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Supplementary figure legends

Supplemental Figure 1. RPPA heat map of H460 cells treated with varying doses of radiation and ganetespib. Supplemental Figure 2. RPPA heat map of A549 cells treated with varying doses of radiation and ganetespib. Supplemental Figure 3. Western blot validation of some of the altered proteins seen on RPPA analysis in H460 cells. Supplemental Figure 4. The apoptosis indicated by Caspase cleavage in RPPA and western blot were confirmed by flow cytometry Annexin V analysis. Supplemental Figure 5. H460 xenograft recapitulated in vitro DNA damage and apoptosis results.

Purpose: Evidence suggests that PD-L1 can be induced with radiotherapy and may be an immune escape mechanism in cancer. Monitoring this response is limited, as repetitive biopsies during therapy are impractical, dangerous, and miss tumor stromal cells. Monitoring PD-L1 expression in both circulating tumor cells (CTCs) and circulating stromal cells (CStCs) in blood-based biopsies might be a practical alternative for sequential, noninvasive assessment of changes in tumor and stromal cells.Experimental Design: Peripheral blood was collected before and after radiotherapy from 41 patients with lung cancer, as were primary biopsies. We evaluated the expression of PD-L1 and formation of RAD50 foci in CTCs and a CStC subtype, cancer-associated macrophage-like cells (CAMLs), in response to DNA damage caused by radiotherapy at the tumor site.Results: Only 24% of primary biopsies had sufficient tissue for PD-L1 testing, tested with IHC clones 22c3 and 28-8. A CTC or CAML was detectable in 93% and 100% of samples, prior to and after radiotherapy, respectively. RAD50 foci significantly increased in CTCs (>7×, P < 0.001) and CAMLs (>10×, P = 0.001) after radiotherapy, confirming their origin from the radiated site. PD-L1 expression increased overall, 1.6× in CTCs (P = 0.021) and 1.8× in CAMLs (P = 0.004): however, individual patient PD-L1 expression varied, consistently low/negative (51%), consistently high (17%), or induced (31%).Conclusions: These data suggest that RAD50 foci formation in CTCs and CAMLs may be used to track cells subjected to radiation occurring at primary tumors, and following PD-L1 expression in circulating cells may be used as a surrogate for tracking adaptive changes in immunotherapeutic targets. Clin Cancer Res; 23(19); 5948–58. ©2017 AACR.

Supplementary Figure 1. Individual and expanded images of DAPI, Cytokeratin and CD45 from Figure 2. Supplementary Figure 2. Determining the thresholds for scoring PD-L1 expression in circulating cells. Supplementary Figure 3. Average PD-L1 and RAD50 changes in each individual patient before and after induction of radiotherapy, separated by cell type. Supplementary Figure 4. Confocal imaging of RAD50 within the nuclei of a cluster of EMTCTCs from Figure 1, imaged top to bottom. Supplementary Figure 5. Distribution of RAD50 foci in cells by stage and before treatment or after treatment. Supplementary Figure 6. Percent change in pixel intensity from the T0 time point to the T1 time point. Supplementary Table 1. Patient population overview Supplementary Table 2. Tracking the longevity of PD-L1 expression levels after completion of radiotherapy.

Purpose: HSP90 inhibition is well known to sensitize cancer cells to radiation. However, it is currently unknown whether additional radiosensitization could occur in the more clinically relevant setting of chemoradiation (CRT). We used the potent HSP90 inhibitor ganetespib to determine whether it can enhance CRT effects in NSCLC.Experimental Design: We first performed in vitro experiments in various NSCLC cell lines combining radiation with or without ganetespib. Some of these experiments included clonogenic survival assay, DNA damage repair, and cell-cycle analysis, and reverse-phase protein array. We then determined whether chemotherapy affected ganetespib radiosensitization by adding carboplatin–paclitaxel to some of the in vitro and in vivo xenograft experiments.Results: Ganetespib significantly reduced radiation clonogenic survival in a number of lung cancer cell lines, and attenuated DNA damage repair with irradiation. Radiation caused G2–M arrest that was greatly accentuated by ganetespib. Ganetespib with radiation also dose-dependently upregulated p21 and downregulated pRb levels that were not apparent with either drug or radiation alone. However, when carboplatin–paclitaxel was added, ganetespib was only able to radiosensitize some cell lines but not others. This variable in vitro CRT effect was confirmed in vivo using xenograft models.Conclusions: Ganetespib was able to potently sensitize a number of NSCLC cell lines to radiation but has variable effects when added to platinum-based doublet CRT. For optimal clinical translation, our data emphasize the importance of preclinical testing of drugs in the context of clinically relevant therapy combinations. Clin Cancer Res; 22(23); 5876–86. ©2016 AACR.

Purpose: The MEK inhibitor trametinib radiosensitizes KRAS-mutant non–small cell lung cancer (NSCLC) and is being tested clinically with chemoradiation. However, variability in response to trametinib suggests that additional pathways are involved. The mechanism of resistance to trametinib radiosensitization is still unknown.Experimental Design: We used a panel of KRAS-mutant NSCLC cells and tested the radiosensitization effects of trametinib by clonogenic survival assay. Then, we investigated the mechanisms underlying the resistance to the combination therapy through several knockout and overexpression systems. Finally, we validated our findings in syngeneic mouse models in a treatment setting that mimicked the standard of care in the clinic.Results: Radiosensitization by trametinib was effective only in KRAS-LKB1–mutated cells with wild-type (WT) p53, and we found that restoring LKB1 expression in those cells blocked that sensitization. Trametinib and radiotherapy both induced senescence in a p53-dependent manner, but in WT LKB1 cells, the combination also activated the AMPK-autophagy pathway to rescue damaged cells from senescence. LKB1-knockout or autophagy inhibition in WT LKB1 cells potentiated trametinib radiosensitization. In syngeneic animal models of Kras-mutant lung tumors, Lkb1-knockout tumors were resistant to trametinib and chemoradiation given separately, but the combination greatly controlled tumor growth and prolonged survival.Conclusions: The LKB1 mutation in KRAS-mutant NSCLC conferred enhanced radiosensitization in combination with trametinib. The WT LKB1 could activate autophagy through AMPK pathway to induce resistance to the combination of trametinib and radiation. The KRAS-LKB1 mutation could potentially be a biomarker to select patients for trametinib and radiotherapy combination therapy. Clin Cancer Res; 24(22); 5744–56. ©2018 AACR.

Figure Legends for supplemental figure 1-5

Oligos for pLentiCRISPRv2 construction

We report radiatively coupled arrayed gold nanodisks on invisible substrate (AGNIS) as a cost-effective, high-performance platform for nanoplasmonic biosensing. By substrate undercut, the electric field distribution around the nanodisks has been restored to as if the nanodisks were surrounded by a single medium, thereby provides analyte accessibility to otherwise buried enhanced electric field. The AGNIS substrate has been fabricated by wafer-scale nanosphere lithography without the need for costly lithography. The LSPR blue-shifting behavior synergistically contributed by radiative coupling and substrate undercut have been investigated for the first time, which culminates in a remarkable refractive index sensitivity increase from 207 nm/RIU to 578 nm/RIU. The synergy also improves surface sensitivity to monolayer neutravidin-biotin binding from 7.4 nm to 20.3 nm with the limit of detection (LOD) of neutravidin at 50 fM, which is among the best label-free results reported to date on this specific surface binding reaction. As a potential cancer diagnostic application, extracellular vesicles such as exosomes excreted by cancer and normal cells were measured with a LOD within 112–600 (exosomes/ $\mu \text{L}$ ), which would be sufficient in many clinical applications. Using CD9, CD63, and CD81 antibodies, label-free profiling has shown increased expression of all three surface antigens in cancer-derived exosomes. This work demonstrates, for the first time, strong synergy of arrayed radiative coupling and substrate undercut can enable economical, ultrasensitive biosensing in the visible light spectrum where high-quality, low-cost silicon detectors are readily available for point-of-care applications.

