
==== Front
Hum Genomics
Hum Genomics
Human Genomics
1473-9542
1479-7364
BioMed Central London

668
10.1186/s40246-024-00668-8
Brief Report
Multi-regional genomic and transcriptomic characterization of a melanoma-associated oral cavity cancer provide evidence for CASP8 alteration-mediated field cancerization
Chakravarty Shouvik 12
Ghosh Arnab 12
Das Chitrarpita 1
Das Subrata 1
Patra Subrata 1
Maitra Arindam 1
Ghose Sandip sanindra1967@gmail.com

3
Biswas Nidhan K nkb1@nibmg.ac.in

12
1 grid.410872.8 0000 0004 1774 5690 Biotechnology Research and Innovation Council, National Institute of Biomedical Genomics (BRIC-NIBMG), Kalyani, 741251 India
2 grid.502122.6 0000 0004 1774 5631 Biotechnology Research and Innovation Council-Regional Centre for Biotechnology (BRIC- RCB), Faridabad, India
3 https://ror.org/021aj5d10 grid.414131.2 0000 0004 1801 040X Dr R Ahmed Dental College and Hospital, Kolkata, 700014 India
7 9 2024
7 9 2024
2024
18 9629 6 2024
24 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Background

Precancerous and malignant tumours arise within the oral cavity from a predisposed “field” of epithelial cells upon exposure to carcinogenic stimulus. This phenomenon is known as “Field Cancerization”. The molecular genomic and transcriptomic alterations that lead to field cancerization and tumour progression is unknown in Indian Oral squamous cell carcinoma (OSCC) patients.

Methods

We have performed whole exome sequencing, copy-number variation array and whole transcriptome sequencing from five tumours and dysplastic lesions (sampled from distinct anatomical subsites - one each from buccal anterior and posterior alveolus, dorsum of tongue–mucosal melanoma, lip and left buccal mucosa) and blood from a rare OSCC patient with field cancerization.

Results

A missense CASP8 gene mutation (p.S375F) was observed to be the initiating event in oral tumour field development. APOBEC mutation signatures, arm-level copy number alterations, depletion of CD8 + T cells and activated NK cells and enrichment of pro-inflammatory mast cells were features of early-originating tumours. Pharmacological inhibition of CASP8 protein in a CASP8-wild type OSCC cell line showed enhanced levels of cellular migration and viability.

Conclusion

CASP8 alterations are the earliest driving events in oral field carcinogenesis, whereas additional somatic mutational, copy number and transcriptomic alterations ultimately lead to OSCC tumour formation and progression.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40246-024-00668-8.

Keywords

Field cancerization
OSCC
CASP8
APOBEC
CD8 + T cells
Immune checkpoint
http://dx.doi.org/10.13039/501100001407 Department of Biotechnology, Ministry of Science and Technology, India ICMR, Govt of IndiaNational Supercomputing Mission, Govt of Indiaissue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcIntroduction

Oral squamous cell carcinoma (OSCC) is one of the topmost health threats in India [1]. Under the influence of carcinogenic stimulants (smokeless tobacco) [2], normal oral epithelia develop dysplasia and over a period of time accumulate pro-tumorigenic alterations. This gives rise to a localized cancerous “patch” (field cancerization). In head and neck squamous cell cancer (HNSCC), the cancerized field may stretch over an area having a diameter of up to 7 cm. In a clinico-pathological study by Slaughter et al. [3]. involving 783 OSCC cases, ~ 20% of the patients harbored a secondary lesion (precancerous or malignant) in addition to their primary tumours. Due to morphologically indistinguishable changes, histopathological characterization is often inadequate in field cancerization for predicting cancer risk [4]. Other studies had indicated that ~ 25% of patients with HNSCC develop local recurrence even with clinically negative surgical margins. Secondary HNSCC tumours often arise in the same area after the primary tumor is surgically removed [5].

