpose/Objective(s): Ionizing radiation (IR) is a known mutagen and can cause cancers, for example second malignant neoplasms (SMNs); SMNs are therapy-induced malignancies and severe late complications that develop in cancer survivors. The mutational landscape of fractionated IRinduced tumorigenesis is not well characterized on a genome level and but may reveal biological mechanisms that specifically contribute to the development of SMNs. We previously developed mouse models of SMNs by delivering focal, fractionated irradiation to wild type and Nf1 mutant mice. The goal of this study was to characterize the mutational profile of tumors induced by ionizing radiation modeling radiation therapy delivered to patients. Materials/Methods: Whole-exome sequencing was performed on 25 IRinduced malignancies arising in wild type and Nf1 mutant mice. Indexed paired-end libraries were prepared using a NGS target enrichment system, and the sequencing was performed high-density microarray. Alignments
International Journal of Radiation Oncology Biology Physics and somatic variants were identified using established procedures. Each exome was sequenced to a minimum of 5 Gb. Results: Seven thousand five hundred sixty-six somatic mutations were identified, of which 5 187 were non-synonymous. Tumors had an average mutation rate of 200 mutations (range, 31e594). Most nucleotide substitutions were C->T or G->A transitions. We analyzed the immediately flanking sequence context for each somatic variant using non-negative matrix factorization, and extracted 3 stable and distinctive mutational signatures. These signatures were detectable in multiple types of radiationinduced histologies, were uniquely distinguishable from mutational signatures of other well-recognized mutagens such as UV, and were similarly present in tumors arising in wild type mice as well as Nf1 mutant mice, suggesting that the IR mutational signature persists in genetic backgrounds either resistant or susceptible to IR-induced tumorigenesis. Copy number alterations and involved biological pathways differed between the genetic backgrounds. Conclusion: Ionizing radiation-induced malignancies possess distinguishable and unique mutational signatures characterized by base substitutions occurring in very specific and unique sequence contexts. This analysis suggests that IR and genetic background influence the mutational landscape of tumors in very specific and discrete ways that are distinguishable from other cancer-promoting processes. In conjunction with this work, radiation-induced SMNs from pediatric cancer survivors are being sequenced and similarly analyzed for mutational signatures. One implication of these findings is that the identification of mutational signatures might be exploited to develop assays to screen cancer survivors for SMN risk. Author Disclosure: J.L. Nakamura: None. A. Sherborne: None. P. Davidson: None. K. Yu: None. A. Nakamura: None.
163 Insertional Mutagenesis in HPV Cancers Identifies That Paradoxical Notch1 Activity Promotes Tumorigenesis M.T. Spiotto,1 R. Zhong,2 R. Bao,2 P. Faber,2 J. Bechill,2 and M. Lingen2; 1 University of Chicago, Chicago, IL, 2The University of Chicago, Chicago, IL Purpose/Objective(s): Human Papillomavirus (HPV) cancers possess mutations in multiple cellular genes including Notch1, which is believed to act as a tumor suppressor; however, the functional significance of these cellular mutations remains unclear. To identify cellular changes that impacted HPV oncogenesis, we used transposon-based insertional mutagenesis as an in vivo functional screen for genes that accelerated HPV tumorigenesis. Materials/Methods: Using a tamoxifen-regulated Cre recombinase localized to the skin, we conditionally induced HPV16 E6 and E7 oncogene expression alone (KH mice) or with an activated Sleeping Beauty transposition (KH-Onc mice). We identified genes significantly enriched for transposon insertion sites using microarray high-throughput sequencing followed by Poisson distribution analysis. Significant genes were validated in transgenic mice developing primary HPV oral tumors that also possessed a mutant KrasG12D allele (KHR mice). We modeled Notch1 gain or loss in oral tumors using KHR mice possessing an activated Notch1 transgene (KHR-NICD) or a targeted Notch1 allele (KHR-Notch1-/-), respectively. Results: Transposon-based insertional mutagenesis accelerated the development of primary squamous cell cancers in HPV-Onc mice compared to control HPV mice or Onc mice (P < .0001). After sequencing and mapping 273 054 unique transposon insertion sites, we identified 39 significant genes, including Notch1 (P Z 9.1 x 10-32). Analysis of transposon orientations and gene expression in HPV-Onc tumors suggested that gain or loss of Notch1 promoted carcinogenesis. Expressing the activated Notch1 transgene, KHR-NICD mice developed autochthonous HPV-positive oral tumors that grew faster and had increased tumor cell proliferation compared to control KHR mice (P < .02). Similarly, Notch1 loss in KHRNotch1-/- mice accelerated oral tumor growth compared to KHR mice.
