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H. Kadara
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MINI 21 - Novel Targets (ID 133)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:B.P. Levy, D.S. Tan
- Coordinates: 9/08/2015, 16:45 - 18:15, Mile High Ballroom 2a-3b
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MINI21.07 - Oncogenic EZH2 Is an Actionable Target in Patients with Adenocarcinoma of the Lung (LUAD) (ID 3169)
17:20 - 17:25 | Author(s): H. Kadara
- Abstract
- Presentation
Background:
The methyltransferase enhancer of zeste homolog 2 (EZH2) belongs to the polycomb repressive 2 complex (PRC2). EZH2 is upregulated in several malignancies including prostate, breast and lung cancer. The EZH2 protein forms one of the critical protein complexes of PRC2 by partnering with EED (embryonic ectoderm development) protein. This EED/EZH2 complex has been shown to interact with histone deacytelase (HDAC). This interaction is highly specific and HDAC does not interact with any other PRC2 protein complexes. In the present study, we investigated the link between EZH2 and HDAC in lung cancer cell lines and in human tumor tissue microarrays (TMAs). We also further investigated EZH2 as a marker for response to HDAC inhibitors.
Methods:
We analyzed EZH2 and HDAC1 mRNA expression in two lung adenocarcinoma datasets (MDACC n=152, and TCGA n=308), and correlated the gene expression with tumors’ clinico-pathological characteristics and patients’ outcome. To study the association of EZH2 and HDAC1 expression with response to the HDAC1 inhibitor suberanilohydroxamic acid (SAHA), we examined mRNA and protein expression by RT-PCR and Western blot, respectively, in twelve lung adenocarcinoma (LUAD) cell lines at baseline and after overexpression or knock-down of EZH2 or HDAC1 gene expression using siRNA. Response to (SAHA) in cell lines was measured by MTT assay and correlated with protein and mRNA expression levels of EZH2 and HDAC1.
Results:
Direct and positive correlation was found between EZH2 and HDAC1 expression NSCLC cell lines (P <0.0001). This correlation was confirmed in NSCLC specimens from MDACC (Spearman’s correlation r=0.416; p < 0.0001) and TCGA datasets (r=0.221; p <0.0001).Patients with high EZH2 and high HDAC1 expression in stage I NSCLC specimens of MDACC and TCGA datasets had lowest survival compared to the patients who had either or both low expressions. Overall survival in the univariate analysis (MDACC dataset; Hazard Ratio (HR)=2.97; p=0.031 and TCGA dataset; HR=2.6 and p=0.041) and multivariate analysis (MDACC; HR=2.92 and p=0.034 and TCGA; HR=3.17 p=0.016). When EZH2 expression was knock down, there was a significant reduction in HDAC1 expression; conversely, when HDAC1 was knocked down EZH2 expression was also decreased. These concordant change in expression was seen both at the protein and mRNA level. Importantly, while all 8 cell lines with high EZH2 protein expression responded to SAHA treatment with average inhibition rate reaching 73.1%, three out of four cell lines with low EZH2 expression had a significantly lower response rate to SAHA inhibition with average inhibition rate 43.2% (P<0.0001). Additionally, altering the expression of EZH2 concordantly altered the sensitivity to SAHA i. e. forced increased expression of EZH2 increased the response to SAHA and vice versa.
Conclusion:
Our data suggest that EZH2 and HDAC expression are correlated in LUAD cell lines in human tissue microarrays and overexpression of both is a negative prognostic indicator. Additionally we show that increased EZH2 expression predicts for response to HDAC inhibitors and thus could serve as a biomarker for selecting LUAD patients with HDAC inhibitors.
