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Purpose: Patients with advanced non–small cell lung cancer (NSCLC) harboring activating EGFR mutations are initially responsive to tyrosine kinase inhibitors (TKI). However, therapeutic resistance eventually emerges, often via secondary EGFR mutations or EGFR-independent mechanisms such as epithelial-to-mesenchymal transition. Treatment options after EGFR-TKI resistance are limited as anti-PD-1/PD-L1 inhibitors typically display minimal benefit. Given that IL6 is associated with worse outcomes in patients with NSCLC, we investigate whether IL6 in part contributes to this immunosuppressed phenotype. Experimental Design: We utilized a syngeneic genetically engineered mouse model (GEMM) of EGFR-mutant NSCLC to investigate the effects of IL6 on the tumor microenvironment and the combined efficacy of IL6 inhibition and anti-PD-1 therapy. Corresponding in vitro studies used EGFR-mutant human cell lines and clinical specimens. Results: We identified that EGFR-mutant tumors which have oncogene-independent acquired resistance to EGFR-TKIs were more mesenchymal and had markedly enhanced IL6 secretion. In EGFR-mutant GEMMs, IL6 depletion enhanced activation of infiltrating natural killer (NK)- and T-cell subpopulations and decreased immunosuppressive regulatory T and Th17 cell populations. Inhibition of IL6 increased NK- and T cell–mediated killing of human osimertinib-resistant EGFR-mutant NSCLC tumor cells in cell culture. IL6 blockade sensitized EGFR-mutant GEMM tumors to PD-1 inhibitors through an increase in tumor-infiltrating IFNγ+ CD8+ T cells. Conclusions: These data indicate that IL6 is upregulated in EGFR-mutant NSCLC tumors with acquired EGFR-TKI resistance and suppressed T- and NK-cell function. IL6 blockade enhanced antitumor immunity and efficacy of anti-PD-1 therapy warranting future clinical combinatorial investigations.

IL-6 modulates expression of NK receptor and their ligands in EGFR mutant tumors.

IL-6 modulates activation state of infiltrating T cells in EGFR mutant tumors.

Diet, nutritional status, and certain dietary supplements are postulated to influence the development and progression of prostate cancer. Angiogenesis and inflammation are central to tumor growth and progression, but the effect of diet on these processes remains uncertain. We explored changes in 50 plasma cytokines and angiogenic factors (CAF) in 145 men with prostate cancer enrolled in a preoperative, randomized controlled phase II trial with four arms: control (usual diet), low-fat (LF) diet, flaxseed-supplemented (FS) diet, and FS+LS diet. The mean duration of dietary intervention was 30 to 31 days. Among the individual arms, the largest number of significant changes (baseline vs. preoperative follow-up) was observed in the LF arm, with 19 CAFs decreasing and one increasing (P < 0.05). Compared with the control arm, 6 CAFs—including proangiogenic factors (stromal-cell derived-1α) and myeloid factors (granulocyte-colony-stimulating factor, macrophage colony-stimulating factor)—all decreased in the LF arm compared with controls; three and four CAFs changed in the FS and FS+LF arms, respectively. Weight loss occurred in the LF arms and significantly correlated with VEGF decreases (P < 0.001). The CAFs that changed in the LF arm are all known to be regulated by NF-κB, and a pathway analysis identified NF-κB as the most likely regulatory network associated with these changes in the LF arm but not in the FS-containing arms. These results suggest that a LF diet without flaxseed may reduce levels of specific inflammatory CAFs and suggests that the NF-κB pathway may be a mediator of these changes. Cancer Prev Res; 4(10); 1590–8. ©2011 AACR.

EGFR-mutant NSCLC tumor cells with acquired resistance to an EGFR-TKI display augmented IL-6 gene expression.

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Supplementary Tables 1-3 and Table A-1, Figure 1, Appendix and Information from The BATTLE Trial: Personalizing Therapy for Lung Cancer

We previously showed that low-dose fenretinide (200 mg/d) had limited activity in retinoid-resistant oral leukoplakia (34% response rate) possibly because serum drug levels were insufficient to induce retinoid receptor–independent apoptosis. Therefore, we designed the single-arm phase II trial reported here to investigate whether higher-dose fenretinide would improve leukoplakia response over that of our previous study. Leukoplakia patients received fenretinide (900 mg/m2 twice daily) in four 3-week cycles (1 week on drug followed by 2 weeks off). At week 12, clinical responses were determined and blood samples were collected for serum drug level assessments. A planned interim futility analysis led to early trial closure after the initial 15 (of 25 planned) patients because only 3 (20%) had a partial response (stopping rule: ≤4 responses in first 16 patients). Fenretinide was well tolerated—only one grade 3 adverse event (diarrhea) occurred. Serum fenretinide levels changed from 0 (baseline) to 0.122 ± 0.093 μmol/L (week 12). In correlative in vitro studies, high-dose fenretinide inhibited the growth of head and neck cancer cells more and oral leukoplakia cells less than did lower doses of fenretinide. This result is consistent with our clinical finding that high-dose fenretinide did not improve on the historical response rate of lower-dose fenretinide in our previous oral leukoplakia trial.

Perspective on this Article from High-Dose Fenretinide in Oral Leukoplakia

Perspective on this Article from Cyclic AMP-Responsive Element Binding Protein– and Nuclear Factor-κB–Regulated CXC Chemokine Gene Expression in Lung Carcinogenesis

Development of IL-6 knockout EGFR-mutant GEMM tumors.

EGFR-mutant NSCLC EGFR-independent tumor model is refractory to EGFR-TKI treatment.

Supplementary tables S1-S3. Supplementary Table S1: Institutional Tumor Genotyping Strategies. Supplementary Table S2: T790M Genotype by Modality. Supplementary Table S3: EGFR Primary Mutation Detection.

Supplementary Fig. S3 from Algorithmic guided screening of drug combinations of arbitrary size for activity against cancer cells

Supplementary Fig. S1 from Algorithmic guided screening of drug combinations of arbitrary size for activity against cancer cells

Supplementary Methods from Algorithmic guided screening of drug combinations of arbitrary size for activity against cancer cells

Mutation list

Purpose:Malignant pleural mesothelioma (MPM) is considered an orphan disease with few treatment options. Despite multimodality therapy, the majority of MPMs recur and eventually become refractory to any systemic treatment. One potential mechanism underlying therapeutic resistance may be intratumor heterogeneity (ITH), making MPM challenging to eradicate. However, the ITH architecture of MPM and its clinical impact have not been well studied.Experimental Design:We delineated the immunogenomic ITH by multiregion whole-exome sequencing and T-cell receptor (TCR) sequencing of 69 longitudinal MPM specimens from nine patients with resectable MPM, who were treated with dasatinib.Results:The median total mutation burden before dasatinib treatment was 0.65/Mb, similar with that of post-dasatinib treatment (0.62/Mb). The median proportion of mutations shared by any given pair of two tumor regions within the same tumors was 80% prior to and 83% post-dasatinib treatment indicating a relatively homogenous genomic landscape. T-cell clonality, a parameter indicating T-cell expansion and reactivity, was significantly increased in tumors after dasatinib treatment. Furthermore, on average, 82% of T-cell clones were restricted to individual tumor regions, with merely 6% of T-cell clones shared by all regions from the same tumors indicating profound TCR heterogeneity. Interestingly, patients with higher T-cell clonality and higher portion of T cells present across all tumor regions in post-dasatinib–treated tumors had significantly longer survival.Conclusions:Despite the homogeneous genomic landscape, the TCR repertoire is extremely heterogeneous in MPM. Dasatinib may potentially induce T-cell response leading to improved survival.

