Chi-Wan Chow

Whole-exome sequencing and immune profiling of early-stage lung adenocarcinoma with fully annotated clinical follow-up

Background Lung adenocarcinomas (LUADs) lead to the majority of deaths attributable to lung cancer. We performed whole-exome sequencing (WES) and immune profiling analyses of a unique set of clinically annotated early-stage LUADs to better understand the pathogenesis of this disease and identify clinically relevant molecular markers. Methods We performed WES of 108 paired stage I-III LUADs and normal lung tissues using the Illumina HiSeq 2000 platform. Ten immune markers (PD-L1, PD-1, CD3, CD4, CD8, CD45ro, CD57, CD68, FOXP3 and Granzyme B) were profiled by imaging-based immunohistochemistry (IHC) in a subset of LUADs (n = 92). Associations among mutations, immune markers and clinicopathological variables were analyzed using ANOVA and Fishers exact test. Cox proportional hazards regression models were used for multivariate analysis of clinical outcome. Results LUADs in this cohort exhibited an average of 243 coding mutations. We identified 28 genes with significant enrichment for mutation. SETD2-mutated LUADs exhibited relatively poor recurrence- free survival (RFS) and mutations in STK11 and ATM were associated with poor RFS among KRAS-mutant tumors. EGFR, KEAP1 and PIK3CA mutations were predictive of poor response to adjuvant therapy. Immune marker analysis revealed that LUADs in smokers and with relatively high mutation burdens exhibited increased levels of immune markers. Analysis of immunophenotypes revealed that LUADs with STK11 mutations exhibited relatively low levels of infiltrating CD4+/CD8+ T-cells indicative of a muted immune response. Tumoral PD-L1 was significantly elevated in TP53 mutant LUADs whereas PIK3CA mutant LUADs exhibited markedly down-regulated PD-L1 expression. LUADs with TP53 or KEAP1 mutations displayed relatively increased CD57 and Granzyme B levels indicative of augmented natural killer (NK) cell infiltration. Conclusion(s) Our study highlights molecular and immune phenotypes that warrant further analysis for their roles in clinical outcomes and personalized immune-based therapy of LUAD.BACKGROUND Lung adenocarcinomas (LUADs) lead to the majority of deaths attributable to lung cancer. We performed whole-exome sequencing (WES) and immune profiling analyses of a unique set of clinically annotated early-stage LUADs to better understand the pathogenesis of this disease and identify clinically relevant molecular markers. METHODS We performed WES of 108 paired stage I-III LUADs and normal lung tissues using the Illumina HiSeq 2000 platform. Ten immune markers (PD-L1, PD-1, CD3, CD4, CD8, CD45ro, CD57, CD68, FOXP3 and Granzyme B) were profiled by imaging-based immunohistochemistry (IHC) in a subset of LUADs (n = 92). Associations among mutations, immune markers and clinicopathological variables were analyzed using ANOVA and Fishers exact test. Cox proportional hazards regression models were used for multivariate analysis of clinical outcome. RESULTS LUADs in this cohort exhibited an average of 243 coding mutations. We identified 28 genes with significant enrichment for mutation. SETD2-mutated LUADs exhibited relatively poor recurrence- free survival (RFS) and mutations in STK11 and ATM were associated with poor RFS among KRAS-mutant tumors. EGFR, KEAP1 and PIK3CA mutations were predictive of poor response to adjuvant therapy. Immune marker analysis revealed that LUADs in smokers and with relatively high mutation burdens exhibited increased levels of immune markers. Analysis of immunophenotypes revealed that LUADs with STK11 mutations exhibited relatively low levels of infiltrating CD4+/CD8+ T-cells indicative of a muted immune response. Tumoral PD-L1 was significantly elevated in TP53 mutant LUADs whereas PIK3CA mutant LUADs exhibited markedly down-regulated PD-L1 expression. LUADs with TP53 or KEAP1 mutations displayed relatively increased CD57 and Granzyme B levels indicative of augmented natural killer (NK) cell infiltration. CONCLUSION(S) Our study highlights molecular and immune phenotypes that warrant further analysis for their roles in clinical outcomes and personalized immune-based therapy of LUAD.