BACKGROUND: Various methods of liquid biopsy through the sampling of blood in cancer patients allow access to minuscule amounts of tumor that can easily be sampled repeatedly throughout therapy. Circulating tumor cells (CTCs) represent shed tumor cells that can be characterized by imaging or molecular techniques using an amenable enrichment platform. Here we validate the Hitachi Chemical Micro Cavity Array (MCA) for the enrichment of CTCs from the blood of patients diagnosed with stage III non-small cell lung cancer (NSCLC). MCA is a semi-automated filtration system that enriches CTCs on the basis of size and membrane deformability rather than a biased selection of surface antigens. METHODS: CTCs were enriched from the peripheral blood of 38 patients diagnosed with stage III NSCLC at the start of chemoradiation. Two tubes of EDTA blood were collected from each patient and processed through MCA in parallel. In the first tube, CTCs were identified as pan-cytokeratin (CK)+ CD45− nucleated cells and enumerated. The second tube was depleted of leukocytes using CD45 antibody-coated magnetic microbeads before enrichment by MCA, followed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) to interrogate CTC-enriched lysates for expression of 16 target mRNAs from a panel of epithelial, mesenchymal, stem-like, and cancer signaling-related genes. CTC-enriched lysates from similarly prepared peripheral blood samples from 18 healthy donors were used to define positive gene expression. RESULTS: CTCs were identified by imaging in 30 of 38 patient samples (79%). At least 1 target gene was positively expressed in 23 of 25 (92%) patient samples that was subjected to molecular characterization. A CTC count of ≥7 was associated with poor progression-free survival (PFS) [hazard ratio (HR) 4.24, 95% confidence interval (CI), 1.73–10.40, P=0.020] and poor overall survival (HR 8.17, 95% CI, 2.87–23.26, P

Purpose: Cancer extracellular vesicles (EVs) are involved in cellular communication, tumor growth, progression, and metastasis in cancer. The origins of EVs, their formation, and potential clinical use as biomarkers are not well understood. Recently, budding of extracellular structures on Cancer Associated Macrophage-Like Cells [CAMLs] a specific subtype of phagocytic circulating stromal cells has been observed in metastatic non-small cell lung carcinoma (mNSCLC) patients. In this prospective analysis of n=40 mNSCLC samples, we enumerated EV budding on CAMLs to determine if their formation had an effect on clinical outcomes. These preliminary results suggest that EV budding from a specific subtype of circulating tumor associated macrophage prognosticates for worse clinical outcome which may serve as the mechanism for cancer EV formation and spread throughout the body. Patients and Methods: We initiated a single blind prospective pilot study to evaluate extracellular budding on the CAMLs of mNSCLC patients from blood samples obtained prior to therapy to determine their prevalence and clinical utility. Anonymized blood was procured and filtered to isolate CAMLs and stained for cytokeratin, CD45, CD31 and PD-L1. EV budding was observed as small (≤1 µm) bulbous protrusions from the cell periphery. EVs were quantified and compared against patient progression free survival (PFS) and overall survival (OS) with hazard ratios (HRs) at 24 months by censored univariate analysis. The imaged EVs were also characterized by their PD-L1 biomarker expression. Results: CAMLs were identified in 88% (n=35/40) of all samples, with EV budding identified in 60% (n=21/35) of CAMLs. These EVs appeared with tumor positive proteins (i.e. CD31, CD45, PD-L1 and cytokeratin). With a minimum of 24 months of follow-up, it was determined that the presence of EV budding in patients’ CAMLs was associated with significantly worse PFS (HR=4.00, 95%CI=1.4-12, p=0.0251) and borderline significant OS (HR=3.57, 95%CI=1.1-12, p=0.0747). Conclusions: EV budding found on phagocytic stromal cells found in the blood appear with tumor positive biomarkers and predict poor survival. These findings suggest that CAMLs are an origin cell for some cancer EVs. Larger validation studies and cross comparison of PD-L1 on EVs as it related to immunotherapy response is ongoing. Citation Format: Jillian A. Moran, Steven H. Lin, Martin J. Edelman, Pablo Lopez, Jianzhong He, Yawei Qiao, Ting Xu, Zhongxing Liao, Kirby P. Gardner, Cha-Mei Tang, Daniel L. Adams. Extracellular vesicles budding from stromal macrophages in the blood of metastatic non-small cell lung cancer patients correlates with poor survival [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 3310.

Circulating tumor DNA (ctDNA) molecular residual disease (MRD) following curative-intent treatment strongly predicts recurrence in multiple tumor types, but whether further treatment can improve outcomes in patients with MRD remains unclear. We applied CAPP-Seq ctDNA analysis to 218 samples from 65 patients receiving chemoradiation therapy (CRT) for locally advanced NSCLC, including 28 patients receiving consolidation immune checkpoint inhibition (CICI). Patients with undetectable ctDNA after CRT had excellent outcomes whether or not they received CICI. Among such patients, one died from CICI-related pneumonitis, highlighting the potential utility of only treating patients with MRD. In contrast, patients with MRD after CRT who received CICI had significantly better outcomes than patients who did not receive CICI. Furthermore, the ctDNA response pattern early during CICI identified patients responding to consolidation therapy. Our results suggest that CICI improves outcomes for NSCLC patients with MRD and that ctDNA analysis may facilitate personalization of consolidation therapy.

Purpose The combination of cytotoxic chemotherapy with radiation therapy (CRT) has resulted in significant improvements in clinical outcomes for patients with many locally advanced unresectable cancers. Only a small proportion of patients achieve pathologic complete responses to CRT; combination of CRT with targeted agents offers the promise of further improving treatment responses. However, numerous clinical trials have failed to show an improvement in clinical outcomes with the addition of targeted agents. To increase the accessibility of our screening method and accelerate the pace at which novel combinations with CRT are identified and incorporated into standard practices for treatments, we report details on screening method optimization, data generation, and downstream data analysis. Methods In part, the gap in translation to large, expensive, and ultimately unsuccessful clinical trials reflects the shortcomings of inconsistently designed, executed, and reported preclinical data on which these studies are based. In an effort to standardize the selection of agents for future clinical testing, we have designed, optimized and validated a high throughput, high content, clonogenic assay platform for step-wise progression of preclinical studies from in vitro to in vivo in non-small cell lung cancer and pancreatic ductal adenocarcinoma. Results This highly stable in vitro method was standardized for identification of the most promising CTEP drugs that could best be combined with CRT from among as screen of multiple agents tested in an unbiased manner using 96-well plates. The methodology lends itself to seamless testing of multiple agents in a similar fashion allowing cross-comparisons, evaluation of CRT, or radiation therapy alone, and testing multiple concentrations of test agents sequenced at different times before and after radiation. The method identified Trametinib as a strong CRT sensitizer in KRAS-mutant non-small cell lung cancer and pancreatic ductal adenocarcinoma cell lines. This platform has enabled the screening and identification of several chemoradiation sensitizers.Conclusions: High throughput, high content clonogenic drug screening assay allows for the rapid identification of targets and agents to be translated to the clinic to help improve the effectiveness of current standard of care CRT in various solid tumors.