Supplementary Table S1 from Algorithmic guided screening of drug combinations of arbitrary size for activity against cancer cells

Supplementary Fig. S2 from Algorithmic guided screening of drug combinations of arbitrary size for activity against cancer cells

Purpose: Resistance to VEGFR inhibitors is a major obstacle in the treatment of non–small cell lung cancer (NSCLC). We investigated the cellular mechanisms mediating resistance of NSCLCs to VEGFR tyrosine kinase inhibitors.Experimental Design: We generated murine models of human NSCLC and performed targeted inhibition studies with the VEGFR TKIs cediranib and vandetanib. We used species-specific hybridization of microarrays to compare cancer (human) and stromal (mouse) cell transcriptomes of TKI-sensitive and -resistant tumors. We measured tumor microvascular density and vessel tortuosity to characterize the effects of therapy on the tumor vascular bed. Circulating cytokine and angiogenic factor levels in patients enrolled in VEGFR TKI trials were correlated with clinical outcomes.Results: Murine xenograft models of human lung adenocarcinoma were initially sensitive to VEGFR TKIs, but developed resistance to treatment. Species-specific microarray analysis identified increased expression of stromal-derived hepatocyte growth factor (HGF) as a candidate mediator of TKI resistance and its receptor, c-MET, was activated in cancer cells and tumor-associated stroma. A transient increase in hypoxia-regulated molecules in the initial response phase was followed by adaptive changes resulting in a more tortuous vasculature. Forced HGF expression in cancer cells reduced tumor sensitivity to VEGFR TKIs and produced tumors with tortuous blood vessels. Dual VEGFR/c-MET signaling inhibition delayed the onset of the resistant phenotype and prevented the vascular morphology alterations. In patients with cancer receiving VEGFR TKIs, high pretreatment HGF plasma levels correlated with poorer survival.Conclusions: HGF/c-MET pathway mediates VEGFR inhibitor resistance and vascular remodeling in NSCLC. Clin Cancer Res; 23(18); 5489–501. ©2017 AACR.

The standard treatment for most advanced cancers is multidrug therapy. Unfortunately, combinations in the clinic often do not perform as predicted. Therefore, to complement identifying rational drug combinations based on biological assumptions, we hypothesized that a functional screen of drug combinations, without limits on combination sizes, will aid the identification of effective drug cocktails. Given the myriad possible cocktails and inspired by examples of search algorithms in diverse fields outside of medicine, we developed a novel, efficient search strategy called Medicinal Algorithmic Combinatorial Screen (MACS). Such algorithms work by enriching for the fitness of cocktails, as defined by specific attributes through successive generations. Because assessment of synergy was not feasible, we developed a novel alternative fitness function based on the level of inhibition and the number of drugs. Using a WST-1 assay on the A549 cell line, through MACS, we screened 72 combinations of arbitrary size formed from a 19-drug pool across four generations. Fenretinide, suberoylanilide hydroxamic acid, and bortezomib (FSB) was the fittest. FSB performed up to 4.18 SD above the mean of a random set of cocktails or “too well” to have been found by chance, supporting the utility of the MACS strategy. Validation studies showed FSB was inhibitory in all 7 other NSCLC cell lines tested. It was also synergistic in A549, the one cell line in which this was evaluated. These results suggest that when guided by MACS, screening larger drug combinations may be feasible as a first step in combination drug discovery in a relatively small number of experiments. [Mol Cancer Ther 2009;8(3):521–32]

Supplementary Figure 1: Heatmap of the 68 non-small cell lung cancer cell lines with EGFR wild-type used to train the Sorafenib Sensitivity Signature; Supplementary Figure 2: Heatmap of the 37 patients with EGFR mutant tumors using the Sorafenib Sensitivity signature; yes: disease control at 8 weeks; no: no disease control at 8 weeks.

Supplementary Figure S1-10 and Table S1

Purpose: The T790M gatekeeper mutation in the EGFR is acquired by some EGFR-mutant non–small cell lung cancers (NSCLC) as they become resistant to selective tyrosine kinase inhibitors (TKI). As third-generation EGFR TKIs that overcome T790M-associated resistance become available, noninvasive approaches to T790M detection will become critical to guide management.Experimental Design: As part of a multi-institutional Stand-Up-To-Cancer collaboration, we performed an exploratory analysis of 40 patients with EGFR-mutant tumors progressing on EGFR TKI therapy. We compared the T790M genotype from tumor biopsies with analysis of simultaneously collected circulating tumor cells (CTC) and circulating tumor DNA (ctDNA).Results: T790M genotypes were successfully obtained in 30 (75%) tumor biopsies, 28 (70%) CTC samples, and 32 (80%) ctDNA samples. The resistance-associated mutation was detected in 47% to 50% of patients using each of the genotyping assays, with concordance among them ranging from 57% to 74%. Although CTC- and ctDNA-based genotyping were each unsuccessful in 20% to 30% of cases, the two assays together enabled genotyping in all patients with an available blood sample, and they identified the T790M mutation in 14 (35%) patients in whom the concurrent biopsy was negative or indeterminate.Conclusions: Discordant genotypes between tumor biopsy and blood-based analyses may result from technological differences, as well as sampling different tumor cell populations. The use of complementary approaches may provide the most complete assessment of each patient's cancer, which should be validated in predicting response to T790M-targeted inhibitors. Clin Cancer Res; 22(5); 1103–10. ©2015 AACR.

Supplemental Figures and Tables

Purpose: To report the clinical efficacy of sorafenib and to evaluate biomarkers associated with sorafenib clinical benefit in the BATTLE (Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination) program.Patients and Methods: Patients with previously treated non–small cell lung cancer (NSCLC) received sorafenib until progression or unacceptable toxicity. Eight-week disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) were assessed. Prespecified biomarkers included K-RAS, EGFR, and B-RAF mutations, and EGFR gene copy number. Gene expression profiles from NSCLC cell lines and patient tumor biopsies with wild-type EGFR were used to develop a sorafenib sensitivity signature (SSS).Results: A total of 105 patients were eligible and randomized to receive sorafenib. Among 98 patients evaluable for eight-week DCR, the observed DCR was 58.2%. The median PFS and OS were 2.83 [95% confidence interval (CI), 2.04–3.58] and 8.48 months (95% CI, 5.78–10.97), respectively. Eight-week DCR was higher in patients with wild-type EGFR than patients with EGFR mutation (P = 0.012), and in patients with EGFR gene copy number gain (FISH-positive) versus patients FISH-negative (P = 0.048). In wild-type EGFR tumors, the SSS was associated with improved PFS (median PFS 3.61 months in high SSS vs. 1.84 months in low SSS; P = 0.026) but not with eight-week DCR. Increased expression of fibroblast growth factor-1, NF-κB, and hypoxia pathways were identified potential drivers of sorafenib resistance.Conclusion: Sorafenib demonstrates clinical activity in NSCLC, especially with wild-type EGFR. SSS was associated with improved PFS. These data identify subgroups that may derive clinical benefit from sorafenib and merit investigation in future trials. Clin Cancer Res; 19(24); 6967–75. ©2013 AACR.

Extracellular vesicles (EVs) are known effectors of cell signaling and exchange implicated in cancer development and progression, as well as remodeling and immunomodulation of the tumor microenvironment. EVs are detected in various biofluids and are considered to convey signature features of the cells from which they originate. Clinical assessment of plasma-derived EV molecular contents therefore offers promise of “liquid biopsies” for detection or subtyping of cancers as well as dynamic reporting of tumor status during and after treatment. To date, there have been no comprehensive analyses aimed at interrogating the diverse repertoire of EV protein cargoes in small cell lung carcinoma (SCLC). Using plasmas from a cohort of 18 SCLC cases, 16 lung adenocarcinoma (LUAD) cases and 34 controls matched based on smoking status, age, and sex, EVs were enriched via affinity capture using magnetic beads functionalized with phosphatidyl serine binding protein TIM4 in addition to single-step density flotation ultracentrifugation to deplete abundant protein background. In-depth proteomic profiling was performed on enriched EVs via nano liquid chromatography / high-resolution ion-mobility mass spectrometry. Profiles were intersected with proteomic profiles generated from intratumor EVs dissociated from 12 SCLC patient-derived xenograft (PDX) mouse models as well as EVs isolated from cell-conditioned medias of a panel of 17 SCLC cell lines. A total of 496 proteins were quantified in TIM4pos EVs. Unsupervised hierarchical clustering and principal component analyses showed that TIM4pos EV protein cargoes differentiate SCLC from LUAD and matched controls. Differential analyses identified 117 proteins that were elevated (AUC> 0.60) in TIM4pos SCLC EVs compared to controls. Of these 117 features, 101 (86%) were also elevated (AUC> 0.60) in TIM4pos SCLC EVs compared to TIM4pos LUAD EVs. Intersection of proteins elevated in TIM4pos SCLC EVs with proteomic profiles of intratumor EVs from SCLC PDX models and conditioned media-derived EVs from SCLC cell lines yielded 67 and 92 overlapping proteins, respectively. Overlapping proteins were characterized by YAP1-associated signatures of cytoskeletal re-arrangement and epithelial-to-mesenchymal transition and included a prevalence of neuronal tubulin and actin family members, YWHA family members, and annexins. We developed and applied an optimized EV isolation and proteomics workflow to profile EV protein cargoes and establish circulating EV-associated protein signatures of SCLC with biomarker potential. Our integrated analyses identified novel EV-associated proteins for detection of SCLC that are associated with oncogenic drivers. These studies demonstrate that EVs harbor cancer cell disseminated signatures and that continued efforts to query the EV proteome towards discovery of novel SCLC biomarkers are warranted. Citation Format: Jody Vykoukal, Taketo Kato, Hiroyuki Katayama, Allison Stewart, Ehsan Irajizad, Yining Cai, Fu-Chung Hsiao, Jennifer B. Dennison, Edwin J. Ostrin, Hai T. Tran, Carl M. Gay, Lauren A. Byers, Johannes F. Fahrmann, Samir M. Hanash. Cancer cell derived extracellular vesicles convey protein signatures of small cell lung carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P078.

