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Supplementary File 4 shows transcriptional pathways associated with radioresponse within distinct tissue types.

Supplementary Figure from Tumor-Naïve Multimodal Profiling of Circulating Tumor DNA in Head and Neck Squamous Cell Carcinoma

Supplementary File 2 shows transcriptional pathways associated with alpha or beta from the linear-quadratic formalism.

The Supplementary Data file contains 10 supplementary figures and 3 supplementary tables.

A schematic of tumor/host combinations showing Gal-1 WT and null C57Bl6/mice subcutaneous injection with Wt and shGal-1 LLC1 cells, time line of cell injection, tumor irradiation and blood collection time points. The relative plasma Gal-1 levels for each treatment group is also shown.

Supplementary materials and methods and figure legends from Galectin-1 Mediates Radiation-Related Lymphopenia and Attenuates NSCLC Radiation Response

Gal-1 inhibition increases tumor radiation response in vitro and in vivo.

Supplementary Data from HPV Sequencing Facilitates Ultrasensitive Detection of HPV Circulating Tumor DNA

Supplementary Figure from HPV Sequencing Facilitates Ultrasensitive Detection of HPV Circulating Tumor DNA

Supplementary Table 1. Patient characteristics of 20 NSCLC patient receiving SABR. Supplementary Table 2. Patient characteristics of 24 HNSCC patients receiving radiation with cetuximab or cisplatin.

Radiotherapy is integral to the care of a majority of patients with cancer. Despite differences in tumor responses to radiation (radioresponse), dose prescriptions are not currently tailored to individual patients. Recent large-scale cancer cell line databases hold the promise of unravelling the complex molecular arrangements underlying cellular response to radiation, which is critical for novel predictive biomarker discovery. Here, we present RadioGx, a computational platform for integrative analyses of radioresponse using radiogenomic databases. We fit the dose–response data within RadioGx to the linear-quadratic model. The imputed survival across a range of dose levels (AUC) was a robust radioresponse indicator that correlated with biological processes known to underpin the cellular response to radiation. Using AUC as a metric for further investigations, we found that radiation sensitivity was significantly associated with disruptive mutations in genes related to nonhomologous end joining. Next, by simulating the effects of different oxygen levels, we identified putative genes that may influence radioresponse specifically under hypoxic conditions. Furthermore, using transcriptomic data, we found evidence for tissue-specific determinants of radioresponse, suggesting that tumor type could influence the validity of putative predictive biomarkers of radioresponse. Finally, integrating radioresponse with drug response data, we found that drug classes impacting the cytoskeleton, DNA replication, and mitosis display similar therapeutic effects to ionizing radiation on cancer cell lines. In summary, RadioGx provides a unique computational toolbox for hypothesis generation to advance preclinical research for radiation oncology and precision medicine.Significance:The RadioGx computational platform enables integrative analyses of cellular response to radiation with drug responses and genome-wide molecular data.

Supplementary Data from Tumor-Naïve Multimodal Profiling of Circulating Tumor DNA in Head and Neck Squamous Cell Carcinoma

Purpose:Circulating tumor DNA (ctDNA) enables personalized treatment strategies in oncology by providing a noninvasive source of clinical biomarkers. In patients with low ctDNA abundance, tumor-naïve methods are needed to facilitate clinical implementation. Here, using locoregionally confined head and neck squamous cell carcinoma (HNSCC) as an example, we demonstrate tumor-naïve detection of ctDNA by simultaneous profiling of mutations and methylation.Experimental Design:We conducted CAncer Personalized Profiling by deep Sequencing (CAPP-seq) and cell-free Methylated DNA ImmunoPrecipitation and high-throughput sequencing (cfMeDIP-seq) for detection of ctDNA-derived somatic mutations and aberrant methylation, respectively. We analyzed 77 plasma samples from 30 patients with stage I–IVA human papillomavirus–negative HNSCC as well as plasma samples from 20 risk-matched healthy controls. In addition, we analyzed leukocytes from patients and controls.Results:CAPP-seq identified mutations in 20 of 30 patients at frequencies similar to that of The Tumor Genome Atlas (TCGA). Differential methylation analysis of cfMeDIP-seq profiles identified 941 ctDNA-derived hypermethylated regions enriched for CpG islands and HNSCC-specific methylation patterns. Both methods demonstrated an association between ctDNA abundance and shorter fragment lengths. In addition, mutation- and methylation-based ctDNA abundance was highly correlated (r > 0.85). Patients with detectable pretreatment ctDNA by both methods demonstrated significantly worse overall survival (HR = 7.5; P = 0.025) independent of clinical stage, with lack of ctDNA clearance post-treatment strongly correlating with recurrence. We further leveraged cfMeDIP-seq profiles to validate a prognostic signature identified from TCGA samples.Conclusions:Tumor-naïve detection of ctDNA by multimodal profiling may facilitate biomarker discovery and clinical use in low ctDNA abundance applications.

Tumor irradiation effect on T lymphocytes, common lymphoid progenitor cells (CLPs) and Gal-1 expression in bone marrow cells.

Supplementary File 1 shows transcriptional pathways associated with the radioresponse indicators SF2 or AUC.

Radiation-related lymphopenia is associated with worse distant, local and regional progression free survival in NSCLC, in similar between radiation and cetuximab or cisplatin treated HNSCC and its severity trends with relapse in HNSCC. A higher rise in plasma Gal-1 was also seen with relapse.

Purpose: Radiotherapy can result in lymphopenia, which has been linked to poorer survival. Here, we test the hypothesis that radiotherapy-induced lymphopenia is mediated by a tumor-secreted factor, Galectin-1 (Gal-1), which possesses T-cell proapoptotic activities.Experimental Design: Matched Gal-1 wild-type (WT) or null mice were implanted with Lewis lung carcinoma (LLC-1) that either expressed Gal-1 or had Gal-1 stably downregulated. Tumors were irradiated locally and circulating Gal-1 and T cells were measured. Tumor growth, lung metastasis, intratumoral T-cell apoptosis, and microvessel density count were quantified. Thiodigalactoside (TDG), a Gal-1 inhibitor, was used to inhibit Gal-1 function in another group of mice to validate the observations noted with Gal-1 downregulation. Lymphocyte counts, survival, and plasma Gal-1 were analyzed in cohorts of radiotherapy-treated lung [non–small cell lung cancer (NSCLC)] and head and neck cancer patients.Results: LLC irradiation increased Gal-1 secretion and decreased circulating T cells in mice, regardless of host Gal-1 expression. Inhibition of tumor Gal-1 with either shRNA or thiodigalactoside ablated radiotherapy-induced lymphopenia. Irradiated shGal-1 tumors showed significantly less intratumoral CD8+ T-cell apoptosis and microvessel density, which led to marked tumor growth delay and reduced lung metastasis compared with controls. Similar observations were made after thiodigalactoside treatment. Radiotherapy-induced lymphopenia was associated with poorer overall survival in patients with NSCLC treated with hypofractionated radiotherapy. Plasma Gal-1 increased whereas T-cell decreased after radiation in another group of patients.Conclusions: Radiotherapy-related systemic lymphopenia appeared to be mediated by radiotherapy-induced tumor Gal-1 secretion that could lead to tumor progression through intratumoral immune suppression and enhanced angiogenesis. Clin Cancer Res; 20(21); 5558–69. ©2014 AACR.