Cancerized fields can evolve over time, accumulating genomic alterations and transcriptomic dysregulation. Histopathological diagnoses need to be correlated with genome-wide molecular features to properly classify patients based on risk. Previously, we analyzed precancerous lesions and tumours (two sites of oral cavity) from 28 oral cancer patients to identify molecular features of tumour progression [6]. Past studies have identified driver genomic, transcriptomic and epigenomic alterations using mainly a single tissue subsite from OSCC patients [7, 8]. Omics-driven characterization of multiregional biopsies may provide key information on the trajectory of tumour evolution in field cancerization setting which has not been attempted yet on Indian OSCC patients.

We have performed whole exome sequencing (WES), genome-wide copy-number profiling and transcriptome sequencing from an Indian OSCC patient with histopathologically verified multiregional tumours and dysplastic lesions from five different subsites. The genomic finding derived from the patient in respect to initiation of oral field cancerization was further studied in-vitro with genomically characterized OSCC cell line. Characterization of driver events and in-vitro validation reveals that early CASP8 alteration is a key factor in initiating field carcinogenesis. The inferences derived from this study provides key indications for understanding OSCC evolution in field cancerization background.

Methods

Patient recruitment and biospecimen collection

A Female patient was diagnosed with multiple oral tumours and dysplastic lesions at Dr. R. Ahmed Dental College and Hospital, Kolkata (RADCH), India. The tumours and dysplastic lesions were located in the anterior alveolus and vestibule (hereafter referred to as OC1), posterior alveolus (OC2), dorsal surface of tongue (OC3), lip (OC4) and left buccal mucosa (OC5); (Fig. 1a). With signed informed consent, tumour tissue was preserved in RNAlater (Invitrogen) and 2 ml of blood was collected in EDTA-coated tubes (BD). The study was conducted with the approval of Institutional Review Boards (IRB) of RADCH and BRIC-NIBMG, Kalyani and part of the ongoing oral cancer progression study.

Whole-exome sequencing, copy-number array data generation, identification of somatic and rare germline mutations and copy-number alteration events.

WES was performed (~ 100X) depth using Illumina Novaseq 6000. quality control, alignment to reference genome, post-alignment QC, somatic and germline variant calling, variant filtering and annotation were performed according to community-standard best practice guidelines (Supplementary methods). Genome-wide genotyping array (2.5 million SNP markers) data was generated for blood and tumour tissues and processed using standard tools. Details of analytical procedures outlined in Supplementary methods.

Results

Somatic alterations of CASP8 is one of the early hallmarks of oral field cancerization.

Histopathological characterization of the multiregional tumours and dysplastic lesions were– infiltrating squamous cell carcinoma (OC1 and OC2), malignant mucosal melanoma with pagetoid growth of atypical melanocytes (OC3), epithelial hyperplasia with low-grade dysplasia (OC4) and epithelial hyperplasia with high grade dysplasia (OC5, clinically normal appearing) (Fig. 1a, Supplementary methods and results). Following AJCC 8th edition convention guidelines [9], the pathological staging and grading of the tumours were following – (i) OC1 : T2 and well- differentiated, (ii) OC2 : T2 and well-differentiated, (iii) OC3 (Melanoma): T4IIB. The OC1 and OC2 tumors showed a worse pattern of Invasion [10] (WPOI) score of 3. All tumours and lesions were Human Papilloma Virus (HPV)-negative (Supplementary methods). On an average, 204 somatic mutations (coding, nonsynonymous = 116, non-coding = 88) were identified after WES profiling (Fig. 1b) in the malignant tissues: OC1 (total = 171; coding = 93), OC2 (355; 205), OC3 (149; 97), OC4 (237; 124) and OC5 (107; 59) (Supplementary Table 1).Twelve nonsynonymous somatic mutations were shared among the five multi-regionally sequenced tissues (Fig. 1c, Supplementary Table 2), and a median of 76 non-silent somatic mutations were unique (private) to each subsite (OC1: 19 unique mutations, OC2: 127, OC3: 76, OC4: 95, OC5: 29). A total 12 somatic mutations (one in known OSCC driver gene, rest in passenger genes) were found to be common among all tissue subsites. Within any two subsites, the highest number of coding somatic mutations that were shared was between the anterior and posterior alveolus tumours (56 mutations, 60% and 27% of the total coding non-silent mutations in OC1 and OC2, respectively). Fourteen mutations were shared between the OC4 and OC5 dysplastic lesions (Fig. 1c). The patient harbored somatic mutations in known oral cancer driver genes- CASP8, PIK3CA, NOTCH1, FBXW7, HRAS, FAT1, AJUBA, HLA-A, HLA-B and TGFBR2. 13 somatic mutations in these known driver genes were identified in the multiregionally sampled tumours and dysplastic lesions. A fraction of these driver mutations (4 mutations, 31%) were present at least at two sites. The OC3 tumour harbored the highest number of somatic mutations in known OSCC driver genes − 7 mutations, followed by OC4 (5 mutations), OC2 (4 mutations each) and OC5 and OC1 (3 mutations each).