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ORAL 21 - Biology - Moving Beyond the Oncogene to Oncogene-Modifying Genes (ID 118)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:A. Katz, M.S. Tsao
- Coordinates: 9/08/2015, 10:45 - 12:15, Mile High Ballroom 4a-4f
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ORAL21.02 - Landscape and Functional Significance of KRAS Co-Mutations in Lung Adenocarcinoma (LUAC) (ID 3224)
10:56 - 11:07 | Author(s): H. Kadara
- Abstract
- Presentation
Background:
The biological heterogeneity of KRAS-mutant LUAC represents a major impediment to the successful implementation of targeted therapeutic strategies for this clinically challenging group of lung cancer patients. Through integrative, multi-platform analysis of large scale omics data we recently identified three major subsets of KRAS-mutant LUAC defined on the basis of co-occurring genomic alterations in STK11/LKB1 (KL subgroup), TP53 (KP) and CDKN2A/B (KC), the latter coupled with low expression of the TTF1 transcription factor. We further demonstrated subset-specific molecular dependencies, patterns of immune system engagement and therapeutic vulnerabilities. Here, we extend these findings through comprehensive analysis of a wide panel of KRAS co-mutations and assess the impact of key co-mutations on facets of the malignant phenotype including flux through the MAPK and PI3K/AKT pathways and heterotypic interactions with the host immune system.
Methods:
Our datasets consisted of 431 tumors from TCGA (122 KRAS-mutant), 41 additional chemo-naive KRAS-mutant LUACs (PROSPECT dataset) and 36 platinum-refractory KRAS-mutant LUACs from the BATTLE-2 clinical trial. Significant KRAS co-mutations were identified on the basis of a P value threshold of ≤0.05 (Fisher’s exact test) coupled with a baseline prevalence of ≥3%. RNASeq data were downloaded directly from the TCGA site. Expression profiling of PROSPECT tumors was performed using the Illumina Human WG-6 v3 BeadChip Array whereas BATTLE-2 tumors were profiled using the GeneChipâHuman Gene 1.0 ST Array from Affymetrix. Generation of MAPK and PI3K proteomic scores, based on Reverse Phase Protein Array (RPPA) data, has been previously reported.
Results:
Our analysis identified somatic mutations in 31 genes as significantly co-mutated with KRAS in LUAC samples. Among them, co-mutations in STK11/LKB1 (P=0.00011) and ATM (P=0.0004) predominated. Somatic mutations in ERBB4 (P=0.0059), encoding a member of the ErbB family of receptor tyrosine kinases and MAP3K4 (P=0.0017) were also enriched in KRAS-mutant LUAC. We assessed the impact of KRAS co-mutations on the amplitude and directionality of signaling downstream of mutant KRAS using the proteomic “MAPK score“ and “PI3K score” as surrogates of effector pathway activation. Interestingly, co-mutations in ERBB4 were associated with significantly suppressed flux through the MAPK pathway (P=0.0024, t-test). Somatic mutations in other genes, including CAMSAP2, were associated with suppressed signaling through both the MAPK (P=0.00876, t-test) and PI3K-AKT (P=0.0032, t-test) cascades. Finally, within KRAS-mutant tumors, co-mutations in NLRC5, a master transcriptional regulator of MHC Class I molecules were associated with reduced mRNA expression of several of its classical target genes. In addition, low mRNA expression of NLRC5 correlated strongly with reduced expression of key components of the antigen presentation pathway across multiple independent datasets of chemotherapy naïve and platinum refractory KRAS-mutant tumors and cell lines. Thus, in addition to cell autonomous effects, co-mutations can also impinge on the reciprocal relationship between malignant cells and their immune microenvironment.
Conclusion:
Our work identifies a compendium of KRAS co-mutations that impact classical and emerging cancer hallmarks, including evasion of the host immune response. Systematic interrogation of the functional impact of prevalent KRAS co-mutations is essential for the development of personalized treatment approaches for this heterogeneous group of tumors.
