Virtual Library

Start Your Search

P.C. Ma



Author of

  • +

    MINI 08 - Prognostic/Predictive Biomarkers (ID 106)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
    • +

      MINI08.01 - Quantitative Mass Spectrometry Proteomics Identifies FRalpha and GARFT as Predictive Biomarkers in NSCLC Patients Treated With Pemetrexed (ID 1685)

      16:45 - 18:15  |  Author(s): P.C. Ma

      • Abstract
      • Presentation
      • Slides

      Background:
      Lung cancer remains the leading cause of cancer mortality in United States and globally. Pemetrexed combined with platinum chemotherapy is specifically indicated for treatment of non-squamous non-small cell lung cancer (non-sq NSCLC). Pemetrexed is a folate-analog metabolic inhibitor that disrupts folate-dependent processes essential for cell replication. Pemetrexed inhibits thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT), which are folate-dependent enzymes involved in the de novo biosynthesis of thymidine and purine nucleotides. Folate receptor alpha (FRalpha) is a folate/antifolate transporter protein that is overexpressed by a number of epithelial tumors. The purpose of this study is to identify proteomic biomarkers predictive of response to pemetrexed-based chemotherapy in non-sq NSCLC.

      Methods:
      Patients with advanced non-sq NSCLC who received pemetrexed-based chemotherapy at West Virginia University from 2009 to 2014 were retrospectively identified. Formalin-fixed, paraffin-embedded tumor biopsies were laser microdissected, solubilized, enzymatically digested and subjected to quantitative proteomic analysis. A multiplexed, selected reaction monitoring (SRM) mass spectrometry (MS) assay was used to determine the absolute levels of 46 different candidate proteomic markers, including those in the folate receptor pathway. The Kaplan-Meier method and log-rank test were used in statistical analysis of overall survival (OS) and progression-free survival (PFS).

      Results:
      The 74 patients included in the study had a median follow-up of 26 months, a median OS of 16.6 months (95%CI: 11.6 - 43.4), and a median PFS of 9.61 months (95%CI: 8.43, 12.98). There were 65 patients who received pemetrexed-based regimen as a first line therapy and 9 patients as subsequent salvage treatment. In a comparison between patients who survived >24 months and < 8 months, there were no significant differences between the two groups in terms of sex, age, ECOG performance status, TNM stage at diagnosis, and smoking history. Among the 37 patients with sufficient tumor specimens available for multiplexed proteomic analysis, 30 biomarkers were detected with varying levels of expression. Sixteen additional biomarkers were undetectable. TS protein expression was detected in only 2 patients. Patients whose tumors expressed low levels of GARFT protein (≤900 amol/µg; n=7) had statistically significantly longer median PFS than those whose tumors expressed high levels of GARFT (>900 amol/µg; n=30) (40.6 vs. 11.4 months; p= 0.014). Patients with high FRalpha protein expression (>1510 amol/µg, n=9) had significantly longer median PFS than those with low FRalpha expression (≤1510 amol/µg; n=28) (>50 vs. 11.4 months; p= 0.021). Moreover, the 23 patients with both high GARFT expression (>900 amol/µg) and low FRalpha expression (≤1510 amol/µg) faired considerably worse than the remainder of patients (median PFS 10.1 vs. 40.6 months; p=0.0003).

      Conclusion:
      Multiplexed mass spectrometry-based proteomics offers a feasible and promising approach for tumor biomarker profiling and quantification to predict therapeutic response. Of note, our results show that FRalpha and GARFT protein expression may be predictive of response to pemetrexed-based treatment in patients with non-sq NSCLC. Further investigation is needed to validate the utility of these biomarkers for guiding personalized treatment decisions in clinical practice.

      Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.

      Only Active Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login or select "Add to Cart" and proceed to checkout.

  • +

    MINI 09 - Drug Resistance (ID 107)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
    • +

      MINI09.02 - Transcriptome-Metabolome Reprogramming of EGFR-Mutant NSCLC Contributes to Early Adaptive Drug-Escape via BCL-xL Mitochondrial Priming (ID 3085)

      16:45 - 18:15  |  Author(s): P.C. Ma

      • Abstract
      • Slides

      Background:
      Precision therapy using EGFR small molecular inhibitors is the current standard-of-care in treatment of advanced non-small cell lung cancer (NSCLC) patients (pts) with EGFR mutations. Nonetheless, emergence of acquired resistance to therapy invariably occurs despite effective initial response. Classical rebiopsy studies of EGFR-mutant pts at clinical tumor progression based on RECIST criteria have identified diverse resistance mechanisms involving T790M-EGFR, MET amplification or activation and AXL upregulation. Tumor cells within minimal or microscopic residual disease during drug response may constitute founder cells for future disease relapse. The mechanisms of molecular changes intrinsic to these early therapeutic survivors are not yet well-understood. Our studies focus on tumor cells adaptation early during therapy to map the initial course of molecular drug resistance emergence and evolution.