Fig. 1 Clinico-histopathological and mutational profile of an OSCC patient with five multiregional field tumours and dysplastic lesions. a Tissue subsite of origin and histopathological characterization of multiregional tumours and dysplastic lesions, b Multi-omics profiling and analysis strategy (High-depth WES, genome-wide genotyping array and whole transcriptomic sequencing data was generated and analyzed using best practice community standard pipelines), c Upset plot showing the profile of shared and private coding somatic mutations among the five field tumours and lesions from distinct anatomical subsites., d Distribution of variant allele frequencies (VAF) at coding somatic mutational loci in five multiregionally sampled tumours and dysplastic lesions. Presence of the same CASP8 missense mutation (S375F) at varying VAF was indicated in blue font, e Somatic mutational signature profile in multiregional oral tumours and dysplastic lesions, f Profile of genome-wide somatic copy-number alteration signatures in multiregional oral tumours and dysplastic lesions. Defective Non-homologous end joining (NHEJ)-associated copy-number alteration signatures (marked in green) are only present in the lesions arising later in field tumorigenesis (OC3, OC4 and OC5)

Other than known OSCC drivers, the OC4 lesion had highest number (n = 8) of somatic mutations in other pan-cancer driver genes [11], followed by, OC2 and OC3 (5 mutations each) and OC1 and OC5 (3 mutations each). Among the 12 mutations shared among all tissue subsites, a coding missense somatic mutation in the known OSCC driver gene CASP8 (p.S375F; fitCon rankscore : 0.93) was present, indicative of driving founder (initiating) event of tumourigenesis in this patient. Oncogenic hotspot mutation in PIK3CA (p.H1047R) was present in four sites (except tongue), likely to be the second important event during field formation. The OC1 and OC2 tumours harbored oncogenic FBXW7 and HRAS mutations. The driver gene alteration profile of the tongue tumour (melanoma) was distinct from the other oral subsites, having frame-shift insertion/deletion mutations in AJUBA, FAT1 and HLA-A genes and missense NOTCH1 mutations. Somatic mutations in 9 genes that were common amongst all multiregional tumours and dysplastic lesions (includes CASP8) occurred at a higher clonal fraction in the OC2 tumour compared to other field lesions (Supplementary Table 2). Therefore, driver initiating CASP8 mutation and subsequent mutational hits in other cancer drivers are key molecular events for oral field tumour development in this patient. Pharmacological CASP8 blockade with pan-caspase inhibitor Emricasan (5 µM/L) showed significant increase in cellular viability in a CASP8-Wild type OSCC cell line in response to treatment with apoptosis ligand TRAIL (one-tailed t test p = 0.0001). Transwell migration assay revealed enhanced cellular migration in CASP8-WT cells in response to Emricasan treatment (Supplementary methods and results) – providing support to the hypothesis that tumour-field initiating CASP8 alteration promote tumour progression by regulating cellular migration.

