Whole Transcriptome Analysis of Pre-invasive and Invasive Early Squamous Lung Carcinoma in Archival Laser Microdissected Samples

Abstract

BackgroundPreinvasive squamous cell cancer (PSCC) are local transformations of bronchial epithelia that are frequently observed in current or former smokers. Their different grades and sizes suggest a continuum of dysplastic change with increasing severity, which may culminate in invasive squamous cell carcinoma (ISCC). As a consequence of the difficulty in isolating cancerous cells from biopsies, the molecular pathology that underlies their histological variability remains largely unknown.MethodTo address this issue, we have employed microdissection to isolate normal bronchial epithelia and cancerous cells from low- and high-grade PSCC and ISCC, from paraffin embedded (FFPE) biopsies and determined gene expression using Affymetric Human Exon 1.0 ST arrays. Tests for differential gene expression were performed using the Bioconductor package limma followed by functional analyses of differentially expressed genes in IPA.ResultsExamination of differential gene expression showed small differences between low- and high-grade PSCC but substantial changes between PSCC and ISCC samples (184 vs 1200 p-value <0.05, fc ±1.75). However, the majority of the differentially expressed PSCC genes (142 genes: 77%) were shared with those in ISCC samples. Pathway analysis showed that these shared genes are associated with DNA damage response, DNA/RNA metabolism and inflammation as major biological themes. Cluster analysis identified 12 distinct patterns of gene expression including progressive up or down-regulation across PSCC and ISCC. Pathway analysis of incrementally up-regulated genes revealed again significant enrichment of terms related to DNA damage response, DNA/RNA metabolism, inflammation, survival and proliferation. Altered expression of selected genes was confirmed using RT-PCR, as well as immunohistochemistry in an independent set of 45 ISCCs.ConclusionsGene expression profiles in PSCC and ISCC differ greatly in terms of numbers of genes with altered transcriptional activity. However, altered gene expression in PSCC affects canonical pathways and cellular and biological processes, such as inflammation and DNA damage response, which are highly consistent with hallmarks of cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0496-3) contains supplementary material, which is available to authorized users.

Altered Cell-Cycle Control, Inflammation, and Adhesion in High-Risk Persistent Bronchial Dysplasia

“…Additionally, although an extensive set of bronchoscopy derived specimens are available in the Colorado SPORE tissue bank, the availability of frozen tissue from sites with informative follow-up sampling was limited and thus might also have contributed to false negatives regarding changes in the expression of certain genes. One other, recently published study has employed gene expression to assess alterations associated with degree of histologic atypia in precursor lesions of squamous cell carcinoma of the lung (55). Unlike our analysis, this study included gene expression from invasive squamous cell carcinomas as part of the spectrum of progression.…”

Stress Reactivity, Susceptibility to Hypertension, and Differential Expression of Genes in Hypertensive Compared to Normotensive Patients

“…Using the PubMed database [ 69 ], we collected all the DEGs (of hypertensive compared with normotensive patients) that we could find ( Table 5 ). The total number of hypertension-related human DEGs found was 7865, as cited in the rightmost column of Table 5 [ 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 ].…”

“…In this work, we analyzed all the publicly available independent experimental RNA-Seq datasets—on transcriptomes from the tissues of hypertensive versus normotensive patients [ 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 ], hypertensive versus normotensive animals [ 7 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 ], and domestic versus wild animals [ 72 , 176 , 177 , 178 , 179 , 180 , 181 , 182 , 183 , 184 , 185 , 186 , 187 , 188 , 189 , 190 , 191 , 192 , 193 ].…”

“…In this work, we analyzed all the publicly available independent experimental RNA-Seq datasets-on transcriptomes from the tissues of hypertensive versus normotensive patients [26][27][28][29][30][31][32][33][34][35][36][37][38][39], hypertensive versus normotensive animals [7,[40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57], and domestic versus wild animals [72,[176][177][178][179][180][181][182][183][184][185][186][187][188][189][190][191][192][193].…”

“…Parents of half of the hypertensive patients had hypertension, but all the known (>60) hypertension-related whole-genome human loci explained only 3% of this heritability of hypertension [ 25 ]. Maybe this is why mainstream transcriptome-profiling studies on hypertensive versus normotensive patients [ 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 ] and animals [ 7 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 ] are focused on the differentially expressed genes (DEGs) that are specific to gender, age, and the stage of hypertension development. This is needed in predictive preventive personalized participatory (4P) medicine [ 58 ] to estimate where, how, why, and when hypertension might occur in a given patient depending on his/her genetic background.…”