Early-stage Lung Squamous Cell Research Articles

Transcriptomic Profiling for Prognostic Biomarkers in Early-Stage Squamous Cell Lung Cancer (SqCLC).

Squamous cell lung carcinoma (SqCLC) is associated with high mortality and limited treatment options. Identification of therapeutic targets and prognostic biomarkers is still lacking. This research aims to analyze the transcriptomic profile of SqCLC samples and identify the key genes associated with tumorigenesis, overall survival (OS), and a profile of the tumor-infiltrating immune cells. Differential gene expression analysis, pathway enrichment analysis, and Gene Ontology analysis on RNA-seq data obtained from FFPE tumor samples (N = 23) and healthy tissues (N = 3) were performed (experimental cohort). Validation of the results was conducted on publicly available gene expression data using TCGA LUSC (N = 225) and GTEx healthy donors' cohorts (N = 288). We identified 1133 upregulated and 644 downregulated genes, common for both cohorts. The most prominent upregulated genes were involved in cell cycle and proliferation regulation pathways (MAGEA9B, MAGED4, KRT, MMT11/13), while downregulated genes predominately belonged to immune-related pathways (DEFA1B, DEFA1, DEFA3). Results of the survival analysis, conducted on the validation cohort and commonly deregulated genes, indicated that overexpression of HOXC4 (p < 0.001), LLGL1 (p = 0.0015), and SLC4A3 (p = 0.0034) is associated with worse OS in early-stage SqCLC patients. In contrast, overexpression of GSTZ1 (p = 0.0029) and LILRA5 (p = 0.0086) was protective, i.e., associated with better OS. By applying a single-sample gene-set enrichment analysis (ssGSEA), we identified four distinct immune subtypes. Immune cell distribution suggests that the memory T cells (central and effector) and follicular helper T cells could serve as important stratification parameters.

942P Camrelizumab combined with albumin paclitaxel and platinum in perioperative treatment of resectable squamous cell lung cancer: A single-arm, open-label, phase II clinical trial

Several studies have shown that the combination of programmed cell death 1 (PD-1) inhibitor and chemotherapy is efficacious for resectable non-small-cell lung cancer (NSCLC). However, there are few studies on neoadjuvant immunotherapy for early stage lung squamous cell carcinoma (LUSC). This phase II study aimed to evaluate the efficacy and safety of adding camrelizumab to standard neoadjuvant chemotherapy in early LUSC. In this single-arm phase II trial (ChiCTR2100044645), eligible patients were aged 18-70 years with previously untreated, histologically confirmed stage IIB-IIIB LUSC. Patients received camrelizumab (200 mg on day 1), albumin paclitaxel (90 mg/m2 on days 1 and 8) and a platinum agent (carboplatin area under the curve of 5 or cisplatin 75 mg/m2 on day 1) intravenously every 3-week cycle for two to four cycles, followed by surgery after 3-4 weeks. After surgery, patients received adjuvant treatment with camrelizumab monotherapy for 1 year. The primary endpoint was major pathological response (MPR). From March 2021 to March 2022, 26 patients were enrolled. As of April 8, 2022, all 26 patients received neoadjuvant therapy and 22 (22/26, 84.6%) had completed prescribed treatment, of whom 17 (17/22, 77.3%) underwent surgical resection. Among all the patients regardless of resection, the MPR rate was 38.5% (10/26, 95% CI 20.2%-59.4%), and the pathological complete response rate was 19.2% (5/26, 95% CI 7.6%-39.4%). Treatment-related adverse events (TRAEs) occurred in 10 patients (10/26, 38.4%) and two (2/26, 7.6%) experienced grade 3 or 4 TRAEs. The most common TRAEs were rash (15.4%), myelosuppression (11.5%), hemoptysis (7.7%), immune dermatitis (3.8%) and diarrhea (3.8%). Of the 17 patients who had surgery, eight (47.1%) patients reported surgery-related complications, of whom six (35.3%) had intraoperative blood loss > 400 ml and five (29.4%) had postoperative indwelling catheter duration ≥ 7 days. Neither unexpected safety signals nor treatment-related death occurred. The addition of camrelizumab to neoadjuvant chemotherapy showed promising antitumor activity and manageable toxicity for resectable LUSC.

