Circulating Tumor DNA Quantitation for Early Response Assessment of Immune Checkpoint Inhibitors for Lung Cancer

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International Journal of Radiation Oncology  Biology  Physics

Author Disclosure: V. Bernard: None. D.U. Kim: None. J. Castillo: None. F.A. San Lucas: None. B. Stephens: None. F. Mulu: None. J.M. Herman: None. C.M. Taniguchi: None. E.J. Koay: Research Grant; MD Anderson Cancer Center, PANCAN-AACR, Philips, Radiological Society of North America. Patent/License Fees/Copyright; Pending Patent. G.R. Varadhachary: None. M.H. Katz: None. H.A. Alvarez: None. A. Maitra: None.

induces the Notch-1 signaling pathway required for crypt cell differentiation alleviate GI symptoms, preserve and replenish gastrointestinal mucosal integrity and function, and improve survival after acute radiation exposure. Author Disclosure: P.K. Singh: None. X. Li: None. J. Zhang: None. G. Liu: None. S. Bhattarai: None. M. Ahmad: None. M. Bodd: None. A.S. Chadha: None. R.C. Tailor: None. S. Krishnan: Research Grant; Cancer Prevention and Research Institute of Texas, Celgene, DoD, Dunn Foundation, NIH. Royalty; Taylor and Francis Group.; RTOG.

41 Development of Wnt/Notch Activators as Mitigators of Acute Radiation Gastrointestinal Syndrome P.K. Singh,1 X. Li,2 J. Zhang,1 G. Liu,3 S. Bhattarai,1,4 M. Ahmad,5 M. Bodd,1 A.S. Chadha,1 R.C. Tailor,6 and S. Krishnan1; 1Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, 2Department of Geriatric Gastroenterology, Affiliated Hospital with Nanjing Medical University, Nanjing, China, 3 Zhejiang Cancer Hospital, Hangzhou, China, 4Department of Biomedical Engineering, University of Houston, Houston, TX, 5College of Science and Mathematics, Houston Baptist University, Houston, TX, 6Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, Houston, TX Purpose/Objective(s): We hypothesize that both proliferation and differentiation of surviving crypt stem cells are necessary for recovery from gastrointestinal (GI) radiation injury. The purpose of the current study was to evaluate the role of Wnt/Notch activators in mitigating acute radiation GI syndrome. Materials/Methods: C57BL/6 male mice were exposed to 10 Gy of total body 137Cs- gamma radiation. Twenty-four hours later mice (n Z 8 each) received (a) PBS (100 ml I.P.), (b) Valproic acid (VPA) (300 mg/kg, I.P.), (c) recWnt3 (100 mg/kg I.V.), and (d) recWnt3 (100 mg/kg I.V.) and VPA (300 mg/kg, I.P.). Animals were monitored for 30 days. Mice were treated identically for histopathological analysis of jejunal sections on day 4 (n Z 4) and day 8 (n Z 4). To evaluate the intestinal permeability on day 4, mice were fed with FITC-dextran (500 mg/kg BW; molecular mass 4 kDa) orally 4 hours before their serum was harvested. Another set of mice received 100 mg/kg of 5-bromo-2-deoxyuridine (BrdU) intraperitoneally 1.5 h prior to jejunum harvest on day 8 for evaluating the crypt cell proliferation. Jejunal tissue sections were analyzed for crypts and villus histopathology (H&E), apoptosis (TUNEL, activated caspase-3), crypt regeneration (BrdU, PCNA) and crypt differentiation (Goblet cell: periodic acideSchiff (PAS); Paneth cell: lysozyme; enterocyte: alkaline phosphatase; enteroendocrine cell: anti-synaptophysin). Results: Combination treatment of recWnt3 and VPA was found to significantly mitigate the acute effects of radiation with respect to the vehicle control extending the life of mice by 7 days or either group alone, extending the life of mice by 7 days. Mice treated with recWnt3 and VPA showed higher number of surviving crypts per circumference on day 4 and 8 in comparison to vehicle control, or either group alone. The number of surviving crypts was significantly higher on day 8 in recWnt3+VPA group suggesting better GI recovery. The combination treatment also enhanced the number of mitotic figures per high power field and mean villus length of the irradiated mice. Higher number of proliferating crypt and lower number of apoptotic cells were observed in the combination treatment group as compared to the irradiated control. Low serum dextran levels were observed in the combination treatment group showing better intestinal mucosal integrity. In the recWnt3+VPA group significantly higher number of differentiated cells was observed as compared to the irradiated control group. Conclusion: Treatment with recWnt3, the natural ligand for Wnt receptor and a potent activator of Wnt signaling, and valproic acid (VPA), which