Chemoradiation (CRT) is widely used to treat locally advanced non-small cell lung cancer (NSCLC) patients, but only around 30% of patients achieve pathological complete response. Here, we utilized an in vitro high-content clonogenic survival screening method to identify CRT sensitizers from the NCI Cancer Therapy Evaluation Program (CTEP) portfolio. Talazoparib (PARPi) was identified among the top candidates to show potent CRT sensitization effects in both KRAS mutant and TP53 mutant NCSLC cell lines. We validated the CRT sensitization effect of PARPi on lung tumors in vivo with cell line xenografts mouse models. To further investigate alterations in the tumor microenvironment (TME) induced by PARPi, we utilized syngeneic mouse models and showed that the combination of radiotherapy, PARPi, and PD-L1 blockade achieved superior tumor control effects. By applying single cell RNA sequencing technologies to the mouse tumors, we found that PARPi combined with CRT or immunotherapy significantly reprogrammed the tumor cells and TME, resulting in the increase of tumor-infiltrating T cells which elicit potent tumor control effects. Our data showed that PARPi sensitizes NSCLC tumors to CRT and immunotherapy through a combination of tumor-intrinsic and extrinsic phenomenon. Citation Format: Rui Ye, Kaile Wang, Yawei Qiao, Zhenna Xiao, Yun Yan, Jianzhong He, Nan Li, Yifan Wang, Min Hu, Shanshan Bai, Emi Sei, Nicholas Navin, Steven Lin. Chemoradiation sensitization effect of PARPi in non-small cell lung cancer [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 3308.

Background Cancer Associated Macrophage-Like cells (CAMLs) are polynucleated circulating stromal cells found in the bloodstream of numerous solid-tumor malignancies. Variations within CAML size have been associated with poorer progression free survival (PFS) and overall survival (OS) in a variety of cancers; however, no study has evaluated their clinical significance in esophageal cancer (EC). Methods To examine this significance, we ran a 2 year prospective pilot study consisting of newly diagnosed stage I-III EC patients (n = 32) receiving chemoradiotherapy (CRT). CAML sizes were sequentially monitored prior to CRT (BL), ~ 2 weeks into treatment (T1), and at the first available sample after the completion of CRT (T2). Results We found CAMLs in 88% (n = 28/32) of all patient samples throughout the trial, with a sensitivity of 76% (n = 22/29) in pre-treatment screening samples. Improved 2 year PFS and OS was found in patients with CAMLs p = 0.004) and OS (HR = 9.0, 95%CI = 1.9–43.5, p = 0.019). Conclusions Tracking CAML sizes throughout CRT as a minimally invasive biomarker may serve as a prognostic tool in mapping EC progression, and further studies are warranted to determine if presence of these cells prior to treatment suggest diagnostic value for at-risk populations.

Purpose: The MEK inhibitor trametinib radiosensitizes KRAS-mutant non–small cell lung cancer (NSCLC) and is being tested clinically with chemoradiation. However, variability in response to trametinib suggests that additional pathways are involved. The mechanism of resistance to trametinib radiosensitization is still unknown.Experimental Design: We used a panel of KRAS-mutant NSCLC cells and tested the radiosensitization effects of trametinib by clonogenic survival assay. Then, we investigated the mechanisms underlying the resistance to the combination therapy through several knockout and overexpression systems. Finally, we validated our findings in syngeneic mouse models in a treatment setting that mimicked the standard of care in the clinic.Results: Radiosensitization by trametinib was effective only in KRAS-LKB1–mutated cells with wild-type (WT) p53, and we found that restoring LKB1 expression in those cells blocked that sensitization. Trametinib and radiotherapy both induced senescence in a p53-dependent manner, but in WT LKB1 cells, the combination also activated the AMPK-autophagy pathway to rescue damaged cells from senescence. LKB1-knockout or autophagy inhibition in WT LKB1 cells potentiated trametinib radiosensitization. In syngeneic animal models of Kras-mutant lung tumors, Lkb1-knockout tumors were resistant to trametinib and chemoradiation given separately, but the combination greatly controlled tumor growth and prolonged survival.Conclusions: The LKB1 mutation in KRAS-mutant NSCLC conferred enhanced radiosensitization in combination with trametinib. The WT LKB1 could activate autophagy through AMPK pathway to induce resistance to the combination of trametinib and radiation. The KRAS-LKB1 mutation could potentially be a biomarker to select patients for trametinib and radiotherapy combination therapy. Clin Cancer Res; 24(22); 5744–56. ©2018 AACR.

Microdroplet single-cell ATAC-seq is widely used to measure chromatin accessibility, however, highly scalable and simple sample multiplexing procedures are not available. Here, we present a transposome-assisted single nucleus barcoding approach for ATAC-seq (SNuBar-ATAC) that utilizes a single oligonucleotide adaptor for multiplexing samples during the existing tagmentation step and does not require a pre-labeling procedure. The accuracy and scalability of SNuBar-ATAC was evaluated using cell line mixture experiments. We applied SNuBar-ATAC to investigate treatment-induced chromatin accessibility dynamics by multiplexing 28 mice with lung tumors that received different combinations of chemo, radiation, and targeted immunotherapy. We also applied SNuBar-ATAC to study spatial epigenetic heterogeneity by multiplexing 32 regions from a human breast tissue. Additionally, we show that SNuBar can multiplex single cell ATAC and RNA multiomic assays in cell lines and human breast tissue samples. Our data show that SNuBar is a highly accurate, easy-to-use, and scalable system for multiplexing scATAC-seq and scATAC and RNA co-assay experiments.

Purpose: Evidence suggests that PD-L1 can be induced with radiotherapy and may be an immune escape mechanism in cancer. Monitoring this response is limited, as repetitive biopsies during therapy are impractical, dangerous, and miss tumor stromal cells. Monitoring PD-L1 expression in both circulating tumor cells (CTCs) and circulating stromal cells (CStCs) in blood-based biopsies might be a practical alternative for sequential, noninvasive assessment of changes in tumor and stromal cells. Experimental Design: Peripheral blood was collected before and after radiotherapy from 41 patients with lung cancer, as were primary biopsies. We evaluated the expression of PD-L1 and formation of RAD50 foci in CTCs and a CStC subtype, cancer-associated macrophage-like cells (CAMLs), in response to DNA damage caused by radiotherapy at the tumor site. Results: Only 24% of primary biopsies had sufficient tissue for PD-L1 testing, tested with IHC clones 22c3 and 28-8. A CTC or CAML was detectable in 93% and 100% of samples, prior to and after radiotherapy, respectively. RAD50 foci significantly increased in CTCs (>7×, P < 0.001) and CAMLs (>10×, P = 0.001) after radiotherapy, confirming their origin from the radiated site. PD-L1 expression increased overall, 1.6× in CTCs (P = 0.021) and 1.8× in CAMLs (P = 0.004): however, individual patient PD-L1 expression varied, consistently low/negative (51%), consistently high (17%), or induced (31%). Conclusions: These data suggest that RAD50 foci formation in CTCs and CAMLs may be used to track cells subjected to radiation occurring at primary tumors, and following PD-L1 expression in circulating cells may be used as a surrogate for tracking adaptive changes in immunotherapeutic targets. Clin Cancer Res; 23(19); 5948–58. ©2017 AACR.