Purpose: Resistance to VEGFR inhibitors is a major obstacle in the treatment of non–small cell lung cancer (NSCLC). We investigated the cellular mechanisms mediating resistance of NSCLCs to VEGFR tyrosine kinase inhibitors. Experimental Design: We generated murine models of human NSCLC and performed targeted inhibition studies with the VEGFR TKIs cediranib and vandetanib. We used species-specific hybridization of microarrays to compare cancer (human) and stromal (mouse) cell transcriptomes of TKI-sensitive and -resistant tumors. We measured tumor microvascular density and vessel tortuosity to characterize the effects of therapy on the tumor vascular bed. Circulating cytokine and angiogenic factor levels in patients enrolled in VEGFR TKI trials were correlated with clinical outcomes. Results: Murine xenograft models of human lung adenocarcinoma were initially sensitive to VEGFR TKIs, but developed resistance to treatment. Species-specific microarray analysis identified increased expression of stromal-derived hepatocyte growth factor (HGF) as a candidate mediator of TKI resistance and its receptor, c-MET, was activated in cancer cells and tumor-associated stroma. A transient increase in hypoxia-regulated molecules in the initial response phase was followed by adaptive changes resulting in a more tortuous vasculature. Forced HGF expression in cancer cells reduced tumor sensitivity to VEGFR TKIs and produced tumors with tortuous blood vessels. Dual VEGFR/c-MET signaling inhibition delayed the onset of the resistant phenotype and prevented the vascular morphology alterations. In patients with cancer receiving VEGFR TKIs, high pretreatment HGF plasma levels correlated with poorer survival. Conclusions: HGF/c-MET pathway mediates VEGFR inhibitor resistance and vascular remodeling in NSCLC. Clin Cancer Res; 23(18); 5489–501. ©2017 AACR.

Background: We have previously demonstrated that the presence of a proinflammatory peripheral cytokine milieu correlates with high levels of circulating neutrophils at the time of surgery and reduced overall survival and recurrence-free survival. In this study, we hypothesized that functional immune features or states in circulation may indicate early tumor recurrence when assessed longitudinally. We also investigated whether potential deficiencies in peripheral immune functionality at the time of lung cancer resection could identify correlates with subsequent outcome. Methods: We performed flow cytometry and luminex profiling of blood samples collected from patients with stage I-IIIA resected NSCLC (n=150) and enrolled on the Immunogenomic profiling of NSCLC (ICON) prospective protocol. Patient characteristics include 75 adenocarcinomas, 30 squamous and 12 mixed or other histologies. Only patients who underwent primary cancer resection without neoadjuvant therapy were included. At a median follow up of 18.2 months, 37 patients had disease recurrence. Blood was collected at the time of primary lung cancer resection, at 4 weeks, and 4 months thereafter with PBMCs utilized for flow cytometry and plasma for cytokine assessment. Changes in cytokines were assessed by normalizing to baseline levels. Results: Larger tumors as well as advanced clinical and pathological stages were associated with higher frequencies of proliferating Ki67+CD4+ and Ki67+CD8+ T cells in circulation at time of resection, suggesting an activated circulating immune response. We identified novel strong correlations in the plasma between soluble BTLA and Tim3 (r= 0.87, p=1.74e-140), PD1 and CD80 (r=0.72, p=6.41e-74) and moderate correlations between soluble PD1 and PDL1 (r=0.39, p=8.41e-18). Efforts are ongoing to determine the association of specific circulating immune states with the presence of these soluble receptors. CD8+Tim3+ T cells (FC=5.2%, p=0.028) and CTLA4+NK cells (FC=47.7%, p=0.002) as well as CTLA4+Tregs (FC=25.2%, p=0.05) were found to be increased in circulation at pre-recurrence time points (either 4 weeks or 4 months) relative to resection. This suggests the emergence of a suppressive cell type as well as induction of specific checkpoint receptors on effector cells that correlate with tumor recurrence. Finally, we identified a cytokine signature associated with recurrence by testing three sets within the ICON cohort with a training set AUC = 0.76 and the test set AUC=0.72, which was validated in a third set of patients yielding an AUC = 0.76. Conclusions: We identified circulating immune features associated with initial tumor size and overall stage as well as unique associations among soluble proteins. Increased presence of potentially inhibited or suppressed CD8+ T cells and NK cells as well as Tregs are suggestive of mechanisms of immune suppression relative to tumor recurrence. Citation Format: Younghee Lee, Daniel McGrail, Hai Tran, Mayra E. Vasquez, Carlos Ramos, Alexandre Reuben, Ara A. Vaporciyan, Annikka Weissferdt, Chantale Bernatchez, Tina Cascone, Ignacio I. Wistuba, Jianjun Zhang, John Heymach, Marcelo V. Negrao, Don L. Gibbons, Boris Sepesi, Cara L. Haymaker. Circulating biomarkers are associated with recurrence following complete resection of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1670.

Purpose: VEGF pathway inhibitors have been investigated as therapeutic agents in the treatment of non–small cell lung cancer (NSCLC) because of its central role in angiogenesis. These agents have improved survival in patients with advanced NSCLC, but the effects have been modest. Although VEGFR2/KDR is typically localized to the vasculature, amplification of KDR has reported to occur in 9% to 30% of the DNA from different lung cancers. We investigated the signaling pathways activated downstream of KDR and whether KDR amplification is associated with benefit in patients with NSCLC treated with the VEGFR inhibitor vandetanib. Methods: NSCLC cell lines with or without KDR amplification were studied for the effects of VEGFR tyrosine kinase inhibitors (TKI) on cell viability and migration. Archival tumor samples collected from patients with platinum-refractory NSCLC in the phase III ZODIAC study of vandetanib plus docetaxel or placebo plus docetaxel (N = 294) were screened for KDR amplification by FISH. Results: KDR amplification was associated with VEGF-induced activation of mTOR, p38, and invasiveness in NSCLC cell lines. However, VEGFR TKIs did not inhibit proliferation of NSCLC cell lines with KDR amplification. VEGFR inhibition decreased cell motility as well as expression of HIF1α in KDR-amplified NSCLC cells. In the ZODIAC study, KDR amplification was observed in 15% of patients and was not associated with improved progression-free survival, overall survival, or objective response rate for the vandetanib arm. Conclusions: Preclinical studies suggest KDR activates invasion but not survival pathways in KDR-amplified NSCLC models. Patients with NSCLC whose tumor had KDR amplification were not associated with clinical benefit for vandetanib in combination with docetaxel. Clin Cancer Res; 22(8); 1940–50. ©2015 AACR.

Purpose: The T790M gatekeeper mutation in the EGFR is acquired by some EGFR-mutant non–small cell lung cancers (NSCLC) as they become resistant to selective tyrosine kinase inhibitors (TKI). As third-generation EGFR TKIs that overcome T790M-associated resistance become available, noninvasive approaches to T790M detection will become critical to guide management. Experimental Design: As part of a multi-institutional Stand-Up-To-Cancer collaboration, we performed an exploratory analysis of 40 patients with EGFR-mutant tumors progressing on EGFR TKI therapy. We compared the T790M genotype from tumor biopsies with analysis of simultaneously collected circulating tumor cells (CTC) and circulating tumor DNA (ctDNA). Results: T790M genotypes were successfully obtained in 30 (75%) tumor biopsies, 28 (70%) CTC samples, and 32 (80%) ctDNA samples. The resistance-associated mutation was detected in 47% to 50% of patients using each of the genotyping assays, with concordance among them ranging from 57% to 74%. Although CTC- and ctDNA-based genotyping were each unsuccessful in 20% to 30% of cases, the two assays together enabled genotyping in all patients with an available blood sample, and they identified the T790M mutation in 14 (35%) patients in whom the concurrent biopsy was negative or indeterminate. Conclusions: Discordant genotypes between tumor biopsy and blood-based analyses may result from technological differences, as well as sampling different tumor cell populations. The use of complementary approaches may provide the most complete assessment of each patient's cancer, which should be validated in predicting response to T790M-targeted inhibitors. Clin Cancer Res; 22(5); 1103–10. ©2015 AACR.