Mutation profiles in early-stage lung squamous cell carcinoma with clinical follow-up and correlation with markers of immune function.

Background Lung squamous cell carcinoma (LUSC) accounts for 20–30% of non-small cell lung cancers (NSCLCs). There are limited treatment strategies for LUSC in part due to our inadequate understanding of the molecular underpinnings of the disease. We performed whole-exome sequencing (WES) and comprehensive immune profiling of a unique set of clinically annotated early-stage LUSCs to increase our understanding of the pathobiology of this malignancy. Methods Matched pairs of surgically resected stage I-III LUSCs and normal lung tissues (n = 108) were analyzed by WES. Immunohistochemistry and image analysis-based profiling of 10 immune markers were done on a subset of LUSCs (n = 91). Associations among mutations, immune markers and clinicopathological variables were statistically examined using analysis of variance and Fisher’s exact test. Cox proportional hazards regression models were used for statistical analysis of clinical outcome. Results This early-stage LUSC cohort displayed an average of 209 exonic mutations per tumor. Fourteen genes exhibited significant enrichment for somatic mutation: TP53, MLL2, PIK3CA, NFE2L2, CDH8, KEAP1, PTEN, ADCY8, PTPRT, CALCR, GRM8, FBXW7, RB1 and CDKN2A. Among mutated genes associated with poor recurrence-free survival, MLL2 mutations predicted poor prognosis in both TP53 mutant and wild-type LUSCs. We also found that in treated patients, FBXW7 and KEAP1 mutations were associated with poor response to adjuvant therapy, particularly in TP53-mutant tumors. Analysis of mutations with immune markers revealed that ADCY8 and PIK3CA mutations were associated with markedly decreased tumoral PD-L1 expression, LUSCs with PIK3CA mutations exhibited elevated CD45ro levels and CDKN2A-mutant tumors displayed an up-regulated immune response. Conclusion(s) Our findings pinpoint mutated genes that may impact clinical outcome as well as personalized strategies for targeted immunotherapies in early-stage LUSC.

Expression Profiling Stratifies Mesothelioma Tumors and Signifies Deregulation of Spindle Checkpoint Pathway and Microtubule Network with Therapeutic Implications

BACKGROUND Malignant pleural mesothelioma (MPM) is a lethal neoplasm exhibiting resistance to most treatment regimens and requires effective therapeutic options. Though an effective strategy in many cancer, targeted therapy is relatively unexplored in MPM because the therapeutically important oncogenic pathways and networks in MPM are largely unknown. MATERIALS AND METHODS We carried out gene expression microarray profiling of 53 surgically resected MPMs tumors along with paired normal tissue. We also carried out whole transcriptomic sequence (RNA-seq) analysis on eight tumor specimens. Taqman-based quantitative Reverse-transcription polymerase chain reaction (qRT-PCR), western analysis and immunohistochemistry (IHC) analysis of mitotic arrest deficient-like 1 (MAD2L1) was carried out on tissue specimens. Cell viability assays of MPM cell lines were carried out to assess sensitivity to specific small molecule inhibitors. RESULTS Bioinformatics analysis of the microarray data followed by pathway analysis revealed that the mitotic spindle assembly checkpoint (MSAC) pathway was most significantly altered in MPM tumors with upregulation of 18 component genes, including MAD2L1 gene. We validated the microarray data for MAD2L1 expression using quantitative qRT-PCR and western blot analysis on tissue lysates. Additionally, we analyzed expression of the MAD2L1 protein by IHC using an independent tissue microarray set of 80 MPM tissue samples. Robust clustering of gene expression data revealed three novel subgroups of tumors, with unique expression profiles, and showed differential expression of MSAC pathway genes. Network analysis of the microarray data showed the cytoskeleton/spindle microtubules network was the second-most significantly affected network. We also demonstrate that a nontaxane small molecule inhibitor, epothilone B, targeting the microtubules have great efficacy in decreasing viability of 14 MPM cell lines. CONCLUSIONS Overall, our findings show that MPM tumors have significant deregulation of the MSAC pathway and the microtubule network, it can be classified into three novel molecular subgroups of potential therapeutic importance and epothilone B is a promising therapeutic agent for MPM.