42 Circulating Tumor DNA Quantitation for Early Response Assessment of Immune Checkpoint Inhibitors for Lung Cancer D.J. Merriott, A.A. Chaudhuri, M. Jin, J.J. Chabon, A. Newman, H. Stehr, C. Say, J.N. Carter, S. Walters, H.C.R. Becker, M. Das, S. Padda, B.W. Loo Jr, H.A. Wakelee, J.W. Neal, A.A. Alizadeh, and M. Diehn; Stanford University School of Medicine, Stanford, CA Purpose/Objective(s): Immune checkpoint inhibitors (ICIs) are commonly used for treatment of advanced stage non-small cell lung cancer (NSCLC) and clinical studies are examining combinations of ICIs with radiotherapy (RT). Standard-of-care response assessment is CT imaging, which is typically performed no earlier than 2-3 months after treatment initiation due to delayed radiographic responses and occasional pseudoprogression. Improved methods of response assessment would be useful clinically and in trials combining ICIs and RT. Here we explored if CAPP-Seq ctDNA analysis might allow early identification of metastatic NSCLC patients who will benefit from immune checkpoint inhibitors. Materials/Methods: We enrolled 14 patients with metastatic NSCLC (adenocarcinoma n Z 10, squamous cell carcinoma n Z 2, large cell carcinoma n Z 2) treated with nivolumab (n Z 13) or pembrolizumab (n Z 1), 9 of whom had received prior RT. Nivolumab was administered every 2 weeks and pembrolizumab every 3 weeks. CT imaging and CAPPSeq ctDNA quantitation were performed for each patient pre-treatment. Response assessment was performed by surveillance imaging every w2 months and CAPP-Seq ctDNA analysis at w6 weeks. CT scans were scored by RECIST v1.1 criteria using MINT Lesion software and blinded to the ctDNA results. Patients were classified as having durable clinical benefit (DCB; partial response or stable disease for
4 months) or no durable benefit (NDB) based on CT imaging. Results: Median follow-up was 15.6 months and median progression free survival (PFS) was 2.0 months. Four patients had DCB and 10 had NDB. Average time from treatment start to ctDNA response assessment was 41 days (range 14-71), and to imaging response was 60 days (range 37-83), which did not differ between patients with DCB vs. NDB. Median PFS in patients with DCB and NDB was 8.8 and 1.8 months, respectively. There was no significant difference in pre-treatment ctDNA allele fraction (AF) between those with DCB and NDB (average 4.8% vs. 6.0%; PZ0.8). However, patients with DCB had significantly larger ctDNA drop compared to those with NDB (average post:pre fold-change þ/-SEM: 0.005þ/-0.005 vs. 1.3þ/-0.3, PZ0.001). Evaluable patients with undetectable post-treatment ctDNA all had DCB whereas no patients with NDB had undetectable post-treatment ctDNA. Another DCB patient with detectable post-treatment ctDNA displayed a 100-fold decrease in ctDNA (1% pre-treatment vs. 0.01% post-treatment). Response classification by ctDNA analysis occurred on average 19 days earlier than imaging. Evaluation of earlier time-points is ongoing. Conclusion: Our findings suggest that analysis of ctDNA potentially allows early identification of NSCLC patients who will have DCB from ICIs. Response assessment by ctDNA may therefore be useful in clinical studies examining combinations of ICIs and radiotherapy.