Background: Tumor genomic information from tissue has been the standard of practice for identifying actionable molecular alterations. The same genomic profiling is also widely available by a non-invasive blood test from circulating tumor DNA (ctDNA). We hypothesized that patients with advanced non-small cell lung cancer (NSCLC) with actionable oncogenic driver mutations with either low or high variant allelic frequency (%VAF) identified by ctDNA would have similar clinical benefits after treatment with targeted therapies. Methods: In this retrospective study (UTMDACC IRB-approved protocol PA16-0061), patients with a targetable mutation identified by ctDNA NGS assay who received an FDA-approved therapy were identified in our GEMINI database. All potential patients were individually reviewed and information confirmed with review of their electronic medical records. Blood based genomic profiling was completed by NGS of ctDNA with a panel of up to 70 cancer-related genes at a CLIA-certified lab (Guardant360, Guardant Health, Redwood City, CA). The %VAF was reported for each mutation identified. We defined low group with VAF of 0.01% to 0.99% and high group with VAF > 1%. Tumor measurements were assessed using RECIST V1.1. For comparisons analysis, Kaplan-Meier estimator was used to estimate progression free survival (PFS) with Mantel-Cox and un-paired t-test was used for tumor responses. Results: Eighty-nine patients with advanced NSCLC treated with an FDA approved therapy based on ctDNA genomic profiling were identified and the results are summarized in table below: LOW %VAFHIGH %VAFP valuegroup (n=39)group (n=50)Gender (Female/Male)20/1928/22Age (median, range)54(27-85)63(29-89)Targetable mutations:EGFR2246ALK114MET exon 1420ROS120BRAF20Line of therapy (median, range)2(1-7)2(1-7)ALL PatientsTumor response (mean, range)-37.23% (-100 to +38)-31.08% (-100 to +59)0.42PFS (median, range)215 (27 – 848) days280 (43 – 894) days0.70EGFR onlyTumor response (mean, range)-24.86% (-100 to +16)-23.54% (-100 to +59)0.88PFS (median, range)189 (57 – 848) days293 (43-894) days0.23 Conclusions: In this analysis of patients who were treated with an FDA approved therapy based on genomic results from ctDNA profiling, there were no differences in either tumor response (P value = 0.42) or PFS (P value = 0.70) between those patients with low and high ctDNA as assessed by %VAF of identified targetable mutation. Similar findings were also observed in the analysis of the EGFR only subgroup. This study reveals patients with low %VAF received comparable clinical benefits as those with high %VAF and no deleterious harm was observed from treating patients with low %VAF with appropriate targeted therapy. Citation Format: Hai T. Tran, Vincent K. Lam, Mayra Vasquez, Yasir Elamin, Victoria Raymond, Lingzhi Hong, George Blumenschein, Brett Carter, Richard Lanman, Mehmet Altan, Ann Tsao, Lauren Byers, Don Gibbons, Jianjun Zhang, John V. Heymach. Comparable clinical benefits between low and high variant allelic frequency in NSCLC patients treated with targeted therapies based on molecular alterations from ctDNA NGS [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 787.

Purpose: To report the clinical efficacy of sorafenib and to evaluate biomarkers associated with sorafenib clinical benefit in the BATTLE (Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination) program. Patients and Methods: Patients with previously treated non–small cell lung cancer (NSCLC) received sorafenib until progression or unacceptable toxicity. Eight-week disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) were assessed. Prespecified biomarkers included K-RAS, EGFR, and B-RAF mutations, and EGFR gene copy number. Gene expression profiles from NSCLC cell lines and patient tumor biopsies with wild-type EGFR were used to develop a sorafenib sensitivity signature (SSS). Results: A total of 105 patients were eligible and randomized to receive sorafenib. Among 98 patients evaluable for eight-week DCR, the observed DCR was 58.2%. The median PFS and OS were 2.83 [95% confidence interval (CI), 2.04–3.58] and 8.48 months (95% CI, 5.78–10.97), respectively. Eight-week DCR was higher in patients with wild-type EGFR than patients with EGFR mutation (P = 0.012), and in patients with EGFR gene copy number gain (FISH-positive) versus patients FISH-negative (P = 0.048). In wild-type EGFR tumors, the SSS was associated with improved PFS (median PFS 3.61 months in high SSS vs. 1.84 months in low SSS; P = 0.026) but not with eight-week DCR. Increased expression of fibroblast growth factor-1, NF-κB, and hypoxia pathways were identified potential drivers of sorafenib resistance. Conclusion: Sorafenib demonstrates clinical activity in NSCLC, especially with wild-type EGFR. SSS was associated with improved PFS. These data identify subgroups that may derive clinical benefit from sorafenib and merit investigation in future trials. Clin Cancer Res; 19(24); 6967–75. ©2013 AACR.

Fibroblast growth factor (FGF) signaling regulates cell proliferation, differentiation, survival, angiogenesis, and wound healing. Compelling evidence for deregulated FGF signaling in tumorigenesis continues to emerge, and a growing body of research suggests that FGF may also play an integral role in the resistance to anti-VEGF therapy. Although agents targeting FGF signaling are early in development, the potential to target both the VEGF and FGF pathways may translate into improvements in the clinical care of cancer patients. Clin Cancer Res; 17(19); 6130–9. ©2011 AACR.

Diet, nutritional status, and certain dietary supplements are postulated to influence the development and progression of prostate cancer. Angiogenesis and inflammation are central to tumor growth and progression, but the effect of diet on these processes remains uncertain. We explored changes in 50 plasma cytokines and angiogenic factors (CAF) in 145 men with prostate cancer enrolled in a preoperative, randomized controlled phase II trial with four arms: control (usual diet), low-fat (LF) diet, flaxseed-supplemented (FS) diet, and FS+LS diet. The mean duration of dietary intervention was 30 to 31 days. Among the individual arms, the largest number of significant changes (baseline vs. preoperative follow-up) was observed in the LF arm, with 19 CAFs decreasing and one increasing (P < 0.05). Compared with the control arm, 6 CAFs—including proangiogenic factors (stromal-cell derived-1α) and myeloid factors (granulocyte-colony-stimulating factor, macrophage colony-stimulating factor)—all decreased in the LF arm compared with controls; three and four CAFs changed in the FS and FS+LF arms, respectively. Weight loss occurred in the LF arms and significantly correlated with VEGF decreases (P < 0.001). The CAFs that changed in the LF arm are all known to be regulated by NF-κB, and a pathway analysis identified NF-κB as the most likely regulatory network associated with these changes in the LF arm but not in the FS-containing arms. These results suggest that a LF diet without flaxseed may reduce levels of specific inflammatory CAFs and suggests that the NF-κB pathway may be a mediator of these changes. Cancer Prev Res; 4(10); 1590–8. ©2011 AACR.