Clinique et maternite´ Sainte-Elisabeth, Namur, Belgium, 2NARILIS, Namur, Belgium, 3UCL-CHU Dinant-Godinne, Yvoir, Belgium, 4University of Namur, Namur, Belgium, 5CH Mouscron, Mouscron, Belgium Purpose/Objective(s): Due to a risk of 18% to 45% of occult metastases among the clinically free of nodes head and neck squamous cell carcinoma (HNSCC) patients, prophylactic neck irradiation is often mandatory. Based on international guidelines for the selection of the neck node levels, this prophylactic treatment still leads to unnecessary large irradiation of normal tissues because bilateral drainage is the rule in only 30% to 50% of individuals, and 15% to 30% of the tumors drain in unpredicted nodal basins. Sentinel lymph node (SLN) single-photon emission computerized tomography (SPECT/CT) lymphoscintigraphy makes it possible to individually predict cervical subregions requiring prophylactic irradiation in cN0 patients. This ongoing prospective phase 2 study analyzes its oncological safety. Materials/Methods: Twenty-one patients with newly diagnosed cN0 SCC of the oral cavity, oropharynx, larynx, or hypopharynx were included. All patients were imaged with SPECT/CT after 99mTc nanocolloid injection around the tumor. The neck levels containing up to 4 hottest nodes were identified and selected for prophylactic irradiation (CTVn-LS) by volumetric modulated arc therapy. A comparative virtual planning was performed with volumes selected according to international guidelines (CTVn-IG). Results: Migration was observed in all of the 21 patients (1 with gamma probe only) with an average of 2.7 sentinel nodes detected per patient. CTVn-LS was totally encompassed by CTVn-IG in all patients but 2 with an unpredicted drainage in retropharyngeal levels. On average, CTVn-LS and related planning target volume (PTV) were 2 times smaller than IG ones. This led to significant dose decrease in identified organs at risk as well as remaining volume at risk. With a median follow-up of 14 months, no regional relapse was observed, while 2 patients had a local one (9%). Currently, 3 patients have died (1 patient from geriatric degradation and 2 experienced fatal local relapse). Conclusion: SPECT/CT lymphoscintigraphy of SLN allows individualization of prophylactic node CTV in cN0 HNSCC patients eligible for definitive radiation therapy. Both CTV and PTV are significantly reduced, which results in a significant dose decrease in all identified organs at risk. At a median follow-up of 14 months, no regional relapse was observed, but further follow-up and recruitment are necessary to ensure the oncological safety. Author Disclosure: E. Longton: None. G. Lawson: None. J. Installe´: None. B. Bihin: None. M. Laloux: None. T. Vander Borght: None. I. Mathieu: None. K. Thevissen: None. J. Daisne: None.