Purpose:Human papillomavirus (HPV) DNA offers a convenient circulating tumor DNA (ctDNA) marker for HPV-associated malignancies, but current methods, such as digital PCR (dPCR), provide insufficient accuracy for clinical applications in patients with low disease burden. We asked whether a next-generation sequencing approach, HPV sequencing (HPV-seq), could provide quantitative and qualitative assessment of HPV ctDNA in low disease burden settings.Experimental Design:We conducted preclinical technical validation studies on HPV-seq and applied it retrospectively to a prospective multicenter cohort of patients with locally advanced cervix cancer (NCT02388698) and a cohort of patients with oropharynx cancer. HPV-seq results were compared with dPCR. The primary outcome was progression-free survival (PFS) according to end-of-treatment HPV ctDNA detectability.Results:HPV-seq achieved reproducible detection of HPV DNA at levels less than 0.6 copies in cell line data. HPV-seq and dPCR results for patients were highly correlated (R2 = 0.95, P = 1.9 × 10–29) with HPV-seq detecting ctDNA at levels down to 0.03 copies/mL plasma in dPCR-negative posttreatment samples. Detectable HPV ctDNA at end-of-treatment was associated with inferior PFS with 100% sensitivity and 67% specificity for recurrence. Accurate HPV genotyping was successful from 100% of pretreatment samples. HPV ctDNA fragment sizes were consistently shorter than non–cancer-derived cell-free DNA (cfDNA) fragments, and stereotyped cfDNA fragmentomic patterns were observed across HPV genomes.Conclusions:HPV-seq is a quantitative method for ctDNA detection that outperforms dPCR and reveals qualitative information about ctDNA. Our findings in this proof-of-principle study could have implications for treatment monitoring of disease burden in HPV-related cancers. Future prospective studies are needed to confirm that patients with undetectable HPV ctDNA following chemoradiotherapy have exceptionally high cure rates.

Increases in gal-1 secretion after tumor cell irradiation in vitro.

Lymphocytes from Gal-1 WT and null mice exhibiting similar sensitivity to ionizing radiation and recombinant Gal-1.

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PDF file - 52K, Clinical information for BCA patient-derived xenografts.

PDF file - 581K, Breast TICs are enriched in the CD24HiCD49fHi subpopulation (S1); Expression of HER receptors and ligands in breast cancer cell lines (S2); HER2 inhibition reduces the EpCAM- TIC-like subpopulation of SUM149 cells (S3); Breast TICs are enriched in the EPCAM+CD49f+ subpopulation of a triple-negative patient-derived xenograft breast tumor BCA70 (S4); Dual inhibition of HER2 and EGFR targets breast TICs in patient-derived xenograft BCA71 (S5).

Introduction: Clinical benefit from Immune Checkpoint Blockade (ICB) is a function of local T-cell specificity for tumor-associated antigens. However, overcoming local T-cell dysfunction necessitates systemic immunity engagement. Therefore, studying the dynamics of both local and peripheral T-cell repertoires in response to ICB is required to identify features of T-cell repertoires associated with pathological response. Methods: We conducted TCRβ-sequencing on tumor-residing T-cells (n=59), Peripheral Blood Mononuclear Cells (PBMCs) (n=306) and cell-free DNA (cfDNA) (n=73) from pre- and multiple on-ICB timepoints collected from patients enrolled in the pan-cancer INvestigator-initiated Phase II Study of Pembrolizumab Immunological Response Evaluation (INSPIRE; NCT02644369) trial. To assess specificity-agnostic shifts in TCR repertoires, we first compared TCR diversity and clonal expansion in longitudinal tumor and PBMC samples. Then, to temporally track the specificity-associated features of local and systemic TCR repertoires, we leveraged a Graph Neural Network (GNN) model that took in unique TCRβ chains as nodes. The connectivity between the nodes was defined by multi-relational edges that represented VJ-gene usage and GLIPHII-identified (Grouping Lymphocyte Interactions by Paratope Hotspots) specificities derived from a compendium of TCR sequences with empirically confirmed specificities. Results: While absolute diversity and clonal expansion values in baseline tumor (n=33) were not associated with response to ICB, changes in these values were informative between pre- and on-ICB tumors. All patients (n=4) with low baseline tumor TCR diversity and lack of clonotypic re-structuring in tumor TCR repertoire on-ICB had either progressive or short-term stable disease. Furthermore, pairwise comparison of pre- and on-ICB tumors for each patient (n=17) revealed that all the patients, irrespective of their pathological response, experienced emergence of new TCR clonotypes (i.e., clonal replacement) in response to ICB, suggesting only a minority of these TCRs might consist of tumor-associated clonotypes. Patients with clinical benefit also had higher degree of GLIPHII-identified clustering at baseline tumor, highlighting the role of both specificity-agnostic and specificity-centric TCR analysis in determining the response to ICB. Analysis of TCR sequences in blood plasma found cfDNA contains a small number of TCR sequences (median 32, range 12-89) enriched for TCRs found in matched tumor tissues, suggesting that cfDNA TCR repertoire may provide an indirect measurement of tumor-residing T-cells. Conclusions: TCR diversity and functional clonal annotation are emerging biomarkers of ICB response and cfDNA TCR repertoire can potentially be exploited for clinical diagnostics and monitoring. Citation Format: Shirin Soleimani, Ben X. Wang, Stephanie Pedersen, Jenna Eagles, Jacob Brick, Marcus O. Butler, Scott V. Bratman, Lillian L. Siu, Pamela S. Ohashi, Trevor J. Pugh. Pan-cancer assessment of tumour and peripheral T-cell receptor repertoire dynamics in patients treated with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6667.

Research objectives: To conduct a prospective observational study of patient and tumor-related factors in women with high risk hormone receptor (HR)+/HER2- breast cancer (BC) following at least 5 years of adjuvant hormonal therapy, in order to identify risk factors for imminent recurrence. Rationale: Many of the life-threatening BC recurrences in women with HR+HER2- BC take place more than 5 years post-diagnosis, often after completion of adjuvant hormonal therapy. The identification of a biomarker(s) for late BC recurrence could lead to interventional trials to evaluate preventive therapies. We will evaluate whether the presence of blood-based biomarkers [(i) Circulating Tumor Cells (CTCs), (ii) circulating tumor DNA (ctDNA), (iii) tumor markers (CA 15-3, CEA)] and patient factors may predict BC recurrence. Trial design: A prospective cohort of eligible women with previously treated HR+HER2- BC who have not experienced a distant recurrence will be enrolled; patient and circulating factors will be measured annually until distant recurrence or study completion. Host factors (including BMI, lifestyle, medical illness, surgery, trauma and stress, as well as circulating PlGF, VEGF-1 and inflammatory markers) that may contribute to exit of BC cells from dormancy will also be assessed. The primary outcome is distant BC recurrence. Any BC event, including loco-regional recurrence, new breast or other primary cancer will be evaluated as a secondary endpoint. Outcomes will be ascertained by regular self-report (via annual telephone calls) and/or physician report and confirmed by medical record review. Key eligibility criteria: i) Diagnosis of ER and/or PR positive (either or both 10% positive), HER2 negative invasive BC, ii) predicted >1.5-2% annual risk of recurrence (T2, T3 or T4 with any N+;T1 N2+; T2N0 or T1 N1 cancers with high risk genomic scores), iii) receipt of adjuvant endocrine therapy for at least 4 years, with discontinuation planned in the next 12 months or completion of endocrine therapy within the last 5 years, iv) prior adjuvant chemotherapy, targeted therapy and bone targeted therapies are allowed provided they have been completed. Specific aims: 1) Determine if the presence of (i) CTCs, (ii) ctDNA, (iii) CA15-3 and CEA are associated with imminent risk (within 1-2 years) of distant recurrence in the study population. 2) Identify host factors associated with these blood-based biomarkers, as well as clinical outcomes. Statistical methods: A matched case control design (matching for time since completion of adjuvant hormone therapy, baseline T, N and grade) will be used to investigate associations of key study variables with imminent risk of distant recurrence within the next 1-2 years. Measurements of patients who do versus do not recur will be compared over the 1-2 years prior to relapse. Each variable will be allocated one third of a study-wide type one error of 0.05 (2-sided). ROC analyses and multivariable modelling will be used to optimize sensitivity, specificity, PPV and NPV. Available questionnaire data will be summarized at all time-points to generate descriptive survivorship data. Accrual: Starting in August 2018, we plan to recruit 1,000 patients over 2 years at selected Canadian cancer centres. Citation Format: Jerzak KJ, Cescon DW, Chia SK, Bratman S, Ennis M, Stambolic V, Chang M, Dowling R, Goodwin PJ. Exploration of factors associated with imminent risk of late recurrence in hormone receptor positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT1-12-01.