The standard treatment for most advanced cancers is multidrug therapy. Unfortunately, combinations in the clinic often do not perform as predicted. Therefore, to complement identifying rational drug combinations based on biological assumptions, we hypothesized that a functional screen of drug combinations, without limits on combination sizes, will aid the identification of effective drug cocktails. Given the myriad possible cocktails and inspired by examples of search algorithms in diverse fields outside of medicine, we developed a novel, efficient search strategy called Medicinal Algorithmic Combinatorial Screen (MACS). Such algorithms work by enriching for the fitness of cocktails, as defined by specific attributes through successive generations. Because assessment of synergy was not feasible, we developed a novel alternative fitness function based on the level of inhibition and the number of drugs. Using a WST-1 assay on the A549 cell line, through MACS, we screened 72 combinations of arbitrary size formed from a 19-drug pool across four generations. Fenretinide, suberoylanilide hydroxamic acid, and bortezomib (FSB) was the fittest. FSB performed up to 4.18 SD above the mean of a random set of cocktails or “too well” to have been found by chance, supporting the utility of the MACS strategy. Validation studies showed FSB was inhibitory in all 7 other NSCLC cell lines tested. It was also synergistic in A549, the one cell line in which this was evaluated. These results suggest that when guided by MACS, screening larger drug combinations may be feasible as a first step in combination drug discovery in a relatively small number of experiments. [Mol Cancer Ther 2009;8(3):521–32]

Non–small cell lung cancer (NSCLC) cells with somatic mutations in K-ras recruit to the tumor a variety of cell types (hereafter collectively termed “stromal cells”) that can promote or inhibit tumorigenesis by mechanisms that have not been fully elucidated. Here, we postulated that stromal cells in the tumor microenvironment alter the tumor cell secretome, including those proteins required for tumor growth and dissemination, and we developed an in vitro model to test this hypothesis. Coculturing a murine K-ras mutant lung adenocarcinoma cell line (LKR-13) with a murine lung stromal cell (macrophage, endothelial cell, or fibroblast) enhanced stromal cell migration, induced endothelial tube formation, increased LKR-13 cell proliferation, and regulated the secretion of proteins involved in angiogenesis, inflammation, cell proliferation, and epithelial-to-mesenchymal transition. Among these proteins, CXCL1 has been reported to promote NSCLC development, whereas interleukin-18 (IL-18) has an undefined role. Genetic and pharmacologic strategies to inhibit CXCL1 and IL-18 revealed that stromal cell migration, LKR-13 cell proliferation, and LKR-13 cell tumorigenicity required one or both of these proteins. We conclude that stromal cells enhanced LKR-13 cell tumorigenicity partly through their effects on the secretome of LKR-13 cells. Strategies to inhibit tumor/stromal cell interactions may be useful as therapeutic approaches in NSCLC patients. [Cancer Res 2008;68(17):7237–45]

Purpose: A phase I trial of BMS-214662, a selective farnesyltransferase inhibitor with significant preclinical antitumor activity in which drug was given as a weekly 1-hour infusion for four of six weeks, was conducted to evaluate the tolerability, pharmacokinetics, and pharmacodynamic effect on farnesyltransferase activity in peripheral blood mononuclear cells. Experimental Design: BMS-214662 was given to 27 patients with solid tumors at 10 escalating dose levels (28-220 mg/m2) allowing intrapatient dose escalation; pharmacokinetics and pharmacodynamics were done at the first seven dose levels. Results: Grade 4 neutropenia (four patients) was the most common dose-limiting toxicity followed by aminotransferase elevation (grade 3 alanine aminotransferase and grade 4 aspartate aminotransferase) and grade 3 dehydration. Most frequent toxicities were neutropenia in 11 (14%), anemia in 15 (19%), fatigue in 9 (12%), and nausea and diarrhea in 6 (8%) of courses, respectively. One minor response lasting 18 weeks in a patient with non–small cell lung cancer, serum calcitonin level reduction accompanied by disease stabilization in two of four patients with medullary thyroid carcinoma, and stable disease in 16 of 25 evaluable patients was seen. No correlation was observed between dose and Cmax, total body clearance (mean, 26.15 ± 10.88 L per hour per m2), volume of distribution at steady state (mean, 39.51 ± 17.91 L/m2), or half-life (mean, 2.63 ± 1.81 hours); a moderate correlation existed between dose given and systemic drug exposure (AUC). Substantial inhibition of peripheral blood mononuclear cell farnesyltransferase activity but near complete recovery by 24 hours was seen. Conclusion: BMS-214667 was well tolerated as a weekly 1-hour i.v. infusion for four of six weeks with evidence of pharmacodynamic effect. The study was terminated before maximum tolerated dose was reached. Alternative schedules of drug administration might result in improved pharmacodynamic profile.

PURPOSE: Host immune activation is believed to be a major player of radiation pneumonitis (RP). Several cytokine and angiogenic factors (CAFs) have been studied as correlative markers and therapeutic targets for RP; however, studies are sparse in the setting of prospective trials and high-grade toxicity (Grade≥3) endpoints. We examined the predictive value of several CAFs in combination with clinical factors for RP in the context of a Phase II, prospective randomized study of intensity modulated RT (IMRT) versus proton therapy for locally advanced NSCLC (n=178) as well as a prospective laboratory cohort (n=43). METHODS: From July 2009 to April 2014, 221 patients were treated with proton therapy or IMRT to doses of 60-74 Gy equivalents with concurrent platinum-doublet chemotherapy. Primary endpoints were symptomatic RP (Grade≥2) and RP requiring intervention (Grade≥3). Plasma circulating levels of IL-6, IL-10, IL-8, IL-17, IL-2, IL-1β, and IL-1α were measured at baseline and post-treatment (median time 48 days post-RT) by multiplex electrochemiluminescence immunoassay. Clinical characteristics were evaluated for associations with Grade≥3 RP: histology, age, smoking status, mean lung dose (MLD), chemotherapy, stage, performance status, and RT technique. CAFs with >50% undetectable samples were removed from analysis, and samples below the lower limit of quantitation (LLQ) were transformed to the mean of zero and the LLQ. Predictive values of clinical and cytokine factors were evaluated in univariate and multivariate analyses via Cox proportional hazards modeling. RESULTS: Of the 221 patients, 42 (19.0%) developed Grade 2 RP, and 22 (9.95%) developed Grade≥3 RP. Both IL-1α and IL-1β had >50% of undetectable samples and were removed from analysis. Pre-treatment level of IL-6 ≥4.00 pg/mL was associated with Grade≥3 RP (HR 2.487; p=.048), as was post-treatment level of IL-10 ≥0.50 pg/mL (HR 2.931; p=.020). On multivariate analysis, the factors associated with Grade≥3 RP development were post-treatment level of IL-10 ≥0.50 pg/mL (HR 3.332; p=.015) and MLD ≥20 Gy (HR 5.864; p CONCLUSIONS: In this preliminary analysis of prospectively collected CAFs, post-radiation IL-10 level was significantly associated with Grade≥3 RP and, if validated, may serve as a predictor of this toxicity in combination with MLD. IL-10 plays an important role in the inflammatory response and is potentially targetable, warranting further investigation. Citation Format: Matthew S. Ning, Ting Xu, Daniel Gomez, Hai T. Tran, Ritsuko Komaki, Xiong Wei, Steven H. Lin, Stephen Hahn, Eric Prado, Jianjun Zhang, John V. Heymach, Zhongxing Liao. Circulating cytokine levels predictive of Grade≥3 radiation pneumonitis: Preliminary secondary analysis of a randomized controlled trial of definitive chemoradiation in locally advanced NSCLC [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 4745. doi:10.1158/1538-7445.AM2017-4745

Introduction: We present serum correlative analysis from a phase II randomized trial to identify markers associated with progression free survival (PFS) in NSCLC. Methods: NSCLC patients presenting with oligometastatic disease (≤3 metastases) were enrolled on a multi-institution phase II randomized trial (NCT01725165). Patients who did not progress after ≥3 months of first-line systemic therapy were randomized to upfront local consolidative therapy (LCT arm) to all sites of disease or at later progression (no-LCT arm). For correlative analyses, peripheral blood was collected from consenting patients after completion of initial systemic therapy but prior to randomization (baseline) and at each follow up. Serum cytokine and angiogenesis factors (CAFs) were analyzed via magnetic bead-based multiplex assay (Merck Millipore). Peripheral blood T cell receptor (TCR) repertoire was profiled by immunoSEQ (Adaptive Biotechnologies). Peripheral cell-free DNA (cfDNA) was subjected to next generation sequencing of a panel of cancer genes. Comparisons of PFS, the primary endpoint of this study, were conducted via log-rank test. Results: After enrollment of 74 patients with a median follow up of 19 months, this study was closed early after the Data Safety Monitoring Committee review determined efficacy in the LCT arm. Of 49 randomized patients, correlative blood draws were conducted on 33 patients. Considering all patients, high baseline EGF (cutpoint 50 pg/ml; median PFS: 5.7 vs 19.8 mo), Eotaxin (cutpoint 110 pg/ml; median PFS: 5.7 vs 21 mo), TNF alpha (cutpoint 8 pg/ml; median PFS: 3.6 vs 17.9 mo), or low baseline GRO (cutpoint 600 pg/ml; median PFS: 17.9 vs 3.6 mo) were associated with longer PFS (all P Conclusions: In this exploratory analysis, we identified baseline CAFs associated with PFS. These finding are being validated in a larger cohort of patients. If validated, CAFs could be a biomarker to select patients with oligometastatic NSCLC for aggressive local treatment. Citation Format: Chad Tang, John V. Heymach, Alexandre Reuben, Hai Tran, James Welsh, Jennifer Wargo, Zhongxing Liao, Anne Tsao, Eric Prado, Stephen Swisher, Jianjun Zhang, Daniel Gomez. Correlative analysis of systemic markers from a randomized trial assessing local consolidative therapy for non-small cell lung cancer (NSCLC) patients with oligometastatic disease [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 2774. doi:10.1158/1538-7445.AM2017-2774