      Methods:
      Drug-sensitive model studies of EGFR-mutant lung cancer were performed using HCC827 and PC-9 cells (exon 19 deletions EGFR) under erlotinib, and H1975 (T790M/L858R-EGFR) cells under CL-387,785 inhibition. Affymetrix microarray profiling was performed in triplicate at 0h, 8h, 9d and 9d tyrosine kinase inhibitor (TKI) followed by 7d drug-washout. Both in vitro and in vivo xenograft analyses, immunofluorescence, immunohistochemistry, time-lapse video microscopy analysis were conducted. Mass-spectrometry based global metabolomics profiling was also conducted under similar conditions as in gene expression profiling.

      Results:
      We identified an early adaptive and reversible drug-escape within EGFR-mutant cells that could emerge as early as 9 days during course of effective therapy with molecular drug resistance. Principal component analysis (PCA) of gene expression profiling data identified distinct transcriptome signatures in each cell state. Of note, the prosurvival cell state was independent of MET pathway activation, and had a TKI cytotoxicity escape at 100x higher IC50. The drug resistant cell state was associated with reversible cellular quiescence, suppressed Ki-67 expression, and profoundly inhibited cellular motility and migration. Transcriptome gene expression profiling revealed a remarkable adaptive genome-wide signature reprogramming, centered on the autocrine TGFβ2 cascade that involved pathways of cell adhesion, cell cycle regulation, cell division, glycolysis, and gluconeogenesis. Global metabolomic profiling of cellular metabolites in HCC827 cells under erlotinib inhibition also revealed a concurrent adaptive reprogramming of cellular metabolism during the early drug-resistant cell state, with suppression of glycolysis, TCA cycle, amino acids metabolism, and lipid bioenergetics. Our studies identified a direct link of TGFβ2 within the drug escaping cells, with the metabolic-bioenergetics quiescence, reverse Warburg metabolism and mitochondrial BCL-2/BCL-xL priming. Furthermore, this adaptive drug-resistant cell state also displayed an increased EMT and cancer stem cell signaling as adaptation to the drug treatment and that could be overcome by broad BCL-2/BCL-xL BH3 mimetic ABT-263, but not BCL-2 only mimetic ABT-199.

      Conclusion:
      We identified and characterized the emergence of early adaptive drug-escape within EGFR-mutant NSCLC cells amid an overall precision therapy excellent response, through a MET-independent mechanism. The profoundly drug-resisting prosurvival cell state undertook remarkable cellular transcriptome-metabolome adaptive reprogramming coorindated through autocrine TGFβ2 signaling augmentation. Our study results have important implications in lung cancer drug-resistant minimal/microscopic disease and future therapeutic remedies in precision therapy.

      Only Active Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login or select "Add to Cart" and proceed to checkout.

  • +

    MINI 26 - Circulating Tumor Markers (ID 148)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
    • +

      MINI26.07 - Circulating Tumor Cells (CTC) Enrichment as Liquid-Biopsy for Molecular and Genomic Characterization in ALK-Rearranged (ALK+) Lung Cancer (ID 3132)

      16:45 - 18:15  |  Author(s): P.C. Ma

      • Abstract
      • Presentation
      • Slides

      Background:
      Precision therapy with tyrosine kinase inhibitor (TKI) crizotinib and ceritinib against EML4-ALK (ALK+) non-small cell lung cancer (NSCLC) has advanced rapidly in recent years as a new paradigm in personalized cancer therapy. However, acquired drug resistance despite initial response still remains the rule, necessitating further investigations into mechanisms of resistance and novel therapies to overcome progressive resistant disease. Liquid-biopsy using peripheral blood circulating tumor cells (CTCs) as a minimally-invasive tool to determine patient’s disease status, tumor cells molecular-genomic make-up and evolution during therapies, is highly desirable. There is still an unmet need to develop affordable and robust technology platforms to empower such liquid-biopsy assay of CTC. There are relative advantages and pitfalls with various CTC platforms and a method to capture CTC in an unbiased fashion without pre-definition would be beneficial.