A Novel Pyroptosis-Related Gene Signature for Early-Stage Lung Squamous Cell Carcinoma.

BackgroundDiagnosis of early stage lung squamous cell carcinoma (LUSC) has improved; however, a comprehensive analysis of prognostic signatures is needed.PurposeTo identify, establish, and validate a signature model based on pyroptosis-related genes for prognostic predictions of early stage LUSC.Patients and MethodsTwo independent cohorts were included. RNA-seq transcriptome data from patients with early stage LUSC were obtained from The Cancer Genome Atlas (TCGA) database. Thirty-three pyroptosis-related genes were analyzed between early stage LUSC and normal lung tissues. Cox regression analysis, random survival forest, and least absolute shrinkage and selection operator algorithms established a three-gene signature. Kaplan–Meier survival and receiver-operating characteristic curves assessed the prognostic efficacy of the model. Single-sample gene set enrichment analysis (ssGSEA) assessed the relationship between pyroptosis and immune cells. Patients with early stage LUSC from the GSE74777 dataset were used for validation. Pyroptosis-related genes were verified by RT-qPCR and Western blotting.ResultsTwenty-three differentially expressed pyroptosis-related genes were identified in the LUSC and adjacent normal tissues. Three differentially expressed pyroptosis-related genes were identified as hub genes in early stage LUSC. Patients with early stage LUSC in the TCGA cohort were classified into low- and high-risk subgroups according to the risk score. Overall survival (OS) was significantly short in the high-risk subgroup versus the low-risk subgroup. A similar result was found for the GSE74777 dataset. ssGSEA of immune cells and immune-related pathways between the low- and high-risk subgroups may explain the different OS for patients with early-stage LUSC. IL-6 expression was upregulated, which was inconsistent with the bioinformatic analysis. NOD1 and CASP4 were downregulated in LUSC (all P < 0.05) versus normal lung tissues.ConclusionDifferentially expressed pyroptosis-related genes may be involved in early stage LUSC. Pyroptosis-related genes are important in tumor immunity and may be potential prognostic predictors for early stage LUSC.

A Novel Immune-Related Seventeen-Gene Signature for Predicting Early Stage Lung Squamous Cell Carcinoma Prognosis.

With the increasingly early stage lung squamous cell carcinoma (LUSC) being discovered, there is an urgent need for a comprehensive analysis of the prognostic characteristics of early stage LUSC. Here, we developed an immune-related gene signature for outcome prediction of early stage LUSC based on three independent cohorts. Differentially expressed genes (DEGs) were identified using CIBERSORT and ESTMATE algorithm. Then, a 17-immune-related gene (RPRM, APOH, SSX1, MSGN1, HPR, ISM2, FGA, LBP, HAS1, CSF2, RETN, CCL2, CCL21, MMP19, PTGIS, F13A1, C1QTNF1) signature was identified using univariate Cox regression, LASSO regression and stepwise multivariable Cox analysis based on the verified DEGs from 401 cases in The Cancer Genome Atlas (TCGA) database. Subsequently, a cohort of GSE74777 containing 107 cases downloaded from Gene Expression Omnibus (GEO) database and an independent data set consisting of 36 frozen tissues collected from National Cancer Center were used to validate the predictive value of the signature. Seventeen immune-related genes were identified from TCGA cohort, which were further used to establish a classification system to construct cases into high- and low-risk groups in terms of overall survival. This classifier was still an independent prognostic factor in multivariate analysis. In addition, another two independent cohorts and different clinical subgroups validated the significant predictive value of the signature. Further mechanism research found early stage LUSC patients with high risk had special immune cell infiltration characteristics and gene mutation profiles. In conclusion, we characterized the tumor microenvironment and established a highly predictive model for evaluating the prognosis of early stage LUSC, which may provide a lead for effective immunotherapeutic options tailored for each subtype.

Stabilization of Recurrent Respiratory Papillomatosis with Pembrolizumab Therapy: A Case Report

Recurrent respiratory papillomatosis (RRP) is a benign neoplastic disease of the respiratory tract that is caused by human papilloma virus (HPV). The current standard of care is surgical excision with adjuvant treatment as needed. Multiple adjuvant treatments have been used with some success, but long-term control of disease remains difficult. We report on a case of a patient with a long history of RRP who had stabilized true vocal fold disease while on pembrolizumab for concurrent early stage lung squamous cell carcinoma.