Introduction: Small cell lung cancer (SCLC) is a deadly disease and patients often suffer from recurrent disease. Biologic mechanisms of recurrence are unclear. Epigenetic mechanisms, like DNA methylation, may be operant. SCLC is rarely resected; therefore, the SCLC methylome is understudied due to scarce tumor tissue. Methods: In this study, we examined the pre-treatment methylome of 72 SCLC patients at our institution through cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq) on plasma cell-free DNA (cfDNA) and on sheared genomic DNA from paired peripheral blood leukocytes (PBLs) (n = 72) to increase tumor signal specificity. cfMeDIP-seq was also performed on an independent cohort of healthy non-cancer controls (n = 20) and on tumors from circulating tumor cell-derived xenograft (CDX) models (n = 12 CDXs of 72 patients). cfMeDIP-seq libraries were sequenced at a depth of 100 million reads, paired-end, on the NovaSeq 6000. For all bioinformatic analyses, chromosomes 1-22 were binned into 300-bp windows (n = 9.6e6 genome-wide windows); reads from cfMeDIP-seq were tallied per bin. To filter out noise from non-tumor cells, ENCODE blacklist regions (n = 1e6 windows) were removed and CG-rich (>5 CGs per window), PBL-associated windows with MeDEStrand-converted beta-values < 0.2 were kept (n = 190,769 windows). Subsequent cfDNA analyses were performed using these 190,769 windows. Results: 33 (45.8%) and 39 (54.2%) of the 72 patients had limited-stage (LS) and extensive-stage (ES) SCLC, respectively. Most were current or former smokers (65/72, 90.3%). Using filtered windows (n = 190,769), methylated cfDNA from SCLC were distinguished from healthy controls: SCLC cfDNA was enriched for hypermethylated CpG islands and shore regions, whereas controls had more methylated open-sea regions. SCLC cohort by consensus clustering of the top 5000 most variant windows revealed two distinct cfDNA methylation patterns. Cluster A (9 ES, 22 LS) and Cluster B (33 ES, 11 LS) were significantly different by stage (X2(1) = 13.79, p < 0.001). There was no significant difference in sex (p = 0.81). Patients in Cluster A had a lower concentration of cfDNA (median = 7.2ng/ml) compared to Cluster B (median = 15.9ng/ml) but not statistically significant (p = 0.33). High concordance between genome-wide methylome profiles was found between paired patient cfDNA and CDX tumor, demonstrating that cfDNA is representative of tumor methylation (Pearson's correlation median r = 0.86). Conclusion: We identified stage-specific methylation patterns in the plasma of SCLC patients using the cfMeDIP-seq assay that may reveal novel epigenetic and biologic mechanisms of SCLC disease progression. Moreover, CDXs recapitulate the methylome of SCLC cell-free patient samples highlighting their utility in future work as representative models for methylome analysis. Citation Format: Sami Ul Haq, Sabine Schmid, Mansi K. Aparnathi, Katrina Hueniken, Luna J. Zhan, Danielle Sacdalan, Janice J.N. Li, Devalben Patel, Dangxiao Cheng, Vivek Philip, Geoffrey Liu, Scott V. Bratman, Benjamin H. Lok. Identification of small cell lung cancer stage-specific DNA methylation in patients using liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3396.

Introduction: Durvalumab immunotherapy has rapidly emerged as standard treatment for stage 3 NSCLC patients following definitive chemoradiotherapy (CRT). Multiple novel immunotherapeutic strategies are in development to enhance the chance of cure in this setting as well. There exists a critical need to identify blood-based biomarkers capable of predicting clinical benefit from adjuvant immunotherapy as well selecting patients at high-risk of relapse for further drug development. Cell-free DNA (cfDNA)-based analysis of both minimal residual disease (MRD) and T-cell receptor (TCR) clonality have immense potential to predict and monitor response to adjuvant immunotherapy. In this study, we have combined innovative cfDNA measures of MRD (CAPPseq), TCR clonality (CapTCR-seq) and methylation (cfMeDIPseq) as potential predictive biomarkers of disease progression in stage 3 NSCLC patients treated with CRT and durvalumab. Methods: Stage 3 NSCLC patients undergoing CRT and durvalumab were recruited prospectively to undergo serial blood collections at baseline, pre- and post- durvalumab. CAPPseq and cfMeDIPseq were performed as measures of MRD. TCR repertoire analysis (CapTCR-seq) was performed on cfDNA using hybrid-capture TCR sequencing and TCR diversity/clonality was estimated using the Shannon's index. Correlations between MRD, TCR clonality, response and progression-free survival (PFS) were examined using logistic/cox regression. Results: 79 stage 3 NSCLC patients have been prospectively recruited and undergone serial blood collection. CAPPseq, cfMeDIPseq and capTCR-seq have been completed in 22 patients (5 primary progression on CRT, 17 received durvalumab). Tumor cfDNA was detectable by CAPPseq at baseline in 14 patients. High correlation between tumor cfDNA detected by CAPPseq and cfMeDIPseq was found (R=0.68, p Conclusions: Failure to clear MRD and decreasing TCR clonality as assessed by cfDNA was highly correlated with increased risk of recurrence and reduced PFS with consolidation durvalumab. This innovative approach has significant potential to define a new biomarker for the use and development of adjuvant immunotherapy. Citation Format: Sally CM Lau, Shirin Soleimani, Jinfeng Zou, Justin Burgener, Shelley Kuang, Stephanie WY Wong, Malcolm Ryan, Ben X. Wang, Stephanie Pedersen, Devalben Patel, Penelope A. Bradbury, Geoffrey Liu, Natasha Leighl, Ming S. Tsao, Pamela S. Ohashi, Scott V. Bratman, Trevor Pugh, Frances A. Shepherd, Adrian G. Sacher. cfDNA-based analysis of minimal residual disease and T-cell receptor clonality as predictors of relapse in stage 3 NSCLC treated with chemoradiotherapy and durvalumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 563.