Additional file 1. Supplementary tables.

Introduction: Despite evidence that a subset of patients with locoregionally advanced non-small cell lung cancer (NSCLC) can be cured with radiation doses less than 60 Gy, there are currently no validated approaches to identify patients that could benefit from radiation dose de-escalation. Normal tissue changes including inflammation and fibrosis can be difficult to distinguish from residual disease on standard imaging during and following chemoradiation therapy (CRT), making assessment of treatment response and identification of favorable responders challenging. We hypothesized that circulating tumor DNA (ctDNA) kinetics during CRT could be used as a surrogate of response to identify genomic predictors of rapid response to treatment. Methods: We applied cancer personalized profiling by deep sequencing (CAPP-Seq) ctDNA analysis to 61 patients treated with CRT for Stage II-III NSCLC. We quantified ctDNA concentrations pre-CRT and a median of 21 days into CRT (mid-CRT) to determine the log-fold change in ctDNA concentration and identify “rapid responders.” The association between ctDNA log-fold change as a continuous variable with progression-free survival (PFS) was analyzed using univariable and multivariable regression, including gender, age, and stage as co-variables. The prevalence of driver gene single nucleotide variants in rapid responders versus slow responders was compared for each gene using Fisher’s exact tests with P-values adjusted using the Benjamini-Hochberg procedure. Results: Mid-CRT ctDNA log-fold change was significantly associated with progression-free survival as a continuous variable on both univariable (P=0.02) and multivariable analysis (P=0.03). Among patients whose ctDNA log-fold change was more negative than -2.15, 10/11 (91%) did not recur within the radiation field. We defined ctDNA rapid responders as the 10 patients with the largest decrease in ctDNA concentration mid-CRT without local progression. Compared with slow responders, ctDNA rapid responders had a trend towards more TP53 mutations (P=0.12), but no driver mutations were significantly enriched in rapid responders. Notably, mutations in common driver genes KEAP1, NFE2L2, KRAS, and EGFR were observed in 36% of slow responders and 0% of rapid responders (P=0.03). Conclusions: Our results suggest that ctDNA kinetics during CRT can identify patients responding favorably to treatment. Additional molecular characterization of ctDNA rapid responders may enable identification of patients who could benefit from treatment de-escalation. Citation Format: Everett J. Moding, Yufei Liu, Angela B. Hui, Jianzhong He, Yawei Qiao, Ting Xu, Luyang Yao, Saumil Gandhi, Zhongxing Liao, Millie Das, Kavitha J. Ramchandran, Sukhmani K. Padda, Joel W. Neal, Heather A. Wakelee, Billy W. Loo, Steven H. Lin, Ash A. Alizadeh, Maximilian Diehn. Circulating tumor DNA kinetics to identify genomic predictors of rapid response to chemoradiation in non-small cell lung cancer [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-069.

Background: Circulating stromal cells (CStCs) have been found to be common in the peripheral blood of cancer patients and hypothesized to be a blood based biomarker for monitoring cancer treatment. It has previously been described that the dynamic changes of PD-L1 expression during chemoradiotherapy (CRT) could be tracked using circulating stromal cells. However, how these changes relate to PD-L1/PD-1 immunotherapy (IMT) response is unstudied. We prospectively monitored PD-L1 expression in 2 cell types found in circulation (Circulating Tumor Cells [CTCs] and Cancer Associated Macrophage-like Cells [CAMLs]) in locally advanced non-small cell lung cancer (NSCLC) patients (pts) treated with Atezolizumab (Atezo) after definitive CRT (n=39) or in pts with CRT alone (n=40). Methods: A 2 year single blind prospective study was undertaken in pts with locally advanced NSCLC in 40 patients treated with CRT alone, or from a phase II DETERRED trial (NCT02525757) where Atezo was added for 1 year after completing CRT (n=10), or concurrently and after CRT (n=30). Samples from 39 of 40 pts from the DETERRED study were available for analysis. Baseline blood samples (7.5 ml) were drawn prior to start of CRT (T0), and a second sample was drawn ~1 month after completing CRT (T1), but prior to induction of Atezo. Blood was processed by CellSieve™ microfilters; stained for cytokeratin/PDL1/CD45 to identify CTCs and CAMLs. PD-L1 intensity was measured and grouped by 4 scores: 0-negative, 1-low, 2-medium, & 3-high. PD-L1 levels from circulating cells were used to evaluate PFS and OS. Significance was assessed by log-rank testing. Results: At least one CTC and/or CAML was found in 91% of available T0 samples and 96% of available T1 samples. In the 40 patients that received CRT alone, 35 pts had measurable cells at T0 and PD-L1 expression in CStCs was low (0-1) in 20 pts and high (2-3) in 15 pts. Further, at T1 PD-L1 expression in CStCs was low in 12 pts and high in 28 pts, with no relationship to PFS (T0 HR=1.1, 95%CI 0.5-2.8, p=0.96 and T1 HR=0.8, 95%CI 0.3-2.0, p=0.82) or OS (T0 HR=0.8, 95%CI 0.2-2.8, p=0.99 and T1 HR=1.1, 95%CI 0.3-3.5, p=0.84). In the patient arm that received Atezo at T0, PD-L1 expression in CStCs was low in 21 pts and high in 17 pts, with no relationship to PFS (HR=0.5, 95%CI 0.2-1.7, p=0.18) or OS (HR=1.6, 95%CI 0.4-6.0, p=0.75). However at T1, pts with high PD-L1 had significantly improved PFS response to Atezo (HR 5.4, 95%CI 1.7-17.0, p=0.009), and improved OS (HR 21.5, 95%CI 4.5-91.9, p Conclusions: PD-L1 expression in tissue is often not used in IMT treatment decisions due to limited correlation with clinical responses. However, it has been suggested that sequential monitoring of PD-L1 expression in circulating stromal cells in blood may predict for patients who will respond to IMT. These data suggests that CRT altered PD-L1 expression, and monitoring dynamic changes of PD-L1 in CStCs may predict immunotherapy effectiveness in NSCLC after CRT. Citation Format: Daniel L. Adams, Jianzhong He, Yawei Qiao, Kirby P. Gardner, John V. Haymach, Anne S. Tsao, Ashvathi Raghavakaimal, Cha-Mei Tang, Alexander Augustyn, Steven H. Lin. Expression of pd-l1 on circulating stromal cells predicts immunotherapy response in unresectable non-small cell lung carcinoma after definitive chemoradiotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3297.