Abstract 4550: Correlations between tumor mutation burden, inflammatory profile and histological characteristics of tumor microenvironment in early-stage squamous cell lung carcinoma

Abstract Background: Anti-PD1/PD-L1 immunotherapy has demonstrated success in the treatment of advanced non-small cell lung cancer (NSCLC). Clinical data have shown that both the expression of PD-L1 in patient tumors and high tumor mutation burden (TMB) predicts the likelihood of a positive response to anti-PD-1/PD-L1 immunotherapy. Also, tumor microenvironment (TME) is the constitutive element in cancer immunity, in which analysis of characteristics reflects the potential existing immune reaction. Method: Histologic sections from 150 squamous cell lung carcinoma (SqCLC) were evaluated by two pathologists independently for percentage and character of intratumoral inflammatory cells and percentage and character of para-tumoral infiltrate. The ratios of infiltrating inflammatory cells to tumor cells were estimated in 10% increments by microscopic inspection. The proportions of immune cell populations were deconvulated using the CIBERSORT method based on Affymetrix gene expression profiles. PD-L1 protein expression by IHC was evaluated using the Dako PD-L1 22C3 pharmDx kit and scoring was determined according to the Dako tumor proportion score (TPS). Tumor Mutation Burden (TMB) was calculated based on data from targeted genome sequencing. CD4 and CD8 mRNA levels were determined from Affymetrix gene expression data from frozen specimens. Results: The infiltrates could be divided into intratumoral and paratumoral patterns according to their location in relation to microscopic tumor cell nests. Using the CIBERSORT assay, we confirmed our histological findings by microscopic examination that the SqCLC cohort can be subtyped into plasma cell dominant (74.8%) or other immune infiltrates dominant (such as macrophages), based on the proportions of immune cell populations. We found by regression analysis that TMB had a negative correlation with the percentage of intratumoral inflammatory cells (P=0.014), but did not significantly correlate with paratumoral infiltrates. The TMB demonstrated a significant negative correlation with CD4 mRNA level (P=0.017), but not with CD8 mRNA level. No correlation was determined for TMB and the immune cells dominant subgroup. Interestingly, we didn’t find any association for PD-L1 protein expression with the percentage of intra- or para-tumoral infiltrates, plasma cells dominant group and CD4 and CD8 mRNA levels. Conclusions: TMB was negatively correlated with the percentage of intratumoral inflammatory cells and CD4 mRNA level, which indicate that high TMB may promote an immune suppression environment. In addition, we did not find any association of PD-L1 expression with characteristics of TME in this early-stage SqCLC cohort. Further studies are needed to verify these interesting results. Citation Format: Hui Yu, Daniel T. Merrick, Ming-Sound Tsao, William G. Richards, Lucian R. Chirieac, Mark A. Watson, Christopher J. Rivard, David H. Harpole, Raphael Bueno, Adrie van Bokhoven, Aik-Choon Tan, Fred R. Hirsch, Wilbur A. Franklin. Correlations between tumor mutation burden, inflammatory profile and histological characteristics of tumor microenvironment in early-stage squamous cell lung carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4550.

Correlation of PD-L1 Expression with Tumor Mutation Burden and Gene Signatures for Prognosis in Early-Stage Squamous Cell Lung Carcinoma