Cancer survival rates are significantly improved when detected at early stages, particularly when the tumor is still localized to the tissue of origin. However, effective screening tools for early cancer detection is currently limited to a subset of cancer types. The early development of human malignancies are difficult to observe as cancers are often detected once it becomes symptomatic, as such many cancer biomarker and evolution studies to date have primarily examined the genomics from solid tumor or liquid biopsies following a diagnosis. Investigating early tumor evolution in the pre-diagnosis context could allow us to better understand how to prevent or detect cancers in the earliest stage when survival rates are significantly higher, however this requires application of new technologies to biologics collected prior to a cancer diagnosis. Here, we leverage blood samples collected from participants in the Canadian Partnership for Tomorrow Project (CPTP), a longitudinal population cohort, prior to the onset of a cancer. Specifically, we utilize hybrid capture approaches to enrich for and characterize early mutations and methylation changes in circulating tumor DNA (ctDNA) of pre-cancer plasma samples collected from patients several months to years prior to clinical diagnosis. Here, we identify the earliest detectability of aberrant genetic and epigenetic events in ctDNA and describe the molecular evolution of these events at various stages prior to clinical detection of cancers. Further, we develop molecular biomarkers and implement machine learning tools to classify individuals with early cancers, and to develop risk scores from survival analyses predictive of cancer development up to 5 years prior to diagnosis. In our current study, we focus specifically on breast, prostate, lung and pancreatic cancer cases, and are extending this to pan-cancer applications in subsequent studies. Citation Format: Nicholas Cheng, Kimberly Skead, David Soave, Jocelyn Meng, Elias Gbeha, Ilinca Lungu, Bernard Lam, Scott Bratman, Daniel De Carvalho, Philip Awadalla. Leveraging cell-free methylome markers for early cancer detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2602.

Background: Radiotherapy is a mainstay of curative treatment for squamous cell carcinomas (SCC) and is frequently combined with radiosensitizing drugs to improve efficacy. Outcomes for HPV-negative SCCs remain heterogeneous, in part due to variable resistance to ionizing radiation (IR) and modest benefit of existing radiosensitizing drugs. Hypothesis driven testing of novel radiosensitizing drugs has previously been limited due to a lack of robust, efficient, and reproducible methods to assess drug radiation combinations. Aims: To improve the throughput of preclinical studies on radiosensitizers, we have developed an in vitro system for assessing drug/radiation combinations, validated using the known radiosensitizing chemotherapeutic drug cisplatin. We extended the assay to assess the effect of ML-385, a NRF2 inhibitor that increases cellular reactive oxygen species (ROS), and abemaciclib and palbociclib, two CDK4/6 inhibitors predicted to radiosensitize cancer cells by inhibiting entry into more resistant phases of the cell cycle. Experimental Methods: We adapted the 9-day viability assay of Abazeed et al. (Cancer Res., 2013) to enable drug/radiation efficacy assessments. We first validated the assay’s ability to recapitulate clonogenic survival among 19 HPV-negative SCC cell lines. Drug/radiation experiments were performed in the 6 most radioresistant SCC cell lines. ROS levels after 4Gy IR was used to confirm NRF2 suppression. Flow cytometry was used to evaluate cell cycle phase. AKT inhibitor GSK690693 was used in combination with a NRF2 shRNA knockdown cell line to investigate the mechanism of sensitivity. Western blot was used to detect p16. Cellular response to multiple IR doses was summarized using an area-under-the-curve (AUC) metric. The delta AUC across drug doses was used to evaluate radiosensitization. Results: Among the 19 cell lines, AUCs with the clonogenic and 9-day viability assays were strongly correlated (Pearson r=0.74, p=3.00 × 10–4). Six (32%) of the cell lines were reproducibly radioresistant (AUC >3.5) using both assays. All of these cell lines saw sensitization with cisplatin. None of these 6 cell lines harboured mutations in the canonical NRF2 pathway, whereas all 6 harboured either CCND1 amplification, CDKN2A mutation, or both. Only one cell line showed radiosensitization with the NRF2 inhibitor ML-385, an effect that was abrogated by PIK3CA or AKT pathway inhibition. In contrast, 5 of the 6 cell lines showed reproducible radiosensitization following CDK4/6 inhibition; with robust p16 expression detected in the lone unaffected cell line. Conclusion: The 9-day viability assay allows efficient evaluation of putative radiosensitizers. Our tests of putative radiosensitizing drugs in a cohort of radioresistant cell lines have identified CDK4/6 inhibitors to be of interest for further investigation in biomarker-selected populations. Citation Format: Meghan Lambie, Rita Gill, Laurie Ailles, Scott V. Bratman. Selective radiosensitization in preclinical models of HPV-negative squamous cell carcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-086.

While radiation therapy is an integral method for cancer treatment, clinical choices are not currently informed by the genetic and molecular profile of a patient’s tumor. Though it has been shown that genetic features implicate variability, the exact relationship between these features and radiosensitivity is poorly understood. Our work focuses on predicting radiosensitivity of squamous cell carcinoma from molecular features. First, we employ RadioGx, a computational platform for integrative analysis of radiogenomic datasets, to identify a gene expression signature predictive of radiosensitivity. To mitigate the high-dimensionality of gene expression data, we employ pathway-level transcriptomic modeling methods to identify cellular programs associated with radiation sensitivity. Our pancancer radiosensitivity model achieves a Pearson’s correlation of 0.47 with measured radiosensitivity from a radiogenomic dataset using only high level transcriptomic features. Second, we use that gene expression signature to query the Connectivity Map via the PharmacoGx resource and identify candidate radiosensitizers - compounds that may induce the radiosensitivity signature. Further validation is necessary to evaluate the efficacy of these compounds at improving radiosensitivity in both model systems and patient contexts. These results implicate cellular processes in radiosensitivity and advance an approach for identifying radiosensitizing agents through integration of large scale datasets. Citation Format: Ian Smith, Rachel Bell, Meghan Lambie, Benjamin Haibe-Kains, Scott Bratman. Characterizing transcriptomic indicators of radiosensitivity in cancer and identifying sensitizing therapeutic agents [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-071.

Radiomics involves the use of high-dimensional quantitative imaging features for predictive purposes. However, studies showed that these radiomic features are sensitive to the variability of imaging parameters (e.g., scanner model). One of the major challenges in radiomics lies in improving the robustness of quantitative features against the variation in the imaging dataset in multi-center studies. Here, we assess the impact of scanner choice on the computed tomography (CT)-derived radiomic features to predict association of oropharyngeal squamous cell carcinoma with human papillomavirus (HPV), which has a well-established impact on CT-derived radiomic features. This experiment was performed on CT image datasets acquired with two different scanner types. We demonstrate strong scanner dependency by developing a machine learning model to classify HPV status from radiological images. These experiments revealed the effect of scanner type on the robustness of the radiomic features, and the extent of this dependency is reflected on the performance of HPV prediction models. The result of this study highlighted the importance of implementing an appropriate approach to reduce the impact of the imaging domain radiomic features and consequently on the machine learning models. Citation Format: Reza Reiazi, Collin Arrowsmith, Farnoosh Abbas-Aghababazadeh, Christopher Eles, Aria Rezaie, Scott V. Bratman, Andrew J. Hope, Benjamin Haibe-Kains. The impact of the variation of CT scanner on the prediction of HPV status in head & neck cancer patients [abstract]. In: Proceedings of the AACR Virtual Special Conference on Artificial Intelligence, Diagnosis, and Imaging; 2021 Jan 13-14. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(5_Suppl):Abstract nr PO-033.