162 Mutational Analysis of Ionizing Radiation Induced Neoplasms J.L. Nakamura,1 A. Sherborne,2 P. Davidson,3 K. Yu,2 and A. Nakamura3; 1 University of California, San Francisco, San Francisco, CA, 2University of California San Francisco, San Francisco, CA, 3University of Alberta, Edmonton, AB, Canada Purpose/Objective(s): Ionizing radiation (IR) is a known mutagen and can cause cancers, for example second malignant neoplasms (SMNs); SMNs are therapy-induced malignancies and severe late complications that develop in cancer survivors. The mutational landscape of fractionated IRinduced tumorigenesis is not well characterized on a genome level and but may reveal biological mechanisms that specifically contribute to the development of SMNs. We previously developed mouse models of SMNs by delivering focal, fractionated irradiation to wild type and Nf1 mutant mice. The goal of this study was to characterize the mutational profile of tumors induced by ionizing radiation modeling radiation therapy delivered to patients. Materials/Methods: Whole-exome sequencing was performed on 25 IRinduced malignancies arising in wild type and Nf1 mutant mice. Indexed paired-end libraries were prepared using a NGS target enrichment system, and the sequencing was performed high-density microarray. Alignments
International Journal of Radiation Oncology Biology Physics and somatic variants were identified using established procedures. Each exome was sequenced to a minimum of 5 Gb. Results: Seven thousand five hundred sixty-six somatic mutations were identified, of which 5 187 were non-synonymous. Tumors had an average mutation rate of 200 mutations (range, 31e594). Most nucleotide substitutions were C->T or G->A transitions. We analyzed the immediately flanking sequence context for each somatic variant using non-negative matrix factorization, and extracted 3 stable and distinctive mutational signatures. These signatures were detectable in multiple types of radiationinduced histologies, were uniquely distinguishable from mutational signatures of other well-recognized mutagens such as UV, and were similarly present in tumors arising in wild type mice as well as Nf1 mutant mice, suggesting that the IR mutational signature persists in genetic backgrounds either resistant or susceptible to IR-induced tumorigenesis. Copy number alterations and involved biological pathways differed between the genetic backgrounds. Conclusion: Ionizing radiation-induced malignancies possess distinguishable and unique mutational signatures characterized by base substitutions occurring in very specific and unique sequence contexts. This analysis suggests that IR and genetic background influence the mutational landscape of tumors in very specific and discrete ways that are distinguishable from other cancer-promoting processes. In conjunction with this work, radiation-induced SMNs from pediatric cancer survivors are being sequenced and similarly analyzed for mutational signatures. One implication of these findings is that the identification of mutational signatures might be exploited to develop assays to screen cancer survivors for SMN risk. Author Disclosure: J.L. Nakamura: None. A. Sherborne: None. P. Davidson: None. K. Yu: None. A. Nakamura: None.
163 Insertional Mutagenesis in HPV Cancers Identifies That Paradoxical Notch1 Activity Promotes Tumorigenesis M.T. Spiotto,1 R. Zhong,2 R. Bao,2 P. Faber,2 J. Bechill,2 and M. Lingen2; 1 University of Chicago, Chicago, IL, 2The University of Chicago, Chicago, IL Purpose/Objective(s): Human Papillomavirus (HPV) cancers possess mutations in multiple cellular genes including Notch1, which is believed to act as a tumor suppressor; however, the functional significance of these cellular mutations remains unclear. To identify cellular changes that impacted HPV oncogenesis, we used transposon-based insertional mutagenesis as an in vivo functional screen for genes that accelerated HPV tumorigenesis. Materials/Methods: Using a tamoxifen-regulated Cre recombinase localized to the skin, we conditionally induced HPV16 E6 and E7 oncogene expression alone (KH mice) or with an activated Sleeping Beauty transposition (KH-Onc mice). We identified genes significantly enriched for transposon insertion sites using microarray high-throughput sequencing followed by Poisson distribution analysis. Significant genes were validated in transgenic mice developing primary HPV oral tumors that also possessed a mutant KrasG12D allele (KHR mice). We modeled Notch1 gain or loss in oral tumors using KHR mice possessing an activated Notch1 transgene (KHR-NICD) or a targeted Notch1 allele (KHR-Notch1-/-), respectively. Results: Transposon-based insertional mutagenesis accelerated the development of primary squamous cell cancers in HPV-Onc mice compared to control HPV mice or Onc mice (P < .0001). After sequencing and mapping 273 054 unique transposon insertion sites, we identified 39 significant genes, including Notch1 (P Z 9.1 x 10-32). Analysis of transposon orientations and gene expression in HPV-Onc tumors suggested that gain or loss of Notch1 promoted carcinogenesis. Expressing the activated Notch1 transgene, KHR-NICD mice developed autochthonous HPV-positive oral tumors that grew faster and had increased tumor cell proliferation compared to control KHR mice (P < .02). Similarly, Notch1 loss in KHRNotch1-/- mice accelerated oral tumor growth compared to KHR mice.