Radiation Sensitivity and Resistance Theodore S. Hong, MD Director, - PowerPoint PPT Presentation

Radiation Sensitivity and Resistance Theodore S. Hong, MD Director, Gastrointestinal Radiation Oncology Massachusetts General Hospital Co-Leader, Gastrointestinal Malignancies Progam Dana-Farber/Harvard Cancer Center Associate Professor of Radiation Oncology Harvard Medical School

Disclosures • Novartis- Research Funding • Taiho- Research Funding • Astra-Zeneca- Research Funding • Bristol Meyers-Squibb- Research Funding • Clinical Genomics- Advisory Board

“Radiosensitive” or “Radioresistant”

Multiple mechanisms of cell death/toxicity after radiation • Mitotic catastrophe • Stimulation of apoptosis (or autophagy, or necrosis) • Irreversible senescence • Toxic oxidative modification of biomolecules • Redox balance alterations; metabolic derangements Courtesy of Christie Eyler

Gold Standard Radiosensitivity Assay: Colony Formation Assay • Benefits: – Incorporates multi- generational cell death and clonogenicity/self- renewal – Accounts for senescence – Accounts for delayed cell death/cell arrest • Drawbacks: – Artificial, in vitro system – Time and effort investment Eyler CE, unpublished

Gold Standard Radiosensitivity Assay: Colony Formation Assay • Benefits: 0.5 Ln(Surviving Fraction) 0 – Incorporates multi- 0 2 4 6 8 10 -0.5 generational cell death -1 and clonogenicity/self- -1.5 renewal -2 – Incorporates senescence -2.5 -3 – Accounts for delayed cell -3.5 death/cell arrest -4 • Drawbacks: -4.5 Dose Radiation (Gy) – Artificial, in vitro system – Time and effort investment Eyler CE, unpublished

(Time given for one plate, usually process 10+ per experiment) Crowley, LC et al. CSHL

Short term cell viability and proliferation rates do not reflect radiosensitivity 3 Day Viability Assay Colony Formation Assay Radiation Dose (Gy) 120 1 -2 3 8 RT-Naive 100 ** Surviving Fraction Percent Surviving 80 % of 0Gy 0 RT- 60 ** % of 0Gy 2 Exposed % of 0Gy 10 40 0.1 20 ** 0 Naïve RT exposed ** 0.01 Eyler CE, unpublished

Biomarkers for Radiation Oncology Association with Biological Examples of Candidate Potential Intervention(s) Current Clinical Status Radioresistance or Parameter Biomarkers Radiosensitivity Higher baseline number of Tumor volume is a surrogate Number of Higher radiation dose or Cell surface markers such as clonogenic cells or CSCs for CSC number, (should) Clonogenic Tumor radiosensitizer for high CSC CD44 being studied correlates with impact RT dosing in clinical Cells or CSCs number radioresistance practice Accelerated repopulation of Shortening of overall HNSCC histology has been Accelerated Tumor EGFR expression being clonogenic tumor cells or treatment time limits number used as surrogate in clinical Cell Repopulation studied CSCs during RT causes of clonogenic cells that need practice to guide accelerated radioresistance to be sterilized by RT fractionation schemes. Breast or prostate histology No candidate markers Some tumors are associated Tumor Sensitivity to Hypofractionation used as surrogate in clinical currently exist to predict α / β with high sensitivity to RT RT Fraction Size (> 2 Gy daily fraction size) practice to guide hypo- fraction size (low α / β <10 Gy) of individual tumors fractionation schedules Tumor hypoxia reduces Combination of RT with PET/MRI-based imaging radiation damage to DNA, hypoxic radio-sensitizer or Not yet used in clinical Tumor Hypoxia markers, hypoxia gene practice thereby increasing dose increase to hypoxic signatures radioresistance tumor parts HPV infection causes De-intensified treatment to HPV16 DNA or p16 HPV Status Treatment de-intensification radiosensitivity, likely through reduce toxicity in HPV+ expression interfering with DNA repair HNSCC in clinical trials DSB repair gene mutations, Variations in ability of tumor Intrinsic altered expression, DNA cells to cope with radiation Treatment de-intensificaation Not yet used in clinical damage foci (eg. γ -H2AX), Radiosensitivity or intensification, respectively practice damage may cause RSI/GARD, ctDNA ,et cetera radiosensitivity or -resistance Tumor mutation status may Mutations in cancer genes correlate with radiosensitivity Treatment de-intensification Not yet used in clinical Tumor Genotype such as KRAS, BRAF, or intensification, respectively practice or -resistance through EGFR, KEAP1, NRF2 several mechanisms 9 Adapted from Kirsch et al., JNCI in press

KRAS mutation has long been known to affect cellular radioresistance - but no impact on clinical management yet! Bernhard EJ, et al. Direct Evidence for the Contribution of Activated N-ras and K-ras Oncogenes to Increased Intrinsic Radiation Resistance in Human Tumor Cell Lines. Cancer Research 2000;60:6597-6600. 10 Courtesy of Henning Willers