Osteoradionecrosis (ORN), or bone death, is a multifactorial late complication caused by radiotherapy (RT), that diminishes the bone’s ability to withstand trauma and avoid infection. In head and neck cancer (HNC), ORN most commonly manifests in the mandible. It is influenced by RT-induced factors and patient-related parameters. The most effective way to limit RT-induced ORN is to reduce mandibular volumes receiving high RT doses. However, this strategy may also result in a reduced dose to the tumor due to its proximity to the mandible, and therefore must be restricted to only the patients deemed most vulnerable to mandibular ORN. The objective of this study was to design a prognostic model based on RT-planning CT-derived radiomic features extracted from mandible contours along with the patient’s clinical features to predict the probability of mandibular ORN from the end of RT to the onset of ORN in HNC patients. We hypothesized that these features are related to mandibular ORN and that incorporating them into a prediction model will help to identify patients at risk of mandibular ORN after HNC RT. Patient data was retrospectively collected from the Princess Margaret Cancer Centre, University Health Network and based on the following inclusion criteria: patients had (1) had status regarding radiation-induced bone toxicity, (2) the time to the toxicity event was recorded, and (3) head RT-planning CT images in addition to mandible contours. Then, the patient’s history was reviewed by radiation oncologists to collect clinical features. Quantitative image features were then extracted from the segmented mandible for each patient. Finally, multivariable models, a binary classifier, and a regressor were independently trained on three sets of features (radiomic, demo-clinical, and both) to predict the patient’s risk of ORN and the time between the end of RT and the start of ORN respectively. In total, we analyzed CT images from 92 OPC patients with known ORN status (55 positive, 37 negative). We extracted a total of 1877 radiomic features from the manually-segmented Mandible from each patient. Initially, cases were labelled with their status regarding presence and time to radiation toxicity. Top 50, most relevant and least redundant, features were with mRMRe were used for both binary classification and regression models to predict time to the mandibular ORN. Model training upon radiomics and clinical features resulted in the higher accuracy value of 0.93 (AUCROC) compared to the only radiomics features (0.92) and clinical model (0.74). The distribution of AUC values was significantly wider in models trained on clinical features than radiomics or radiomics plus clinical features. Citation Format: Reza Reiazi, Mattea Welch, Scott V. Bratman, Andrew J. Hope, Benjamin Haibe-Kains. The prediction of mandibular osteoradionecrosis in head and neck cancer patients using CT-derived radiomics features [abstract]. In: Proceedings of the AACR Virtual Special Conference on Artificial Intelligence, Diagnosis, and Imaging; 2021 Jan 13-14. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(5_Suppl):Abstract nr PO-034.

Head and neck squamous cell carcinoma (HNSCC) comprises 3% of all cancer cases worldwide. Despite intensive multimodal therapies, HNSCC patient outcomes remain heterogeneous with minimal improvements in survival. Utilization of fluid-based biomarkers for prognostication, risk stratification, and disease surveillance may improve patient outcomes by enabling more effective treatment decisions. Here, we describe the performance of comprehensive mutation and methylome analysis for highly sensitive detection of circulating tumor (ct)DNA in HNSCC plasma. To detect HNSCC-specific mutations and aberrant methylation in ctDNA, we conducted CAPP-Seq (CAncer Personalized Profiling by deep Sequencing) and cfMeDIP-seq (cell-free Methylated DNA ImmunoPrecipitation sequencing), respectively. For CAPP-Seq, we developed a HNSCC-specific hybrid capture panel and applied it to plasma DNA and peripheral blood leukocyte (PBL) genomic DNA from a cohort of HNSCC patients (n=32) and healthy controls (n=20). Single-nucleotide variants (SNVs) were identified by integrated digital error suppression (iDES). For cfMeDIP-seq, hypermethylated differentially methylated regions within regions of low PBL methylation were identified by DESeq2. A median of 3 mutations (range: 1-10) were detected by CAPP-Seq within plasma DNA of 20/32 (62.5%) HNSCC patients. Mean mutant allele frequency ranged from 0.1–5% (median: 0.92%) and correlated with tumor stage. Among these 20 patients, cfMeDIP-seq identified 860 DMRs that were enriched (hypermethylated) in HNSCC plasma DNA compared with healthy controls. These hypermethylated DMRs (hyper-DMRs) were over-represented by CpG islands and gene promoters and showed significant overlap with HNSCC-specific methylated regions in TCGA. When applied to all 32 HNSCC patients, hyper-DMR abundance was positively correlated with mutation-based ctDNA abundance (R=0.87; p=1e−16). Hyper-DMRs were capable of accurate discrimination of HNSCC patients and healthy controls (AUC=0.85). The median DNA fragment size within these hyperDMRs was lower in HNSCC patients compared to healthy controls—a characteristic of ctDNA described in previous studies—and correlated with both hyperDMR-based and mutation-based ctDNA abundance. We have conducted the first comparative analysis of genetic and epigenetic profiling approaches for ctDNA detection in HNSCC. Both CAPP-Seq and cfMeDIP-seq have the potential to detect ctDNA in patient plasma without prior knowledge of patient-specific tumor aberrations. With CAPP-seq, ctDNA was detectable at levels as low as 0.1%. Plasma methylome profiling using cfMeDIP-seq revealed hyper-DMRs with tumor-related features and that correlated with mutation-based ctDNA abundance. Future analysis will validate patient-specific mutations and methylation signals in tumor tissue and in longitudinally collected samples from this cohort. This abstract is also being presented as Poster B47. Citation Format: Justin M. Burgener, Jinfeng Zou, Zhen Zhao, Shu Y. Shen, Daniel D. De Carvalho, Scott V. Bratman. Comprehensive detection of ctDNA in localized head and neck cancer by genome- and methylome-based analysis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR13.

Background: Human papillomavirus (HPV)-associated cancers often present with locoregionally confined disease and are treated with curative intent. An emerging treatment paradigm includes radical therapy (i.e., chemoradiotherapy or surgery) followed by adjuvant treatment. Accurate detection of minimal residual disease (MRD) following radical therapy could enable personalized use of adjuvant treatment. The HPV genome offers a convenient circulating tumor (ct)DNA marker for HPV-associated cancers, but current methods such as digital (d)PCR provide insufficient accuracy for accurate MRD detection and for other clinical applications in patients with low disease burden. We asked whether a next-generation sequencing approach (HPV-seq) could provide quantitative and qualitative assessment of HPV ctDNA in low-disease-burden settings. Methods: We conducted preclinical technical validation studies on HPV-seq using cervix cancer cell lines. We then applied HPV-seq retrospectively to a prospective multicenter cohort study of locally advanced cervix cancer patients accrued from 2015 to 2016 (NCT02388698). Patients were treated with radical chemoradiotherapy with blood obtained at baseline, end of treatment, and 3 months post-treatment. Median follow-up was 27.5 months. In 38 plasma samples from 17 patients, HPV-seq results were compared with dPCR. The primary outcome was progression-free survival according to end-of-treatment HPV ctDNA detectability. Results: HPV-seq achieved reproducible detection of HPV DNA at levels Conclusions: HPV-seq is a quantitative method for ctDNA detection that outperforms dPCR. HPV-seq also reveals qualitative information about ctDNA fragments such as HPV genotype and ctDNA fragment length distribution. Our findings will have implications for MRD detection in HPV-related cancers. Future prospective studies are needed to confirm that patients with undetectable HPV ctDNA following chemoradiotherapy have exceptionally high cure rates. Citation Format: Eric Leung, Kathy Han, Jinfeng Zou, Zhen Zhao, Yangqiao Zheng, Ting Ting Wang, Lillian L. Siu, Trevor J. Pugh, Scott V. Bratman. HPV sequencing facilitates ultrasensitive detection of HPV circulating tumor DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A45.

