Plasmonic nano-aperture label-free imaging of single small extracellular vesicles for cancer detection.

BACKGROUND: Small extracellular vesicle (sEV) analysis can potentially improve cancer detection and diagnostics. However, this potential has been constrained by insufficient sensitivity, dynamic range, and the need for complex labeling. METHODS: In this study, we demonstrate the combination of PANORAMA and fluorescence imaging for single sEV analysis. The co-acquisition of PANORAMA and fluorescence images enables label-free visualization, enumeration, size determination, and enables detection of cargo microRNAs (miRs). RESULTS: An increased sEV count is observed in human plasma samples from patients with cancer, regardless of cancer type. The cargo miR-21 provides molecular specificity within the same sEV population at the single unit level, which pinpoints the sEVs subset of cancer origin. Using cancer cells-implanted animals, cancer-specific sEVs from 20 µl of plasma can be detected before tumors were palpable. The level plateaus between 5-15 absolute sEV count (ASC) per µl with tumors ≥8 mm3. In healthy human individuals (N = 106), the levels are on average 1.5 ASC/µl (+/- 0.95) without miR-21 expression. However, for stage I-III cancer patients (N = 205), nearly all (204 out of 205) have levels exceeding 3.5 ASC/µl with an average of 12.2 ASC/µl (±9.6), and a variable proportion of miR-21 labeling among different tumor types with 100% cancer specificity. Using a threshold of 3.5 ASC/µl to test a separate sample set in a blinded fashion yields accurate classification of healthy individuals from cancer patients. CONCLUSIONS: Our techniques and findings can impact the understanding of cancer biology and the development of new cancer detection and diagnostic technologies.

Small extracellular vesicles (sEVs) are tiny particles derived from cells that can be detected in bodily fluids such as blood. Detecting sEVs and analyzing their contents may potentially help us to diagnose disease, for example by observing differences in sEV numbers or contents in the blood of patients with cancer versus healthy people. Here, we combine two imaging methods ­ our previously developed method PANORAMA and imaging of fluorescence emitted by sEVs­to visualize and count sEVs, determine their size, and analyze their cargo. We observe differences in sEV numbers and cargo in samples taken from healthy people versus people with cancer and are able to differentiate these two populations based on our analysis of sEVs. With further testing, our approach may be a useful tool for cancer diagnosis and provide insights into the biology of cancer and sEVs.

Monitoring PD-L1 Expression on Circulating Tumor-Associated Cells in Recurrent Metastatic Non-Small-Cell Lung Carcinoma Predicts Response to Immunotherapy With Radiation Therapy.

PURPOSE: Current diagnostic methods to determine programmed death 1 (PD-1) receptor and its ligand (PD-L1)/PD-1 immunotherapy (immune checkpoint inhibitor [ICI]) efficacy in recurrent or metastatic non-small-cell lung carcinoma (rmNSCLC) are imprecise. Although previously shown that patients with high tumor PD-L1 (≥ 50%) demonstrate clinical benefit in the form of disease reduction and improved survival, patients with low PD-L1 (< 50%) sometimes benefit from treatment. Since the PD-L1/PD-1 pathway is dynamic, monitoring PD-L1 levels during treatment may be more accurate than a static baseline tumor biopsy; however, rebiopsying the primary or metastatic disease is rarely feasible. Liquid biopsies that measure the upregulation of PD-L1 on tumor-associated cells (TACs), ie, cancer-associated macrophage-like cells and circulating tumor cells, have been performed, but their predictive value for ICI therapy efficacy is unknown. MATERIALS AND METHODS: We initiated a single-blind prospective study to evaluate TAC PD-L1 expression changes in rmNSCLC from blood samples before (T0) and after (T1) treatment with ICI (ICI, n = 41) or without ICI (no ICI, n = 41). Anonymized blood was filtered to isolate TACs, which were then quantified for high/low PD-L1 expression. Progression-free survival (PFS) or overall survival (OS) hazard ratios (HRs) were evaluated at 18 and 24 months by censored univariate analysis. RESULTS: Increased TAC PD-L1 expression between T0 and T1 in patients who were not treated with ICI had no relationship with PFS or OS. However, increased TAC PD-L1 expression between T0 and T1 in patients treated with ICI had significantly better PFS (HR, 3.49; 95% CI, 1.5 to 8.3; P = .0091) and OS (HR, 3.058; 95% CI, 1.2 to 7.9; P = .0410). CONCLUSION: Blood-based monitoring of dynamic changes in PD-L1 in TACs appears to identify patients with rmNSCLC who may benefit from ICI.

Exploring the synergy of radiative coupling and substrate undercut in arrayed gold nanodisks for economical, ultra-sensitive label-free biosensing.

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/µL), 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.

Simple oligonucleotide-based multiplexing of single-cell chromatin accessibility.

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.

High-Content Clonogenic Survival Screen to Identify Chemoradiation Sensitizers.

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.

Giant Circulating Cancer-Associated Macrophage-Like Cells Are Associated With Disease Recurrence and Survival in Non-Small-Cell Lung Cancer Treated With Chemoradiation and Atezolizumab.