      Methods:
      We conducted pilot studies with adoption of two different CTC detection and enrichment platforms. First, we used the CellSearch[®] “positive-selection” platform through EpCAM immunomagnetic separation to profile 11 pts with ALK(+) NSCLC who were treated with crizotinib prospectively. Blood samples were collected (i) pretreatment, (ii) on TKI with CR/PR/SD, and (iii) at disease progression. Second, we evaluated a novel “negative-selection” CTCs capture-isolation platform, based on unbiased immunomagnetic removal of pan-leukocyte marker CD45+ cells coupled with RBC lysis, to enable CTC isolation without predefined CTC criteria. Pilot studies utilizing ALK+ H3122 cell line and ALK+ patients’ blood samples were performed for assay optimization and comparison. Whole genome sequencing using Illumina HiSeq x TEN was performed after whole genome amplification of the CTC tumor gDNA with paired-normal germline DNA for genomic interrogation.

      Results:
      Using ALK+ NSCLC patients’ peripheral blood samples, we demonstrated the presence of the EML4-ALK fusion (variant 1) in QPCR assay from the enriched CTC isolated using the CellSearch® platform. Also, CellSearch[®] enumeration in our pilot ALK+ cohort revealed a trend of correlation between the CTC numbers and disease status. The spike-in experiment in “negative selection” CTC platform enriched the spiked H3122 cells by 10,000 fold from the nucleated blood cells. Applying our “negative-selection” CTC assay to a patient with known EML4-ALK variant 1 (EML4-ex13/ALK-ex20) fusion lung cancer during disease progression on crizotinib, we detected the specific EML4-ALK variant 1 fusion in QPCR assay from the CTC enriched “eluate” fraction, but not in the “feed” fraction, whether the CellSearch® platform revealed any CTCs or not. In our index case of ALK-rearrangement NSCLC, we successfully performed whole genome sequencing analysis on the pretreatment negative-selection CTCs in comparison with the germline DNA and pretreatment lymph node tumor biopsied tissue tumor DNA. Our preliminary WGS results revealed similar genomic landscapes between the CTC and the biopsied tumor tissues.

      Conclusion:
      Taken together, our pilot CTC study results support the high sensitivity of the unbiased “negative selection” enrichment platform and its potential to empower molecular and genomic determinations in lung cancer. We also demonstrated the feasibility of the negative-selection CTC liquid-biopsy platform to achieve whole genome sequencing analysis of the captured CTCs.

      Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.

      Only Active Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login or select "Add to Cart" and proceed to checkout.

  • +

    MINI 29 - Meta Analyses and Trial Conduct (ID 156)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Treatment of Advanced Diseases - NSCLC
    • Presentations: 1
    • +

      MINI29.13 - Safety and Clinical Implications of Repeat Tumor Biopsy (RTB) in Patients with Advanced Lung Cancer: A Retrospective Institutional Study (ID 3146)

      18:30 - 20:00  |  Author(s): P.C. Ma

      • Abstract
      • Presentation
      • Slides

      Background:
      Clinical management of lung cancer and personalized cancer therapy have undergone major advances and paradigm shifts in recent years. Repeat tumor biopsy (RTB) at disease progression is not only used for diagnostic confirmation in lung cancer but also has recently been increasingly adopted to profile tumor biomarkers and identify drug resistance mechanisms. However, information on safety and clinical consensus on the use of RTB remain lacking.

      Methods:
      The aim of this study is to review RTB patterns and safety in advanced lung cancer patients at Cleveland Clinic and its impact on treatment (Rx) decisions. Patients who were diagnosed and underwent RTB for suspected progressive disease between 2007-2013 were included in this retrospective study. Statistical analysis is primarily descriptive.