Abstract B37: Investigating the potential of miR-203 as a therapeutic candidate and its role in the pathobiology of malignant pleural mesothelioma (MPM)

Background: MPM is a lethal neoplasm of the pleural layer of cells surrounding lungs and is in dire need of newer therapeutic treatments. microRNAs (miRs) play a critical role in the pathobiology of many cancers but there are few reports investigating their function in MPM. Recently many studies have highlighted the potential of miRs as therapeutic agents in cancer. We completed a microarray profiling strategy to discover miRs of therapeutic and biological importance in MPM. Methods: We extracted total RNA from 53 frozen resected tumor tissue specimens, comprised of 39 epitheloid, 7 sarcomatoid and 7 biphasic histotypes, along with paired normal tissue. The RNA was labeled and hybridized to Agilent v3 Human miR microarrays. These were scanned and the data was processed using the AgiMicroRna “R” package involving background correction, quantile normalization and summarization. The microarray results were validated by quantitative reverse transcription polymerase chain reaction (qRTPCR) using Taqman assays on the ABI 7300 platform. Also qRT-PCR was employed to determine the levels of miR-203 expression in a panel of 26 mesothelioma cell lines including Met-5A, an SV-40 immortalized pleural mesothelial control cell line. For all qRT-PCR experiments the miR-203 levels were determined relative to endogenous miR-U6 as control using ΔΔCT calculation. Moreover in a previous study RNA from these same tissue specimens were hybridized on Affymetrix U133 plus 2.0 microarray to obtain transcriptomic profiles. Results: The bioinformatic analysis of miR microarray data showed that a number of miRs, including miR-203, are differentially expressed between the tumor and normal samples. A paired T-test conducted on a miRNA-by-miRNA basis and at a highly significant FDR value of 1e-06 showed miR-203 to be down regulated, more than 2 fold, in tumors compared to paired normal tissue. We decided to explore the role of miR-203 in the pathogenesis of MPM since it has been postulated to play a tumor suppressor role in skin and prostate cancer by inhibiting proliferation, metastasis and acting antagonistic to stem cells (1, 2). Using qRT-PCR, we compared levels of expression in 40 pairs of tumor vs. paired normal tissues and demonstrated that miR-203 was down regulated to more than 10 fold in MPM tumors. Also qRT-PCR showed that 70 % of 25 MPM cancer cell lines had lower expression of miR-203 compared to Met-5A cell line. Our previous transcriptomic profiling study had shown differentially expressed transcripts between these same tumors and paired normal specimens. Survivin (BIRC5) message level, which codes for an apoptosis inhibitor protein and reported to be regulated by miR-203 in prostate cancer cell lines (3), was found to be 2.7 fold higher in tumors (p = 2.00e-15) in an expected anti-correlation direction to miR-203 expression levels (i.e. low miR = high target mRNA). Conclusion: Our findings suggest that miR-203 may play a tumor suppressor role in pleural mesothelioma and regulate levels of survivin message. Supported by Grants: DoD W81XWH-07-1-0306 (IW and AT), Fleming Foundation, IASLC Young Investigator Award 2011–2013 (MS).