Evolutionary trajectories of field cancerization are shaped by sequential acquisition of driver somatic events

Shared somatic mutations between multiple oral tumours indicates early origin of those mutations. Variant allele frequency (VAF) is a well-established proxy for timing of mutational events - mutations at higher VAF are expected to occur in higher proportions of cells and are likely arise from clonal selection [12]. Median VAF of all coding non-silent somatic mutations was the highest (0.32) for the OC2 tumour (posterior alveolus), followed by OC1: 0.22, OC4: 0.22, OC5: 0.17 and OC3: 0.14 -indicative of varied level of mutational heterogeneity within the oral tumour field. The highest number of coding somatic mutations were shared among OC1 and OC2 tumours. For the 12 coding somatic mutations shared among all tissue subsites (Supplementary Table 1), mean VAF of these mutations was higher in the OC2 tumour (mean = 0.37) compared with all other tumours - OC1 (mean = 0.23, p = 0.005), OC3 (mean = 0.13, p = 6.86E-05), OC4 (mean = 0.27, p = 0.03) and OC5 (mean = 0.2, p = 0.001) indicating the antiquity of the mutations in OC2 tumour. VAF of the initiating CASP8 mutation p. S375F was the highest in the OC2 tumour (Fig. 1d). Tumour age (methylation of 5-methylcytosine at mutated residues) was estimated through somatic mutation signature analyses. The OC2 tumour had the highest number (121) of age-associated point mutations, followed by OC3 (100 mutations) and (OC1) (72 mutations, ) - indicating an earlier origin of the OC2 tumour (Fig. 1e). The distance of the other field tumour and lesions compared to the early originating OC2 tumour were as follows – (i) OC2 to OC1 : 2.5 cm, (ii) OC2 to OC3 : 3.5 cm, (iii) OC2 to OC4 : 6.5 cm, (iv) OC2 to OC5 : 6 cm. From the melanoma (OC3), distance of other field tumour and lesions were : OC1 : 4.5 cm, OC2 : 3.5 cm, OC4 : 5.5 cm, OC5 : 5 cm. APOBEC mutational signature were present in only the OC1 and OC2 tumours. The OC4 and OC5 dysplastic lesions had fewer age-associated point mutations (64 and 68 respectively), indicating a later origin. Analysis of copy number alterations (CNA) identified arm level CNA events were observed in OC1 (5p, 8q and 9p amplification, 6p deletion) and OC2 (8q, 20p, 20q amplification, 8p deletion) tumours. Non-homologous end joining (NHEJ)-associated copy-number alteration signatures were present only in OC3, OC4 and OC5 (Fig. 1f, Supplementary methods and results). We propose a possible trajectory of initiation and progression of oral tumour field (Fig. 2a, b). The initiating event – CASP8 mutation was found to have highest clonality in OC2 followed by PIK3CA, FBXW7 and HRAS mutations – suggestive of the early origin of OC2 in the field. Sharing of 56 somatic mutations between this tumour and its physically nearest OC1 tumour shows that these tumours have a common origin and are evolutionarily closely related (Fig. 1c). Fourteen mutations were shared between OC4 and OC5 lesions, both acquiring same PIK3CA (missense hotspot) and FAT1 mutations. Thus, followed by initiating CASP8 mutation the OC2 tumour, gradually acquired PIK3CA hotspot mutations as well as HRAS and FBXW7 gene mutations during the course of tumour field evolution. The PIK3CA p.H1047R mutation is shared among the other four tumours and dysplastic lesions except tongue, at a VAF higher than all the other driver genes in these lesions. Thus, multiple non-primary tumors and dysplastic lesions arose after initiating driver genomic alterations in the progenitor OC2 tumour within the patient’s oral cavity.

The OC3 tumour (melanoma) developed following a different evolutionary process after the initial CASP8 mutation, acquiring second hits in known OSCC drivers - FAT1, AJUBA, HLA-A and HLA-B not shared with any of the other field tumours and dysplastic lesions. Tumour mutational heterogeneity (MATH score) was higher in OC1 (47) followed by OC2 and OC5 (33), OC3 (31) and OC4 (29). The average number of clonal non-silent coding mutations detected in the field tumours and dysplastic lesions was 83 (range: 42–153, highest in OC2), as per ABSOLUTE algorithm (Supplementary Methods, Supplementary Table 3). On an average, 33 subclonal coding mutations were identified in the tumours and dysplastic lesions (highest in OC2). Clustering of somatic mutations based on variant allele fractions revealed highest number of mutational clusters (“tumour clones”) in OC2 and OC4 (4 each), followed by OC3 [3], OC1 and OC5 (2 each) (Supplementary Methods, Supplementary Table 7). Our analysis has reconstructed the detailed molecular evolution of field cancerization in a patient initiated by CASP8 alteration, followed by stepwise accumulation of key pathogenic genome alteration events.

