Genomic DNA Copy Number Aberrations, Histological Diagnosis, Oral Subsite and Aneuploidy in OPMDs/OSCCs

Abstract

Oral potentially malignant disorders (OPMDs) characterized by the presence of dysplasia and DNA copy number aberrations (CNAs), may reflect chromosomal instability (CIN) and predispose to oral squamous cell carcinoma (OSCC). Early detection of OPMDs with such characteristics may play a crucial role in OSCC prevention. The aim of this study was to explore the relationship between CNAs, histological diagnosis, oral subsite and aneuploidy in OPMDs/OSCCs. Samples from OPMDs and OSCCs were processed by high-resolution DNA flow cytometry (hr DNA-FCM) to determine the relative nuclear DNA content. Additionally, CNAs were obtained for a subset of these samples by genome-wide array comparative genomic hybridization (aCGH) using DNA extracted from either diploid or aneuploid nuclei suspension sorted by FCM. Our study shows that: i) aneuploidy, global genomic imbalance (measured as the total number of CNAs) and specific focal CNAs occur early in the development of oral cancer and become more frequent at later stages; ii) OPMDs limited to tongue (TNG) mucosa display a higher frequency of aneuploidy compared to OPMDs confined to buccal mucosa (BM) as measured by DNA-FCM; iii) TNG OPMDs/OSCCs show peculiar features of CIN compared to BM OPMDs/OSCCs given the preferential association with total broad and specific focal CNA gains. Follow-up studies are warranted to establish whether the presence of DNA aneuploidy and specific focal or broad CNAs may predict cancer development in non-dysplastic OPMDs.

Fig 1. Relationship between DNA aneuploidy and histological diagnosis in OPMDs/OSCCs.

DNA diploid oral potentially malignant disorders (OPMDs) and oral squamous cell carcinomas (OSCCs) are shown in white stacked bars, while DNA aneuploid OPMDs/OSCCs are shown in black stacked bars. Non-dysplastic oral potentially malignant disorder (ND-OPMD); dysplastic oral potentially malignant disorder (D-OPMD). Significant P-values (P < 0.05) are shown. The FDR q-value method was applied for multiple testing (n = 4) correction; q-values < 0.1 are indicated in bold. N = 331 OPMDs/OSCCs; N = 224 ND-OPMDs; N = 34 D-OPMDs; N = 73 OSCCs.

Fig 2. Relationship between DNA aneuploidy, histological diagnosis in OPMDs/OSCCs and oral subsite TNG or BM.

(A) shows the results of the analysis of oral potentially malignant disorders (OPMDs) and oral squamous cell carcinomas (OSCCs) limited to the tongue (TNG) or buccal mucosa (BM) mucosa; (B) shows the results of the analysis of TNG or BM from patients with OPMDs/OSCCs in multiple oral subsites. Non-dysplastic OPMDs (ND-OPMDs); dysplastic OPMDs (D-OPMDs). DNA diploid oral mucosa sites are shown in white stacked bars, while DNA aneuploid oral mucosa sites are shown in black stacked bars. Significant P-values (P < 0.05) are indicated. The FDR q-value method was applied for multiple testing (n = 4) correction; q-values < 0.1 are indicated in bold. (A) N = 163; N = 22 TNG and N = 82 BM ND-OPMDs; N = 43 TNG and N = 16 BM D-OPMDs/OSCCs. (B) N = 56; N = 18 including TNG and N = 24 including BM ND-OPMDs; N = 7 including TNG and N = 7 including BM D-OPMDs/OSCCs.

Fig 3. Relationship between total CNAs and histological diagnosis in OPMDs/OSCCs.

The bottom and the top of each box show the first and third quartile, respectively, while the line inside the box represents the median (second quartile). Please notice that when the median is not shown, its value = 0. The tips of the whiskers represent the minimum and the maximum data value. Oral potentially malignant disorders (OPMDs); oral squamous cell carcinomas (OSCCs); non-dysplastic oral potentially malignant disorders (ND-OPMDs); dysplastic oral potentially malignant disorders (D-OPMDs). CNAs are referred to as: total focal gains, TFG; total broad gains, TBG; total focal losses, TFL; total broad losses, TBL. Broad gains and broad losses correspond to gains or losses of more than half a chromosome arm, respectively. The boxes corresponding to the number of CNAs detected in ND-OPMD sites are shown in white; the boxes corresponding to the number of CNAs detected in mucosa sites affected by D-OPMDs and OSCCs are shown in gray. Significant MW P-values (P < 0.05) and their corresponding q-values are shown. The FDR q-value method was applied for multiple testing (n = 4) correction; q-values < 0.1 are indicated in bold. N = 94 OPMDs/OSCCs; N = 46 ND-OPMDs; N = 48 D-OPMDs/OSCCs.

Fig 4. Relationship between total CNAs and DNA ploidy in OPMDs/OSCCs.

(A) shows the number of total CNAs detected in oral potentially malignant disorders (OPMDs) and oral squamous cell carcinomas (OSCCs); (B) shows the number of total CNAs detected in non-dysplastic OPMDs (ND-OPMDs); (C) shows the number of total CNAs detected in dysplastic OPMDs (D-OPMDs) and OSCCs. The bottom and the top of each box show the first and third quartile, respectively, while the line inside the box represents the median (second quartile). Please notice that when the median is not shown, its value = 0. The tips of the whiskers represent the minimum and the maximum data value. The boxes corresponding to the number of CNAs detected in DNA diploid sites of oral mucosa are shown in white; the boxes corresponding to the number of CNAs detected in DNA aneuploid sites of oral mucosa are shown in gray. CNAs are referred to as: total focal gains, TFG; total broad gains, TBG; total focal losses, TFL; total broad losses, TBL. Broad gains and broad losses correspond to gains or losses of more than half a chromosome arm, respectively. Significant MW P-values (P < 0.05) and their corresponding q-values are shown. The FDR q-value method was applied for multiple testing (n = 4) correction; q-values < 0.1 are indicated in bold. (A) N = 94 OPMDs/OSCCs; N = 58 DNA diploid; N = 36 DNA aneuploid. (B) N = 46 ND-OPMDs; 35 DNA diploid; 11 DNA aneuploid. (C) N = 48 D-OPMDs/OSCCs; 24 DNA diploid; 24 DNA aneuploid.

Fig 5. Relationship between total CNAs and histological diagnosis in TNG and BM OPMDs/OSCCs.

(A) shows the number of total CNAs detected in oral potentially malignant disorder (OPMD) and oral squamous cell carcinoma (OSCC) in tongue (TNG) and buccal mucosa (BM) sites; (B) shows the number of total CNAs detected in non-dysplastic OPMDs (ND-OPMDs) in TNG and BM sites. (C) shows the number of total CNAs detected in dysplastic OPMDs (D-OPMDs) and OSCC TNG and BM sites. The bottom and the top of each box show the first and third quartile while the line inside the box represents the median (second quartile). Please notice that when the median is not shown, its value = 0. The tips of the whiskers represent the minimum and the maximum data value. CNAs are referred to as: total focal gains, TFG; total broad gains, TBG; total focal losses, TFL; total broad losses, TBL. Broad gains and broad losses correspond to gains or losses of more than half a chromosome arm, respectively. The boxes corresponding to the number of CNAs detected in TNG OPMDs/OSCCs are shown in white bars, whereas those of CNAs detected in BM OPMDs/OSCCs are shown in gray bars.