Volume 99  Number 2S  Supplement 2017 Author Disclosure: D.J. Merriott: None. A.A. Chaudhuri: Consultant; Oscar Health. M. Jin: None. J.J. Chabon: None. A. Newman: Consultant; Roche Sequencing. Stock; CiberMed. H. Stehr: None. C. Say: None. J.N. Carter: None. S. Walters: None. H.R. Becker: None. M. Das: Employee; Stanford University. Research Grant; CelGene. S. Padda: Consultant; Clovis Oncology. B.W. Loo: Research Grant; RaySearch, Varian Medical Systems Inc. Stock; TibaRay, Inc.; American College of Radiology, National Comprehensive Cancer Network, TibaRay, Inc. H.A. Wakelee: Research Grant; BMS, Celgene, Clovis, Exelixis, Lilly, Novartis, Pfizer, Pharmacyclics, Roche/Genentech, Xcovery. Consultant; Peregrine. Advisory Board; Gilead, Pfizer; International Thymic Malignancies Interest Group. J.W. Neal: Research Grant; Genentech/Roche, Merck, ArQule, Novartis, Exelixis, Boehringer Ingelheim, Nektar. Consultant; Clovis Oncology, CARET, Nektar, Boehringer Ingelheim, ARMO BioSciences, ARIAD, Lilly. A.A. Alizadeh: Research Grant; Celgene. Honoraria; Roche Sequencing. Consultant; Roche Sequencing, Celgene, Gilead Sciences. Stock; CiberMed. Member of 45-person board with leadership responsibilities; Lymphoma Research Foundation. M. Diehn: Employee; Kaiser Permanente. Consultant; Roche. Stock; CiberMed. Stock Options; Quanticel Pharmaceuticals.

43 Plasma HPV DNA as a Biomarker of Response in Patients with Locally Advanced Cervical Cancer Treated With Definitive Chemoradiation Therapy K. Han,1 E.W. Leung,2 and S.V. Bratman1; 1Radiation Medicine Program, Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada, 2Department of Radiation Oncology, Odette Cancer Centre, Sunnybrook Health Sciences Centre, Toronto, ON, Canada Purpose/Objective(s): Despite recent advances, a significant proportion of women with locally advanced cervical cancer still experience relapse. Post-therapy fluorodeoxyglucose positron emission tomography (FDG PET) at 3 months is associated with survival, but accuracy for detecting relapse is limited. The majority of cervical cancer is caused by HPV, but there is limited data on the utility of measuring circulating HPV DNA for response assessment. We hypothesize that plasma HPV DNA may identify patients with (minimal) residual disease following concurrent chemoradiation (CRT) and improve on the accuracy of 3-month FDG PET for detecting relapse. Materials/Methods: This prospective multi-center study accrued 20 patients with HPV-positive stage IB-IVA cervical cancer treated with definitive CRT from 2015-2016. Patients underwent phlebotomy at baseline, end of, and 3 months post CRT for the measurement of plasma HPV DNA levels, along with 3-month FDG PET. A pre-treatment cervical swab was collected for HPV genotyping. Plasma HPV DNA levels were assessed from 10 ng cell-free DNA (cfDNA) by digital polymerase chain reaction using TaqMan assays targeting the E6 and E7 open reading frames from the appropriate HPV genotype for each patient. Progression-free survival (PFS) was estimated using the Kaplan-Meier method and compared using the log rank test and Cox proportional hazards model. Results: Of the 19 analyzable patients (one did not complete CRT), 26% were stage IB, 63% IIB, 5% IIIB, 5% IVA; 95% squamous cell carcinoma and 5% adenocarcinoma. Median age was 43. Median follow-up was 16 (range 6-20) months. Sensitivity for detecting plasma HPV DNA at baseline was 100% (range 2 - 66880 copies of E6 or E7/10ng of cfDNA; 15 HPV16, 2 HPV33, 1 HPV45 and 1 HPV52). Six patients (32%) had detectable plasma HPV DNA at the end of CRT, and 3 of them developed distant metastases by 3 months (follow-up ongoing). Of the 13 patients (68%) with undetectable plasma HPV DNA at the end of CRT, to date only 1 has developed recurrence (1.2 cm lung metastasis 12.1 months post CRT). Seven of those patients had indeterminate FDG uptake in the cervix

