Chromosomal Alterations and Gene Expression Changes Associated with the Progression of Leukoplakia to Advanced Gingivobuccal Cancer.
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Abstract
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Chromosomal Alterations and Gene Expression Changes Associated with the Progression of Leukoplakia to Advanced Gingivobuccal Cancer
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Abstract
We present an integrative genome-wide analysis that can be used to predict the risk of progression from leukoplakia to oral squamous cell carcinoma (OSCC) arising in the gingivobuccal complex (GBC). We find that the genomic and transcriptomic profiles of leukoplakia resemble those observed in later stages of OSCC and that several changes are associated with this progression, including amplification of 8q24.3, deletion of 8p23.2, and dysregulation of DERL3, EIF5A2, ECT2, HOXC9, HOXC13, MAL, MFAP5 and NELL2. Comparing copy number profiles of primary tumors with and without lymph-node metastasis, we identify alterations associated with metastasis, including amplifications of 3p26.3, 8q24.21, 11q22.1, 11q22.3 and deletion of 8p23.2. Integrative analysis reveals several biomarkers that have never or rarely been reported in previous OSCC studies, including amplifications of 1p36.33 (attributable to MXRA8), 3q26.31 (EIF5A2), 9p24.1 (CD274), and 12q13.2 (HOXC9 and HOXC13). Additionally, we find that amplifications of 1p36.33 and 11q22.1 are strongly correlated with poor clinical outcome. Overall, our findings delineate genomic changes that can be used in treatment management for patients with potentially malignant leukoplakia and OSCC patients with higher risk of lymph-node metastasis.
Introduction
Oral cancer starts with an oral pre-invasive lesion (OPL) that progresses from hyperplasia through dysplasia, and finally develops into invasive oral squamous cell carcinoma (OSCC). Leukoplakia is the most predominant pre-invasive lesion [1], [2], [3], [4], however the ability to predict the malignant potential from histopathological data is limited. Moreover, the 5-year overall survival in OSCC has not substantially improved in recent decades [5], and early diagnosis and primary prevention remain the best approaches for OSCC management. To this end, the major challenge in early diagnosis is identifying pre-invasive lesions that are at high risk of malignant transformation [6], [7]. However, OSCC is frequently diagnosed in advanced stages, which negatively influences prognosis. The most important prognostic factors that determine mortality and morbidity in OSCC patients are lymph node involvement and locoregional recurrence.
The histopathological evaluation of oral cancers is often not sufficient to predict disease aggressiveness and clinical outcome [8]. Multiple genetic and epigenetic events occur before tissue changes are microscopically detectable. The number of acquired genetic alterations increases with disease advancement from squamous hyperplasia through dysplasia to invasive carcinoma [5]. It is known that copy number alterations (CNAs) ranging from a small number of specific genes to entire chromosomes are significantly associated with OSCC development and progression [9], [10]. These alterations are presumed to alter the expression level of single genes or gene clusters mapping within CNA regions [11]. Therefore, analyses that integrate CNA data with gene expression (GE) data may identify predictive DNA-based markers applicable in clinical prognosis [12].
Molecular profiles of oral cancers are largely influenced by the site of tumor development and associated etiological agents, implying divergent pathways for oral cancer development [13], [14], [15], [16], [17]. India is an interesting location to study the genomics of tobacco-associated OSCC, due to the fact that in India there is a high incidence of oral cancers associated with the abuse of smokeless tobacco, most of which are negative for human papilloma virus (HPV) [18].
This is the first comprehensive study combining genomic profiling and integrative analyses of HPV-negative gingivobuccal complex (GBC) leukoplakia and OSCC of different stages from a large set of Indian patients. We identified signatures associated with the progression of pre-invasive lesions to invasive OSCC and found candidate driver alterations unique to primary tumors with lymph node metastasis and related to patient survival.
Materials and Methods
Tissue Specimen Collection
The study was approved by the Institutional Local Ethics Committee of Tata Memorial Hospital (TMH) and Nair Hospital Dental College, Mumbai, India. Written informed consent was obtained from all the study participants. Paraffin blocks and frozen tissue samples of leukoplakia, neo-primary oral tumor tissues, and non-inflamed gingivobuccal mucosa tissues from clinically healthy individuals with no previous personal history of cancer were recruited from Nair Hospital and TMH, respectively. Patients received neither radiation nor chemotherapy before surgery. Histopathologically confirmed leukoplakia and tumor tissues were subjected to DNA–RNA extraction as detailed in Supplementary Information. Screening for the presence of HPV was done as described in [18]. Details regarding the numbers of samples used in the test and validation sets, as well as the clinicopathologic and demographic characteristics of patients, are provided in Table 1 and Figure S1.
Table 1
The Clinicopathologic and Demographic Characteristic of the Study Patients.