Abstract 142: Mutation and immune profiles in early-stage lung squamous cell carcinoma

PURPOSE: Lung squamous cell carcinoma (LUSC) accounts for 20-30% of non-small cell lung cancers (NSCLCs). There are limited treatment strategies for LUSC in part due to our inadequate understanding of the molecular underpinnings of the disease. We sought to perform whole-exome sequencing (WES), comprehensive immune profiling and clinicopathological analysis of early-stage LUSCs to increase our understanding of the pathobiology of this malignancy. METHODS: Matched pairs of surgically resected stage I-III LUSCs and normal lung tissues (n = 108) were analyzed by WES. Immunohistochemistry and image analysis-based profiling of 10 immune markers was done on a subset of LUSCs (n = 91). Associations among mutations, immune markers and clinicopathological variables were statistically examined using ANOVA and Fisher tests. Cox proportional hazards regression models were used for statistical analysis of clinical outcome. RESULTS: This early-stage LUSC cohort displayed an average of 209 exonic mutations per tumor. Fourteen genes exhibited significant enrichment for mutation: TP53, MLL2, PIK3CA, NFE2L2, CDH8, KEAP1, PTEN, ADCY8, PTPRT, CALCR, GRM8, FBXW7, RB1 and CDKN2A. Among mutated genes associated with poor recurrence-free survival, MLL2 mutations predicted poor prognosis in both TP53 mutant and wild type LUSCs. We also found that in treated patients, FBXW7 and KEAP1 mutations were associated with poor response to adjuvant therapy, particularly in TP53-mutant tumors. Analysis of mutations with immune markers revealed that ADCY8 and PIK3CA mutations were associated with markedly decreased tumoral PD-L1 expression, LUSCs with PIK3CA mutations exhibited elevated CD45ro levels and CDKN2A-mutant tumors displayed an up-regulated immune response. CONCLUSION: Our findings pinpoint mutated genes that may impact clinical outcome as well as personalized strategies for targeted immunotherapies in early-stage LUSC. Citation Format: Murim Choi, Humam Kadara, Jiexin Zhang, Edwin Parra Cuentas, Jaime Rodriguez Canales, Stephen G. Gaffney, Zi-Ming Zhao, Carmen Behrens, Junya Fujimoto, Chi-Wan Chow, Neda Kalhor, Cesar Moran, David Rimm, Stephen Swisher, Don L. Gibbons, John V. Heymach, Edward Kaftan, Jeffrey Townsend, Thomas J. Lynch, Joseph Schlessinger, J. Jack Lee, Richard Lifton, Roy S. Herbst, Ignacio I. Wistuba. Mutation and immune profiles in early-stage lung squamous cell carcinoma. [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 142.

Abstract 89: Whole-exome sequencing and immune profiling of early-stage lung adenocarcinoma

PURPOSE: Lung adenocarcinomas (LUADs) lead to the preponderance of deaths attributable to lung cancer. We performed whole-exome sequencing (WES), comprehensive immune profiling and clinicopathological analysis of LUADs to better understand the molecular pathogenesis of this disease and identify clinically relevant molecular markers. METHODS: We performed WES of 108 paired surgically resected stage I-III LUADs and normal lung tissues using the Illumina HiSeq 2000 platform. Additionally, ten immune related markers (PD-L1, PD-1, CD3, CD4, CD8, CD45ro, CD57, CD68, FOXP3 and Granzyme B) were profiled by imaging-based immunohistochemistry in a subset of LUADs (n = 92). Associations among mutations, immune markers and clinicopathological variables were analyzed using ANOVA and Fishers Exact tests. Cox proportional hazards regression models were employed for multivariate analysis of clinical outcome. RESULTS: LUADs in this cohort exhibited an average of 243 coding mutations per tumor. We identified 28 genes with significant enrichment for mutation. SETD2-mutant LUADS exhibited relatively poor recurrence-free survival (RFS) and mutations in STK11 and ATM were associated with poor RFS in KRAS-mutant tumors. EGFR, KEAP1 and PIK3CA mutations were predictive of poor response to adjuvant therapy. Immune marker analysis demonstrated that PD-L1 expression was increased in smoker compared to non-smoker LUADs and, along with other immune markers, was positively correlated with somatic mutation burden. Moreover, immune marker levels including PD-L1 were elevated in TP53-mutant LUADs. In contrast, STK11 and U2AF1 mutant tumors exhibited a suppressed immune response and LUADs with PIK3CA mutations exhibited markedly decreased tumoral PD-L1 expression. CONCLUSION: Our study highlights mutations that may impact clinical outcome and personalized strategies for immune-based therapy of early-stage LUAD patients. Citation Format: Humam Kadara, Murim Choi, Jiexin Zhang, Edwin Parra Cuentas, Jaime Rodriguez Canales, Stephen Gaffney, Zi-Ming Zhao, Carmen Behrens, Junya Fujimoto, Chi-Wan Chow, Neda Kalhor, Cesar Moran, David Rimm, Stephen G. Swisher, Don L. Gibbons, John V. Heymach, Edward Kaftan, Jeffrey Townsend, Thomas J. Lynch, Joseph Schlessinger, J. Jack Lee, Richard Lifton, Ignacio I. Wistuba, Roy S. Herbst. Whole-exome sequencing and immune profiling of early-stage lung adenocarcinoma. [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 89.