Oral Scientific Sessions

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(SUVmax2.5 - 4) on the PET scan, with no definite residual disease on subsequent MRI or clinical examination to date, and 5 of these 7 had undetectable plasma HPV DNA at 3 months. Patients with undetectable plasma HPV DNA at the end of CRT had significantly higher 1-year PFS than those with detectable plasma HPV DNA (100% vs 50%, P Z 0.01; Cox hazard ratio 11, P Z 0.04). Conclusion: Early results from this prospective multi-center study suggest that detectable plasma HPV DNA at the end of CRT predates the clinical diagnosis of distant metastases, and at 3-months provides greater accuracy for detecting relapse compared with FDG PET. The study will accrue more patients to confirm the prognostic significance of detectable plasma HPV DNA following CRT. Author Disclosure: K. Han: None. E.W. Leung: None. S.V. Bratman: Patent/License Fees/Copyright; Inventor on patent.

44 A Subtype-Independent Signature Predictive of Early Versus Late Recurrence After Radiation Treatment for Breast Cancer That May Inform the Biology of Early, Aggressive Recurrences C. Speers,1 S.L. Chang,1 S.G. Zhao,1 B. Chandler,1 E. Olsen,1 M. Liu,1 F.Y. Feng,2 and L.J. Pierce1; 1University of Michigan, Ann Arbor, MI, 2 UCSF Department of Urology, San Francisco, CA Purpose/Objective(s): An unmet clinical need in breast cancer management includes the identification of patients (pts) at high risk to fail locally despite standard therapy and understanding the biology of these recurrences. We previously reported1a RT response signature and here extend those studies to identify a signature predictive of timing of recurrence after completion of RT (before or after 3 years). Materials/Methods: Two independent pt. cohorts were used for training and validation. The training cohort2 included 119 pts with in-breast tumor recurrence (343 pts total) and the validation cohort3 had 25 pts with recurrences (195 pts total). All received RT after breast-conserving surgery and systemic therapy as appropriate. Initial feature selection used Spearman’s rank correlation to correlate gene expression (14,806 genes) to recurrence time. Genes with sig. correlation and fold change >2 were used to train an elastic net penalized Poisson regression model. This model was locked for validation. Cox regression was used for both univariable and multivariable analyses (UVA and MVA). To identify biological-related concepts, GSEA pathway analysis was performed. Results: Spearman’s correlation identified 485 genes whose expression was sig. associated with recurrence time (early vs. late). Feature reduction further refined the gene list to 41 genes which were retained within the signature and locked for validation. In training, the correlation of score to recurrence time was 0.85; P-value Z 1.3 x 10-34, AUC of 0.91. Application of this early vs late signature to an independent BC validation set accurately identifies pts with early vs. late recurrences (Correlation Z 0.54, P-value < 0.007, AUC Z 0.74, sensitivity Z 0.71, specificity Z 1.0, PPV Z 1.0, NPV Z 0.61). PAM50 analysis demonstrates unique associations of BC intrinsic subtype to timing of local recurrence. In UVA and MVA the early vs. late recurrence signature remained the most significant factor associated with recurrence time. Although independent of intrinsic subtype, GSEA analysis of the 41 genes retained within the signature identifies proliferation and EGFR concepts associated with early recurrences and luminal and ER-signaling pathways associated with late recurrences. Knockdown of genes associated with the early and late recurrences demonstrates novel effects on proliferation and clonogenic survival (respectively). Conclusion: Here, we derive a BC-specific RT signature predictive of early vs. late recurrence with biologic relevance and subtype independence and further validate this signature for prediction of timing of recurrence in an