| Patient Characteristics | Total Study Samples | aCGH & GE Study (n = 121)# | qRT-PCR (n = 207)# | IHC & FISH (n = 370)# | |||
|---|---|---|---|---|---|---|---|
| Normal, Leukoplakia and OSCC | Leukoplakia | OSCC | Leukoplakia | OSCC | Leukoplakia | OSCC | |
| n = 481 | aCGH: n = 24 GE: n = 15 | aCGH: n = 91 GE: n = 61 | n = 37 | n = 138 | n = 108 | n = 185 | |
| Age at diagnosis | |||||||
| Median age | 49 | 42 | 50 | 41 | 50 | 44 | 50 |
| Range (IQR)* | 40–59 | 38–50 | 43–61 | 33–53 | 42–59 | 32–57 | 42–60 |
| Gender | |||||||
| Male | 299 (76.3%) | 21 (87.5%) | 70 (76.9%) | 33 (89.2%) | 102 (75.6%) | 96 (90.6%) | 140 (75.7%) |
| Female | 93 (23.7%) | 3 (12.5%) | 21 (23.1%) | 4 (10.8%) | 33 (24.4%) | 10 (9.4%) | 45 (24.3%) |
| Pathological stage | |||||||
| Stage 1 and 2 (Early stage OSCC) | 82 (35.5%) | NA | 32 (35.2%) | NA | 56 (41.5%) | NA | 67 (36.2%) |
| Stage 3 and 4 (Advanced stage OSCC) | 149 (64.5%) | NA | 59 (64.8%) | NA | 79 (58.5%) | NA | 118 (63.8%) |
| Pathological T classification | |||||||
| T1 | 31 (13.4%) | NA | 7 (7.7%) | NA | 25 (18.5%) | NA | 24 (13%) |
| T2 | 100 (43.3%) | NA | 40 (44%) | NA | 64 (47.4%) | NA | 80 (43.2%) |
| T3 | 10 (4.3%) | NA | 4 (4.4%) | NA | 4 (3%) | NA | 8 (4.3%) |
| T4 | 90 (39%) | NA | 40 (44%) | NA | 42 (31.1%) | NA | 73 (39.5%) |
| Pathological cervical lymph node involvement | |||||||
| Node negative (N0) | 133 (57.6%) | NA | 55 (60.4%) | NA | 79 (58.5%) | NA | 112 (60.5%) |
| Node positive (N+) | 98 (42.4%) | NA | 36 (39.6%) | NA | 56 (41.5%) | NA | 73 (39.5%) |
| Pathological grade | |||||||
| Well | 27 (7.9%) | NA | 8 (8.8%) | NA | 12 (8.9%) | NA | 23 (12.5%) |
| Moderate | 139 (40.9%) | NA | 55 (60.4%) | NA | 87 (64.4%) | NA | 106 (57.6%) |
| Poor | 64 (18.8%) | NA | 28 (30.8%) | NA | 36 (26.7%) | NA | 55 (29.9%) |
| Hyperplasia | 89 (26.2%) | 21 (87.5%) | NA | 31 (86.1%) | NA | 80 (80.8%) | NA |
| Mild dysplasia | 11 (3.2%) | 3 (12.5%) | NA | 3 (8.3%) | NA | 9 (9.1%) | NA |
| Moderate dysplasia | 8 (2.4%) | NA | NA | 2 (5.6%) | NA | 8 (8.1%) | NA |
| Severe Dysplasia | 2 (0.6%) | NA | NA | NA | NA | 2 (2%) | NA |
| Habit profile | |||||||
| No Habit | 9 (3.1%) | 9 (45%) | NA | NA | 3 (2.6%) | NA | 8 (5.3%) |
| Exclusive tobacco users | 157 (54.5%) | 3 (15%) | 63 (77.8%) | 13 (41.9%) | 79 (70%) | 30 (33%) | 98 (64.5%) |
| Exclusive smoker | 18 (6.3%) | NA | 2 (2.5%) | 5 (16.2%) | 2 (2%) | 12 (13.2%) | 4 (2.6%) |
| Exclusive alcohol drinker | 1 (0.3%) | NA | NA | NA | 1 (0.8%) | NA | 1 (0.7%) |
| Mixed habit** | 103 (35.8%) | 8 (40%) | 16 (19.8%) | 13 (41.9%) | 28 (24.7%) | 49 (53.8%) | 41 (27%) |
# Represents total number of samples, including Buccal Mucosa (BM) Normals: n = 6 (GE), n = 32 (qRT-PCR) and n = 77 (IHC); all samples belonged to the gingivobuccal complex region of the oral cavity; T: Tumor classification based on size; N: Tumor classification based on lymph node metastasis; * IQR: Inter quartile range; **Mixed Habit: Tobacco chewing along with bidi/cigarette smoking and/or alcohol users.
Array CGH and Gene Expression Profiling
Whole-genome copy number and gene expression profiling was performed on 2x105K CGH oligonucleotide arrays and Whole Human Genome Microarray 4x44K (Agilent Technologies, USA) respectively. Hybridization and detailed analysis are described in Supplementary Information. The raw aCGH data have been submitted to the Gene Expression Omnibus (GEO) with accession numbers GSE85514 and GSE23831 and accession numbers for GE raw data are GSE85195 and GSE23558.
Validation of Targets
The copy number status of the targets was evaluated by fluorescence in situ hybridization (FISH/nuc ish). Immunohistochemical analysis (IHC) and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) were performed for selected candidate genes found significantly deregulated. Detailed protocol and analysis is provided in Supplementary Information. Details regarding FISH probes, fluorescent TaqMan probes and antibodies used are listed in Tables S1, S2, S3.
Literature Mining
We updated our existing literature-based list of genes related to oral cancer from 277 genes [10] to 562 genes (as of May 2015). The list (Table S4) includes genes that were previously found to be either differentially expressed or copy number altered in oral cancers. The purposes were 1) to place our new results in the context of previous knowledge and 2) to determine the novelty of any gene expression change or CNA that we would choose for targeted validation.
Results
Clinicopathological and Demographic Characteristics of the Study Cohort
Clinicopathological and demographic characteristics of all 481 leukoplakia and OSCC patients, together with follow-up data, are summarized in Tables 1 and S5, while Figure S1 shows how many samples were used in each phase of the study. Most patients were smokeless tobacco users while many had mixed habits (~35%) (chewing, bidi/cigarette smoking or consuming alcohol) and were negative for high-risk HPV [18]. The histopathology of the leukoplakia samples was either hyperplastic (89 samples) or mild dysplastic (11 samples) and 50% of the lesions analyzed for aCGH and GE study either transformed to OSCC or recurred after primary treatment. One hundred forty-nine patients (~65%) had advanced-stage OSCC and 82 patients (~35%) had early-stage OSCC. Approximately 60% cases were negative for lymph node metastasis.