ObjectivesAnti–programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) immunotherapy has demonstrated success in the treatment of advanced NSCLC. Recently, PD-1/PD-L1 blockade also has demonstrated interesting results in small trials of neoadjuvant treatment in stage IB to IIIA NSCLC. In addition, several clinical trials using anti–PD-1/PD-L1 immunotherapy as an adjuvant or neoadjuvant treatment in patients with resectable stage NSCLC are ongoing. However, few analyses of anti–PD-1/PD-L1 immunotherapy–related biomarkers in early-stage squamous cell lung carcinoma (SqCLC) have been reported. In this study, we evaluated PD-L1 protein expression, tumor mutation burden, and expression of an immune gene signature in early-stage SqCLC, providing data for identifying the potential role for patients with anti–PD-1/PD-L1 treatment in early-stage SqCLC. MethodsA total of 255 specimens from patients with early-stage SqCLC were identified within participating centers of the Strategic Partnering to Evaluate Cancer Signatures program. PD-L1 protein expression by immunohistochemistry was evaluated by using the Dako PD-L1 22C3 pharmDx kit on the Dako Link 48 auto-stainer (Dako, Carpinteria, CA). Tumor mutation burden (TMB) was calculated on the basis of data from targeted genome sequencing. The T-effector and interferon gamma (IFN-γ) gene signature was determined from Affymetrix gene chip data (Affymetrix, Santa Clara, CA) from frozen specimens. ResultsThe prevalence of PD-L1 expression was 9.8% at a tumor proportion score cutoff of at least 50%. PD-L1 mRNA and programmed cell death 1 ligand 2 mRNA positively correlated with PD-L1 protein expression on tumor cells (TCs) and tumor-infiltrating immune cells. PD-L1 protein expression on tumor-infiltrating immune cells was correlated with the T-effector and IFN-γ gene signature (p < 0.001), but not with TMB. For TCs, all of these biomarkers were independent of each other and neither PD-L1 protein expression, TMB, or T-effector and IFN-γ gene signatures were independently prognostic for patient outcomes. ConclusionsEvaluation of PD-L1 expression, TMB, and T-effector and IFN-γ gene signatures in the cohort with early-stage SqCLC found them to be independent of each other, and none was associated with overall survival. Our results also support the hypothesis that PD-L1 expression is regulated by an intrinsic mechanism on TCs and an adaptive mechanism on immune cells.

MiR-192 and miR-662 enhance chemoresistance and invasiveness of squamous cell lung carcinoma

ObjectivesOverexpression of miR-192, miR-192* and miR-662 was previously found to correlate with poor prognosis of early-stage squamous cell lung cancer (SCC) patients. In this study, we investigated the relevance of these miRNAs to cancer cell biology and chemoresistance. Materials and methodsMiRNA expression profile was analysed in 10 non-small cell lung cancer (NSCLC) cell lines using RT-qPCR. H520 and H1703 cells were transfected with miRNA inhibitors (anti-miR-192, -192* and -662) for functional studies. Chemoresistance to cisplatin and etoposide was evaluated using MTT colorimetric assay. H520 cells were subjected to 3D soft-agar colony formation assay and H1703 cells to wound healing assay. Whole transcriptome analysis was used to assess the effect of miR-192 and miR-662 inhibition on gene expression. ResultsSCC cell lines, H520 and H1703, differed in miRNA expression and phenotypic features. MiR-192 and miR-662 inhibition decreased clonogenicity and motility of SCC cells. MiR-192 and miR-662 inhibition sensitized SCC cells to etoposide but not to cisplatin. Whole transcriptome analysis revealed genes regulated by miR-192 and miR-662 in SCC, relevant to maintaining chemoresistance, invasiveness, epithelial-mesenchymal transition (EMT) and immune evasion. ConclusionsWe showed for the first time that miR-192 and miR-662 have functional role in SCC cells. Our findings suggest that targeting these miRNAs may impact both chemoresistance and invasiveness of SCC, and add to the evidence linking these aspects of tumour biology. Overexpression of miR-192 and miR-662 might be useful as a marker of resistance to etoposide.

Aligning digital CD8+ scoring and targeted next-generation sequencing with programmed death ligand 1 expression: a pragmatic approach in early-stage squamous cell lung carcinoma.