Fig. 2 a Key multi-omics signatures of oral field tumours.CASP8 protease domain missense mutation (S375F) was found to occur at the highest VAF in OC2 tumour (0.72). This tumour also harboured the most number of tumor-age specific mutations. The OC1 and OC2 tumours had higher copy number alteration burden, presence of APOBEC mutational signature and mutations in PIK3CA, FBXW7 and HRAS genes, indicative of their close evolutionary origin. OC3, OC4 and OC5 followed a different, later trajectory of development.Overall, OC2 was identified as initiator field and OC1,OC3, OC4 and OC5 were identified as non-primary or second field tumours (OC1 and OC3) and dysplastic lesions (OC4 and OC5), (b) Putative construction of tumour evolution path from the profile of VAF and CNAs. Dotted arrow heads indicate that from the progenitor OC2 tumour, tumour development followed three major paths – (i) OC2 ◊ OC1 (shared CASP8 and PIK3CA mutations, arm-level CN changes), (ii) OC2 ◊ OC5 ◊ OC4 (Shared CASP8 and PIK3CA somatic mutations), (iii) OC2 ◊ OC3 (shared CASP8 mutation, additional mutational hits in FAT1, AJUBA, NOTCH1 etc.)

Discussion

Heterogenous tumor microenvironment governing oral tumour field formation influences disease recurrence. Study designs with a single tumour biopsy per patient often fails to capture the full extent of this heterogeneity. Due to difficulty in histologically characterizing field cancerization, residual tumor cells at surgical margins might give rise to multiple metachronous primary tumours [13]. The proper mechanism governing this phenomenon remains elusive in Indian OSCC patients. We have sampled oral tumour and dysplastic tissue from multiple subsites from a patient to understand evolutionary timelines of field cancerization. In specific cases, a single clonal mutation may be the dominant feature driving oral field cancerization phenomenon [14]. In our study, shared somatic mutations among all subsites indicate that these mutations arose early during tumour evolution. Among these mutations, a protease domain mutation of CASP8 gene is present, in agreement with our earlier finding of CASP8 mutations triggering tumourigenesis events in OSCC [6]. The hotspot PIK3CA mutation p.H1047R, is an oncogenic trigger in all tissue subsites (tumor and dysplasia) except tongue (OC3) in the patient. Further support in favor of CASP8 alteration triggering pro-tumourigenic behavior was obtained in vitro, showing enhanced proliferation and migration in CASP8-wild type cells upon CASP8 blocking. CASP8 inhibition have a detrimental effect on Fas-receptor mediated cell death [15]. After acquiring the initiating CASP8 mutation, the tongue tumour did not share any other OSCC driver gene mutation with the other field tumours and dysplastic lesions, suggesting that after initiation of tumourigenesis, the evolution of tongue subsite follows an independent path compared to the evolutionary paths followed by the other oral cavity tumours and lesions. The fact that oral epithelium and melanocytes arise from ectoderm and neural crest (which in turn originates from ectoderm) shows that OSCC and melanoma might have common developmental origin but later follows a divergent evolutionary pattern [16, 17]. Base-excision repair gene TDG was mutated in the OC3 tumor, reported earlier in non-skin cancer melanomas [18]. Multiple lines of evidence - (i) highest number of mutations among all field tumours, (ii) higher number of tumour-age associated point mutations, (iii) median VAF of all coding somatic mutations is highest, (iv) CASP8 mutation occurring at highest VAF, (v) arm level amplification events, show that the posterior alveolus (OC2) tumour is the progenitor of tumourigenesis in the oral cavity. The observed high number of shared coding somatic mutations and the similarities in the VAFs of driver gene mutations indicate that the OC1 and OC2 tumours had a shared, early origin. Depletion of CD8 + T cells, immune checkpoint marker overexpression and enrichment of activated mast cells were observed in early-arising OC1 and OC2 tumours (Supplementary results). Therefore, in a uniquely sampled set of tumours and dysplastic lesions, we found that CASP8 alteration occurs early in oral field initiation, whereas subsequent acquisition of genomic and transcriptomic alterations confirm the basis of field cancerization. We provide genomic and functional evidence in support of CASP8 alteration favoring tumour field formation and cellular migration in a best-case field cancerization scenario.