Genome-Wide copy Number Alterations
Genome-wide analysis of CNA was carried out in 24 leukoplakia, 32 early-stage OSCC, and 59 advanced-stage OSCC cases and revealed recurrent focal regions of amplification and deletion (Figures 1 and S2). We identified 19 alterations in leukoplakia, 32 alterations in early-stage OSCCs and 69 alterations in advanced-stage OSCCs (Table S6). The 10 most frequently amplified regions were 11q13.1 (70% of all patients), 8q24.3 (69%), 11p15.5 (60%), 1p36.33 (59%), 9q34.3 (59%), 8q24.21 (55%), 7q22.1 (54%), 7q11.23 (53%), 16p13.3 (53%) and 3q27.2 (52%). The 10 most frequently deleted regions were 8p23.2 (66% of all patients), 8p11.22 (65%), 3p14.2 (56%), 3p21.1 (54%), 8p22 (54%), 3p11.1 (53%), 3p22.3 (53%), 3p26.3 (53%), 8p23.1 (51%) and 15q11.1 (40%). Previous reports have proposed amplifications in 3q26-qter, 8q24-qter, and 11q13.2-q13.4 to be common in high grade dysplasia, and amplifications in 3q, 7p, 8q, 11q, together with deletions in 3p and 8p to be most common in OSCCs [19].
All amplifications (a) and deletions (b) inferred by GISTIC 2.0 in leukoplakia (A), early-stage OSCC (B), and advanced-stage OSCC (C) samples. Each alteration is assigned a G-score (left axis) by GISTIC 2.0, which considers the amplitude of the alteration, as well as its occurrence across samples.
CNAs associated with disease progression
Alterations associated with disease progression were identified by comparison between the following groups: 1) leukoplakia vs. early-stage OSCC, 2) leukoplakia vs. advanced-stage OSCC, and 3) early-stage OSCC vs. advanced-stage OSCC (Figure S2). Three amplified regions (4q13.2, 6p21.32, 8q24.3) and four deleted regions (8p23.1, 8p11.22, 14q11.2, 15q11.1) are common among the leukoplakia, early-stage OSCC, and advanced-stage OSCC samples (Figures 1 and S2), suggesting their involvement in disease progression. Interestingly, the amplification of 8q24.3 (harboring the candidate genes FBXL6, GPR172A, PTP4A3) was found in almost 60% of the analyzed leukoplakia cases and also in almost 70% of the tumors (Table S6).
We validated the 8q24.3 amplification by nuc ish using a disjoint validation set of 108 leukoplakia and 185 OSCC samples (Figure 2A and Table 1). The 8q24.3 locus-specific probe and a centromere 8 probe hybridized to their target loci and showed no cross reactivity (Figure 2A). The percentage of cells with 8q24.3 amplification increased as disease progressed from leukoplakia to OSCC, and the amplification was associated with disease progression (P <0.001) (Figure 3A). Almost 95% of the advanced-stage OSCC samples used for validation had a weak amplification of 8q24.3 in more than 40% of the tumor cells (Figure 3B). Moreover, this amplification was significantly associated with tumor grade (P =.046) and lymph node metastasis (P =. 0.007). The strong amplification of 8q24.3 in leukoplakia and tumors was only present in 5–20% of cells (Figure 3C). Our validation results confirm that 8q24.3 amplification is an important early event associated with OSCC progression.
Nuc ish (FISH) for validating the amplifications of the regions 8q24.3 and 1p36.33. A) nuc ish for 8q24.3 locus in leukoplakia (b) and OSCC (c). B) nuc ish for 1p36.33 locus with weak amplification (b) and strong amplification (c) in OSCC. The specificities of the 8q24.3 locus probe (red)/chr 8 centromere (CEP) (green) and 1p36.33 (red) locus probe/chr 1 centromere (CEP) (green) were confirmed on the metaphase spreads as represented in A(a) and B(a). In all cases, the magnification was 630X.
Correlation between 8q24.3 amplification and oral cancer progression. A) Percentage of cells with 8q24.3 weak amplification across different groups, correlated with disease progression (P-value calculated using Spearman correlation). B) and C) The increased percentage of tumor cells with 8q24.3 amplification in leukoplakia and OSCC.
CNAs associated with clinical outcome
Chromosomal alterations were also analyzed for their associations with clinicopathologic parameters, including nodal status, grade, and survival. We identified 64 recurrent chromosomal alterations in lymph node metastasis-negative OSCCs and 46 recurrent alterations in lymph node metastasis-positive OSCCs (q-value <0.25, Figure S3). In addition, we found 13 alterations unique to the primary tumors that metastasized to the nodes (Figure S2C and D), including amplifications of 8p23.1, 8q24.21 (harboring the candidate gene MYC), 11q22.1 (MMPs, BIRC2, BIRC3), and deletions of 2q23.3, 3p26.3 (CHL1), 3p12.2, 4q21.3, 7q31.1, 8p23.2 (CSMD1), 9p12, 11q22.3 (ATM, H2AFX), as well as 18q12.1. We hypothesize that these alterations are potential predictive biomarkers of lymph node metastasis. According to previous studies, the amplification of 8q24.21 and the deletion of 3p26.3 are associated with metastasis, invasion, and therapy resistance [20], [21].
We found 25 CNAs associated with recurrence-free survival and 26 CNAs associated with disease specific survival (q-value <0.25, Table 2). For example, the amplifications of 1p36.33, 11q13.3, 11q22.1 and 16p11.2 were associated with poor clinical outcome, whereas the amplification of 22q11.21 was associated with better survival. Additionally, a poor clinical outcome was also associated with the deletions of 2p11.2, 3p14.2, 4q13.2, 9p23 and 11q22.3. Kaplan–Meier survival curves for 1p36.33 and 11q22.1 are shown in Figure 4A. We validated the amplification of 1p36.33 by nuc ish (Figure 2B), and we confirmed that the amplification of 1p36.33 is associated with poor survival in an independent OSCC cohort (Figure 4B). Moreover, we found a strong association between the amplification of 1p36.33 and lymph node metastasis (P < .001).
Kaplan–Meier plots of disease specific patient survival for selected chromosomal alterations based on aCGH analysis (A, 1p36.33 and 11q22.1) and nuc ish validation experiments (B, 1p36.33). Survival in months (X axis) is plotted against the fraction of patients alive (Y axis).