      Results:
      The study involved a total of 184 (56% male) patients. Median age at diagnosis was 65Y (21-87). 100 (54%) were treated initially with single modality (Surgery = 41; Chemo = 33; Radiation = 17; targeted therapy = 9) and 83 (45%) with multimodality Rx (2-modality = 57, 3-modality = 26), 1 (1%) unknown. Number of RTB per patient: 1 in 66.3% (n=122), 2 in 20.1% (n=37), 3 in 11.4% (n=21), and 4 in 2.2% (n = 4). The most common procedure employed at 1st RTB was bronchoscopy (44.6%, n = 82), followed by CT-guided biopsy (bx) (20.7 %, n=38), surgery (10.3%, n=19), excision bx (8.2%, n=15), fine needle aspiration of skin & lymph node (LN) (7.6%, n=14), ultrasound guided bx (5.9%, n=11) & others (2.7%, n=5). Lung was the most commonly rebiopsied site (46%) followed by LN (15%). Procedure-related complications were reported in 13 of 181 (7.2%) pts at 1st RTB (data missing in 3 pts), 3 of 61 (4.9%) at 2nd RTB, 1 of 25(4%) at 3rd RTB, and 0 of 4 (0%) at 4th RTB. The 17 (6.2%) complications are shown in the table below. Histologic change was seen in 13 cases, including adeno-to-squamous carcinoma (at erlotinib resistance) and vice-versa, and non-small cell to small cell histology. The T790M-EGFR mutation was noted in 6 cases, the PIK3CA mutation in 1, and a change in ALK translocation status in 3. Medical decision-making was impacted in 16% of cases. Further molecular and genomic analysis of selected cases is in progress.

      RTB Complications
      Complications N=17 (%)
      Bleeding without hemodynamic compromise 6 (35)
      Bleeding requiring transfusion 1 (6)
      Pneumothorax 5 (29)
      Hemodynamic instability after premedication 1 (6)
      Cerebral salt wasting 1 (6)
      Tracheaoesophageal fistula 1 (6)
      Severe cough 1 (6)
      Incomplete procedure 1 (6)
      Deaths 0 (0)


      Conclusion:
      Our study results show that RTB can be safely performed in lung cancer patients using standard techniques and can impact lung cancer diagnosis and Rx decision-making.

      Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.

      Only Active Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login or select "Add to Cart" and proceed to checkout.

  • +

    ORAL 22 - Moving Beyond a Smoking Related-Cancer to the Young, Never-smokers and Inherited Disease (ID 117)

    • Event: WCLC 2015
    • Type: Oral Session
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
    • +

      ORAL22.06 - Whole Exome Sequencing to Profile the Genomic Landscapes of Young Patients (pts) Diagnosed with Non-Small Cell Lung Cancer (NSCLC) (ID 3073)

      10:45 - 12:15  |  Author(s): P.C. Ma

      • Abstract
      • Slides

      Background:
      The incidence of NSCLC in pts 45 years of age or younger is ~2% of total cases, with annual newly diagnosed cases reaching 4,500 in the United States alone. Majority of these pts are diagnosed at an advanced stage with poor outcomes. Although several specific genomic alterations have been identified in young NSCLC pts particularly in light-/never smokers, e.g. EGFR mutations, the overarching underlying causative mechanisms in the pathogenesis and progression in these young pts remain largely unknown, and likely are different from older pts. The objective of this study is to examine the genomic landscapes of NSCLC in young pts through comprehensive whole exomic survey with the objective to arrive at novel predictive and prognostic genomic biomarkers and therapeutic opportunities in young NSCLC pts.

      Methods:
      We initially identified a cohort of 20 pts (40% male) diagnosed with NSCLC at an age of ≤45, who underwent surgical resection for the primary tumors or metastatic lesions at Cleveland Clinic from 2000-2012. Matching genomic DNA from FFPE lung tumor samples and paired-normal lung tissue/peripheral blood was subjected to whole exome sequencing using Illumina HiSeq 2000 platform. Exome variant calling was performed using GATK and/or SOAPsnp algorithms to identify somatic mutations in individual tumors. Pathway and protein-protein interaction (PPI) network analysis on mutant genes was performed using KEGG/NCI-PID databases and HotNet suite, respectively. Second cohort of young NSCLC tumor cases (n=50) were identified from the Sun Yat-sen University Cancer Center and genomic analysis is underway.

