PMC

(A) To understand the genomic underpinnings of malignant progression of oral potentially malignant lesion (leukoplakia), oral cancer patients were recruited in whom there was the presence of leukoplakia with cancer. From each patient, tissue samples from tumour, leukoplakia, adjacent normal, and blood were collected. Good‐quality DNA was extracted from tumour, leukoplakia, and blood and sequenced for whole‐exome at about 100X depth; RNA was extracted from tumour, leukoplakia, and adjacent normal tissue and whole transcriptome sequence data were generated (the right‐hand portion represents mRNA expression patterns of differentially expressed genes in leukoplakia and tumour with respect to adjacent normal tissue, described in the supplementary information). (B) Additionally, we recruited 11 individuals diagnosed having oral leukoplakia without the presence of oral tumour. The left‐hand‐side H&E‐stained image showing a leukoplakia lesion in squamous epithelium with features of dysplasia in patients without the presence of tumour in the oral cavity. The middle image represents the histopathology of leukoplakia with the concomitant presence of tumour within the oral cavity, revealing progression from mildly dysplastic epithelium to invasive squamous cell carcinoma with infiltrating dyscohesive neoplastic epithelial cells. And the right‐hand‐side image shows the histopathological features of invasive squamous cell carcinoma with invading neoplastic squamous cells islands in connective tissue stroma. (C) The numbers of somatic mutations in leukoplakia of 11 individuals without tumour in the oral cavity, and in both leukoplakia and tumour in 28 patients with concomitant leukoplakia and cancer are provided. The somatic mutation rate in oral tumours (2.88/Mb) was higher than in leukoplakia (1.21/Mb). The mean number of somatic mutations in tumours (84) was significantly higher than in leukoplakia lesions (44) of the same patient. Additionally, we generated whole‐exome sequence data from the oral leukoplakia tissue of 11 individuals without oral cancer, among whom the mean number of somatic mutations (14) was smaller. (D) The proportion of frameshift deletions in tumours was significantly higher than in leukoplakia tissue. Frameshift deletions in known oral cancer driver genes are provided for each patient.

Five SNPs (studied with leukoplakia, cancer and control samples) of each individual are combined to form super-set genotypes. One hundred fourty three unique genotype-sets are observed of which 18 are specific to each control, leukoplakia and cancer individuals, 12 genotype-sets are present in both control and leukoplakia individuals, 18 genotype-sets are present in both control and cancer individuals, only 6 genotype-sets are common between leukoplakia and cancer individuals and as many as 53 genotype-sets are common to case, control and leukoplakia. The number of occurrences of each particular genotype-set is illustrated through its corresponding edge-width.
The circles in i) red ii) violet iii) yellow iv) prussian blue v) grey vi) orange and vii) blue respectively represents the following groups i) cancer only ii) cancer and control iii) leukoplakia only iv) control only v) control and leukoplakia vi) cancer and leukoplakia and vii) cancer, leukoplakia and control.

Malignant process of (A) a case of a homogenous leukoplakia and (B) a case of a non-homogenous verrucous leukoplakia.

Mutational landscapes of OSCC‐GB patients with the concomitant presence of oral potentially malignant lesions. (A) Histopathological grade of tumour and leukoplakia, clinical information, and location of leukoplakia. (B) Somatic mutations identified in leukoplakia lesion and tumour were grouped as: (1) present in both leukoplakia and tumour, (2) present only in tumour, and (3) present only in leukoplakia. Although none of the mutation signatures associated with tumour age, tobacco smoking, or APOBEC activity showed a statistically significant difference between the leukoplakia lesion and tumour; for the APOBEC mutational signature the difference was high between the leukoplakia lesion (11%) and tumour (21%). The proportion of mutations in a leukoplakia lesion that was shared with the paired tumour was negatively correlated with the angular distance between leukoplakia and tumour (φ). Distant (φ > 80°) leukoplakia lesions (n = 6) did not share any somatic mutation with paired tumours. (C) Nonsynonymous somatic mutations were found in the known driver genes of oral cancer in tumour and leukoplakia. Recurrent somatic mutations in CASP8 were found to be shared among 43% of patients. (D) Amplification of EGFR was found only in the tumour tissue (29%), but not in the leukoplakia lesion. NOTCH1 was deleted in both tumours (29%) and the leukoplakia state (29%). (E) Rare germline mutations in DNA repair pathways —BER (Base Excision Repair) and HR (Homologous Recombination) — in these patients were significantly higher than in the normal healthy Indian population (GenomeAsia100K data).