BACKGROUND & AIMS: Biomarkers are needed to identify patients at risk of tumor progression following chemoradiotherapy for localized esophageal cancer. These could improve identification of patients at risk for cancer progression and selection of therapy. METHODS: We performed deep sequencing (CAPP-Seq) analyses of plasma cell-free DNA collected from 45 patients before and after chemoradiotherapy for esophageal cancer, as well as DNA from leukocytes, and fixed esophageal tumor biopsies collected during esophagogastroduodenoscopy. Patients were treated from May 2010 through October 2015; 23 patients subsequently underwent esophagectomy and 22 did not undergo surgery. We also sequenced DNA from blood samples from 40 healthy individuals (controls). We analyzed 802 regions of 607 genes for single-nucleotide variants previously associated with esophageal adenocarcinoma or squamous cell carcinoma. Patients underwent imaging analyses 6–8 weeks after chemoradiotherapy and were followed for 5 years. Our primary aim was to determine whether detection of circulating tumor DNA (ctDNA) following chemoradiotherapy is associated with risk of tumor progression (growth of local, regional, or distant tumors, detected by imaging or biopsy). RESULTS: The median proportion of tumor-derived DNA in total cell-free DNA before treatment was 0.07%, indicating that ultrasensitive assays are needed for quantification and analysis of ctDNA from localized esophageal tumors. Detection of ctDNA following chemoradiotherapy was associated with tumor progression (hazard ratio, 18.7; P

Background: Cancer Associated Macrophage-Like cells (CAMLs) are recently described circulating stromal cells common in the peripheral blood of cancer patients hypothesized to be a mechanism in cancer pathogenesis. We previously described that treatment naive patients with circulating CAMLs ≥50µm is a significant independent prognostic indicator of shorter progression free survival (PFS) in a variety of cancers. However, the clinical value of CAMLs in specific diseased cohorts for predicting response to treatment has not been evaluated. We present the results of a prospective study on treatment naive lung cancer patients before induction and directly after completion of definitive radiotherapy to determine if CAMLs are predictive of treatment response and cancer progression. Methods: A 2 year single blind prospective study was undertaken, testing the relationship of enlarged CAMLs (≥50µm) to PFS of lung cancer patients before & after induction of definitive radiation therapy. To achieve a 2-tailed 95% power (α=0.05) we recruited a training set of 55 patients, all with pathologically confirmed lung cancer: Stage I (n=13), Stage II (n=7), Stage IIIa (n=10), Stage IIIb (n=18) & Stage IV (n=7). Baseline (BL) blood samples were taken prior to start of therapy. If possible, a second blood sample (T1) was taken after completion of radiotherapy (~60 days), n=46 patients. Blood was filtered by CellSieveTM filtration and CAMLs quantified. Analysis by CAML size of Results: CAMLs were found in 93% of BL samples averaging 3.2 CAMLs/7.5mL. Patients with at least one CAML ≥50 µm had reduced PFS (HR=2.9, 95%CI 1.4-6.0, p=0.010). 46 patients agreed to a follow up blood draw. 13 of the 46 patients had an increase of CAML size to ≥50 µm, but 3 patients had a decrease to Conclusions: Our data suggests that for lung cancer patients undergoing definitive radiation therapy, the presence of enlarged CAMLs appears to predict patients that are resistant to treatment. Furthermore, these circulating stromal cells appear useful for sequential monitoring of patients that may prognosticate who are likely to progress after treatment. Citation Format: Daniel L. Adams, Jianzhong He, Yawei Qiao, Hui Gao, James Reuben, Ignacio I. Wistuba, Cha-Mei Tang, Steven H. Lin. A circulating stromal cell subpopulation that accurately predicts resistance and progression in treatment naïve lung cancer patients receiving definitive radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2915.

Suboptimal cellular DNA repair capacity (DRC) has been shown to be associated with enhanced cancer risk, but genetic variants affecting the DRC phenotype have not been comprehensively investigated. In this study, with the available DRC phenotype data, we analyzed correlations between the DRC phenotype and genotypes detected by the Illumina 317K platform in 1,774 individuals of European ancestry from a Texas lung cancer genome-wide association study. The discovery phase was followed by a replication in an independent set of 1,374 cases and controls of European ancestry. We applied a generalized linear model with single nucleotide polymorphisms as predictors and DRC (a continuous variable) as the outcome. Covariates of age, sex, pack-years of smoking, DRC assay-related variables, and case–control status of the study participants were adjusted in the model. We validated that reduced DRC was associated with an increased risk of lung cancer in both independent datasets. Several suggestive loci that contributed to the DRC phenotype were defined in ERCC2/XPD, PHACTR2, and DUSP1. In summary, we determined that DRC is an independent risk factor for lung cancer, and we defined several genetic loci contributing to DRC phenotype. Cancer Res; 73(1); 256–64. ©2012 AACR.

Purpose: HSP90 inhibition is well known to sensitize cancer cells to radiation. However, it is currently unknown whether additional radiosensitization could occur in the more clinically relevant setting of chemoradiation (CRT). We used the potent HSP90 inhibitor ganetespib to determine whether it can enhance CRT effects in NSCLC. Experimental Design: We first performed in vitro experiments in various NSCLC cell lines combining radiation with or without ganetespib. Some of these experiments included clonogenic survival assay, DNA damage repair, and cell-cycle analysis, and reverse-phase protein array. We then determined whether chemotherapy affected ganetespib radiosensitization by adding carboplatin–paclitaxel to some of the in vitro and in vivo xenograft experiments. Results: Ganetespib significantly reduced radiation clonogenic survival in a number of lung cancer cell lines, and attenuated DNA damage repair with irradiation. Radiation caused G2–M arrest that was greatly accentuated by ganetespib. Ganetespib with radiation also dose-dependently upregulated p21 and downregulated pRb levels that were not apparent with either drug or radiation alone. However, when carboplatin–paclitaxel was added, ganetespib was only able to radiosensitize some cell lines but not others. This variable in vitro CRT effect was confirmed in vivo using xenograft models. Conclusions: Ganetespib was able to potently sensitize a number of NSCLC cell lines to radiation but has variable effects when added to platinum-based doublet CRT. For optimal clinical translation, our data emphasize the importance of preclinical testing of drugs in the context of clinically relevant therapy combinations. Clin Cancer Res; 22(23); 5876–86. ©2016 AACR.

XPA, a DNA binding protein in the nucleotide excision repair (NER) pathway, modulates damage recognition. Recently, a common single-nucleotide polymorphism (A --> G) of unknown function was identified in the 5' non-coding region of the XPA gene. Because a deficiency in NER is associated with an increased risk of lung cancer, we evaluated the role of this polymorphism in 695 lung cancer case patients and 695 age-, sex-, ethnicity- and smoking-matched control subjects. We also studied the effect of this polymorphism on NER capacity in a subset sample for whom the host cell reactivation data were available. The presence of one or two copies of the G allele was associated with a reduced lung cancer risk for Caucasians [adjusted odds ratio (ORadj) = 0.69 [95% confidence interval (CI) = 0.53-0.90]], Mexican-Americans [ORadj = 0.32 (95% CI = 0.12-0.83)] and African-Americans [ORadj = 0.45 (95% CI = 0.16-1.22)]. In Caucasians, ever smokers with one or more copies of the G allele were observed to have a significantly reduced risk of lung cancer. Control subjects with one or two copies of the G allele demonstrated more efficient DRC than did those with the homozygous A allele. Our data suggest that the XPA 5' non-coding region polymorphism modulates NER capacity and is associated with decreased lung cancer risk, especially in the presence of exposure to tobacco carcinogens.