BACKGROUND: Cancer-associated macrophage-like cells (CAMLs) are a potential peripheral blood biomarker for disease progression. This study used data from a phase 2 clinical trial to evaluate prognostic utility of CAMLs for locally advanced non-small-cell lung cancer treated with definitive chemoradiotherapy (CRT) and atezolizumab (DETERRED; ClinicalTrials.gov NCT02525757). PATIENTS AND METHODS: Sample collection occurred at baseline (T0), during CRT (T1), at end of CRT (T2), and at first follow-up (T3). CAMLs were captured and quantified by the CellSieve system using multiplex immunostaining. Giant CAMLs were defined as characteristic CAMLs ≥ 50 µm. Kaplan-Meier methodology estimated progression-free survival, distant failure-free survival, relapse-free survival, and overall survival at 30 months. RESULTS: Thirty-nine patients were evaluated between December 2015 and March 2018. Median follow-up was 27 months. Most disease was stage III (85%) and comprised squamous-cell carcinoma (38%) or adenocarcinoma (59%). In total, 267 blood samples were analyzed. Giant CAMLs were identified in 57%, 60%, 64%, and 63% of patients at T0, T1, T2, and T3, respectively. Patients with giant CAMLs at T3, occurring at a median of 30 days after completion of CRT, had significantly worse distant failure-free survival (hazard ratio [HR] 4.9, P = .015), progression-free survival (HR 2.5, P = .025), recurrence-free survival (HR 2.4, P = .036), and overall survival (HR 3.5, P = .034) compared to patients with small or no CAMLs. CONCLUSIONS: Presence of giant CAMLs after CRT completion was associated with development of metastatic disease and poorer survival despite the use of maintenance immunotherapy. Monitoring CAMLs may help risk-stratify patients for adaptive treatment strategies.

Enumeration and molecular characterization of circulating tumor cells enriched by microcavity array from stage III non-small cell lung cancer patients.

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<0.001). Expression of BCL2 by MCA-enriched CTCs was associated with poor PFS (HR 3.11, 95% CI, 1.18-8.22, P=0.022). Individually, CTC count and expression of BCL2 each remained statistically significant predictors of disease progression and overall survival in multivariate analysis. CONCLUSIONS: This is the first demonstration that lysates of MCA-enriched CTCs are amenable to molecular characterization. CTCs enriched by MCA are an independent prognostic marker in NSCLC.

Cancer associated macrophage-like cells and prognosis of esophageal cancer after chemoradiation therapy.

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 < 50 µm by the completion of CRT over patients with CAMLs ≥ 50 µm; PFS (HR = 12.0, 95% CI = 2.7-54.1, 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.

Circulating Tumor DNA Dynamics Predict Benefit from Consolidation Immunotherapy in Locally Advanced Non-Small Cell Lung Cancer.

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.

Circulating Tumor DNA Analysis for Detection of Minimal Residual Disease After Chemoradiotherapy for Localized Esophageal Cancer.

BACKGROUND & AIMS: Biomarkers are needed to risk stratify after chemoradiotherapy for localized esophageal cancer. These could improve identification of patients at risk for cancer progression and selection of additional therapy. METHODS: We performed deep sequencing (CAncer Personalized Profiling by 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 biopsy samples 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 control individuals. 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) after 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 after chemoradiotherapy was associated with tumor progression (hazard ratio, 18.7; P < .0001), formation of distant metastases (hazard ratio, 32.1; P < .0001), and shorter disease-specific survival times (hazard ratio, 23.1; P < .0001). A higher proportion of patients with tumor progression had new mutations detected in plasma samples collected after chemoradiotherapy than patients without progression (P = .03). Detection of ctDNA after chemoradiotherapy preceded radiographic evidence of tumor progression by an average of 2.8 months. Among patients who received chemoradiotherapy without surgery, combined ctDNA and metabolic imaging analysis predicted progression in 100% of patients with tumor progression, compared with 71% for only ctDNA detection and 57% for only metabolic imaging analysis (P < .001 for comparison of either technique to combined analysis). CONCLUSIONS: In an analysis of cell-free DNA in blood samples from patients who underwent chemoradiotherapy for esophageal cancer, detection of ctDNA was associated with tumor progression, metastasis, and disease-specific survival. Analysis of ctDNA might be used to identify patients at highest risk for tumor progression.

Tankyrase disrupts metabolic homeostasis and promotes tumorigenesis by inhibiting LKB1-AMPK signalling.

The LKB1/AMPK pathway plays a major role in cellular homeostasis and tumor suppression. Down-regulation of LKB1/AMPK occurs in several human cancers and has been implicated in metabolic diseases. However, the precise upstream regulation of LKB1-AMPK pathway is largely unknown. Here, we report that AMPK activation by LKB1 is regulated by tankyrases. Tankyrases interact with and ribosylate LKB1, promoting its K63-linked ubiquitination by an E3 ligase RNF146, which blocks LKB1/STRAD/MO25 complex formation and LKB1 activation. LKB1 activation by tankyrase inhibitors induces AMPK activation and suppresses tumorigenesis. Similarly, the tankyrase inhibitor G007-LK effectively regulates liver metabolism and glycemic control in diabetic mice in a LKB1-dependent manner. In patients with lung cancer, tankyrase levels negatively correlate with p-AMPK levels and poor survival. Taken together, these findings suggest that tankyrase and RNF146 are major up-stream regulators of LKB1-AMPK pathway and provide another focus for cancer and metabolic disease therapies.