Purpose: Rapid release of circulating tumor DNA (ctDNA) during cytotoxic therapy could provide an early indication of treatment efficacy. However, the biologic basis for treatment-induced ctDNA release is poorly understood. To study this phenomenon, we evaluated ctDNA release in head and neck squamous cell carcinoma (HNSCC) treated with radiotherapy (RT) or chemoradiotherapy (CRT). We sought to characterize the kinetics and biologic underpinnings of this process. Methods: 33 HPV+ HNSCC patients treated with RT (n=17) or CRT (n=16) had blood collected at baseline, after the 4th fraction of RT (early-RT), mid-RT, and 3 months post-RT. ctDNA was quantified using digital PCR for HPV-16. Nine HNSCC cell lines (3 HPV+, 6 HPV-) were used for in vitro ctDNA kinetic analysis. Apoptosis was measured by luminescent DEVD cleavage assay, triggered by staurosporine and blocked by z-vad-fmk. Senescence was measured by SA-β-galactosidase activity, triggered by HrasG12V-transduction of fibroblast cell lines and blocked by navitoclax. Three HNSCC cell lines (2 HPV+, 1 HPV-) were grown as xenografts in NRG mice for in vivo analysis. Endpoint xenograft tumors were assessed for necrotic area and for markers of apoptosis (cleaved-caspase 3) and senescence (p21) by immunohistochemistry. ctDNA was quantified from media or mouse plasma by quantitative PCR using human-specific LINE-1 primers. Results: Of 30 patients with detectable HPV-16 ctDNA (90.9%), we observed variable kinetic patterns of release. We grouped patients into three categories: pattern 1, peak at early RT (12, 40%); pattern 2, peak at mid RT (7, 23.3%); pattern 3, no peak (11, 36.7%). Based on these findings, we characterized the mechanisms dictating RT-induced ctDNA release in preclinical models of HNSCC. Following RT, HNSCC cell lines exhibited variable kinetics of ctDNA release in vitro and in vivo with peak release observed after 72-96 hours. RT-induced ctDNA release was not associated with intrinsic radiosensitivity, HPV status, or propensity to undergo apoptosis. Although caspase inhibition resulted in a near-complete reduction in RT-induced caspase activity (-84%±8%), a comparatively minor reduction in ctDNA release (-29%±9%) was observed. There was an inverse association between RT-induced senescence and ctDNA release. HrasG12V-triggered senescence reduced ctDNA release, while navitoclax treatment reversed this effect. HNSCC models with greater RT-induced ctDNA release displayed more necrosis and less senescence in endpoint xenograft tumors. Conclusion: Dynamic changes in HPV-16 ctDNA are observed during treatment, with variable patterns of release. Preclinical HNSCC models demonstrate RT-induced spikes within 3-4 days of treatment, revealing a complex interplay between distinct mechanisms of cell death and ctDNA release. Necrosis and senescence are the major determinants of RT-induced ctDNA release, while apoptosis has a comparatively minor impact. This study will inform future work on implementing ctDNA as a response biomarker. Citation Format: Ariana Rostami, John N. Waldron, Scott V. Bratman. Biologic rationale for radiation-induced release of circulating tumor DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A59.

Purpose: Radiotherapy can result in lymphopenia, which has been linked to poorer survival. Here, we test the hypothesis that radiotherapy-induced lymphopenia is mediated by a tumor-secreted factor, Galectin-1 (Gal-1), which possesses T-cell proapoptotic activities. Experimental Design: Matched Gal-1 wild-type (WT) or null mice were implanted with Lewis lung carcinoma (LLC-1) that either expressed Gal-1 or had Gal-1 stably downregulated. Tumors were irradiated locally and circulating Gal-1 and T cells were measured. Tumor growth, lung metastasis, intratumoral T-cell apoptosis, and microvessel density count were quantified. Thiodigalactoside (TDG), a Gal-1 inhibitor, was used to inhibit Gal-1 function in another group of mice to validate the observations noted with Gal-1 downregulation. Lymphocyte counts, survival, and plasma Gal-1 were analyzed in cohorts of radiotherapy-treated lung [non–small cell lung cancer (NSCLC)] and head and neck cancer patients. Results: LLC irradiation increased Gal-1 secretion and decreased circulating T cells in mice, regardless of host Gal-1 expression. Inhibition of tumor Gal-1 with either shRNA or thiodigalactoside ablated radiotherapy-induced lymphopenia. Irradiated shGal-1 tumors showed significantly less intratumoral CD8+ T-cell apoptosis and microvessel density, which led to marked tumor growth delay and reduced lung metastasis compared with controls. Similar observations were made after thiodigalactoside treatment. Radiotherapy-induced lymphopenia was associated with poorer overall survival in patients with NSCLC treated with hypofractionated radiotherapy. Plasma Gal-1 increased whereas T-cell decreased after radiation in another group of patients. Conclusions: Radiotherapy-related systemic lymphopenia appeared to be mediated by radiotherapy-induced tumor Gal-1 secretion that could lead to tumor progression through intratumoral immune suppression and enhanced angiogenesis. Clin Cancer Res; 20(21); 5558–69. ©2014 AACR.

Currently, only patients with HER2-positive tumors are candidates for HER2-targeted therapies. However, recent clinical observations suggest that the survival of patients with HER2-low breast cancers, who lack HER2 amplification, may benefit from adjuvant therapy that targets HER2. In this study, we explored a mechanism through which these benefits may be obtained. Prompted by the hypothesis that HER2/HER3 signaling in breast tumor-initiating cells (TIC) promotes self-renewal and survival, we obtained evidence that neuregulin 1 (NRG1) produced by TICs promotes their proliferation and self-renewal in HER2-low tumors, including in triple-negative breast tumors. Pharmacologic inhibition of EGFR, HER2, or both receptors reduced breast TIC survival and self-renewal in vitro and in vivo and increased TIC sensitivity to ionizing radiation. Through a tissue microarray analysis, we found that NRG1 expression and associated HER2 activation occurred in a subset of HER2-low breast cancers. Our results offer an explanation for why HER2 inhibition blocks the growth of HER2-low breast tumors. Moreover, they argue that dual inhibition of EGFR and HER2 may offer a useful therapeutic strategy to target TICs in these tumors. In generating a mechanistic rationale to apply HER2-targeting therapies in patients with HER2-low tumors, this work shows why these therapies could benefit a considerably larger number of patients with breast cancer than they currently reach. Cancer Res; 74(1); 341–52. ©2013 AACR.