To study programmed death ligand 1 (PD-L1) expression, tumour-infiltrating T lymphocytes (TILs) and the molecular context in patients with early-stage squamous cell lung carcinomas (SCCs). The study included samples from 40 patients (discovery cohort) and 29 patients (validation cohort) diagnosed with early-stage SCC. PD-L1 immunohistochemistry (IHC) was performed with three commercially available clones (E1L3N, SP263 and SP142). CD8+ TILs were scored with a digital algorithm. All tumours were analysed with targeted next-generation sequencing (NGS). Additionally, TP53 mutations were investigated with direct sequencing. In both cohorts, we observed a significant association between CD8+ TILs density and high PD-L1 IHC expression in tumour cells (TCs). Furthermore, high SP142 PD-L1 expression in immune cells (ICs) was also associated significantly with CD8+ TILs density. Therefore, CD8+ TILs density discriminated between patients with high versus low PD-L1 IHC expression with excellent sensitivity and specificity. Interestingly, the highest percentages of PD-L1-positive TCs with the three antibodies were found in samples with cyclin-dependent kinase 6 (CDK6) amplification, with high amplification of proto-oncogene C-Myc (CMYC) or with cyclin D1-PI3 kinase subunit alpha (CCND1-PIK3CA) co-amplification. High SP142 PD-L1 IHC expression in ICs showed a non-significant correlation with TP53 mutations. Conversely, most cases with fibroblast growth factor receptor 1 (FGFR1) amplification were negative for all PD-L1 clones. Our preliminary results support the use of digital CD8+ TILs scoring and targeted NGS alongside PD-L1 expression. The approach presented herein could help define patients with SCCs candidates to immune checkpoints inhibitors.

P3.05-007 Potential of CYFRA 21-1 and CEA to Predict Adjuvant Chemotherapy Benefit in Early-Stage Squamous Cell Lung Cancer

P3.05-007 Potential of CYFRA 21-1 and CEA to Predict Adjuvant Chemotherapy Benefit in Early-Stage Squamous Cell Lung Cancer

Identification of long noncoding RNAs for the detection of early stage lung squamous cell carcinoma by microarray analysis.

The aberrant expressions of long noncoding RNAs have been reported in numerous cancers, which have facilitated the cancer diagnosis. However, the expression profile of lncRNAs in early stage lung squamous cell carcinoma has not been well discussed. The present study aimed to examine the expression profile of lncRNAs in early stage lung squamous cell carcinoma and identify lncRNA biomarkers for diagnosis. Through high-throughput lncRNA microarray, we screened thousands of aberrantly expressed lncRNAs and mRNAs in early stage lung squamous cell carcinoma tissues compared to their corresponding adjacent nontumorous tissues. Bioinformatics analyses were used to investigate the functions of aberrantly expressed mRNAs and their associated lncRNAs. After that, in order to identify lncRNA biomarkers for early detection, candidate lncRNA biomarkers were selected based on our established filtering pipeline and validated by real-time quantitative polymerase chain reaction on a total of 63 pairs of tumor samples. Five lncRNAs were finally identified which were able to distinguish early stage tumor and normal samples with high sensitivity (92%) and specificity (83%). These results imply that lncRNAs may be powerful biomarker for early diagnosis.

P2.01-064 Molecular Context of Immune Microenvironment in Early-Stage Lung Squamous Cell Carcinoma

Although it has been proposed that the number of somatic mutations, together with high PD-L1 and CD8 expression, defines a type of tumor microenvironment predictive of response to immune checkpoint inhibitors, this data has been challenged because the methodologies are difficult to reproduce (e.g. tissue microarrays, whole exome sequencing or complicated scoring approaches). This situation prompted us to investigate possible alternatives that are easier to reconcile with daily practice. A total of 40 consecutive patients with early stage lung squamous cell carcinoma who underwent surgery at HM Sanchinarro University Hospital were considered. Automated immunohistochemistry (IHC) for PD-L1 expression was performed on whole tissue sections (Benchmark ULTRA, OptiView, Ventana Medical Systems, USA) with three different antibodies: SP142 (Ventana), SP263 (Ventana), and E1L3N (Cell Signaling). PD-L1 IHC was considered positive according to the criteria used in the corresponding clinical trials. P53 aberrant IHC expression was used as a surrogate marker for TP53 mutations. Whole tissue sections were also automatically scored for CD8+ tumor-infiltrating lymphocytes (TILs) density (off-label algorithm on the iScan Coreo). Afterwards, we performed targeted next-generation sequencing (NGS) in 52 genes (Oncomine Focus AssayTM, Life Technologies, USA). The prognostic impact of all these variables was also evaluated. Correlation between E1L3N and SP142 or SP263 was similar when scoring tumor cells (TCs) (0.94). There was a significant association between the intraepithelial and peritumor stromal CD8+ TILs density and overall survival when using the image analysis software (p=0.032). The presence of >247 CD8+ cells/mm2 had a 94% specificity and a 67% sensitivity for identifying patients with at least 5% SP142 PD-L1+ TCs. The highest percentage of PD-L1+ TCs were found in samples with CDK6 (2.6%) or MYC (2.6%) amplifications. Cases with FGFR1 amplification (7.9%) were negative for all PD-L1 antibodies. P53 aberrant expression and PD-L1 expression in TCs also seem to be related. The methodology presented herein could help align the use of targeted NGS and the immune microenvironment assessment to increase the clinical value of immune checkpoint inhibitors. Acknowledgements This study was partially funded by Instituto de Salud Carlos III (ISCIII), Fondo de Investigaciones Sanitarias (FIS), Fondos FEDER-Plan Estatal de I+D+I 2013-2016 (PI14-01176).