Abstract 156: Integrated exome and transcriptome sequencing of primary lung cancers and paired distant metastases

Background: The precise molecular mechanisms underlying metastasis of nonsmall cell lung cancers (NSCLC) are largely unknown. Two recent studies comparing genomic landscapes of primary NSCLC tumors and paired brain metastases suggested branched evolution, where all metastatic and primary tumors shared a common ancestor yet continued to evolve independently. The integrated genomic and transcriptomic profiles of primary NSCLC and metastases have not been studied in any details. Methods: We performed whole exome sequencing (WES) and RNA sequencing (RNA-seq) of surgically resected primary tumors and paired distant metastases from 7 patients with NSCLC. Results: Totally, 6,945 somatic mutations, including 1,702 non-silent (stop-gain, stop-loss, frameshift, splicing site and nonsynonymous) mutations were identified by WES. Metastases trended to have larger mutation burdens compared to paired primary tumors, although the difference was not statistically different (average 595 mutations per tumor in primary tumors versus 852 mutations per tumor in metastases, respectively, p = 0.54). On average, 51% of all mutations (24% to 93%) were shared between primary tumors and metastases. We identified 14 canonical cancer gene mutations in this cohort of patients, defined as mutations that lead to amino acid changes identical to those found previously in cancer genes or disrupting mutations in tumor suppressor genes, all of which were shared between primary tumors and paired distant metastases. In addition, metastases resembled paired primary NSCLC tumors closely in regard to somatic copy number aberration profiles and mutation signatures. Pathway analysis from RNA-seq data demonstrated that 25 of the 35 signal transduction pathways that were significantly down regulated in metastases relative to primary NSCLC tumors were related to immune activation. Validation study with a larger patient cohort is in progress. Conclusions: Although branched evolution is a common phenomenon during metastasis of NSCLC, majority of canonical cancer gene mutations are probably early molecular events likely acquired before metastasis initiates. Mutation mechanism may be determined early during carcinogenesis and preserved during cancer evolution even at the metastatic sites. Immune suppression may be one characteristic feature of cancer cells of metastatic capacity. Citation Format: Jianjun Zhang, Chia-Chin Wu, Jianhua Zhang, Junya Fujimoto, Xingzhi Song, Xizeng Mao, Huadong Sun, Sahil Seth, Rebecca Thornton, Marcus Coyle, Latasha Little, Curtis Gumbs, Carmen Behrens, Chi-Wan Chow, Erik Sulman, Ganesh Rao, Stephen Swisher, Ignacio Wistuba, John Heymach, Andrew Futreal, Daniel Gomez. Integrated exome and transcriptome sequencing of primary lung cancers and paired distant metastases. [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 156.