Purpose: Radiotherapy (RT) is the foundation of curative treatment regimens for many cancer types and is often delivered with drugs to produce synergistic effects. The “precision medicine” paradigm in which genomic biomarkers guide therapeutic decisions has eluded RT. To support preclinical discovery of biomarker-directed RT dose and drug combinations, we built a computational platform for integrating and interrogating radiogenomics data sets. Methods: RadioGx integrates in vitro cancer cell line survival data with multimodal molecular profiles from the NCI-60 and Cancer Cell Line Encyclopedia (CCLE) cell line panels. We fit published radiation response data from both clonogenic and high-throughput viability assays to established radiobiologic models of clonogenic survival. We assessed the concordance of radiation response profiles across different survival endpoints (i.e., surviving fraction at 2Gy [SF2] and Area Under the Curve of the radiobiologic model [AUC]). By comparing radiation response with transcriptomic data, we examined indicators of radiosensitivity at the pathway level. We integrated drug response data from Cancer Therapeutics Response Portal (CTRP v2) using an interface between RadioGx and our previously published pharmacogenomics platform, PharmacoGx. Using the oxygen modification factor in established radiobiologic models, we identified pathways that are enriched under hypoxic conditions. Results: RadioGx includes 600 radiation dose response data, 511 gene expression data sets, and 504 drug dose response data for chemotherapeutic compounds. Goodness-of-fit (given by R2 value) of the linear-quadratic radiobiologic model ranged from 0.927 to 0.99 (median=0.998, n=57) for the clonogenic assay and from 0.043 to 0.99 (median=0.952, n=535) for the viability assay. The concordance between SF2 and AUC using the clonogenic assay and the viability assay was 0.79 (n=12) and 0.82 (n=15), respectively. Among cell lines with survival data from both assays (n=28) across two different studies, the concordance of SF2 and AUC was 0.68 and 0.62, respectively. By interrogating the 504 cell lines that were profiled using the viability assay, we found drugs that behaved similar to RT were enriched in three pharmacologic classes, namely, drugs that impact mitosis, DNA replication, or cytoskeleton. Pathway analysis of radiation response under hypoxia revealed two key DNA repair pathways, namely, DNA double-strand break repair by NHEJ and DNA damage-induced 14-3-3, to be enriched compared to normoxic conditions. Conclusions: Our work is a major step towards analyzing preclinical models of radiation response using in vitro survival and transcriptomic data. RadioGx coupled with PharmacoGx lays the foundation for the identification of synergistic drug-RT combinations, which can further push the boundaries of translational research towards precision radiation medicine. Citation Format: Venkata S. Manem, Meghan Lambie, Petr Smirnov, Mark Freeman, Victor Kofia, Mohamed E. Abazeed, Scott V. Bratman, Benjamin Haibe-Kains. Therapeutic effects of radiotherapy on cancer cell lines using RadioGx computational platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3211.

Purpose: High-dose radiotherapy (RT) is a standard treatment for locally advanced head and neck squamous cell carcinoma (HNSCC). Despite known molecular prognostic biomarkers in HNSCC, such as the presence of human papillomavirus (HPV) within tumor tissues, RT regimens remain one-size-fits-all without any patient-specific individualization. We hypothesize that rapid release of circulating tumor DNA (ctDNA) can act as a biomarker of treatment response in HNSCC by reflecting tumor cell death in response to RT. Methods: Four HNSCC cell lines, two HPV- (Cal33, FaDu) and two HPV+ (HMS-001, 93-Vu147T), were evaluated for ctDNA release following single dose RT in vitro. Quantification of ctDNA was performed by quantitative polymerase chain reaction (qPCR) with primers amplifying the human long interspersed nuclear element 1 (hLINE1). RT-induced apoptosis was measured using a luminometric caspase 3/7 assay, and RT-induced senescence was evaluated using a fluorometric senescence associated β-galactocidase (SA-β-Gal) assay. The pan-caspase inhibitor, z-vad-fmk, was used to block RT-induced apoptosis. Subcutaneous cell-line xenografts were established in Nod-Scid-Gamma (NSG) mice, where plasma from serial blood draws was purified and quantified for ctDNA release using hLINE1 qPCR. Endpoint tumors were evaluated for mechanisms of cell death by histological staining. Results: HNSCC cell lines exhibited variable magnitude and timing of ctDNA release and apoptosis. Maximal ctDNA release occurred between 72 and 144 hours post-RT. The release of ctDNA was not correlated with HPV status nor with apoptosis occurring following RT. To further interrogate the contribution of apoptosis to ctDNA release, we treated cells with a pan-caspase inhibitor and evaluated ctDNA release and caspase activity following RT. Although caspase inhibition resulted in a near complete reduction in RT-induced caspase activity (84.0% ±8.1%), a comparatively minor reduction in ctDNA release (28.9%±8.9%) was observed. To evaluate the impact of senescence on RT-induced ctDNA release, we measured SA-β-Gal activity post-RT. The degree of senescence following RT was inversely associated with ctDNA release. In cell line xenografts, maximal ctDNA release into mouse plasma occurred 96 hours following RT. Staining patterns of endpoint tumors for caspase-3, TUNEL, p21, and Ki67 are currently under analysis. Conclusions: Our results demonstrate a robust and sensitive method for longitudinal evaluation of ctDNA release and cell death post-RT both in vitro and in vivo. Timing of ctDNA release demonstrate peaks around 72-96 hours post-RT, highlighting the potential for ctDNA to depict early response to therapy. Ongoing studies aimed at further investigating ctDNA release in association with mechanisms of cell death in vivo will help further elucidate the potential for ctDNA as a biomarker of treatment response. Citation Format: Ariana Rostami, Caberry Yu, Marco A. Di Grappa, Scott V. Bratman. Rapid release of ctDNA as a biomarker of treatment response in preclinical models of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 583.

Overall outcomes for HPV-negative head and neck squamous cell carcinoma (HNSCC) remain poor with 5-year overall survival rates of 50-60%. Oral squamous cell carcinoma (OSCC), the most common subtype of HPV-negative HNSCC, is typically treated with surgery, and clinico-pathologic features are used to identify patients in need of adjuvant therapies such as radiation therapy (RT), or radiation plus concurrent chemotherapy (CRT). It is clear from the rate of loco-regional or distant failures that more accurate methods of risk stratification would greatly improve outcomes for OSCC patients. This requires biomarkers to identify patients that will benefit from adjuvant RT or CRT but currently there are no validated molecular biomarkers that have been clinically implemented for the personalized treatment of OSCC. In addition to biomarkers for better risk stratification, there is also a need for novel therapeutic strategies leading to improved outcomes. Recently patient-derived xenografts (PDXs) have been shown to faithfully recapitulate human tumor biology and predict drug responses, supporting their relevance as preclinical models for new drug development. Upon subcutaneous implantation of HNSCC specimens into NOD/SCID/IL2Rγ-/- mice, 161 of 243 samples (66%) successfully formed patient-derived xenografts (PDX). Using univariable and multivariable analyses, the ability to form a PDX correlated significantly with adverse clinical outcomes, and specifically, patients with palpable PDX-formation within 8 weeks experienced particularly poor outcomes (hazard ratio for overall survival = 3.0). A cohort of engrafting and non-engrafting samples were sequenced using a targeted sequencing panel designed for both mutational and copy number alteration detection. The overall frequency of somatic genomic alterations detected was similar to The Cancer Genome Atlas cohort and interestingly, successful engraftment correlated to amplification of the CCND1 gene. Twelve PDX models were treated with the CDK4/6 inhibitor, abemaciclib; 5 of 6 models with CCND1 amplifications and/or CDKN2A mutations responded to abemaciclib treatment, whereas only 1 of 6 models lacking these alterations responded. These results demonstrate the potential of using PDX models to identify novel targeted therapies for HNSCC patients who have the poorest outcomes. In the future, PDX avatars could also be exploited to individualize treatment for patients at high risk of relapse following definitive treatment. Citation Format: Christina Karamboulas, Jeffrey P. Bruce, Andrew J. Hope, Jalna Meens, Shao Hui Huang, Jie Su, Fei-Fei Liu, Trevor J. Pugh, Scott V. Bratman, Wei Xu, Laurie E. Ailles. Patient-derived xenografts for prognostication and personalized treatment for head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1033.