Preoperative chemoradiation in neoadjuvant setting is the standard of care for loco-regional esophageal adenocarcinoma (EAC). However, less than 30% of patients develop complete pathological response indicating need for newer therapeutic strategies. Cyclin dependent kinase 9 (CDK9) is found to be over expressed in EAC compared to Barrett’s esophagus. Our previous studies demonstrated strong antitumor effects on inhibition of CDK9 in EAC both in vitro and in vivo. Here we report augmented tumor regression in irradiated xenografts on combination with Flavopiridol, a well-established clinically used CDK inhibitor, compared to single modality treatments. In vitro studies indicate that Flavopiridol could radiosensitize FLO-1 and SKGT4 EAC cells with sustained 53BP1 foci especially in SKGT4 cells. Flavopiridol and BAY1143572, a more selective inhibitor of CDK9, could radiosensitize additional EAC radiation sensitive and resistant cell lines (SKGT4-R, OE-33 and OE-33-R) as analyzed by MTT assay, apoptosis, formation of 53BP1 foci and clonogenic assay. Flavopiridol and BAY1143572 showed a radio-synergistic action by downregulating MCL-1 and Axl. In conclusion, the radio-sensitizing capacities of CDK9 inhibitors presented here suggest that their adjuvant administration might improve EAC therapy. Note: This abstract was not presented at the meeting. Citation Format: Omkara Lakshmi M. Veeranki, Rashmi Dokey, Alicia Mejia, Zhimin Tong, Jianhu Zhang, Yawei Qiao, Pankaj Kumar Singh, Riham Katkhuda, Barbara Minoo, Jaime Rodriguez Canales, Steven Lin, Sunil Krishnan, Dipen Maru. Role of CDK9 inhibition as a sensitizer to radiation in esophageal adenocarcinoma: In vitro and in vivo efficacy study [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 5843. doi:10.1158/1538-7445.AM2017-5843

Background: PD-L1 expression can be induced by radiotherapy (RT) and may be an immune escape mechanism after RT, and has the potential to predict for responsiveness to immune checkpoint blockade immunotherapy. However, repetitive biopsies for monitoring PD-L1 expression in tumor and/or stroma are not feasible. Sequential assessment of PD-L1 expression on cancer associated circulating cells during treatment may be a way to measure the efficacy of combining RT and immunotherapy. RAD50 is a DNA repair gene that can be used in tracking tumor cell response to radiation. We therefore evaluated PD-L1 expression and RAD50 induction in CTCs and Cancer Associated Macrophage-Like Cells (CAMLs) in lung cancer patients (pts) before and during RT to track expression changes of these markers. Methods: Twenty-nine pts with stage I-IV lung cancer were included in this prospective pilot study. Four pts received radiation therapy for stage I disease and 25 other pts received chemoradiation for stage II-IV disease. A baseline blood sample (7.5 ml) was drawn prior to the start of RT (T0), and a second blood sample was drawn at a treatment visit during RT (T1) for a total of 58 samples. Blood was processed using CellSieve™ microfiltration (Creatv MicroTech), stained for cytokeratin 8, 18 & 19 and CD45, and imaged. Using the QUASR (Quench, Underivatize, Amine-Strip and Restain) technique to remove fluoresce signal, all cells were restained for RAD50-AlexaFluor550 and PD-L1-AlexaFluor 488, along with DAPI nuclear stain. The RAD50 foci were quantified within nuclear regions and PD-L1 pixel intensity measured and grouped into 4 IHC groups: 0-negative (pixel average 0-215), 1-low (pixel average 216-300), 2-medium (pixel average 301-750), and 3-high (pixel average 751+). Results: There was at least one cytokeratin positive cell (i.e. CTC or CAMLs) found in all but 2 samples (97%). CTCs were found in 69% of T0 and 72% of T1 samples, and CAMLs in 79% of T0 and 97% of T1 samples. PD-L1 expression ranged from 34-2552 pixel intensity, with an average of 285 at T0 and 525 at T1 (p = 0.07). Interestingly, 9 pts had no PD-L1 expression at T0 but an increase to 2 to 3+ at T1, 12 pts with low/no PD-L1 expression remained low at T1, and 8 pts had high PD-L1 expression that remained high or decreased at T1. RAD50 foci ranged from 0-16 per cell, with an average of 0.56 at T0 that increased to 4.68 at T1 (p Conclusions PD-L1 expression and RAD 50 foci were quantifiable in both CTCs and CAMLs, and a correlative response to radiotherapy +/- chemotherapy was quantified. This data suggests that sequential tracking of CTCs and immune-related cells from the primary lung tumor is feasible using microfiltration and may potentially serve as a predictive biomarker for cancer therapy sensitivity. Citation Format: Daniel L. Adams, Martin J. Edelman, Penny Fang, Wen Jiang, Jianzhong He, Ting Xu, Hui Gao, James M. Reuben, Yawei Qiao, Steven Hahn, Ritsuko Komaki, Zhongxing Liao, Cha-Mei Tang, Steven H. Lin. Sequential tracking of PD-L1 expression and RAD50 induction in CTCs and circulating stromal cells of lung cancer patients during treatment with radiotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4990.

Purpose: To examine the role of suboptimal DNA repair capacity (DRC) for UV light–induced DNA damage in the development of nonmelanoma skin cancer (NMSC) and tumor progression.Experimental Design: We conducted a hospital-based case-control study of 255 patients with newly diagnosed NMSC [146 with basal cell carcinoma (BCC) and 109 with squamous cell carcinoma (SCC)] and 333 cancer-free controls. We collected information on demographic variables and risk factors from questionnaires, tumor characteristics from medical records, and lymphocytic DRC phenotype by the host-cell reactivation assay. Multivariable logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (95% CI).Results: Overall, there was a relative 16% reduction in DRC in NMSC patients compared with controls (P < 0.001 for BCC and for SCC, respectively). DRC below the controls' median value was associated with increased risk significantly for BCC (OR, 1.62; 95% CI, 1.07-2.45) but borderline for SCC (OR, 1.63; 95% CI, 0.95-2.79) after adjustment for age, sex, and other assay-related covariates. When the highest tertile of controls' DRC was used as the reference, the intermediate and low DRC were associated with a statistically significant trend for increasing risk for both BCC (Ptrend = 0.007) and SCC (Ptrend = 0.020). However, patients with aggressive or multiple SCC tended to have a higher DRC than those with nonaggressive or single SCC.Conclusions: Reduced DRC is an independent risk factor for BCC and single or nonaggressive SCC but not for multiple primaries, local aggressiveness, or recurrence of NMSC.