PIK3CA mutations as prognostic factor in squamous cell lung carcinoma

ObjectivesMutation in the PIK3CA gene is reported frequent in squamous cell carcinomas of the lung, but its potential prognostic role is still obscure. We have studied the prognostic importance of PIK3CA mutations as well as the relation to other markers in a large number of early stage lung cancers of squamous carcinoma subtype. Patients and methodsTumour tissue was obtained from 308 consecutively operated lung cancer patients with squamous cell carcinoma in the period 2003–2013. DNA was isolated according to standard procedures, and mutation analysis was done with either the SnapShot method and/or using PIK3CA specific primers in the Cobas system. PD-L1-expression was analysed with immunohistochemistryAfter thorough follow-up (median 67.6 months), overall survival and time to relapse was calculated. ResultsTumour tissue from 102 females and 206 males were analysed. 167 (54.2%) were in stage I, 96 (31.2%) in stage II and 45 (14.6%) in stage III. PIK3CA mutation was found in 35 (11.4%) patients, most frequently in exon 20. There were no differences in sex, stage or smoking behaviour between mutated and non-mutated cases. Patients with PIK3CA mutations had a significantly longer overall survival (p=0.042) and time to relapse (p=0.030) than non-mutated cases, and the difference in time to relapse was also retained in stage I-cases (p=0.044). PD-L1-expression was less frequent among mutated cases. ConclusionOur results indicate that PIK3CA mutations may confer a survival advantage in early stage squamous cell lung cancers, but further work is needed to confirm this finding.

Plasma miR-324-3p and miR-1285 as diagnostic and prognostic biomarkers for early stage lung squamous cell carcinoma.

BackgroundSpecific biomarkers for early detection and outcome prediction of lung squamous cell carcinoma (LSCC) are still lacking. This study assessed the differentially expressed miRNAs as potential biomarkers for early stage LSCC.ResultsBase on the results of multi-phase study, we found that miR-324-3p was significantly up-regulated, whereas mir-1285 was significantly down-regulated in plasma of stage I LSCC patients compared to healthy controls. ROC analysis showed that AUC of miR-324-3p and miR-1285 were 0.79 and 0.85, respectively. The combination of these two miRNAs could further improve the diagnostic accuracy (AUC = 0.89). The multivariate analysis revealed that plasma miR-324-3p level was an independent prognostic predictor for early stage LSCC.Methods395 patients and 195 healthy controls were enrolled in this study. We screened the differentially expressed plasma miRNAs using TaqMan Low Density Arrays (TLDA) followed by three-phase qRT-PCR validation. We also evaluated the association of candidate miRNAs with overall survival of early stage LSCC patients. Finally, the target genes of the candidate miRNAs were analyzed using public available databases and bioinformatics methods.ConclusionsThe current study suggests that plasma miR-324-3p and miR-1285 levels could serve as LSCC early detection markers while miR-324-3p may serve as a prognostic marker for LSCC patients.

Defining a FISH chromosomal aneusomy panel for predicting lung cancer in the setting of CT-detected lung nodules.