Abstract 1597: Transcriptomic architecture of the field of cancerization in the adjacent normal-appearing airway: Early mechanisms in lung carcinogenesis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Increasing our understanding of early events in the pathogenesis of lung cancer is crucial for identification of new targets for prevention and treatment of this malignancy. Earlier work has shown that seemingly normal cells adjacent to the tumor carry specific molecular alterations that are characteristic of the tumor itself suggestive of a field of cancerization. By sampling and studying normal-appearing tissue, the molecular field of cancerization provides biological insights into early phases in cancer development. In this study, we sought to characterize molecular field effects in the normal-appearing airway that are most representative of the nearby lung tumor, and thus, are most likely to denote early events in lung carcinogenesis. To achieve this, we performed genome-wide expression profiling of resected field cancerization specimens (n=20 patients) comprised of matched early-stage non-small cell lung cancers (NSCLCs), cytologically normal airways with varying spatial distance from the tumors and distant (relative to location of tumors) normal lung tissues (n=194 samples). Using ordinal logistic regression, we identified 422 genes that were progressively modulated in expression in normal-appearing airways by spatial distance from tumors. Notably, when examined in paired NSCLC and normal lung tissues, these genes were found to recapitulate tumor expression profiles. We then sought to examine the role of lysosomal protein transmembrane 4 beta (LAPTM4B), a putative oncogene that was found to be up-regulated in airways by shorter spatial distance from tumors, in lung oncogenesis. LAPTM4B was significantly elevated in NSCLC tissues compared to paired distant normal lung and was predictive of poor survival in lung adenocarcinoma. Moreover, LAPTM4B promoted anchorage-dependent and -independent lung cancer cell growth and was crucial for cellular survival and the autophagy response under nutrient- and serum-deprived conditions. In addition, pathways analysis of a LAPTM4B-dependent gene expression profile revealed decreased activation of the canonical nuclear factor erythroid 2-like 2 (NRF2)-mediated pathway following LAPTM4B knockdown. Further, we found that LAPTM4B augmented the expression and nuclear translocation of the NRF2 transcription factor following serum deprivation pointing to the probable role of the novel LAPTM4B/NRF2 signaling axis in promoting lung cancer cell survival. All in all, our study points to molecular field of cancerization profiles in the normal-appearing airway that highly signify the nearby lung tumor and comprise early mechanisms (e.g. LAPTM4B) in lung carcinogenesis. Citation Format: Yuho Maki, Junya Fujimoto, Suk-Young Yoo, Melinda Garcia, Adam Gower, Li Shen, Chi-Wan Chow, Carmen Behrens, Neda Kalhor, Cesar Moran, Jing Wang, Avrum Spira, Kevin R. Coombes, Ignacio I. Wistuba, Humam Kadara. Transcriptomic architecture of the field of cancerization in the adjacent normal-appearing airway: Early mechanisms in lung carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1597. doi:10.1158/1538-7445.AM2014-1597

Abstract 2367: Transcriptomic architecture of the airway field cancerization in early-stage non-small cell lung cancer .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Earlier work has identified in lung cancer a field cancerization (FC) phenomenon in which tumors and adjacent normal appearing tissues share specific molecular abnormalities (e.g., loss of heterozygosity) that may be highly pertinent to cancer pathogenesis. We sought to characterize the global molecular airway FC adjacent to early-stage non-small cell lung cancer (NSCLC) in an attempt to unravel profiles that may help to explain the development of the disease. We performed whole-transcript expression profiling of a set of resected early-stage NSCLC specimens (n=20 patients) with matched histologically normal airways of varying distance from the tumor and paired uninvolved normal lung tissue (n=194 samples). Using linear mixed-effects models, we derived FC profiles signifying genes concordantly differentially expressed between tumors and airways compared to normal lung tissues. Gene set enrichment analysis demonstrated that a subset of the genes (n=299) was significantly and congruently modulated between large airways of smokers with and without lung cancer. We then questioned whether the airway FC exhibits site from tumor-dependent expression patterns. Ordinal regression analysis identified airway profiles (n=422 genes) that were significantly progressively expressed by distance from tumors and topologically organized into canonical cancer-associated pathways, such as eukaryotic initiation factor, p70S6K kinase, polo-like kinase and mammalian target of rapamycin signaling (all p<0.001). In addition, the site-dependent airway profiles recapitulated NSCLC expression patterns and were concordantly modulated between tumors and uninvolved normal lung tissues pinpointing their probable roles in lung cancer pathogenesis. Quantitative real-time PCR (QRTPCR) analysis confirmed the differential expression of FC markers selected by both pathways analysis and statistical criteria. Notably, lysosome associated protein transmembrane 4 beta (LAPTM4B), a putative oncogene with no known role in lung carcinogenesis, was among the top 5 site-dependent FC markers and was significantly elevated in NSCLC and immortalized bronchial epithelial cell lines compared to normal cells. Furthermore, transient or stable knockdown of LAPTM4B by RNA interference decreased NSCLC cell growth as well as anchorage-dependent and -independent colony formation. In conclusion, our efforts in understanding the adjacent molecular FC in NSCLC unraveled airway profiles that 1) are, in part, relevant to lung cancer detection; 2) are modulated by distance from corresponding tumors; 3) recapitulate NSCLC expression patterns and 4) harbor markers engaged in mediating the lung malignant phenotype. Profiling the adjacent airway FC in conjunction with tumors, may provide additional insights into the molecular pathology of NSCLC. Funded in part by Department of Defense award W81XWH-10-1-1007. Citation Format: Yuho Maki, Junya Fujimoto, Suk-Young Yoo, Adam Gower, Li Shen, Melinda M. Garcia, Mohamed Kabbout, Chi-Wan Chow, Waun Ki Hong, Neda Kalhor, Jing Wang, Cesar Moran, Avrum Spira, Kevin R. Coombes, Ignacio I. Wistuba, Humam Kadara. Transcriptomic architecture of the airway field cancerization in early-stage non-small cell 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 2367. doi:10.1158/1538-7445.AM2013-2367