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-066 - Programmed Cell Death Ligand 1 (PD-L1) Overexpression and Low Immune Infiltrate Score Correlate with Poor Outcome in Lung Adenocarcinoma (ID 776)
09:30 - 09:30 | Author(s): H. Kadara
- Abstract
Background:
PD-L1 is a key immunoregulatory checkpoint which suppresses cytotoxic immune response in a variety of physiologic and pathologic conditions. Thus, inhibition of PD-L1 can lead to reactivating tumor immunity and assist to cancer therapy. PD-L1 overexpression in the tumor cells has been correlated to a lessened immune response and consequent worse prognosis in a variety of cancers. To better understand the immune profiling of PD-L1 expression and its interplay with immune cells, we analyzed the correlation between image analysis-based immunohistochemical (IHC) expression of PD-L1 and tumor infiltrating immune cells density in surgically resected non-small cell lung carcinomas (NSCLC), and the correlation with clinical and pathological features, including patient outcome.
Methods:
IHC for PD-L1, PD-1, CD3, CD4, CD8, CD45RO, CD57, CD68, Granzyme B and FOXP3 were performed in 254 surgical resected stages I-III NSCLC, Adenocarcinoma (ADC=146) and Squamous cell Carcinoma (SqCC=108) from formalin-fixed and paraffin-embedded tissues. PD-L1 membrane expression on tumor cells and density of inflammatory cells were quantified using image analysis in intra-tumoral (IT) and peri-tumoral (PT) compartments. H-score > 5 was used as a cut-off for positive PD-L1 expression and an immune-score (IMS) using CD8/CD4/CD68 was devised. PD-L1 expression and inflammatory cells were correlated with clinico-pathologic features and patient outcomes.
Results:
Positive PD-L1 expression was seen in 26.84% (n=69) of the entire cohort, 23.29% (n=34) of 146 ADC and 23.40% (n=35) of 115 SqCC. In ADC, higher levels of PD-L1 expression were detected in tumors with solid histology pattern compared with other histology patterns (P=0.034), and in lifetime smokers compared with non-smokers (P<0.0001). In SqCC PD-L1 expression was positive correlation with tumor size (Rho=0.19471, P=0.0435). In overall, PD-L1 expression correlated positively with inflammatory cell density in both IT and PT compartments in ADC and SqCC. Patients with KRAS mutation (P=0.00058), solid tumor (P<0.0001) or smoker (P = 0.0446) were more likely to have positive PD-L1 expression tumor cells in ADC. No correlation was detected between EGFR mutation and immune markers. Using PD-L1 and CD8/CD4/CD68 IMS expression levels, in ADC and SqCC, we identified 4 groups of tumors (Table 1). Multivariate Cox proportional hazard regression analysis demonstrated that tumors with high PD-L1 expression and low IMS in ADC exhibited significantly poor recurrence-free (HR=4.299; P=0.0101) and overall survival (HR=5.632; P=0.0010).Table 1. Summary of the correlation between PD-L1 expression levels and immune-score (IMS=CD8/CD4/CD68) in adenocarcinoma (ADC) and squamous cells carcinoma (SQCC).
PDL-1 H-score (ADC) IMS (Low) IMS (High) Total <5 61 (41.78%) 51 (34.93%) 112 (76.71%) ≥5 8 (5.48%) 26 (17.81%) 34 (23.29%) Total 69 (47.26%) 77 (52.74%) 146 (100.0%) PDL-1 H-score (SqCC) <5 37 (34.30%) 36 (33.30%) 73 (67.60%) ≥5 17 (15.70%) 18 (16.70%) 35 (32.40%) Total 54 (50.00%) 54 (50.00%) 108 (100.0%)
Conclusion:
Higher PD-L1 expression is associated with solid pattern in adenocarcinoma and higher level of tumoral immune infiltrate. We developed an immune score which when combined with PD-L1 expression significantly correlates with patient outcome in surgically resected ADCs. (Supported by grants UT-Lung SPORE P50CA70907 and CPRIT RP120713).