Therapeutic strategies to target EGFR tyrosine kinase inhibitor (TKI) resistance mediated by mechanisms other than T790M are a major clinical challenge. Studies have implicated IL-6 as a mediator of EGFR TKI resistance, and IL-6 is known to be regulated by adrenergic receptors (AR) in some cancers. We investigated whether adrenergic pathways can promote T790M-independent EGFR TKI resistance in preclinical models and in clinical studies. We found that β2-AR was highly expressed in our panel of 119 cell lines and in NSCLC clinical specimens. Activation of β-ARs by stress hormones such as norepinephrine (NE) induced a dramatic rise in IL-6, and this occurred through β2-ARs. β-AR inhibitors (i.e. propranolol), but not α-AR inhibitors, blocked IL-6 induction. Analysis of downstream signaling pathways revealed that β2-ARs induced IL-6 expression through activation of adenylyl cyclase, p90RSK and CREB. To identify novel signaling pathways modulated by ARs, we stimulated NSCLC cell lines with NE and analyzed protein lysates by RPPA to detect expression and activation of >100 proteins. β-AR signaling inactivated the tumor suppressor LKB1 through phosphorylation of S428 and subsequently increased mTOR activity. LKB1 inactivation was critical for IL-6 induction. This finding is important as LKB1 loss is known to be a driver of NSCLC resistance and metastasis. Moreover, we found that β2-AR activation promoted EGFR TKI resistance in cell lines and in mouse models of EGFR mutant NSCLC. The effect of β-AR on EGFR TKI resistance was blocked by the addition of the beta blocker propranolol or IL-6 antibodies, in vitro and in vivo. Consistent with our preclinical studies, in the phase III ZEST clinical study testing erlotinib vs vandetanib, we found that high plasma levels of IL-6 was associated with a worse PFS and OS in the erlotinib arm. In addition, we found that circulating levels of IL-6 were significantly lower in NSCLC patients incidentally receiving beta blockers in the BATTLE trial. Finally, we analyzed the influence of incidental beta blocker use in the LUX-Lung3 study testing afatinib vs chemotherapy in EGFR mutant NSCLC patients. In patients not receiving beta blockers, the median PFS was 11.1 and 6.9 months for afatinib and chemotherapy, respectively, and afatinib improved PFS with a hazard ratio (HR) of 0.60. In patients receiving beta blockers, the median PFS was 13.6 and 2.5 for afatinib and chemotherapy, respectively, and afatinib improved PFS with a HR of 0.25. In conclusion, our preclinical and clinical data provide evidence that β2-AR activation can upregulate IL-6 in EGFR mutant+ NSCLC, modulate the LKB1/AMPK/mTOR axis, and promote EGFR TKI resistance. Moreover, EGFR mutant+ patients using beta blockers had greater relative PFS benefit from afatinib vs chemotherapy compared with those not using beta blockers, supporting future clinical testing of EGFR TKIs in combination with beta blockers. Citation Format: Monique B. Nilsson, Huiying Sun, Lixia Diao, Pan Tong, Youhong Fan, Hai Tran, Diane Liu, Guillermo Armaiz Pena, Jing Wang, Phil Rowe, Alan Webster, Jack Lee, Daniel Gomez, Waun Ki Hong, Ignacio Wistuba, Anil Sood, John Heymach. Beta blockers abrogate EGFR TKI resistance induced by adrenergic receptor-mediated upregulation of IL-6 and modulation of the LKB1/AMPK/mTOR axis. [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 4662.

Background: The presence of circulating tumor cells (CTCs) has been established as a prognostic indicator in a few metastatic cancers. Although technically challenging, isolation and characterization of CTCs at the protein or nucleic acid level has significant potential to enable the development of minimally invasive methods to detect molecular alterations for personalized cancer targeted therapies. The goal of this study was to demonstrate the ability to enrich phenotypically heterogeneous population of CTCs and detect EGFR and KRAS mutations in isolated CTCs obtained from peripheral blood of patients with Non-small cell lung cancer (NSCLC) using ApoStream®, an antibody-independent CTC capture device based on dielectrophoresis technology. Methods: For condition optimization, blood samples from a small cohort of cancer patients (n = 8) were collected under an IRB approved protocol and processed with multiple ApoStream® operating frequencies (45, 55, 65 and 85 kHz). To evaluate clinical feasibility, blood samples (8 mL) from 35 additional NSCLC patients (with or without targetable “driver” mutations) and 10 normal, healthy donor controls were collected and processed using the optimized ApoStream® conditions. CTC enumeration was performed using a multiplex phenotyping panel targeting hematopoietic (CD45/CD15), epithelial (EpCAM/CK/E-Cadherin) and mesenchymal (Vimentin/N-cadherin/Twist-1) markers. In addition, mutation detection assays were validated for EGFR (E746_A750, L858R, and T790M) and KRAS (G12C, and G12S) with mutant DNA spiked into wild type DNA at multiple dilutions using the droplet-based RainDrop® digital PCR system. Results: An ApoStream® operating frequency of 85 kHz resulted in higher CTC counts and hence was selected for processing samples for CTC enumeration assay and a lower operating frequency of 65 kHz was selected to provide higher purity for processing samples for mutation analysis. Preliminary data demonstrated detectable CTCs (above healthy control baseline of 4 CTCs per mL blood) in 7 out of 10 patient samples processed to date (mean = 24; median = 14 and range = 5-100 CTCs per mL blood). CTCs isolated from all 7 patients exhibited mesenchymal phenotype. In addition, two patients also showed CTCs of epithelial phenotype demonstrating that the ApoStream® platform enriches phenotypically heterogeneous CTCs. Sensitivity of mutation detection determined from dilution series was found to be assay dependent; limit of quantitation (LOQ) for EGFR and KRAS mutations were found to be 0.01% or higher while the linearity for all the assays demonstrated a linear regression coefficient (R2) greater than 0.95 except for EGFR L858R which requires further analysis. Preliminary data from 8 NSCLC patient samples demonstrated the ability to detect mutations in ApoStream® enriched CTCs. Enumeration and mutation analysis is in progress for the remaining patient samples. Conclusion: We demonstrated the ability to isolate, enumerate and define epithelial and mesenchymal phenotypes of CTCs and to detect oncogenic mutations in CTCs isolated from NSCLC patient samples using ApoStream®. Funded by National Cancer Institute under Contract No. HHSN261201300070C. Citation Format: Priya Balasubramanian, Vishal Gupta, David Hasegawa, Carmen Y. Lam, Ran An, Swetha Pratyusha Gunturu, Hai T. Tran, John V. Heymach, Darren W. Davis. Epithelial and mesenchymal phenotypic characterization and mutation detection in circulating tumor cells isolated from peripheral blood of non-small cell lung cancer patients with ApoStream® technology. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C18.