Targeting cyclin-dependent kinase 9 by a novel inhibitor enhances radiosensitization and identifies Axl as a novel downstream target in esophageal adenocarcinoma.

Cyclin-dependent kinase 9 (CDK9) transcriptionally regulates several proteins and cellular pathways central to radiation induced tissue injury. We investigated a role of BAY1143572, a new highly specific CDK9 inhibitor, as a sensitizer to radiation in esophageal adenocarcinoma. In vitro synergy between the CDK9 inhibitor and radiation was evaluated by clonogenic assay. In vivo synergy between the CDK9 inhibitor and radiation was assessed in multiple xenograft models including a patient's tumor derived xenograft (PDX). Reverse phase protein array (RPPA), western blotting, immunohistochemistry, and qPCR were utilized to identify and validate targets of the CDK9 inhibitor. The CDK9 inhibitor plus radiation significantly reduced growth of FLO-1, SKGT4, OE33, and radiation resistant OE33R xenografts and PDXs as compared to the cohorts treated with either single agent CDK9 inhibitor or radiation alone. RPPA identified Axl as a candidate target of CDK9 inhibition. Western blot and qPCR demonstrated reduced Axl mRNA (p = 0.02) and protein levels after treatment with CDK9 inhibitor with or without radiation in FLO-1 and SKGT4 cells. Axl protein expression in FLO-1 xenografts treated with combination of CDK9 inhibitor and radiation was significantly lower than the xenografts treated with radiation alone (p = 0.003). Clonogenic assay performed after overexpression of Axl in FLO-1 and SKGT4 cells enhanced radiosensitization by the CDK9 inhibitor, suggesting dependency of radiosensitization effects of the CDK9 inhibitor on Axl. In conclusion, these findings indicate that targeting CDK9 by BAY1143572 significantly enhances the effects of radiation and Axl is a novel downstream target of CDK9 in esophageal adenocarcinoma.

Mutant LKB1 Confers Enhanced Radiosensitization in Combination with Trametinib in KRAS-Mutant Non-Small Cell Lung Cancer.

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.

Sequential Tracking of PD-L1 Expression and RAD50 Induction in Circulating Tumor and Stromal Cells of Lung Cancer Patients Undergoing Radiotherapy.

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.

Hsp90 Inhibitor Ganetespib Sensitizes Non-Small Cell Lung Cancer to Radiation but Has Variable Effects with Chemoradiation.

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.

Genome-wide association study reveals novel genetic determinants of DNA repair capacity in lung cancer.

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.

Reduced DNA repair capacity for removing tobacco carcinogen-induced DNA adducts contributes to risk of head and neck cancer but not tumor characteristics.

PURPOSE: Although cigarette smoking and alcohol use are known risk factors for squamous cell carcinoma of head and neck (SCCHN), only a few exposed individuals develop this disease, suggesting an individual susceptibility. In this study, we investigated the associations between genetically determined DNA repair capacity (DRC) for removing tobacco-induced DNA adducts and risk of SCCHN and tumor characteristics. EXPERIMENTAL DESIGN: We measured DRC in cultured T lymphocytes using the host-cell reactivation assay in a hospital-based case-control study of 744 SCCHN patients and 753 age-, sex-, and ethnicity-matched cancer-free controls recruited from The University of Texas M.D. Anderson Cancer Center. RESULTS: Patients with SCCHN had significantly lower mean DRC (8.84% +/- 2.68%) than controls (9.97% +/- 2.61%; P < 0.0001), and the difference accounted for approximately 2-fold increased risk of SCCHN [adjusted odds ratio (OR), 1.91; 95% confidence interval (CI), 1.52-2.40] after adjustment for other covariates. Compared with the highest DRC quartile of controls, this increased risk was dose dependent (second highest quartile: OR, 1.40; 95% CI, 0.99-1.98; third quartile: OR, 1.87; 95% CI, 1.34-2.62; and fourth quartile: OR, 2.76; 95% CI, 1.98-3.84, respectively; P(trend) < 0.0001). We also assessed the performance of DRC in risk prediction models by calculating the area of under the receiver operating characteristic curve. The addition of DRC to the model significantly improved the sensitivity of the expanded model. However, we did not find the association between DRC and tumor sites and stages. CONCLUSION: DRC is an independent susceptibility biomarker for SCCHN risk but not a tumor marker.

Repair capacity for UV light induced DNA damage associated with risk of nonmelanoma skin cancer and tumor progression.

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 (P(trend) = 0.007) and SCC (P(trend) = 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.

A modified host-cell reactivation assay to measure repair of alkylating DNA damage for assessing risk of lung adenocarcinoma.

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.