Table 2
Univariate Cox Proportional Hazards Regression Analysis of Single Predictors for Recurrence-Free and Disease Specific Survival.
| Cytoband | Alteration | Disease Specific Survival | Recurrence-Free Survival | ||
|---|---|---|---|---|---|
| BH Corrected P-Value | CPH Coef. | BH Corrected P-Value | CPH Coef. | ||
| 1p36.33* | Amplification | 0.0327 | 1.0292 | 0.013 | 0.9185 |
| 1q23.2 | Amplification | 0.0269 | 0.8763 | 0.0934 | 0.574 |
| 3q27.2 | Amplification | 0.2467 | 0.436 | 0.2171 | 0.3823 |
| 11q13.3 | Amplification | 0.1232 | 0.4237 | 0.2142 | 0.2804 |
| 16p13.3 | Amplification | 0.0655 | 0.6641 | 0.0658 | 0.5375 |
| 16p11.2 | Amplification | 0.0497 | 0.8665 | 0.0549 | 0.6726 |
| 16q12.2 | Amplification | 0.0607 | 0.6662 | 0.0308 | 0.6497 |
| 2p11.2 | Deletion | 0.0473 | 1.0051 | 0.0844 | 0.777 |
| 2q22.1 | Deletion | 0.0147 | 1.0214 | 0.0623 | 0.6329 |
| 2q34 | Deletion | 0.0644 | 0.7772 | 0.0281 | 0.745 |
| 3p14.2 | Deletion | 0.1976 | 0.5665 | 0.2202 | 0.4236 |
| 4q13.2 | Deletion | 0.0582 | 0.8036 | 0.1775 | 0.4836 |
| 4q22.1 | Deletion | 0.0998 | 0.7098 | 0.2113 | 0.4591 |
| 9p23 | Deletion | 0.0969 | 0.711 | 0.1339 | 0.5327 |
| 11q22.3 | Deletion | 0.0829 | 0.7902 | 0.099 | 0.6467 |
| 6p21.1 | Amplification | 0.2198 | 0.4606 | - | - |
| 11q22.1 | Amplification | 0.1543 | 0.5204 | - | - |
| 18p11.31 | Amplification | 0.2227 | 0.5128 | - | - |
| 22q11.21 | Amplification | 0.1832 | −0.6038 | - | - |
| 1q31.3 | Deletion | 0.2371 | 0.5055 | - | - |
| 3p11.1 | Deletion | 0.0809 | 0.7957 | - | - |
| 4q13.2 | Deletion | 0.09 | 0.7457 | - | - |
| 5p14.3 | Deletion | 0.112 | 0.7583 | - | - |
| 7q31.1 | Deletion | 0.1624 | 0.598 | - | - |
| 13q21.32 | Deletion | 0.2166 | 0.5221 | - | - |
| 21q21.3 | Deletion | 0.1385 | 0.6243 | - | - |
| 2q37.3 | Amplification | - | - | 0.2185 | 0.3978 |
| 5p15.33 | Amplification | - | - | 0.223 | 0.3284 |
| 11p15.5 | Amplification | - | - | 0.1793 | 0.4287 |
| 13q21.33 | Amplification | - | - | 0.2436 | −0.7795 |
| 16q21 | Amplification | - | - | 0.1975 | 0.4048 |
| 19p13.3 | Amplification | - | - | 0.1465 | 0.3602 |
| 22q11.23 | Amplification | - | - | 0.1986 | −0.4272 |
| 9p21.3 | Deletion | - | - | 0.2007 | 0.4494 |
| 14q11.2 | Deletion | - | - | 0.1116 | 0.5358 |
| 17p13.1 | Deletion | - | - | 0.0346 | 0.7927 |
BH: Benjamini-Hochberg multiple testing correction method; CPH coef.: Cox Proportional Hazard coefficient. A positive regression coefficient means that the hazard is higher, thus the prognosis is worse; * Targets selected for validation; − represents not applicable.
Gene Expression and Integrative Analyses
Transcriptome-wide analysis was performed on 6 buccal mucosa normal tissues, 15 leukoplakia, 27 early-stage OSCC, and 34 advanced-stage OSCC. Principal component analysis of 3805 differentially expressed genes (log fold change of 2 and q-value ≤0.01) revealed two separate clusters of normal and OSCC samples, while the leukoplakia samples displayed changes overlapping with both these groups (Figure S4). We identified 849 genes differentially expressed (395 up-regulated and 454 down-regulated) in leukoplakia, 1813 (805 up-regulated and 1008 down-regulated) in early-stage OSCC, and 1924 (798 up-regulated and 1056 down-regulated) in advanced-stage OSCC (Figure S5). Up-regulation of NELL2, MFAP5, CA2, FLRT3, HOXC9, CDH16, LRP12, PTPRZ1, TNNT1, WDR66, NEXN, EGR2, HOXC13, E2F7, ECT2, EIF5A2 and down-regulation of KRT19, DERL3, CD177, PSCA, FAM3B, ALOX12, MUC20, CXCL13, KRT4, KRT3, CD19, KRT81, CLDN7, FCRL5, POU2AF1, CD79A, TMPRSS2, MAL, TNFRSF17, FCN1, PNOC, CXCL17, CEACAM1, FUT6, CLCA4, PITX1, DACT2, MEI1, GPX3 were observed in all three groups, revealing their role in disease progression from pre-invasive to cancerous lesions. A higher number of genes were dysregulated in OSCC compared to leukoplakia, including CXCL10, MMP10, INHBA, GBP5, CXCL11, MMP3, FST, BATF2, SPP1, SH2D5, CXCL9, IFIT3, SERPINE1, GALNT6, FOXL2, PDPN, ITGA3, VEGFC, STAT1, LY6K, KLF7, SOX9 and CD274. Among all the differentially expressed genes identified in this study, 61 have been previously reported to be involved in leukoplakia and 188 in oral or head and neck cancers, including ECT2, INHBA, SERPINE1, GBP5, MMP10, MMP3, LY6K, SPP1, PDL1, PTHLH, KRT4, KRT76 and MAL[10], [22], [23], [24], [25], [26]. The novel oral cancer driving genes identified here include DERL3, EIF5A2, HOXC9, HOXC13, MFAP5, NELL2, CD274, DHRS2, FST and GPX3. The top dysregulated genes in leukoplakia and OSCC are represented in Figure 5 and listed in Table S7.
