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#4054 Gene expression Difference (GED) Revealed Immune Function Gene UP- or Down-regulation as Tumor-associated Inflammatory Cell (TAIC) Infiltration in Microenvironment in Non-small Cell Lung Cancer (NSCLC)
Ximing Tang1, Wei Lu1, Jiexin Zhang2, Carmen Berhens1, Edwin Roger Parra1, John Wineman3, Jianjun Zhang4, Don Gibbons4, Mark Koeppel3, BJ Kerns3, Mark Stern3, Boris Sepesi5, Jack J. Lee6, and Ignacio I. Wistuba1
1 Translational Molecular Pathology, 2 Bioinformatics & Comp Biology, 4 Thoracic / Head & Neck Medical Oncology, 5 Surgery, 6 Biostatistics, Departments of, UT MD Anderson Cancer Center; 3 HTG Molecular
Background
Figure 7. Numbers gene with high expression are associated to genotypes of EGFR, KRAS, TP53 and STK11 in ADC
EGFR and KRAS TP53 and KRAS STK11 and KRAS
Figure 1.The heatmap of the immune response-related gene expression profile. Black column label represents ADC (N=125), blue represents SCC (N=74). -2
Advances in technologies of molecular analyses of tumor tissues based on high-throughput can be a better prediction of disease prognosis and the efficacy of immunotherapies. Gene expression difference (GED) between tumor features was analyzed in a large cohort of NSCLC.
GED
Figure 3. GED between smokers and non-smokers in ADC. >0, higher expression in smokers; <0 higher in non-smokers
Materials & Methods
Using low-input, high-throughput HTG EdgeSeq assay, we examined expression of a panel immune response-related genes (IRRGs) (N=549, 28 related to immune function, 19 to B-cell,16 to T-cell, 5 to NK cell function) in 226 RNA samples extracted from surgically resected FFPE NSCLC tumor tissues (stage I to III,137 adenocarcinomas [ADC] and 89 squamous cell carcinomas [SqCC]). The patients have not received neoadjuvant therapy and annotated clinical, pathological characteristics. Genotype data were available on mutations of EGFR and KRAS (n=126), STK11, PIK3CA and TP53 by whole exome sequencing (n=180), and density of TAICs (CD3, CD4, CD68, CD8, FOXP3, GZB, PD-L1 and PD-1) detected by IHC (n=145) were available. By associating the clinical and molecular features to the gene expression data after globalized normalization, using false discover rate (FDR) 5% in multi-testing correction we identified the differentially expressed genes (DEGs) related to histology, clinical and molecular pathologic variables, and TAIC density in tumor field and, recurrence free survival (RFS) and overall survival (OS) in the patients having or having not received adjuvant therapy.
Figure 4. Number of DEGs in the group of immune-, B cell-, T cell and NK cell function having up- or down regulated in the tumor with high density of TAIC infiltration in ADC (top) and SqCC (button). >0, the gene number upregulated; <0, the gene number down regulated.
Number of DEGs
Gene function group
Table. The DEGs identified with effects on overall and recurrence free survival for NSCLC
ADC
SCC
Adjuvant-yes
Adjuvent-no OS
RFS
Total gene 49 35 25 27 14 8 12
Worse effect
22 (5*)
15 (2) 7 2
Protective effect
27 (10) 23
10 (1) 15 6 4
* passed 5% FDR test
Figure 8. Representatives of K-M plots of the genes with effects on survival of ADC patients treated or untreated with adjuvant chemotherapy
Figure 5. GED between the tumor with high and low density of TAICs in ADC and SqCC respectively. >0 show higher expression and <0 show lower expression in the tumor with high density of TAICs.
High TAIC density in ADC
High TAIC density in SqCC
GED
Results
Among the 549 genes, 234 DEGs in ADC and SqCC (Figure 1) were identified. Parts of genes [82 higher in ADC and 89 higher in SqCC (P≤0.001)] were shown in figure 2.
In ADC, more DEGs were down regulated than upregulated in smokers (8 up- and 15 down-regulated) (Figure 3).
DEGs were identified to be associated to TAIC high density in tumor. The ratios of up/down regulated DEGs are 63/11 and 39/7 in ADC and SqCC respectively (Figure 4, 5):
A number of DEGs with up- or down regulation were identified to be associated with mutations of EGFR, KRAS, STK11, PIK3CA and P53 genes in ADC (Figure 6, 7).
The DEGs related to overall or recurrent-free survival for the patients with treated or untreated with adjuvant therapy were identified respectively (table). Cox proportional hazard model using FDR 5% in multi-testing showed in ADC treated with adjuvant chemotherapy 10 genes having a protective effect and 5 genes having worse effect on OS; and in ADC untreated with adjuvant therapy, 1 gene having protective and 2 having worse effect on OS (Figure 8).
Figure 2. Representatives of immune-response related genes (IRRGs) with GED (P≤0.001) between ADC and SqCC. <0, higher expression in SqCC; >0, higher in ADC
GED
Conclusion
Number of DEGs
Using low-input, high-throughput HTG EdgeSeq assay in FFPE RNA, we identified differentially expressed genes (DEGs) of IRRGs associating to histologies, smoking history, tumor driving gene genotypes, infiltration density of TAICs in the tumor field and patients’ outcome in NSCLC.
Gene function group
Figure 6. Number of immune, B cell, T cell and NK cell function genes up- or down regulated in the tumor with mutation of EGFR, KRAS, STK11 and P53. None gene expression up- or down regulated was detected in SqCC tumor with mutation of PIK3CA. >0, the gene number upregulated; <0, the gene number down regulated.