Background: A variety of methods for capture of rare CTCs of epithelial origin are available; most employ antibodies to epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK). Using a classic phenotypic definition, a CTC is a nucleated, CK (+), CD45(-) cell. However, some CTCs may elude capture as they originate from primary tumor cells that have undergone epithelial-mesenchymal transition (EMT). We report here the use of ApoStream™, a novel dielectrophoresis field-flow-assisted, antibody-free method to isolate CTCs from blood. Methods: Blood was collected from consented NSCLC patients and processed using ApoStream™. For CTC enumeration comparison, the CellSearch® FDA-approved kit was used. Isolated cells were evaluated with a multiplexed immunofluorescent assay and laser scanning cytometry was applied to identify multiple combinations of positive and/or negative staining for CK/CD45/DAPI and EpCAM. To determine specific EGFR mutations from captured CTCs, samples were analyzed using Improved and Complete Enrichment with CO-amplification at Lower Denaturation temperature (ICE COLD-PCR). Results: Blood samples from 40 NSCLC patients and 12 healthy volunteers were processed. In the normal, healthy volunteers, ApoStream™ isolated 0-1 CK(+)/CD45(-) cells and 0-33 CK(+)/CD45(+) cells. From the 38 of 40 NSCLC patients, ApoStream™ identified 0 to 65 CK(+)/CD45(-) CTCs, 2 samples failed in processing. Additionally, ApoStream™ recovered 37-3536 CK(-)/CD45(-) and 4-10702 CK(+)/CD45(+) cells. EpCAM expression was detected in 7-100% of CK(+)/CD45(-) and 0-5% of CK(-)/CD45(-) cells, and 18-100% of CK(+)/CD45(+) cells. In comparison, CellSearch® isolated 0 to 13 EpCAM(+)/CK(+)/CD45(-) CTCs in 7 patient samples tested. From our whole-blood spiked cancer cell (H1600, H1975) experiments, CTC recovery ranged from 13% to 60% with detection of EGFR mutations in as low as 10 recovered cells by ICE COLD PCR. From the 35 patients where CTCs were isolated by ApoStream, ICE COLD PCR correctly identified mutation status in 12 cases with EGFR exon 19 deletions (5), exon 21 - L858R(2) and wild type in 5 cases. There were 6 cases with either exon 18 or 20 mutation from tissue anlaysis that tested negative for exon 19 and 21 by ICE COLD PCR; exon 18 and 20 were not tested at this time. Mutation status was not detected in 16 cases when compared to tumor tissue analysis by Sanger sequencing. In 1 case, tissue revealed exon 19 - 15bp deletion meanwhile, CTC-ICP detected mutation at exon 21. Mutations were not tested in 5 CTC samples. Conclusions: The ApoStream™ platform enriched EpCAM(+) and EpCAM(-) CTCs from the blood of NSCLC patients demonstrating utility in recovering cancer cells with multiple phenotypes. From a subset of samples, higher number of CK(+)/CD45(-) cells were recovered by ApoStream™ than CellSearch®. Furthermore, from recovered CTCs, detection of EGFR mutations was possible indicating the clinical relevance and utility of CTCs as an alternative to tissue biopsy. Citation Format: Hai T. Tran, Tsao S. Anne, Katherine Richardson, Ben Legendre, Asifa Haider, Darren Davis, John Heymach. The use of an antibody independent method, ApoStream™, to isolate circulating tumor cells (CTCs) isolated from non-small cell lung cancer patients and identification of EGFR mutations. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C16.

Background: A variety of methods for capture of rare CTCs of epithelial origin are available; most employ antibodies to epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK). Using a classic phenotypic definition, a CTC is nucleated, CK(+), CD45(-) cell. However, some CTCs may elude capture as they originate from primary tumor cells which have undergone epithelial-mesenchymal transition (EMT). We report here the use of ApoStream™, a novel dielectrophoresis field-flow-assisted, antibody-free method to isolate CTCs from blood. Methods: Blood was collected from consented NSCLC patients and processed using ApoStream™. For CTC enumeration comparison, the CellSearch® FDA-approved kit was used. Isolated cells were evaluated with a multiplexed immunofluorescent assay and laser scanning cytometry was applied to identify multiple combinations of positive and/or negative staining for CK/CD45/DAPI and EpCAM. To determine specific EGFR mutations from captured CTCs, samples were analyzed using Improved and Complete Enrichment with CO-amplification at Lower Denaturation temperature (ICE COLD-PCR). Results: Blood samples from 37 NSCLC patients and 8 healthy volunteers were processed and the results of the cell populations isolated using ApoStream™ are summarized in table. EGFR mutations [exon 19 deletion and exon 21 L858R] were determined and found to be concordant when compared to tumor tissue analysis by Sanger sequencing. Conclusions: The ApoStream™ platform enriched EpCAM(+) and EpCAM(-) CTCs from the blood of NSCLC patients demonstrating utility in recovering cancer cells with multiple phenotypes. From recovered CTCs, detection of EGFR mutations was possible indicating the clinical relevance and potential utility of CTCs as an alternative to tissue biopsy. Additional sensitivity and complete mutation analysis will be presented. (a portion of this data was presented at 2013 ASCO Annual Meeting, Chicago, IL, USA) Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C29. Citation Format: Hai T. Tran, Vladislava Melnikova, Anne S. Tsao, Frank V. Fossella, Faye M. Johnson, Vali Papadimitrakoupoulou, Katherine Richardson, Marcia E. Lewis, Ben Legendre, Kenna L. Anderes, Darren W. Davis, John V. Heymach. Characterization and identification of specific EGFR mutations in circulating tumor cells (CTCs) isolated from non-small cell lung cancer patients using an antibody independent method, ApoStream™: An update report. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C29.

Targeted therapies designed to inhibit the vascular endothelial growth factor (VEGF) pathway have been extensively evaluated in the treatment of malignancies including Non-Small Cell Lung Cancer (NSCLC). VEGF pathway inhibitors such as bevacizumab, or the multitargeted receptor tyrosine kinase inhibitors (TKIs) vandetanib and sorafenib, have been shown to prolong progression-free survival (PFS) and/or overall survival (OS). These benefits, however, have been modest, seen only in subsets of patients. Thus, predictive markers for identifying which patients are likely to benefit are critically needed. Although expression of VEGF receptor-2 (VEGFR-2, also known as KDR) was initially thought to primarily occur in endothelial cells, VEGFR-2 has been detected on malignant cells, including lung cancer cells, and in NSCLC, overexpression of VEGFR-2 on tumor cells is associated with a poor clinical outcome. Amplification of KDR has been detected in lung cancer specimens at a relatively high frequency (9% and 32%). The consequences of KDR copy number gains (CNGs) are not yet understood. Recently, we have shown that NSCLC cell lines with KDR copy number gains (CNGs) were associated with in vitro resistance to platinum chemotherapy, and KDR CNG predicted worse overall survival in patients who received platinum adjuvant therapy but not in untreated patients. We investigated the hypothesis that NSCLC tumor cells with KDR CNG display increased sensitivity to VEGFR TKIs compared to tumor cells without KDR CNG. In tumor cell lines with KDR CNG, treatment with exogenous VEGF ligand enhanced cell motility and this was inhibited by VEGFR blockade with TKIs. Multiple receptor tyrosine kinases have been shown to drive HIF-1α levels, and NSCLC cells with KDR CNG express elevated levels of HIF-1α in normoxic conditions compared to NSCLC cell lines without KDR CNG. Here, we show that in NSCLC cell lines with KDR CNG, VEGFR TKIs decreased protein levels of HIF-1α and HIF-1α- regulated proteins. Furthermore, we report a clinical case in which a NSCLC patient with KDR CNG as determined by SNP array had a partial response to VEGFR inhibition with sorafenib. Citation Format: Monique B. Nilsson, Tina Cascone, Jayanthi Gudikote, Emily Roarty, Lixia Diao, Andrew Koo, Sumankalai Ramachandran, Erick Riquelme, Hai Tran, Ignacio Wistuba, David P. Carbone, John Heymach. KDR amplification in NSCLC is associated with sensitivity to VEGFR tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 374. doi:10.1158/1538-7445.AM2013-374

Purpose: The main objectives of this exploratory analysis were to identify biomarkers in blood associated with clinical benefit from bevacizumab, and to assess CAF changes related to nephrectomy (Nx) in mRCC. Methods: Plasma samples were obtained from 44 pts participating in a frontline presurgical study of bevacizumab in clear cell mRCC (22 pts treated with BEV and 22 pts with BEV and erlotinib) (Jonasch et al. J Clin Oncol 2009), before treatment (BL), after 8 weeks of therapy/pre-nephrectomy (Nx), after 16 weeks of therapy/postNx, and every 4 weeks thereafter. Multiplex bead suspension arrays and ELISA were used to measure concentrations of 58 CAFs including VEGF, VEGF-C, placental growth factor (PlGF), soluble CAIX (sCA9), hepatocyte growth factor (HGF), osteopontin (OPN), matrix metalloproteinase-9 (MMP9), transforming growth factor (TGF)b1, endoglin, type IV collagen (ColIV), and multiple chemokines and interleukins (IL). CAFs were correlated with progression-free survival (PFS), overall survival (OS), and best response in primary and metastatic disease (% relative to BL). Results: Of 58 CAFs available at BL, high (above median) concentrations of TRAIL and ColIV were associated with longer PFS (7.6 v 2.8 and 8.1 v 2.9 months [mos], respectively; both P=0.001) and OS (27.9 v 13.2 and 32.8 v 14.3 mos, respectively; P=0.023 and P=0.025). In addition to high TRAIL, low levels of VEGF and PlGF also showed a significant association with enhanced response both in primary disease and metastases (all P Conclusions: We identified TRAIL, ColIV, VEGF and PlGF as candidate CAFs associated with clinical benefit from BEV in clear cell mRCC. The combination of BEV with erlotinib resulted in distinct on treatment CAF changes. The integration of these data with CAF profiling studies in pts with non-metastatic RCC could help define CAF sets useful to assist in risk stratification of RCC pts and provide insights into the biology of RCC progression. Citation Format: Alper Yetil, Eric Jonasch, Xuemei Wang, Christopher Wood, Surena Matin, Hai T. Tran, Nizar M. Tannir, John V. Heymach, Amado J. Zurita. Plasma cytokine and angiogenic factor (CAF) profiling for identification of markers associated with clinical benefit from bevacizumab (BEV) and with primary tumor presence in a phase II presurgical study of metastatic renal cell carcinoma (mRCC) patients [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4645. doi:10.1158/1538-7445.AM2013-4645