Heatmap of the top 20 differentially expressed genes in leukoplakia and OSCC. A) Differentially expressed genes in early-stage OSCC (pink) and advanced-stage OSCC (magenta) when compared to normal (green). B) Differentially expressed genes in leukoplakia (chartreuse) when compared to normal (green). Blue genes are up-regulated, while down-regulation is represented in red. All expression values were scaled across samples.
Integrative analysis of gene expression and CNAs
We integrated the GE and CNA datasets to identify genes whose expression and copy number status were correlated. We found 3q26.31, 6p21.1, 7p11.2, 8q24.21, 8q24.3, 9p24.1, 11q13.3, 12q13.2, 16q24.2 and 17p13.1 as chromosomal hotspots for copy number-dependent gene overexpression, while 1q44, 2q34, 3p26.3, 3p21.1, 10p11.21, 11q22.3, 17p13.1 and 21q21.3 were identified as regions of copy number-dependent gene down-regulation (Table 3). In particular, 3q26.31 and 12q13.2, spanning the genes ECT2, EIF5A2, HOXC9, HOXC13 and MUCL1, showed a strong correlation between copy number amplifications and gene over-expression. The deletion of 11q22.33 was correlated with a few genes with significant copy number-dependent underexpression, including CRYAB, POU2AF1, EXPH5, MPZL2 and ARHGAP32. The opposite direction of expression change (amplification of down-regulated genes and deletion of up-regulated genes) was observed for a few genes, including MMP3, MMP10, FEZ1, CTSC, CHEK1, PANX1 and PAFAH1B2. Interestingly, 16p11.2, 17p13.1 and 22q11.23 were significantly amplified, however, the majority of the genes located in these three regions were down-regulated, e.g., CD19, GDPD3, NUPR1, SPN, CLDN7 and DERL3 (Table 3), potentially a consequence of epigenetic regulation.
Table 3
Associations Between DNA Copy Number Alterations and Differentially Expressed Transcripts in Leukoplakia and OSCC.
DE: Differential expression; Red font indicates amplified loci or up-regulated genes, and Blue font indicates deleted loci or down-regulated genes. The majority of genes showed consistent changes on both the DNA and the RNA levels, and are depicted here in black font (up-regulated genes in amplified regions and down-regulated genes in deleted regions). Few genes showed opposite direction expression changes and are colored respectively (genes in blue font were down-regulated, but located in amplified regions, while genes in red font were up-regulated, but located in deleted regions). * Targets selected for validation.
Validation of dysregulated transcripts
To confirm the results of the GE analysis, real-time qRT-PCR (TaqMan assays) was performed in 32 normal, 37 leukoplakia, and 138 OSCC samples. We selected 10 genes for validation based on either novelty or on published studies implicating these genes in OSCC development (Table S4): seven up-regulated genes DVL1, EIF5A2, FUS, HOXC9, INHBA, LY6K, and MFAP5, two down-regulated genes DERL3 and MAL, and the unchanged gene SLC4A1AP, along with RNA18S5 as endogenous control. All the validation targets that were found to be differentially expressed in the GE analysis were confirmed to display significant differences in expression between normal, leukoplakia, and tumors (Figure 6), and no significant difference was found in the expression of the unchanged gene SLC4A1AP. Specifically, HOXC9, MFAP5, and INHBA showed very high expression changes in leukoplakia, early and advanced tumors versus normal (P<.01), while EIF5A2 and LY6K were significantly overexpressed only in early and advanced-stage OSCCs (P<.0001 and P =.03, respectively). The log2 fold change in expression of DVL1 and FUS was approximately 1 across the three groups, consistent with the microarray data.
qRT-PCR expression changes of the 10 target genes (2 down-regulated, 7 up-regulated and 1 unchanged) in normal, leukoplakia and early and advanced-stage OSCC samples. The P-value (P) was calculated using the Spearman correlation test.
We analyzed the associations between the validated target genes and clinicopathologic parameters (Table 4). Most targets (EIF5A2, HOXC9, MFAP5, LY6K, INHBA and DVL1) showed a positive correlation between their expression changes and OSCC progression from pre-invasive lesions to cancer. The expressions of DERL3 and MAL, which are down-regulated, were negatively correlated with disease progression. EIF5A2, HOXC9, INHBA, and MFAP5 were associated with disease advancement from early-stage OSCC to advanced-stage OSCC, and EIF5A2, HOXC9, INHBA, FUS and DVL1 were significantly associated with lymph node metastasis. IHC was performed to validate the protein overexpression of EIF5A2, ECT2, HOXC9, HOXC13, MFAP5, and NELL2. IHC analysis revealed strong protein expression of all the six targets in leukoplakia (n = 108) and OSCC (n = 185) versus normal (n = 77) (Figure 7). Further analyses reinforced the associations of these markers with disease progression, except NELL2 (Figure S6 and Table S8).
IHC analysis for target validation in normal, leukoplakia, and OSCC samples. NELL2 showed cytoplasmic staining, EIF5A2, ECT2, HOXC9, HOXC13 showed cytoplasmic and nuclear expression, while MFAP5 was localized predominantly into the matrix. Respective isotype controls for all the cases had no staining (images not shown). The rightmost panel shows differential localization of each marker. The magnification was 100X for the leftmost three panels, and 200X for the rightmost panel.