Conclusion

Our approach to characterize the genomic and transcriptomic heterogeneity of oral tumour field development revealed that CASP8 somatic alterations, along with specific mutational signatures, large scale copy-number alteration events and dysregulation of certain tumour-infiltrating immune cells shapes oral field cancerization in a stepwise manner. CASP8 inhibition leads to pro-migratory behavior of OSCC cells, supporting the model of tumour field initiation.

Supplementary Information

Supplementary Material 1

Supplementary Material 2

Acknowledgements

SC acknowledges Department of Biotechnology, Govt of India for PhD fellowship (DBT/2019/NIBMG/1225) and BRIC-RCB (RCB/NIBMG-PhD/2019/1011). AG acknowledges ICMR-SRF fellowship (GENOMICS-BMS/2021-10619) and BRIC-RCB (RCB/NIBMG-PhD/2022-23/A/394/1002). We acknowledge Dr Tamima Sultana and Dr Sayema Bashir for helping SG for collection of patient samples. We are grateful to Prof. Partha Majumder, National Science Chair, Govt of India, for providing encouragement and support to initiate this study. We thank the Genomics lab members of BRIC-NIBMG for their help in multi-omics data generation.

Author contributions

SC : Data curation, Visualization, Investigation, Methodology, Formal analysis, Writing – original draft, AG : Investigation, Visualization, Methodology, Formal analysis, CD : Investigation, Visualization, Methodology, Formal analysis, SD : Methodology, SP : Methodology, AM : Supervision, SG : Clinical sample acquisition, Conceptualization, Supervision, Methodology, NKB : Conceptualization, Supervision, Writing – original draft, Writing – review & editing.

Funding

The data on this project was generated using ICGC project grants of the Department of Biotechnology, Govt of India. The analytical pipelines were developed with support from MeiTy (GoI) under National Supercomputing Mission and ICMR.

Data availability

The raw sequence data is submitted to Indian Nucleotide Data Archive (INDA-CA) under accession code PRJINCAA00294.