Abstract A21: Tissue platinum concentration and tumor response in non-small cell lung cancer

Background: Platinum resistance is a major limitation in the treatment of advanced non-small cell lung cancer (NSCLC). Reduced intracellular drug accumulation is one of the most consistently identified features of platinum-resistant cell lines, but clinical data are limited. We assessed effects of tissue platinum concentrations on tumor response, time to recurrence, progression-free survival (PFS) and overall survival (OS) in patients with early-stage NSCLC following neoadjuvant platinum-based chemotherapy. Methods: We measured total platinum concentrations in 44 archived fresh frozen NSCLC specimens from patients who received neoadjuvant platinum-based chemotherapy. Samples were analyzed by flameless atomic absorption spectrophotometry to assess absorbance reading associated with platinum concentration. Four specimens from patients who underwent surgery only and one patient who received pemetrexed as neoadjuvant chemotherapy were analyzed as negative controls. Absorbance value per mg of tissue was correlated with percent reduction in tumor size on post- vs prechemotherapy CT scans. Kaplan-Meier curves and log-rank tests were used to evaluate differences in time to recurrence and survival between two groups divided by median platinum concentration. Results: Platinum absorbance values in 44 neoadjuvant specimens were easily detectable and ranged from 0.6 to 6.7 × 10−3 per mg of tissue while five negative controls demonstrated absorbance readings similar to 0.1N HCL. Platinum absorbance correlated significantly with percent reduction in tumor size (R2 = 0.48, P<0.00001). The same correlations were seen in cisplatin (P = 0.0003), carboplatin (P = 0.0013), adenocarcinoma (P = 0.0003) and squamous cell carcinoma (P = 0.019) groups. Furthermore, there was no significant impact of potential variables such as the type of platinum compound, number of cycles, pre-treatment tumor diameter and time lapse from last chemotherapy on platinum concentration. Patients with lower Pt concentration also had shorter time to recurrence (P = 0.0494), progression free survival (P = 0.0366) and overall survival (P = 0.035). Conclusion: This is the first tissue-based study to establish a relationship between tissue platinum concentrations and response in NSCLC. Reduced intratumoral platinum accumulation may constitute a significant mechanism of platinum resistance even in clinical specimens. Further studies investigating factors that modulate intracellular platinum concentration are warranted (Supported by National Foundation of Cancer Research 90088436, Department of Defense W81XWH-07-1-0306, National Institute of Health CA127263, CA160687 and CA16672).