      Results:
      Majority of the tumors from the first cohort had adenocarcinoma (n=12) or squamous cell (n=4) histology. Six (6) pts were never-smokers, while the others had a median 30 pack-year cigarette smoking history. A significantly higher mutation burden was found in smokers (Median, 3.47/Mb) compared to never-smokers (Median, 0.76/Mb). We also found that the G:C→T:A transversions were more common in smokers, and C:G→T:A transitions more common among never-smokers. Key driver cancer genes such as TP53 (50%) and KRAS (17%) harbored mutations exclusively in smokers, whereas EGFR mutations (14%) were observed specifically in never-smokers. Interestingly, global pathway/PPI analysis of the mutant genes revealed distinct sub-networks associated with cell adhesion and epithelial mesenchymal transition (EMT) processes with a 7-fold enrichment in mutation frequency in these young pts when compared to their overall frequencies in the COSMIC/TCGA lung cancer dataset.

      Conclusion:
      Our study nominated novel candidate genes/pathways especially relating to cell adhesions and EMT processes, that potentially play a key role in early-onset NSCLC. Further analysis and validation of our findings could improve our understanding of lung cancer pathogenesis and eventually lead to precision therapies to benefit younger NSCLC pts.

      Only Active Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login or select "Add to Cart" and proceed to checkout.

  • +

    ORAL 42 - Drug Resistance (ID 160)

    • Event: WCLC 2015
    • Type: Oral Session
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
    • +

      ORAL42.01 - ALK-Rearranged NSCLC Adaptive Cell Plasticity with Early Onset TGFb2 Mediated Precision Drug Escape through PRC-2 Epigenetic Reprogramming (ID 3111)

      18:30 - 20:00  |  Author(s): P.C. Ma

      • Abstract
      • Slides

      Background:
      ALK-tyrosine kinase inhibitor (ALKi) is currently the standard precision therapy for advanced ALK(2p23)-rearranged (ALK+) non-small cell lung cancer (NSCLC), often with impressive primary responses. Nonetheless, acquired clinical resistance even in excellent/complete responders still develops ultimately with time; thus hampering long term benefits. Classic tumor rebiopsy studies that deciphered drug-resistance mechanisms focused on the “late phase” resistance at time of clinical progression in treated ALK+ NSCLC. These studies identified diverse pattern of drug-resistance mechanisms, including numerous non-dominant secondary drug-resistant ALK kinase mutations (e.g. C1156Y and L1196M), bypass signaling pathways (e.g. EGFR, KIT signaling), ALK gene amplification, and overexpression of microenvironmental factors (e.g. EGF, TGF-α, HGF). The mechanisms underlying the initial and early emergence of drug-resistance under precision therapy are poorly understood.

      Methods:
      EML4-ALK(+) H3122 and patient-derived ALKi acquired resistant biopsied-lung tumor tissue cells were used to investigate drug-escape mechanisms. Stem cell transcription factors QPCR array and RNA-sequencing profiling were performed on H3122 cells under ALKi up to day 14, compared with untreated and drug-washout controls. MTS cell viability assays using ALKis, in vitro and in vivo tissues QPCR assays, as well as in vivo xenograft IHC analyses were also performed. Patient-derived bronchoscopic biopsied NSCLC tissues (Ma0083) during ALKi resistance was procured and propagated in cell culture in accordance with approved institutional protocols.

      Results:
      We identified that H3122 cells displayed cell plasticity and can escape ALKi’s (TAE-684, crizotinib) remarkably early after precision therapy initiation, with augmented prosurvival signaling via upregulated autocrine TGFβ2 signaling, but not TGFβ1 or β3, as early as day 14 post-treatment. We validated using both in vitro and in vivo models the upregulated cascade of tumoral TGFβ2-HOXB3-mitochondrial priming during adaptive drug-escape. The early onset drug-resistant cells were marked by reversible autocrine TGFβ2-mediated transcriptome reprogramming with reversibly enhanced EMT-ness and cancer stemness. Moreover, RNA-seq findings strongly suggest a “reverse Warburg” cell state during adaptive drug-escape. The adaptive cellular plasticity was verified also in patient-derived bronchoscopic biopsied NSCLC tissues (Ma0083) with ALKi resistance. Interestingly, inhibiting mitochondrial priming using dual BCL-2/BCL-xL BH3-mimetics ABT-263 was effective to suppress early drug-escape, but not with the BCL-2-specific agent ABT-199, suggesting BCL-xL is a key target. Importantly, we also identified upregulated HOXB3 expression correlated with the early adaptive drug-resistance cell state, emerged through dynamic remodeling of EZH2/UTX in the polycomb repressive complex-2 (PRC-2). Deregulated EZH2/UTX epigenetic balance impacted the poised chromatin state of HOXB3 promoter H3K27me3/H3K4me3 histone marks. Early drug-escape cell state was correlated with suppressed EZH2 expression, at mRNA and also protein levels, in both in vitro and in vivo models. Finally, our results showed that specific EZH2 inhibitor GSK126 promoted ALKi drug-resistance, while UTX inhibitor GSK-J4 eradicated ALKi adaptive drug-resistance.