10580 Background: Early detection of lung cancer by CT is supported by the National Lung Screening Trial; but while sensitivity of CT is high, specificity is low. Biomarkers to predict the malignant nature of CT detected lung nodules would have great clinical utility. We previously found in a nested case-control study that a 4-target chromosomal aneusomy (CA) FISH panel exhibited sensitivity/specificity of 76%/88% in sputum samples of heavy smokers obtained within 18 months of lung cancer diagnosis. We hypothesized that CA-FISH may be an effective biomarker to assist with clinical decisions in the setting of CT detected lung nodules of indeterminate etiology and attempted to identify a more effective reagent. Methods: Homebrew probes encompassing genomic sequences of EGFR, NXK2-1, PIK3CA, MYC, BRF2, SOX2, PPMID, FGFR1 and the commercial reagent LSI D5S721/D5S23 (Abbott Molecular) were combined in 2-4-target FISH assays to investigate tissue copy number in early stage lung squamous cell carcinoma (SCC, N=19) and adenocarcinoma (AC, N=20). Logistic regression models were used to estimate predictive discrimination [sensitivity, specificity, area under the ROC curve (AUC)]. Results: Copy number gain was largely detected for all markers (mean range 3.39 - PPMID to 4.67 - MYC). Mean copy number of PI3CA, BRF2, SOX2 and FGFR1 were significantly higher in SCC than AC, while NKX2 and MYC were marginally higher in AC than SCC. Gene amplification was detected for all 9 markers, most frequently for SOX2 and FGFR1 (6 and 5 cases) with significant overlap between FGFR1/BRF2 and SOX2/PIK3CA. Based on the AUC results and the existence of targeted inhibitors, the probe set EGFR/FGFR1/MYC/PIK3CA was selected as a candidate for further development as an adjunct to CT screening for early detection. For this selected set,the optimal cutoff based in the linear predictor from logistic regression yields a sensitivity and specificity of 0.85. Conclusions: A highly sensitive/specific CA-FISH probe set was identified which may well complement CT screening in the early diagnosis of lung cancer. This probe set will be tested in the setting of CT detected lung nodules. (Supported by LUNGevity and NCI-Lung Cancer SPORE grants).

A computational index derived from whole-genome copy number analysis is a novel tool for prognosis in early stage lung squamous cell carcinoma

Squamous cell carcinoma of the lung is remarkable for the extent to which the same chromosomal abnormalities are detected in individual tumours. We have used next generation sequencing at low coverage to produce high resolution copy number karyograms of a series of 89 non-small cell lung tumours specifically of the squamous cell subtype. Because this methodology is able to create karyograms from formalin-fixed paraffin-embedded material, we were able to use archival stored samples for which survival data were available and correlate frequently occurring copy number changes with disease outcome. No single region of genomic change showed significant correlation with survival. However, adopting a whole-genome approach, we devised an algorithm that relates to total genomic damage, specifically the relative ratios of copy number states across the genome. This algorithm generated a novel index, which is an independent prognostic indicator in early stage squamous cell carcinoma of the lung.

Early detection of squamous cell lung cancer in sputum by a panel of microRNA markers

Squamous cell carcinoma is a common form of lung cancer, the leading cause of cancer deaths in the world. Identifying early stage lung squamous cell carcinoma patients who would benefit most from effective therapies will reduce the mortality. We have previously shown that microRNAs (miRNAs) were stably present in sputum and potentially useful in diagnosis of lung cancer. The objective of this study was to develop a panel of miRNAs that can be used as a sputum-based test for early stage squamous cell carcinoma of the lungs. This study contained three phases: (1) marker discovery by profiling miRNA expression signatures on 15 lung squamous cell carcinoma and matched normal lung tissue samples with GeneChip miRNA Array; (2) marker optimization by real-time quantitative RT-PCR on sputum of a case–control cohort of 48 stage I lung squamous cell carcinoma patients and 48 healthy individuals; and (3) marker validation on an independent set including 67 lung squamous cell carcinoma patients and 55 healthy subjects. On the surgical tissues, six miRNAs were identified, of which three were overexpressed and three were underexpressed in all 15 tumors. On the sputum samples of the case–control cohort, three (miR-205, miR-210 and miR-708) of the six miRNAs were selected, which in combination produced the best prediction in distinguishing lung squamous cell carcinoma patients from normal subjects with 73% sensitivity and 96% specificity. Validation of the marker panel in the independent populations confirmed the sensitivity and specificity that provided a significant improvement over any single one alone. The sputum markers showed the potential to improve the early detection of lung squamous cell carcinomas.

Prognostic value of three microRNA expression profiles in early-stage squamous cell lung cancer (SqCLC).