DNA repair should occur after cells sense DNA damage signals and undergo cell-cycle arrest to provide sufficient time for DNA repair, and suboptimal DNA repair capacity (DRC) in peripheral lymphocytes has been suggested as a cancer susceptibility marker. Numerous studies showed a functional link between DNA damage sensing, cell-cycle checkpoint, and DNA repair. We hypothesized that in vitro cell-cycle checkpoint-related protein expression levels in stimulated lymphocytes predict DRC levels. To test this hypothesis, we performed the host-cell reactivation assay for DRC by transfecting stimulated peripheral blood lymphocytes from 120 normal donors with transient expression plasmids damaged by benzo[a]pyrene diol epoxide (BPDE). The same cells were assessed for protein expression induction of eight cell-cycle checkpoint-related genes using the reverse-phase protein lysate microarray assay. In multivariate linear regression analysis adjusting for age, sex, blastogenic rate, and sample storage duration, the association between DRC and expression levels of cell-cycle checkpoint-related proteins induced by BPDE-adducts was statistically significant for p27, CCND1, ATM, and MDM2 (P = 0.00, 0.03, 0.03, and 0.03, respectively), borderline for p73 and p21 (P = 0.07 and 0.09, respectively), but not for p53 and p16 (P = 0.13 and 0.18, respectively). Because the relative expression levels of all these eight proteins were highly correlated, we further performed the principal component analysis and identified ATM as the most important predictor of DRC, followed by MDM2 and p27. Our results provide population-based in vitro evidence demonstrating that cell-cycle checkpoint-related proteins play essential roles in regulating DNA repair, at least in unaffected human peripheral blood lymphocytes. Further studies are warranted to investigate the role of interindividual variation in the expression levels of these proteins in cancer susceptibility.

The nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces lung adenocarcinoma through formation of DNA adducts. Our previous research on susceptibility to tobacco-induced carcinogenesis focused on benzo[a]pyrene diol epoxide (BPDE) as the in vitro mutagen for phenotype measurements of DNA repair capacity (DRC) in mammalian cells. Here, we present a modified host-cell reactivation (HCR) assay to measure lymphocytic DRC for alkylating DNA damage as is induced by the tobacco-specific nitrosamine, NNK. We substituted dimethyl sulfate (DMS) to create alkylating damage in pCMVluc plasmid DNA and established the damage-repair dose-response curves in both normal and nucleotide excision repair-deficient lymphoblastoid cell lines and in phytohemagglutinin (PHA)-stimulated primary lymphocytes. We then successfully measured the DRC in PHA-stimulated lymphocytes from 48 patients with lung adenocarcinoma and 45 cancer-free controls and tested our hypothesis that lower DRC for alkylating damage is associated with an increased risk of lung adenocarcinoma. The cases exhibited a lower mean DRC than did the controls. A >3-fold increased risk (odds ratio = 3.21; 95% confidence interval = 1.25-8.21) was found for those with DRC levels below the control median. There was no correlation between the DRC measured with this DMS-HCR assay and that from the parallel BPDE-HCR assay. Interestingly, risk increased to >10-fold for those with sub-optimal DRC measured by both DMS- and BPDE-HCR assays. We conclude that variability in DRC is a risk factor for lung cancer and our results provide proof of principle for a new assay that can assess DRC for NNK-induced DNA damage.

Both reduced DNA repair capacity (DRC) and folate deficiency are associated with increased cancer risk. Furthermore, folate is involved in DNA repair through de novo DNA synthesis and methylation. To determine whether low dietary folate intake is associated with low cellular DRC in humans, we assessed total dietary folate intake using a food frequency questionnaire in 559 non-Hispanic white cancer-free subjects enrolled from 1995 through 2001 as controls for ongoing molecular epidemiological studies from among enrollees in a community-based multispecialty physician practice in the Houston metropolitan area. We assessed cellular DRC using the host-cell reactivation assay that measures nucleotide-excision repair capacity in peripheral blood lymphocytes. The distribution of DRC was approximately normal in this study population. In univariate analysis, subjects in the lowest tertile of total dietary folate intake (170 microg/1000 kcal/day) exhibited a significant reduction (-18%) in DRC compared with those in the upper tertile (225 microg/1000 kcal/day; P0.001). In multivariate linear regression analysis, calorie-adjusted total folate intake remained an independent predictor of DRC (P0.001). Additional stratification analysis indicated that this association was more pronounced in those who did not use folate supplementation (n = 230; P0.001) compared with those who did (n = 329; P = 0.177). Our findings suggest that low dietary folate intake is associated with suboptimal cellular DRC. Once replicated by other investigators, this finding has public health implications by reinforcing the need for folate supplementation or dietary modification for the at-risk population.

BACKGROUND The mechanism underlying the role of UV light exposure from sunlight in the etiology of cutaneous malignant melanoma (CMM) is unclear. Patients with xeroderma pigmentosum, a disease characterized by severe sensitivity to UV radiation and a defect in nucleotide excision repair, have a high incidence of CMM, which suggests that DNA repair capacity (DRC) plays a role in sunlight-induced CMM in the general population as well. METHODS We conducted a hospital-based case-control study of DRC and CMM among 312 non-Hispanic white CMM patients who had no prior chemotherapy or radiation therapy, and 324 cancer-free control subjects who were frequency-matched to case patients on age, sex, and ethnicity. Information on demographic variables, risk factors, and tumor characteristics was obtained from questionnaires and medical records. We used the host-cell reactivation assay to measure the DRC in study subjects' lymphocytes. All statistical tests were two sided. RESULTS Case patients had a 19% lower mean (+/- standard deviation [SD]) DRC (8.5 +/- 3.4%) than control subjects (10.5 +/- 5.1%), a statistically significant difference (P

DNA repair capacity (DRC) plays an important role in genetic susceptibility to cancer. Polymorphisms of a number of DNA repair genes involved in several distinct pathways have been identified. However, their effects on repair function have not been well characterized. We demonstrated previously that DRC for removal of benzo[a]pyrene diol epoxide-induced DNA damage measured by a host-cell reactivation assay was modulated by two XPD/ERCC2 polymorphisms in lung cancer. In this report, we investigated the association between the repair phenotype of ultraviolet (UV)-induced damage and genotypes of three DNA repair genes, XPC and XPD [involved in nucleotide excision repair (NER)] and XRCC1 [involved in base excision repair (BER)]. We measured DRC for removal of UV photoproducts by the host-cell reactivation assay in cryopreserved lymphocytes from 102 healthy non-Hispanic white subjects. We also typed these subjects for five polymorphisms in these three DNA repair genes (at intron 9 of XPC; exons 6, 10 and 23 of XPD and exon 10 of XRCC1). Compared with wild-type homozygotes, subjects homozygous for polymorphisms of the two NER genes consistently had suboptimal DRC. The DRC was consistently lower in subjects homozygous for XPC, XPD or both than in subjects with other genotypes, although the difference was not statistically significant for XPD variants. In contrast, the polymorphic allele of the BER gene, XRCC1, had no consistent effect on DRC. We concluded that these NER polymorphisms may modulate DRC and may be useful biomarkers for identifying individuals at risk of developing cancer.