Table 4
Correlation of qRT-PCR Validation Targets with clinicopathological parameters.
| Targets (Genes) → | EIF5A2 ↑ | HOXC9 ↑ | INHBA ↑ | MFAP5 ↑ | LY6K ↑ | FUS ↑ | DVL1 ↑ | SLC4A1AP ↕ | DERL3 ↓ | MAL ↓ | ||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Clinicopathological Parameters ↓ | N | P-value (Coef*) | P-Value (Coef*) | P-Value (Coef*) | P-Value (Coef*) | P-Value (Coef*) | P-Value (Coef*) | P-Value (Coef*) | P-Value (Coef*) | P-Value (Coef*) | P-Value (Coef*) | |
| Pre invasive stage | Normal Oral Mucosa | 32 | 0.849 (−0.02) | 0.015 (0.31) | 0.05 (0.39) | <0.001 (0.72) | 0.665 (0.58) | <0.001 (0.46) | 0.018 (0.28) | 0.001 (0.43) | <0.001 (−0.48) | <0.001 (−0.51) |
| Leukoplakia | 37 | |||||||||||
| Invasive OSCC | Normal Oral Mucosa | 32 | <0.001 (0.31) | <0.001 (0.27) | <0.001 (0.45) | <0.001 (0.42) | 0.003 (0.23) | <0.001 (0.27) | 0.620 (−0.03) | 0.288 (0.08) | <0.001 (−0.31) | <0.001 (−0.59) |
| OSCC | 138 | |||||||||||
| OSCC progression | Normal Oral Mucosa | 32 | <0.001 (0.39) | <0.001 (0.35) | <0.001 (0.68) | 0.008 (0.19) | <0.001 (0.36) | 0.141 (0.10) | 0.030 (−0.15) | 0.540 (−0.04) | 0.007 (−0.19) | <0.001 (−0.68) |
| Leukoplakia | 37 | |||||||||||
| OSCC | 138 | |||||||||||
| Tumor stage | Stage I/II | 56 | 0.044 (0.17) | <0.001 (0.42) | <0.001 (0.48) | 0.041 (0.18) | 0.001 (0.28) | 0.702 (0.03) | 0.817 (0.09) | 0.130 (0.21) | 0.444 (−0.06) | 0.007 (−0.23) |
| Stage III/IV | 79 | |||||||||||
| Tumor grade | Well | 12 | 0.181 (0.12) | 0.250 (−0.10) | 0.062 (0.16) | 0.620 (0.04) | 0.500 (0.06) | 0.815 (0.02) | 0.144 (−0.12) | 0.980 (−0.02) | 0.418 (0.07) | 0.629 (−0.04) |
| Moderate | 87 | |||||||||||
| Poor | 36 | |||||||||||
| Node metastasis | Node negative | 79 | 0.018 (0.20) | 0.015 (0.21) | <0.001 (0.39) | 0.676 (0.03) | 0.133 (0.13) | 0.029 (0.19) | 0.014 (0.21) | 0.003 (0.25) | 0.369 (0.08) | 0.461 (−0.06) |
| Node positive | 56 | |||||||||||
coef*: Spearman correlation coefficent. Bold font represents significant changes at a significance level of 0.05, ↑: up-regulated gene, ↓: down-regulated gene, ↕: unchanged gene; The association with OSCC progression was computed by comparing expression changes between leukoplakia, early stage OSCC and advance stage OSCC.
Pathway Analyses
To interpret the association of dysregulated genes with biological processes, we used the PANTHER (Protein ANalysis THrough Evolutionary Relationships) classification system [27], [28] (Figure S7). Both leukoplakia and OSCC samples shared a large number of dysregulated processes. However, we identified a higher number of OSCC-related genes as part of dysregulated pathways as compared to genes related to pre-invasive lesions. Among them, we note genes associated with apoptotic processes (FADD, BAX, BAK1, CASP7, INHBA, BIRC5), developmental processes (HOXC9, HOXC13, NELL2, CD274, ETS, STAT1), biological regulation (BATF, HOXC9, TERC), and metabolic processes (RAD51, HOXC13, E2F1, HOXB6, CDK1). The top representative biological processes significantly altered in leukoplakia and OSCC are listed in Table 5.
Table 5
Representative Biological Processes Significantly Altered in Leukoplakia and OSCC.
| GO:BP:ID | P-Value | Odds ratio | Count | Size | Biological process |
|---|---|---|---|---|---|
| Leukoplakia | |||||
| GO:0044691 | 6.57E-05 | 433.0789474 | 2 | 3 | tooth eruption |
| GO:0035116 | 0.000373976 | 24.34222222 | 3 | 30 | embryonic hindlimb morphogenesis |
| GO:0021983 | 0.000879749 | 17.75243243 | 3 | 40 | pituitary gland development |
| GO:0030199 | 0.000879749 | 17.75243243 | 3 | 40 | collagen fibril organization |
| GO:0001568 | 0.001192229 | 3.794054527 | 9 | 556 | blood vessel development |
| GO:0071230 | 0.001591348 | 14.27130435 | 3 | 49 | cellular response to amino acid stimulus |
| GO:0043206 | 0.001656565 | 39.34688995 | 2 | 13 | extracellular fibril organization |
| GO:0040012 | 0.002562823 | 11.97495573 | 3 | 62 | regulation of locomotion |
| GO:0044259 | 0.002720644 | 7.421757892 | 4 | 123 | multicellular organismal macromolecule metabolic process |
| GO:0009888 | 0.002854111 | 2.692398599 | 13 | 1260 | tissue development |
| OSCC | |||||
| GO:0008544 | 1.03E-09 | 5.089295677 | 24 | 307 | epidermis development |
| GO:2,000,145 | 1.90E-08 | 3.377582317 | 33 | 628 | regulation of cell motility |
| GO:0051674 | 3.78E-07 | 2.447114154 | 47 | 1237 | localization of cell |
| GO:0051272 | 4.34E-07 | 3.851913001 | 22 | 361 | positive regulation of cellular component movement |
| GO:0040017 | 6.57E-07 | 3.750171556 | 22 | 370 | positive regulation of locomotion |
| GO:0043207 | 1.47E-06 | 2.622472611 | 36 | 869 | response to external biotic stimulus |
| GO:0060337 | 2.07E-06 | 7.805019305 | 10 | 84 | type I interferon signaling pathway |
| GO:0034340 | 2.31E-06 | 7.70047619 | 10 | 85 | response to type I interferon |
| GO:0018149 | 5.56E-06 | 9.573286052 | 8 | 56 | peptide cross-linking |
| GO:0032496 | 8.14E-06 | 5.32031185 | 12 | 145 | response to lipopolysaccharide |
The top 10 dysregulated pathways (ordered by corrected P-value in Leukoplakia and OSCC), as identified by the Bioconductor package GOStats.