Declarations

Consent for publication

Signed informed consent was obtained from the patient,

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

1. Sung H Ferlay J Siegel RL Laversanne M Soerjomataram I Jemal A Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries CA Cancer J Clin 2021 71 3 209 49 10.3322/caac.21660 33538338
Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71(3):209–49. 10.3322/caac.21660.33538338 10.3322/caac.21660
2. Wiencke JK DNA adduct burden and tobacco carcinogenesis Oncogene 2002 21 48 7376 91 10.1038/sj.onc.1205799 12379880
Wiencke JK. DNA adduct burden and tobacco carcinogenesis. Oncogene. 2002;21(48):7376–91. 10.1038/sj.onc.1205799.12379880 10.1038/sj.onc.1205799
3. Slaughter DP Southwick HW Smejkal W Field cancerization in oral stratified squamous epithelium. Clinical implications of multicentric origin  Cancer 1953 6 5 963 8 13094644
Slaughter DP, Southwick HW, Smejkal W. Field cancerization in oral stratified squamous epithelium. Clinical implications of multicentric origin.  Cancer. 1953;6(5):963–8.13094644
4. Curtius K Wright NA Graham TA An evolutionary perspective on field cancerization Nat Rev Cancer 2018 18 1 19 32 10.1038/nrc.2017.102 29217838
Curtius K, Wright NA, Graham TA. An evolutionary perspective on field cancerization. Nat Rev Cancer. 2018;18(1):19–32. 10.1038/nrc.2017.102.29217838 10.1038/nrc.2017.102
5. Sinjab A Han G Wang L Kadara H Field carcinogenesis in cancer evolution: what the cell is going on? Cancer Res. 2020 80 22 4888 91 10.1158/0008-5472.CAN-20-1956 33023945
Sinjab A, Han G, Wang L, Kadara H. Field carcinogenesis in cancer evolution: what the cell is going on? Cancer Res. 2020;80(22):4888–91. 10.1158/0008-5472.CAN-20-1956.33023945 10.1158/0008-5472.CAN-20-1956
6. Ghosh A Das C Ghose S Maitra A Roy B Majumder PP Integrative analysis of genomic and transcriptomic data of normal, tumour, and co-occurring leukoplakia tissue triads drawn from patients with gingivobuccal oral cancer identifies signatures of tumour initiation and progression J Pathol 2022 257 5 593 606 10.1002/path.5900 35358331
Ghosh A, Das C, Ghose S, Maitra A, Roy B, Majumder PP, et al. Integrative analysis of genomic and transcriptomic data of normal, tumour, and co-occurring leukoplakia tissue triads drawn from patients with gingivobuccal oral cancer identifies signatures of tumour initiation and progression. J Pathol. 2022;257(5):593–606. 10.1002/path.5900.35358331 10.1002/path.5900
7. Maitra A Biswas NK Amin K Kowtal P Kumar S Das S Mutational landscape of gingivo-buccal oral squamous cell carcinoma reveals new recurrently-mutated genes and molecular subgroups Nat Commun 2013 4 1 2873 10.1038/ncomms3873 24292195
Maitra A, Biswas NK, Amin K, Kowtal P, Kumar S, Das S, et al. Mutational landscape of gingivo-buccal oral squamous cell carcinoma reveals new recurrently-mutated genes and molecular subgroups. Nat Commun. 2013;4(1):2873. 10.1038/ncomms3873.24292195 10.1038/ncomms3873
8. Lawrence MS Sougnez C Lichtenstein L Cibulskis K Lander E Gabriel SB Comprehensive genomic characterization of head and neck squamous cell carcinomas Nature 2015 517 7536 576 82 10.1038/nature14129 25631445
Lawrence MS, Sougnez C, Lichtenstein L, Cibulskis K, Lander E, Gabriel SB, et al. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature. 2015;517(7536):576–82. 10.1038/nature14129.25631445 10.1038/nature14129
9. Amin MB Greene FL Edge SB Compton CC Gershenwald JE Brookland RK Meyer L Gress DM Byrd DR Winchester DP The Eighth edition AJCC cancer staging manual: continuing to build a bridge from a population-based to a more personalized approach to cancer staging CA Cancer J Clin 2017 67 2 93 99 10.3322/caac.21388 28094848
Amin MB, Greene FL, Edge SB, Compton CC, Gershenwald JE, Brookland RK, Meyer L, Gress DM, Byrd DR, Winchester DP. The Eighth edition AJCC cancer staging manual: continuing to build a bridge from a population-based to a more personalized approach to cancer staging. CA Cancer J Clin. 2017;67(2):93–9. 10.3322/caac.21388.28094848 10.3322/caac.21388