Radiotherapy is a critical component in the definitive treatment of locally-advanced non-small cell lung cancer (LA-NSCLC), and while concurrent platinum-based chemoradiation is the standard approach, improvements have been minimal. Molecularly-targeted therapy to further enhance radiation effects beyond platinum-sensitization is needed to further improve radiotherapy efficacy. A systematic approach needs to be developed that can rapidly identify novel drugs that can be combined to enhance radiation effects. However, current approaches using traditional clonogenic survival assay (tCSA) and colorimetric assays are inadequate for high throughput drug screens to identify novel radiation sensitizers. To enable screens adapted for radiation drug screening, we have developed a method which we coin the High Throughput Clonogenic Survival Assay (HCSA) that allows rapid screening of drug libraries to identify potent radiation sensitizers. As a proof-of-principle, we used the Kras mutated lung cancer cell line H460, and validated the hits using traditional assays. Drugs at various concentrations or vehicle were added to cells seeded overnight in 96 well plates at low densities, and after 6 hours, plates were irradiated at various doses. After incubation, colonies were stained and imaged on the INCell 6000® (GE Health), and colony count determined using the INCell Developer image analysis software. Drug screens were done using a Custom Clinical Collection of 150 compounds. We compared the HCSA method to tCSA and found that HCSA recapitulates a similar cell survival curve and survival fraction at 2 Gy (SF2) values for several cancer cell types. Drug screens identified several compounds that were cytotoxic to the KRAS mutant H460 cells alone at 1 µM (inhibitors to Src/Abl, IGF1R, ROCK1, AKT, PI3K, c-MET) and at low nM concentrations (HSP90 and HDAC inhibitors). Multiple drugs of the same class were found to be radiation sensitizers and levels of potency seemed to reflect the clinical relevance of these drugs. For instance, several late generation PARP inhibitors were found to be good sensitizers in the HCSA, but the ones that have not made it into clinical development, or in the case of BSI-201, was proven to not even be a PARP inhibitor, do not come out in the screen. We also discovered that several inhibitors of pathways downstream of activated mutant KRAS (PI3K, AKT, mTOR, and MEK1/2) sensitized H460 cells to radiation, which is biologically relevant for this Kras mutant cell line. We believe these results demonstrate the promise of HCSA as a high throughput drug screening platform to identify novel radiation sensitizers. This will greatly expedite the discovery of novel targets and drugs for clinical translation to enhance the effects of radiotherapy in lung cancer. Citation Format: Steven H. Lin, Hui Liu, Yawei Qiao, Uma Giri, Jing Zhang, Clifford Stephan, Mary Sobieski, John V. Heymach, Stephen S. Yoo. High content clonogenic survival assay for drug library screening identifies potent radiation sensitizers for non-small cell lung cancer. [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 4898. doi:10.1158/1538-7445.AM2014-4898

DNA repair is essential in protecting the integrity of the genome. There is considerable inter-individual variation in DNA repair capacity (DRC). We have reported that cellular DRC (measured in lymphocytes using the host-cell reactivation assay with a reporter gene chloramphenicol acetyltransferase [CAT] damaged by an activated tobacco carcinogen, benzo(a)pyrene diol epoxide [BPDE]) modulates susceptibility to lung cancer. To identify genetic polymorphisms predictive of the repair phenotype, we used phenotype and genotype data in a population of 1,474 European ancestry individuals from a Texas lung cancer genome-wide association case-control study using Illumina 317K platform (317498 SNPs). This discovery phase was followed by replication testing of the top 20 SNPs in an independent set of an additional 1,364 European ancestry cases and controls, using Illumina Bead Array Platform. In addition, we performed a replication analysis of all SNPs with P < 0.01 in 145 DNA repair-related genes in BER, NER, MMR, DSBR pathways from the discovery set, and applying a conservative Bonferroni-corrected P -value of 2.37E-5 for the joint P -value, based on the total number of analyzed SNPs in DNA repair genes. The DRC data were divided into quartiles by assigning the top quarter of participants (with highest values of DRC) as the DNA repair proficient group and the bottom quarter of participants with lowest values of DRC as the suboptimal DRC group to enhance the contrast. We used a generalized linear model with SNPs as predictors and DRC as the outcome. The covariates age, gender, smoking status, duration of smoking, number of cigarettes per day, DRC assay-related variables including the sample storage time, baseline reporter gene CAT expression level, blastogenic rate, and the case-control status of the study participants were adjusted in the model. We identified several suggestive loci contributing to the DRC phenotype: rs9390123 in PHACTR2 (phosphatase and actin regulator 2, P = 4.78E-05 in the discovery set and P = 1.08E-05 in the joint analysis); rs4756897 in USH1C (Usher syndrome 1C, P = 5.40E-05 in the discovery set and 8.71E-06 in the joint analysis); rs6785626 in CNTN4 (contactin 4, P = 0.000451 in the discovery set and P = 1.55E-05 in the joint analysis); rs13181 in ERCC2 (excision repair cross-complementing rodent repair deficiency-complementation group 2, joint P = 2.09E-05); rs7443927 in DUSP1 (dual specificity phosphatase 1, joint P =5.04E-05) and rs10878361 in HMGA2 (high mobility group AT-hook 2, joint P =2.54E-4). In conclusion, we identified and replicated a number of SNPs associated with DRC. These genetic determinants of DNA repair phenotype are easier and cheaper to measure, and subject to less misclassification than the phenotype and may be effective biomarkers for risk assessment. (This study was supported by NIH grants CA127219, CA055769, ES011740, CA131274 and CA016672) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4657. doi:10.1158/1538-7445.AM2011-4657

Lung cancer is initiated by tobacco carcinogens-induced DNA damage, such as DNA adducts caused by benzo[a]pyrene diol epoxide (BPDE). These adducts can be removed by an efficient nucleotide excision repair. We have previously reported that DNA repair capacity (DRC) measured by the host-cell reactivation assay is associated with risk of lung cancer in non-Hispanic whites. In the recent analysis with larger sample sizes including minorities, we found that the suboptimal DRC remains associated with increased risk of lung cancer in 1730 cases and 1683 controls of non-Hispanic whites. The mean values of DRC were 8.37% ± 2.8% (SD) in the cases vs. 9.04% ± 3.1% in the controls (P < 0.0001). The odds ratio (OR) was 1.42 (95% confidence interval [CI], 1.24 – 1.64) after adjustment for age, sex, pack-years of smoking, family history of cancer, and other assay-related covariates. The similar results and risk trend have been also found in 127 Mexican-American patients with lung cancer and 176 cancer-free controls with a mean DRC of 8.38% ± 2.7% in the cases vs. 9.04% ± 3.3% in the controls, and an adjusted OR of 1.67 (95% CI, 1.00 – 2.78), although the difference in mean DRC values was borderline significant (P = 0.056) because of the relatively small sample size. However, this difference in DRC for tobacco carcinogen-induced DNA adducts was not significant in 327 African-American patients with lung cancer and 325 cancer-free controls. The mean values of DRC were 8.90% ± 3.1% in the cases vs. 8.66% ± 2.8% in the controls (P = 0.301). The adjusted OR was 0.99 (95% CI, 0.70 – 1.39). The DRC level in African-American controls was non-significantly lower than other ethnic controls (P = 0.144); however, African-American patients had significantly higher DRC than non-Hispanic white patients and Mexican-Americans patients (P = 0.053). The results further confirmed that low DRC is associated with increased risk of lung cancer in non-Hispanic whites and also provided the evidence of this association in Mexican Americans. These findings help us understand the ethnic differences in the development of lung cancer. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A6.