Discussion
We have presented the first comprehensive analysis of genomic and transcriptomic profiles of a large set of tobacco-associated, HPV-negative gingivobuccal leukoplakia and OSCC patients from India. Our main goals were threefold: 1) to identify novel driver events associated with the transformation of pre-invasive lesions to high risk malignant OSCCs, as well as with patient survival; 2) to identify unique driver alterations found in primary tumors with lymph node metastasis; and 3) to identify driver genes with correlated CNA and gene expression profiles. Therefore, our study contributes to a genetic progression model of oral carcinogenesis (Figure 8).
Summary of the predictive genomic and transcriptomic signatures associated with gingivobuccal cancer progression from pre-invasive lesions (leukoplakia) to cancer and lymph node metastasis. (*) represents alterations associated with disease specific survival in OSCC patients. Amp: Amplification (red); Del: Deletion (blue); Up: genes up-regulated (red), Down: genes down-regulated (blue).
The CNA landscape of gingivobuccal cancers is dominated by amplifications of the chromosomal regions 1p36.33, 3q26.31, 6p21.32, 7p11.2, 8q24.21, 8q24.3, 9q34.3, 11q13.1, 11q13.3, 11q22.1, 12q13.2, 16p11.2, and deletions of 3p21.1, 3p14.2, 4q21.3, 8p23.2, 8p11.22, 9p23, 9p21.3, 17p13.1. The amplifications of 3q, 7p, 8q, 9q, 11q, and 12q, as well as the deletions of 3p, 4q, 8p, and 9p were reported at least three times among 12 aCGH studies on primary OSCC tumors [19], with amplifications of 3q26, 11q13 and 11q22.2 being the most reported CNAs in advanced-stage OSCCs [9], [29], [30]. An extensive review by Gollin outlines established associations of most of these alterations in HNSCC [31]. In our data, at the whole-arm level, the amplification of 8q is the most common amplification associated with OSCC progression. At the sub-band level, the amplification of 8q24.3 was observed in 58% of the leukoplakia samples, as well as in 69% of the OSCC samples, while the region 8q24.21 was amplified in 55% of the OSCC samples. PTK2, LY6K, and MYC are prominent candidate oncogenes on 8q [10], [32], [33], [34]. In addition, we observed deletions of multiple regions on 3p (3p26.3, 3p22.3, 3p21.1, 3p14.2, 3p11.1) and 8p (8p23.2, 8p23.1, 8p22, 8p11.22) with high frequency (>52% in OSCCs), in line with literature reports in oral pre-invasive lesions [35], [36]. These alterations can therefore be considered as important events associated with OSCC progression [36].
We observed strong correlations between gene expression and amplifications at 3q26.31 (including the genes ECT2, EIF5A2, KLHL6, GPR160) and 12q13.2 (HOXC9, HOXC13, ERBB3, MUCL1) in both leukoplakia and OSCC. Amplifications at 9p24.1 (CD274), 11q13.3 (ANO1), and 7p11.2 (EGFR) were only identified in OSCCs, indicating their role in disease advancement, rather than their appearance at pre-invasive stages. Additionally, CD274 and its ligand PD1 are important targets of immunotherapy in various cancers, including OSCC [37], [38], [39], [40].
A second hotspot for CNA-dependent gene over-expression was observed on 3q26.31, with ECT2 and the oncogene EIF5A2 over-expressed. Overexpression of EIF5A2 has not been previously reported in leukoplakia or OSCC, even though it has been proposed as a prognosis biomarker and potential therapeutic target for various other human tumors [41], [42], [43], [44]. ECT2 has been previously found to be overexpressed in oral cancers [22], and also be involved in metastasis and angiogenesis of solid tumors [43], [45], [46], [47].
A third interval of interest for amplifications and gene overexpression is 12q13.2, comprising of HOXC9 and HOXC13, genes associated with disease progression in OSCC. The HOX transcriptional regulators family is involved in pattern formation and organogenesis during embryo development [48] and potentially in the maintenance and regulation of cancer stem cells [49]. In particular, HOXC9 has been linked with cell cycle exit and cell invasion in breast cancer and neuroblastoma [48], [50], [51], [52], and HOXC13 plays an important role in maintaining skin homeostasis and in regulating the transcription of cytokeratins genes [53], [54]. Kasiri et al. [55] and Cantile et al. [56] showed that HOXC13 is a key player in tumor cell growth and viability in various human cancers.
Pathare et al. [57] and Bhattacharya et al. [58] demonstrated that specific CNAs are associated with lymph node metastasis. Here, the most frequent such alteration, specific to the lymph node metastatic tumors, was the amplified region 8q24.21 (57%), which includes the gene MYC, whose over-expression is postulated to activate various hallmarks of cancer, such as metastasis, invasion, and therapy resistance [20], [59]. A highly recurrent deletion identified was 3p26.3 (57%), including the gene CHL1, alteration previously reported as a predictor of survival and lymph node metastasis in OSCC, along with loss of 3p14.2 (FHIT) [21], [60]. The deletion of 8p23.2 was the most frequent in our study (68%). Genes in this region have been reported to be involved in lung, head and neck, breast, and skin cancers [61], but further studies are required to delineate its functional role in OSCC progression.
We separately analyzed early-stage and advanced-stage OSCCs to identify distinguishable CNAs with respect to recurrence and survival. For the first time, we report a recurrent amplification on 1p36.33 as significantly associated with clinical outcomes. Literature evidence supports that genes located on 1p, including JUN (1p32–31), TP73 (1p36.3), CASP9 (1p36.21), and NRAS (1p13.2), are important in the initiation and progression of several cancer types [62], [63]. Genes of interest on the 1p36.33 amplicon include MXRA8 and DVL1. Here, we report the copy number dependent up-regulation of MXRA8, previously shown to function in tumor stroma by aiding the recovery of angiogenesis in capillaries [64], [65]. DVL1 belongs to the Wnt signaling pathway known to be involved in growth, progression, and metastasis of various cancer types [66].