Purpose: Radiotherapy (RT) is frequently used either alone or in combination with chemotherapy for curative treatment of squamous cell carcinomas (SCC) originating from the head and neck, lung, esophagus, and cervix. Treatment outcomes for SCCs are heterogeneous, in part due to variable degrees of resistance to ionizing radiation and modest benefit of existing radiosensitizing drugs. Preclinical research into intrinsic cancer cell radiosensitivity biomarkers and novel radiosensitizing drugs has been hampered by a lack of robust high throughput assays of radiation response. We sought to validate a recently published high throughput viability assay as a surrogate of clonogenic survival and extend its use to drug-radiation combinations. Experimental Methods: Clonogenic assays—the gold standard for assessing radiation survival in vitro—and viability assays were performed on 19 SCC cell lines (16 head and neck, 3 esophageal). The clonogenic assay was performed according to published procedures (Franken et al. Nat. Protoc, 2006) with colonies counted using ImageJ. The viability assay was adapted from Abazeed et al. (Cancer Res., 2013). Cells were seeded with a range of densities in 96 well plates then treated with a range of radiation doses (0, 2, 4, and 8 Gy); viability was measured using CellTitre Glo reagent after 9 days. Agreement between clonogenic and viability assays was measured using modified concordance index (C-index) and Pearson correlation. For the addition of drug, cells were allowed to adhere to plates for 2 hours before drug administration. Cells were irradiated 16 hours later again at a range of doses (0, 2, 4, and 6 Gy for drug analysis), and then quantified as above. All assays were performed with technical and biological triplicates. Results: There was a high degree of agreement between the area under the curve with the clonogenic assay and the viability assay (C-index = 0.80, p = 3.03x10-7; Pearson r = 0.70, p = 1.10x10–3). Significant concordance was also seen at 2 Gy and 4 Gy dose points (Pearson r = 0.62 and 0.73 respectively), however not at 8 Gy (Pearson r Conclusion: The proliferative assay recapitulates the clonogenic assay within our cohort of SCC cell lines. Novel higher throughput methods to analyze radiation response will allow for a more efficient measure of drug and radiation combinations. Citation Format: Meghan Lambie, Venkata S. Manem, Benjamin Haibe-Kains, Scott V. Bratman. Validation of a viability assay for assessing radiation response and investigating drug/radiation combinations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 854.

Introduction: Breast cancer patients undergoing adjuvant radiation therapy (RT) commonly experience fatigue and insomnia. We previously reported that this fatigue is associated with a reduction in circulating hematopoetic stem cells (HSCs; CD34+); the role of inflammatory cytokines in mediating this process however, has not been clearly elucidated. Materials and Methods: Breast cancer patients (n=147) undergoing adjuvant RT underwent phlebotomies for analysis of CD34+, CD45+, CBC, as well as 17 inflammatory cytokines using a multiplexed ELISA platform, during 5 time points: prior to RT (D1); after Days 2, 5, during final week of RT (D2, D5, and Df, respectively); as well as one month post RT-completion (M1). At the same time, patients also completed questionnaires for the multidimensional fatigue inventory (MFI-20), hospital anxiety and depression scale (HADS), and insomnia severity index (ISI). Results: General fatigue worsened over the course of RT from D1 to Df, being most severe at the end of treatment (Df), but returned to baseline at M1. This trend was statistically significant (p Conclusions: This study represents one of the most comprehensive longitudinal evaluations of the effects of RT on fatigue and insomnia, demonstrating that this process was associated with increased levels of the pro-inflammatory cytokines MCP-1, TNF-RII and TNF-a, and reductions in circulating HSCs and other hematologic parameters. Further understanding of the roles of these cytokines would provide important insights into both quality of life for patients undergoing cancer therapies, as well as the interactions between RT with immunotherapy. Citation Format: Wei Shi, Kathy Han, Madeline Li, Justin Williams, Megan McCusker, Jie Su, Wei Xu, Scott Bratman, Kenneth Yip, Fei-Fei Liu. Inflammatory cytokines and hematopoietic stem cells are associated with fatigue and insomnia in breast cancer patients undergoing adjuvant radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4752. doi:10.1158/1538-7445.AM2017-4752

Molecular profiles of tumors and tumor-associated cells hold great promise as biomarkers of clinical outcomes. However, existing datasets are fragmented and difficult to analyze systematically. We present a pan-cancer resource and comprehensive meta-analysis of expression signatures from ~18,000 human tumors with overall survival outcomes across 39 malignancies. While a third of prognostic genes were cancer-specific, a FOXM1 regulatory network is a major predictor of adverse outcomes, while favorably prognostic genes largely reflect tumor-associated leukocytes. Using a novel computational approach, we enumerated leukocyte subsets in bulk tumor transcriptomes, revealing complex novel malignancy-specific associations between 22 distinct leukocytes and cancer survival. Tumor-associated neutrophil-like polymorphonuclear cell and plasmacytic cells emerged as significant but opposite predictors of survival for diverse solid tumors, including breast and lung adenocarcinomas. Our results introduce new analytical tools for delineating prognostic genes and leukocytes within and across cancers, and shed light on the impact of tumour heterogeneity on cancer outcomes, with applications for discovering novel biomarkers and therapeutic targets Citation Format: Andrew J. Gentles, Aaron M. Newman, Chih Long Liu, Scott V. Bratman, Weiguo Feng, Dongkyoon Kim, Viswam S. Nair, Xu Yue, Amanda Khuong, Chuong D. Hoang, Maximilian Diehn, Robert B. West, Sylvia K. Plevritis, Ash A. Alizadeh. The prognostic landscape of genes and infiltrating immune cells across human cancers. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr PR09.

Decreased circulating lymphocytes after tumor irradiation has been linked to worse outcomes in several cancers treated with fractionated radiotherapy. The mechanism underlying radiation (RT)-induced lymphopenia is not well understood. Here, we show that lymphopenia also occurs after stereotactic ablative radiotherapy for early stage non-small cell lung cancer, and that it is associated with reduced survival. We provide evidence that RT-induced secretion of Galectin-1 (Gal-1), a b-galactoside binding protein, can potentially explain for this phenomenon. Using matched Gal-1 wild type and null mice together with Lewis lung carcinoma cells stably knocked-down of Gal-1 or scramble control, we show that irradiation of Gal-1 expressing tumor increases Gal-1 secretion in the blood and that it is linked to decreased circulating T-lymphocytes. This was ablated by either genetic down regulation of Gal-1 in the tumor or inhibition of Gal-1 activity by TDG. Moreover, Gal-1 down regulation resulted in enhanced radiation sensitivity in vitro as well as less intratumoral T cell apoptosis and angiogenesis in vivo, resulting in marked tumor growth delay and reduced spontaneous lung metastases when combined with radiotherapy. Similar results were noted when Gal-1 function was inhibited with TDG in vivo. Together, our data identify Gal-1 as an important mediator of RT-related lymphopenia and its proangiogenic and T cell proapototic effect may explain for the poorer outcome associated with RT-related lymphopenia. Gal-1 is therefore a new potential therapeutic target to combine with radiotherapy. Citation Format: Peiwen Kuo, Scott Bratman, David Shultz, Rie von Eyben, Cato Chan, Ziwei Wang, Carmen Say, Aparna Gupta, Bill W. Loo, Amato Giaccia, Albert Koong, Maximilian Diehn, Quynh-Thu Le. Galectin-1 mediates radiation-related lymphopenia in non-small cell lung cancer and attenuates tumor radiation response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 848. doi:10.1158/1538-7445.AM2014-848