The phosphoinositide 3-kinase (PI3K) signaling pathway is the most commonly dysregulated pathway in many human cancers, including non-small cell lung cancer (NSCLC). Therefore, targeting PI3K signaling has become a promising approach in cancer therapy. Although studies have indicated development of resistance to PI3K inhibitors, the molecular mechanism of drug resistance is largely unknown in NSCLC. Here, we sought to identify new potential biomarkers and pathways that confer resistance to PI3K inhibitors in NSCLC. To this end, we used reverse-phase protein array (RPPA) to assess the expression levels and activation status of 137 proteins involved in signaling pathways implicated in lung cancer. Seventy-four NSCLC cell lines were treated with the PI3K inhibitor BAY 80-6946 and lysates were collected for proteomic analysis. First, we evaluted the association between drug sensitivity (IC50) and RPPA expression levels at baseline in sixty NSCLC cell lines. Correlation test was applied to each marker vs drug and Wilcox Rank test was performed to compare the smallest and largest one third of all the cell lines. We found increased phosphorylation and inactivation of pro-apoptotic protein Bad and phospho-LKB1 in the resistant cell lines. An inverse correlation was found for phosphorylation and inactivation of c-Src. Interestingly, activation of EGFR and increased levels of PTCH and PKCα were found in the resistant cell lines. Next, paired t-test was applied to each marker comparing the difference between drug and control treated cells. As expected, treatment with BAY 80-6946, significantly downregulates components of PI3K/mTOR pathway, as illustrated by decreased levels of phospho-Akt (-4.28-fold relative expression) and phospho-S6 ribosomal protein (-9.64-fold), phospho-p70S6K (-3.47-fold) and phospho-mTOR (-1.42-fold). We observed elevated expression of negative regulators of mTOR pathway, LKB1 (1.11-fold), AMPKα (1.12-fold) and phospho-AMPKα (12-fold). On the other hand, our analysis identified new potential pathways of resistance that are activated upon PI3K inhibition. We found increased levels of phospho-EGFR (P Citation Format: Maria A. Cortez, Lauren Averett Byers, You Hong Fan, Lixia Diao, Philip Groth, Julianne Paul, Jing Wang, Uma Giri, Jayanthi Gudikote, Hai Tran, Kevin Coombes, John D. Minna, Ningshu Liu, John V. Heymach. Proteomic analysis reveals Src/Stat and EGFR/MAPK pathways as potential mechanism of resistance to PI3K inhibitors in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2498. doi:10.1158/1538-7445.AM2013-2498

Background: MEK inhibitors such as BAY86-9766 are a new class of agents that show promise in the treatment of non-small cell lung cancer (NSCLC). The downstream effects of MEK inhibition in NSCLC have not been fully elucidated. We performed a broad proteomic analysis to determine which signaling pathways were modulated by BAY86-9766 treatment and how these pathways correlate with sensitivity. Methods: We treated 109 lung cancer cell lines with MEK inhibitor BAY86-9766 at a concentration of 2200 nM. Drug sensitivity was determined by CellTiter-Glo assay and cell lines were classified as sensitive or resistant based on whether their IC50 values were in the highest or lowest 1/3rd of those tested. Using paired t-tests, we compared pre- versus post-treatment protein levels in the overall group and between the sensitive vs. resistant cell lines. Results: MEK inhibitor BAY86-9766 was effective in reducing phosphorylation of direct downstream signaling molecules pMAPK (p Conclusions: We have performed broad proteomic analysis on cell lines treated with MEK inhibitor BAY86-9766 to determine which pathways are modulated by treatment and how they might relate to sensitivity. We conclude that MEK inhibition with BAY86-9766 may exert some of its effects by suppressing mTOR activity via the LKB1/AMPK pathway, and that the degree of modulation of the AMPK pathway correlates with sensitivity. This mechanism may involve decreased activation of p90RSK by MAPK, which leads to decreased degradation and increased activity of LKB1, and thereby increased AMPK activity and decreased mTOR signaling. This works suggests a rational basis for combinations of targeted agents to overcome resistance, such as combinations of MEK inhibitors with mTOR inhibitors or PI3 kinase inhibitors. Citation Format: Kathryn A. Gold, Lauren A. Byers, You Hong Fan, Lixia Diao, Philip Groth, Julianne Paul, Jing Wang, Uma Giri, Jayanthi Gudikote, Hai T. Tran, Kevin R. Coombes, John D. Minna, Ningshu Liu, John V. Heymach. Proteomic analysis of effects of MEK inhibition with BAY86-9766 on LKB1/AMPK and mTOR pathway in lung cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1045. doi:10.1158/1538-7445.AM2013-1045

Background: Circulating tumor cells (CTC) associated with solid tumors are being studied for their diagnostic and prognostic value. In patients with metastatic tumors, CTC presence in the blood has been putatively associated with short survival. Since blood collection is relatively non-invasive, CTC molecular analysis opens up the possibility of monitoring genotypic changes during cancer treatments. Unfortunately, CTCs are not present in large numbers, often at rates as low as one cell per 106-107 leukocytes. Thus, to perform genotypic biomarker analysis on CTCs, methodologies must be developed to using highly specific and sensitive technologies and an enrichment step to increase analytes to detectable levels. Methods: We developed a methodology for detecting mutations in multiple oncogenes and chemotherapy resistance genes in non-small cell lung cancer (NSCLC) CTC specimens using high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) single nucleotide polymorphism (SNP) analysis (MASSarray; Sequenom, Inc.) to determine cancer-associated genetic mutations in lung cancer specimens. This system allows for up to 10 different somatic mutations to be assayed per well in a 384-well format; requires very little DNA; can be used with whole genome amplified (WGA) DNA; and is sensitive enough to use with small samples such as core needle biopsies (CNB), fine needle aspirates (FNA), and CTCs. We developed a lung cancer assay panel of 13 genes/135 mutations (including AKT1, BRAF, CTNNB1, EGFR, ERBB2, KRAS, MEK1, NRAS, PIK3CA, PIK3R1,PTEN, and STK11) to test for somatic mutations in genes representing multiple pathways known to be involved in lung cancer. All assays can detect a mutation in < 25% of a sample. Results: In the effort to analyze CTCs, we first analyzed 57 NSCLC cell lines with known mutations and confirmed known mutation status. Next, we successfully analyzed DNA from 90 frozen and matched FFPE NSCLC resected tissues. Analysis of unamplified and matched WGA cell line DNA quantity CNB and FNA equivalents gave the same mutational status results. Moving to CTC equivalents, we successfully analyzed cell line DNA and matched WGA DNA equivalents of 100–1000 cells with known EGFR L585R and KRAS G34A mutations and negative control DNA (negative for all assays). Next, WGA methodology for direct CTC cell lysate DNA amplification was developed using CTC cell number equivalents (3 – 200 cells) obtained from a typical clinical blood sample CTC preparation. Then, we directly compared unamplified and matched amplified CTC cell equivalents (50, 100, and 200 cells). Analysis of both unamplified and amplified CTC cell equivalents reported identical mutation status results. We have applied this methodology to spiked blood sample and clinical blood sample CTC fractions. Thus, we demonstrated that we are able to study mutations in multiple genes using small amount of DNA from CTC cell numbers in a high-throughput manner. Conclusion: We developed a robust method for accurately determine cancer-associated genetic mutations in NSCLC CTC cell number equivalent lysates using MALDI-TOF MS SNP analysis which can be applied to better understand the molecular characteristics of lung cancer during treatment and progression. As additional clinical NSCLC CTC samples are collected, we will continue applying this methodology to assess CTC mutation status as potential diagnostic and/or prognostic markers.