Additionally, we report copy number independent up-regulation of INHBA, MFAP5 and NELL2 in both leukoplakia and OSCC samples. MFAP5 is a secretory stromal protein overexpressed in leukoplakia and OSCC, possibly playing a role in malignant transformation and as a potential serum biomarker of cancer progression. Reports on ovarian cancer suggest that MFAP5 promotes tumor cell survival and angiogenesis through α5β3 integrin-mediated signaling [67], [68], [69]. We identified few genes in copy number-altered regions that had a significant expression change in the direction opposite to what would be expected (e.g., a down-regulated gene in an amplified region), possibly following epigenetic regulation. One example is the down-regulation of DERL3, located at the 22q11.23 amplicon. Further studies are needed to confirm the significance of the DERL3 in oral tumorigenesis and to understand its gene regulation.
In sum, our study identifies CNAs and gene expression changes related to oral cancer progression. Alterations shared between leukoplakia and OSCC can be considered as important early events that are essential for initial cell transformation and progression. Integrative analysis of CNA and gene expression allows us to identify various novel drivers in oral cancer pathogenesis.
The following are the supplementary data related to this article
Supplementary Figure Caption
The set of all samples (normal, leukoplakia and OSCC) used for array CGH (aCGH), gene expression (GE) and validation analyses (qRT-PCR, FISH and IHC). The numbers of samples are displayed in brackets.
Copy number Amplifications (A and C) and Deletions (B and D) identified in leukoplakia, early-stage OSCC and advanced-stage OSCC (A and B), as well as in node negative (N0) and node positive (N+, C and D) samples.
All amplifications (a) and deletions (b) inferred by GISTIC 2.0 in node negative (N0, A) and node positive (N+, B) OSCC samples. Each aberration is assigned a G-score (left axis) by GISTIC 2.0, which considers the amplitude of the aberration, as well as its occurrence across samples.
PCA plot of the 82 samples analyzed, consisting of 34 advanced and 27 early cancerous lesions, 15 leukoplakia lesions and 6 buccal tissues, acting as controls. The PCA was performed based on the union of all genes differentially expressed (3805 genes), when comparing any two of the four categories of samples plotted here (fold change thresholds of 2 and q-value threshold of 0.01). P-values were corrected for multiple testing using the Benjamini-Hochberg procedure. The percent variance of each of the two components is represented in brackets.
Venn diagrams depicting the numbers of common and unique differentially expressed genes in leukoplakia (orange), early-stage OSCC (blue), and advanced-stage OSCC (pink).
Correlation between protein overexpression and oral cancer progression. A) Significant increase in cytoplasmic and nuclear HOXC9 in leukoplakia and OSCC, as compared to the normal buccal mucosa tissue. B) Significant cytoplasmic overexpression of EIF5A2, as compared to the normal tissue, was more prominent in leukoplakia than in OSCC. C) Increase in the cytoplasmic expression of NELL2 in both leukoplakia and OSCC, however not statistically significant. D) Cytoplasmic overexpression of ECT2 in both leukoplakia and OSCC, and nuclear overexpression of ECT2 only in OSCC. E) Very high overexpression of MFAP5 in the matrix in both leukoplakia and OSCC. F) Increased progressive cytoplasmic expression of HOXC13 between normal tissue, leukoplakia, and OSCC. In all plots, the Y axis represents the H score for protein expression, computed as described in Supplementary Information. P-values (P) were calculated using a Spearman correlation analysis.
Biological processes altered in leukoplakia and OSCC. The plots represent the number of genes associated with dysregulated biological processes, built using the tool PANTHER. “Total # Genes” represents the total number of deregulated genes in either leukoplakia or OSCC, and “Total # process hits” represents the total number of biological process in the PANTHER library.
Enumeration of nuc ish signals. 8q24.3 locus probe (red)/chr 8 centromere (CEP) (green).
Supplementary Table Caption
Table S1: Agilent SureFISH probe details.
Table S2: Taqman assay IDs of genes validated by qRT-PCR.
Table S3: Antibodies used for immunochemistry (IHC) analysis.
Table S4: Literature-based list of genes related to oral cancer.
Table S5: Detailed clinicopathological and demographic characteristics of the (A) leukoplakia and (B) OSCC samples used for the aCGH and gene expression study.
Table S6: Significantly recurrent CNAs identified across the (A) leukoplakia, (B) OSCC, (C) early-stage OSCC, (D) advanced-stage OSCC, (E) node negative, and (F) node positive samples.
Table S7: Top 50 (A) up-regulated and (B) down-regulated genes in leukoplakia and OSCC, ordered by their log 2 fold change (LFC).
Table S8: Correlation of IHC validation targets with clinicopathological parameters.
Grant Support
The work was supported by Grants from the Council of Scientific and Industrial Research (CSIR Scheme No. 27(0271)/12/EMR–II); Department of Biotechnology (DBT - BT/PR3317/MED/12/524/2011) and Annual Scientific Funds (ASF), ACTREC, Tata Memorial Centre. This research was supported in part by the Intramural Research Program of the National Institutes of Health, NLM, and by the Swiss National Science Foundation (Sinergia project 136,247).
Author's Contribution
Conceived and designed the experiments: PGB, MBM. Performed the experiments: PGB, SA. Analyzed the data: PGB, SC, AAS, NB, MBM. Contributed reagents/materials/analysis tools: MBM, NB, AAS, SK, RSD. Wrote the paper: PGB, SC, AAS, NB, MBM. Assessment of clinical annotation, histopathological evaluation, and IHC grading: AMB, AP, RK.
Acknowledgments
The authors thank all participants of the study. ICMR National Tumor Tissue Repository, Tata Memorial Hospital; ACTREC Biorepository and Department of Pathology, Tata Memorial Centre is acknowledged for providing tumor tissue samples. We also thank Mr. P. Chavan and Mr. V. Sakpal from Histology Section, ACTREC, Tata Memorial Centre for their help. We thank Ms. Anjali Manaktala and Ms. Mayuri Inchanalkar for their help in real time PCRs.
References
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