      Conclusion:
      Our study findings provide novel insights into the initial emergence and evolution of ALK precision drug-resistance and highlighted the significance of understanding the role of adaptive tumor cell plasticity in the early drug-escape process with important therapeutic implications. Therapeutic modulation of the coordinated EZH2/UTX balance in the PRC-2 complex can profoundly impact ALKi drug treatment outcome.

      Only Active Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login or select "Add to Cart" and proceed to checkout.

  • +

    P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)

    • Event: WCLC 2015
    • Type: Poster
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
    • +

      P3.04-120 - Quantitative Immunofluorescence Based Expression Analysis in NSCLC Reveals Nuclear EZH2 as Poor Prognostic Biomarker (ID 2396)

      09:30 - 17:00  |  Author(s): P.C. Ma

      • Abstract

      Background:
      EZH2 is a histone-lysine N-methyltransferase enzyme and the key functional enzymatic component of the polycomb repressive complex 2 (PRC2), which is a crucial epigenetic regulator in cancer cell survival. EZH2 methylates histone 3 at lysine 27 (H3K27me/me2/me3) and has been associated with the heterochromatin state, transcriptional repression and activation, hematopoiesis, development, and cell differentiation. Activating EZH2 mutations has been identified in lymphoma, and EZH2 overexpression has recently been reported in solid tumors including melanoma, breast, prostate, and lung cancer. Inhibition of EZH2 is a promising therapeutic strategy and a number of EZH2 targeting drugs are currently in clinical development.

      Methods:
      Multiplexed QIF assay was used to evaluate EZH2 expression using monoclonal antibody (clone D2C9, cell signaling technology) against human EZH2, and cytokeratin (AE1/AE3, Dako) in a retrospective cohort of 298 stages I-IV NSCLC represented in tissue microarray (TMA) format. H3122 NSCLC xenografts and control patient samples were used to determine staining specificity and optimal titer. The association between EZH2 level, clinico-pathological characteristics and survival were studied. The classification and regression tree (CART) analysis was used to determine the optimal cutoff of EZH2 expression to predict survival. Kaplan-Meier and log-rank test were used in statistical analysis of overall survival. Chi-square test was used for clinic-pathologic correlation statistical analysis.

      Results:
      EZH2 protein was detected predominantly in the tumor compartment with nuclear staining pattern. A high EZH2 level was detected in 82% of cases and was correlated with active smoking (92% vs. 64%, P=0.005) and squamous cell histology (94% vs 76%, P=0.013) (Table 1). Elevated tumor nuclear EZH2 expression was significantly associated with worse survival (median survival 53 vs. 104 months; log-rank P=0.002) (Figure 1).

      Table 1: Clinical Correlations of EZH2 expression in NSCLC
      EZH2.in.Nuclear.AQUA.Norm> 570 Chi-square test
      All (%) No (%) Yes (%) p-value
      Age
      <70 120 (56.1) 22(18) 98(82) 0.864
      ≥70 94 (43.9) 19(20) 75(80)
      Gender
      Female 130 (60.7) 25(19) 105(81) 0.885
      Male 84 (39.3) 16(19) 68(81)
      Tobacco.history
      Current Smoker 49 (22.9) 4(8) 45(92) 0.005
      Former 113 (52.8) 21(19) 92(81)
      Never 39 (18.2) 14(36) 25(64)
      unknown 13 (6.1)
      Histology
      Adenocarcinoma 134 (62.6) 32(24) 102(76) 0.013
      Others 26 (12.1) 6(23) 20(77)
      Squamous Cell 54 (25.3) 3(6) 51(94)
      Tumor Size
      <3cm 115 (53.7) 23(20) 92(80) 0.778
      ≥3cm 97(45.3) 17(18) 80(82)
      unknown 2 (1)
      Figure 1



      Conclusion:
      Our study shows that high nuclear EZH2 protein expression is a poor prognostic biomarker in NSCLC, and is correlated with smoking status and squamous cell histology.