Emerging preclinical data on KRAS mutation- dependent pathway of radioresistance A C lo n o g e n ic S u rv iv a l Model: Non-canonical F ra c tio n a fte r IR pathway of EGFR-PKC α mediated radioresistance in KRASmut cells EGFR C B PKC α S u rv iv a l fra c tio n a fte r IR Aurora B P r o b a b i l i t y ( T C P ) ( % ) T u m o r C o n t r o l Radioresistance 11 Wang et al., Cancer Res 2014, 2017; Gurtner et al., unpublished

KRAS mutation associates with radioresistance in clinical cohorts KRAS No. of Cancer Dose End- mut vs Reference Institution genotyped Radiation type (Gy) point wt (%) pts (n) Garcia- Multi- Aguilar et Rectal Preop institutional, 132 50.4 pCR 14 vs 33 al., Ann cancer RT+5FU prospective Surg 2011 Mak et al., DFCI, retro- median 1-yr 9 NSCLC SBRT 57 vs 74 CCR 2015 spective 54 LC Cassidy et Emory, median 2-yr al., Cancer 45 NSCLC SBRT 44 vs 74 retrospective 50 LC 2017 Hong et MGH, Liver SBRT median 1-yr al., JNCI prospective 57 43 vs 72 mets protons 50 LC 2017 phase II 12

Does KRAS status clinically predict response to chemoradiation? • 132 patients – Stage II/III rectal cancer – Treated with standard chemoradiation – Evaluated multiple genes – Used Sanger sequencing – Evaluated 23 genes Garcia-Aguilar J, et al. Ann Surg 2011;254:486-493

Predictors of non-pCR Garcia-Aguilar J, et al. Ann Surg 2014

MGH- Genotypic Predictors of pCR • 47 patients • cT3-4 or N+ • Chemoradiation 50.4 Gy with 5FU • Genotyping across 15 genes evaluating 140 hotspots Russo AL, J Gastroint Canc, 2013

Genotype distribution • KRAS- 43% • APC- 17% • BRAF- 4% • NRAS- 4% • PIK3CA- 4% • TP53-4%

pCR • pCR in WT patients- 23.5% • 1 patient with a mutation had a pCR- 3.3%

KRAS Status as Predictor Biomarker after Lung SBRT Mak et al. Clin Lung Cancer, 2015.

Liver Proton SBRT for Hepatic Metastases • Hepatic metastases • 50 Gy in 5 fractions • 91 patients enrolled, 89 analyzed – 2 did not begin treatment • Median age - 67 (34-88) • Male – 56 (62.9%) Hong TS, et al. JNCI 2017.

Tumor Types Tumor Type N (%) Colorectal 36 (40.4%) Pancreatic 16 (18.0%) Esophagogastric 12 (13.5%) HCC 9 (10.1%) Lung 4 (4.5%) Gallbladder 3 (3.4%) Breast 3 (3.4%) Small bowel/duodenal 3 (3.4%) H&N 2 (2.2%) Anal cancer 1 (1.1%)

Colorectal Metastases

LC by Mutational Status

Whole Exome Sequencing Evaluation of Rectal Samples R Pre-CRT biopsy n=8 Pre- Post-CRT Surgical Analysis operative resection Sequencing resection specimen CRT NR Germline • 5FU and • After 8-11 n=9 • Whole exome RT to 50.4 weeks sequencing Gy • RNA sequencing All FFPE samples ( n=34 ) from 17 patients in our initial cohort were successfully sequenced and passed quality control metrics (out of 175 evaluated) Kamran S, ASTRO 2017

TP53/KRAS mutations in pre-CRT samples R NR Sample TP53 KRAS Sample TP53 KRAS Distribution of mutation mutation mutation mutation KRAS/TP53 RC001 p.M237I - RC002 - p.G12V co-mutations: R 1/8 (12.5%) RC004 p.R248Q p.A146V RC003 p.R175H p.G12D NR 5/9 (55.6%) RC005 p.T125T p.G13D RC007 - p.G12S RC006 p.Y107* p.G12V RC010 - - RC008 p.E287* p.L19F RC012 - p.G13D RC009 - p.G12V RC013 p.R175H - RC011 p.158H p.G12V RC014 p.V274A - RC015 p.R213* - RC017 p.G245S - RC016 p.S241fs -

Co-mutation of TP53 appears to define a particularly radioresistant subset of KRAS mutant cancers A B KRASmut radioresistant tumors C Hong et al., JNCI 2017 Wang et al., Cancer Res 2017 Kamran et al., unpublished 25

EGFR, PKC α , and Chk1 are potential targets for radiosensitization of KRAS mutant cancers A B C AZD7762 (Chk1) R a d is e n s itiz a tio n R a d is e n s itiz a tio n F a c to r (S R F 2 G y ) F a c to r (S R F 2 G y ) Rectal cancer(KRAS mut) Wang et al., Cancer Res 2014, Liu et al., Mol Cancer Res 2015, Kleiman et al., PLoS One 2013 D 26

Expression Based Testing: Radiation Sensitivity Index • Built on 10 hub genes associated with SF2 from 48 cell lines obtained from the NCI Eschrich SA, et al. IJROBP 2009

RSI • Higher score = more radiation resistant

Correlation of RSI with response

RSI • Suggests there may be a way to identify radiation resistant tumors based on expression • Validated in multiple diseases • Does not identify a targeted strategy to radiosensitize