7028 Background: About 50% of NSCLC patients (pts) will develop distant metastases following pulmonary resection. Currently, apart from clinical stage at diagnosis, there are no reliable clinical factors to select high risk pts for adjuvant chemotherapy. We previously demonstrated high prognostic value of selected microRNAs in frozen tissues of early stage SqCLC (Skrzypski et al, J Clin Oncol 27; 15s: 2009). In the present study we evaluated the feasibility and prognostic relevance of this assay in formalin fixed paraffin embedded (FFPE) samples. Methods: FFPE tumor tissue was obtained from 72 stage I-II SqCLC pts. Of those, 30 pts developed distant metastases and 42 had no relapse after a median follow-up of 5.6 years (range, 3.8-7.1 years). MicroRNA was isolated from paraffin blocks after macrodissection of tumor tissue, and extracted with RecoverAll kit (Ambion). Expression of 5 microRNAs was analyzed by RT-PCR assays (Appliedbiosystems). Raw expression data were normalized vs. the expression of U6 RNA and calibrated by ΔΔCt method. After z-score transformation, the risk score was constructed based on the expression of 3 MiRs. Results: MicroRNA yield from tumor tissue was successful in 92% of cases. Expression of MiR 192* and MiR 10b was significantly related to the time to distant metastases (log-rank; p=0.001 and p=0.003, respectively), whereas MiR 532-3p was of borderline significance (p=0.08). The risk score based on the expression of MiRs 192*, 10b and 532-3p was significantly related to the time to distant metastases (log-rank; p=0.00006). With the risk score median as a cut-off value, the test sensitivity for distant relapse prediction was 80% at the specificity level of 72%. Conclusions: Prognostic test based on microRNA assessment in FFPE is feasible and robust. Three-miRNA expression profile: 192*, 10b and 532-3p is strongly related to the risk of distant metastases in operable SqCLC. The results are being validated. No significant financial relationships to disclose.

Prognostic value of microRNAs (miRNAs) profiling in early-stage squamous cell lung cancer (SqCLC)

7594 Background: About 50% of NSCLC patients (pts) will develop distant metastases following pulmonary resection. Currently, apart from clinical stage at diagnosis, there are no reliable clinical factors to select high risk pts for adjuvant chemotherapy. miRNAs profiling is expected to indicate pts with aggressive disease and to provide prognostic information. Confounding effect of NSCLC pathology heterogeneity justifies searching for prognostic miRNAs separately in SqCLC and adenocarcinoma. Methods: Fresh frozen tumor tissue was obtained from 30 SqCLC stage I-II pts during curative pulmonary resection. Of those, 15 pts developed distant metastases and 15 had no relapse after a median follow-up of 5.4 years (range, 3.1–6.9 years). RNA was isolated from tumor sections containing ≥60% of tumor cells per section. Expression of 380 miRNAs was analyzed by RT-PCR arrays (TLDAs-Appliedbiosystems). Raw data were normalized vs. the expression of U6 RNA and calibrated by ΔΔCt method. Results: Expression of 300 miRNAs was detected in tumor tissues. Expression of three miRNAs (193a[5p], 146b[5p] and 10b) differed between groups with and without metastases (t-Student test, p&lt;0.05). Due to the limited number of samples, proportional hazard regression for survival (Cox analysis) could not be performed on the full data set of 300 variables. Cox analysis performed for the top 5 miRNA identified with t-Student test showed significant correlation for 10b (p&lt;0.02). The internal consistency of data sets was tested using PCA, and three samples were excluded. In this modified test set, the top 10 miRNAs (ranked according to p-value of t test) revealed two significantly correlated miRNAs in Cox analysis: 484 and 146b (5p). Similar results were obtained for miRNAs expression defined as continuous or categorized variables. Conclusions: Four miRNAs: 10b, 146b, 193a(5p) and 484 are potentially related to high metastatic propensity of early stage SqCLC. Further validation is necessary to confirm their prognostic relevance in this tumor. No significant financial relationships to disclose.

500 POSTER Real time RT-PCR 3-gene expression signature predicts survival in early-stage squamous cell lung cancer

500 POSTER Real time RT-PCR 3-gene expression signature predicts survival in early-stage squamous cell lung cancer