10. Xu B Salama AM Valero C Yuan A Khimraj A Saliba M Zanoni DK Ganly I Patel SG Katabi N Ghossein R The prognostic role of histologic grade, worst pattern of invasion, and tumor budding in early oral tongue squamous cell carcinoma: a comparative study Virchows Arch 2021 479 3 597 606 10.1007/s00428-021-03063-z 33661329
Xu B, Salama AM, Valero C, Yuan A, Khimraj A, Saliba M, Zanoni DK, Ganly I, Patel SG, Katabi N, Ghossein R. The prognostic role of histologic grade, worst pattern of invasion, and tumor budding in early oral tongue squamous cell carcinoma: a comparative study. Virchows Arch. 2021;479(3):597–606. 10.1007/s00428-021-03063-z.33661329 10.1007/s00428-021-03063-z
11. Bailey MH, Tokheim C, Porta-Pardo E, Sengupta S, Bertrand D, Weerasinghe A et al. Comprehensive Characterization of Cancer Driver Genes and Mutations. Cell [Internet]. 2018;173(2):371–385
12. McGranahan N Favero F de Bruin EC Birkbak NJ Szallasi Z Swanton C Clonal status of actionable driver events and the timing of mutational processes in cancer evolution Sci Transl Med 2015 7 283ra54 283ra54 10.1126/scitranslmed.aaa1408 25877892
McGranahan N, Favero F, de Bruin EC, Birkbak NJ, Szallasi Z, Swanton C. Clonal status of actionable driver events and the timing of mutational processes in cancer evolution. Sci Transl Med. 2015;7(283ra54):283ra54. 10.1126/scitranslmed.aaa1408.25877892 10.1126/scitranslmed.aaa1408
13. Erkal HŞ Mendenhall WM Amdur RJ Villaret DB Stringer SP Synchronous and metachronous squamous cell carcinomas of the head and neck mucosal sites J Clin Oncol 2001 19 5 1358 62 10.1200/JCO.2001.19.5.1358 11230479
Erkal HŞ, Mendenhall WM, Amdur RJ, Villaret DB, Stringer SP. Synchronous and metachronous squamous cell carcinomas of the head and neck mucosal sites. J Clin Oncol. 2001;19(5):1358–62. 10.1200/JCO.2001.19.5.1358.11230479 10.1200/JCO.2001.19.5.1358
14. Franklin WA Gazdar AF Haney J Wistuba II La Rosa FG Kennedy T Widely dispersed p53 mutation in respiratory epithelium. A novel mechanism for field carcinogenesis J Clin Invest 1997 100 8 2133 7 10.1172/JCI119748 9329980
Franklin WA, Gazdar AF, Haney J, Wistuba II, La Rosa FG, Kennedy T, et al. Widely dispersed p53 mutation in respiratory epithelium. A novel mechanism for field carcinogenesis. J Clin Invest. 1997;100(8):2133–7. 10.1172/JCI119748.9329980 10.1172/JCI119748
15. Li C Egloff AM Sen M Grandis JR Johnson DE Caspase-8 mutations in head and neck cancer confer resistance to death receptor-mediated apoptosis and enhance migration, invasion, and tumor growth Mol Oncol 2014 8 7 1220 30 10.1016/j.molonc.2014.03.018 24816188
Li C, Egloff AM, Sen M, Grandis JR, Johnson DE. Caspase-8 mutations in head and neck cancer confer resistance to death receptor-mediated apoptosis and enhance migration, invasion, and tumor growth. Mol Oncol. 2014;8(7):1220–30. 10.1016/j.molonc.2014.03.018.24816188 10.1016/j.molonc.2014.03.018
16. White R Cech J Ratanasirintrawoot S DHODH modulates transcriptional elongation in the neural crest and melanoma Nature. 2011 471 518 522 10.1038/nature09882 21430780
White R, Cech J, Ratanasirintrawoot S, et al. DHODH modulates transcriptional elongation in the neural crest and melanoma. Nature. 2011;471:518–22. 10.1038/nature09882.21430780 10.1038/nature09882
17. Hughes MW Chuong CM A mouthful of epithelial-mesenchymal interactions J Invest Dermatol 2003 121 6 vii viii 10.1111/j.1523-1747.2003.12651.x 14675222
Hughes MW, Chuong CM. A mouthful of epithelial-mesenchymal interactions. J Invest Dermatol. 2003;121(6):vii–viii. 10.1111/j.1523-1747.2003.12651.x.14675222 10.1111/j.1523-1747.2003.12651.x
18. Mancuso P Tricarico R Bhattacharjee V Cosentino L Kadariya Y Jelinek J Thymine DNA glycosylase as a novel target for melanoma Oncogene 2019 38 19 3710 28 10.1038/s41388-018-0640-2 30674989
Mancuso P, Tricarico R, Bhattacharjee V, Cosentino L, Kadariya Y, Jelinek J, et al. Thymine DNA glycosylase as a novel target for melanoma. Oncogene. 2019;38(19):3710–28. 10.1038/s41388-018-0640-2.30674989 10.1038/s41388-018-0640-2
