Genetic and epigenetic predictors of tongue squamous cell carcinoma sensitivity to platinum drugs

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Abstract

Background. Treatment of locally advanced malignant tumors of the tongue at the present stage of development of clinical oncology is a difficult task and is an important problem in oncology. The aim — to screen for genetic and epigenetic predictors of locally advanced tongue cancer sensitivity to platinum therapy. Methods. A comprehensive clinical and molecular genetic examination was carried out in 100 patients with locally advanced malignant tumors of the tongue, as well as 30 donors without oncological pathology. The study used a new method for the treatment of locally advanced squamous cell carcinoma of the tongue using 2-stage superselective embolization as a component of complex treatment. At the first stage, a screening bioinformatics analysis was carried out to form a panel of genetic loci associated with sensitivity to therapy with platinum drugs. The determination of the relative copy number and expression of these loci responsible for repair and for the proliferation regulation and apoptosis was performed by Real-Time qPCR. Results. Integration of RNA-seq datasets, copy number variation data, and relevant clinicopathological information from TCGA and GEO databases allowed us to identify 65 genetic loci associated with sensitivity to platinum therapy. These genes were validated in patient biological material. Laboratory screening showed differential expression of BRCA1, BLM, ERCC1, EXO1, RAD50, PIF1, LIG1, BCL2, ERBB2, MAGEH1, NGFRAP1, and CASP8 genes, correlating with changes in their tissue copy numbers, between groups of patients sensitive and resistant to platinum therapy. The profile of gene copy numbers of BRCA1, BLM, EXO1, RAD50, PIF1, LIG1, CASP8, MAGEH1 and NGFRAP1 was detected in the blood plasma of patients with squamous cell carcinoma of the tongue, which was differential for two groups of patients — sensitive and resistant to platinum therapy. Based on bootstrap models, the final gene panel was obtained: BLM/NGFRAP1, BRCA1/MAGEH1, EXO1/RAD50, BLM/CASP8, BRCA1/RAD50 and BRCA1/EXO1, providing diagnostic sensitivity at the level of 95% and specificity at the level of 90% (AUC 0.98) when dividing patients into a group sensitive and resistant to platinum therapy. Conclusion. Comprehensive bioinformatics analysis of RNA sequencing data, gene copy number data, and clinical and pathological information from the TCGA and GEO databases, as well as laboratory screening of potential predictors, allowed us to obtain a panel of genes — BLM/NGFRAP1, BRCA1/MAGEH1, EXO1/RAD50, BLM/CASP8, BRCA1/RAD50, and BRCA1/EXO1, the combination of which provides high diagnostic sensitivity and specificity in dividing patients into a group susceptible and resistant to platinum therapy.

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About the authors

Oleg I. Kit

National Medical Research Center for Oncology

Author for correspondence.
Email: rnioi@list.ru
ORCID iD: 0000-0003-3061-6108
SPIN-code: 1728-0329

MD, PhD, Professor, Corresponding Member of the RAS

Россия, 63 bldg 12 Line 14, 344037, Rostov-on-Don

Marina A. Engibaryan

National Medical Research Center for Oncology

Email: rnioi@list.ru
ORCID iD: 0000-0001-7293-2358
SPIN-code: 1764-0276

MD, PhD

Россия, 63 bldg 12 Line 14, 344037, Rostov-on-Don

Astanda K. Gvaramiya

National Medical Research Center for Oncology

Email: rnioi@list.ru
ORCID iD: 0000-0003-3546-6014
SPIN-code: 2700-3410

MD

Россия, 63 bldg 12 Line 14, 344037, Rostov-on-Don

Victoria L. Volkova

National Medical Research Center for Oncology

Email: rnioi@list.ru
ORCID iD: 0000-0001-8291-0750
SPIN-code: 8289-6300

MD, PhD

Россия, 63 bldg 12 Line 14, 344037, Rostov-on-Don

Natalia A. Chertova

National Medical Research Center for Oncology

Email: rnioi@list.ru
ORCID iD: 0000-0002-9279-9408
SPIN-code: 7051-4574

MD, PhD

Россия, 63 bldg 12 Line 14, 344037, Rostov-on-Don

Denis S. Kutilin

National Medical Research Center for Oncology

Email: k.denees@yandex.ru
ORCID iD: 0000-0002-8942-3733
SPIN-code: 8382-4460

PhD (Biology), Leading Researcher

Россия, 63 bldg 12 Line 14, 344037, Rostov-on-Don

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Supplementary files

Supplementary Files
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1. JATS XML
2. Fig. 1. Study design

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3. Fig. 2. Volcano raft and PPI DEG network: A — DEGs were selected with a fold change ≥ 2 or ≤ –2 and an adjusted p-value < 0.05. Green dot indicates that gene expression in tumor was half that in normal tissues, while red dot indicates that gene expression in tumor was more than 2-fold higher than that in normal tissues (p < 0.05). Black dots represent genes that were not considered differentially expressed; B — PPI DEG network was constructed using Cytoscape, PPI — protein-protein interaction, DEG — differentially expressed genes

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4. Fig. 3. Analysis of hub genes and their co-expressed genes, and hierarchical clustering of hub genes: A, Hub genes and their co-expressed genes analyzed by cBioPortal. Nodes with thick black outlines represent hub genes, nodes with thin black outlines represent co-expressed genes. Color in circle represents the total gene change in genomic profiles, including up- and downregulation. Color intensity indicates greater change. Blue arrow denotes change in state; green arrow denotes expression control; B, Samples grouped by brown bar are non-malignant samples, and samples grouped by blue bar are tongue cancer samples; C, D, Samples grouped by brown bar are HPV-positive samples, and samples grouped by blue bar are HPV-negative. Red color indicates gene activation, blue color indicates gene repression.

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5. Fig. 4. Genomic position of amplified and deleted regions

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6. Fig. 5. Relative gene copy number in samples of patients with squamous cell carcinoma of the tongue, sensitive and resistant to platinum drugs Note. Statistically significant differences: * — from normal tissue (p < 0.01); ** — between two groups of patients (p < 0.01).

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7. Fig. 6. Peculiarities of relative gene expression in patients with squamous cell carcinoma of the tongue, sensitive and resistant to platinum drugs Note. Statistically significant differences: * — from normal tissue (p < 0.01); ** — between two groups of patients (p < 0.01).

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8. Fig. 7. Peculiarities of relative gene copy number in patients with squamous cell carcinoma of the tongue, sensitive and resistant to platinum drugs, in cfDNA of blood plasma Note. Statistically significant differences: * — from conditionally healthy donors (p < 0.01); ** — between two groups of patients (p < 0.01).

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9. Fig. 8. ROC curves for classification of groups of patients with squamous cell carcinoma of the tongue, sensitive (solid line) and resistant (dashed line) to therapy, based on the gene copy number index. ROC curves with an AUC value ≥ 0.70 are presented

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10. Fig. 9. LASSO-penalized model for logistic regression of relative copy number level of genetic loci in blood plasma cfDNA: A — distribution of regression coefficients in bootstrap datasets; B — importance of variables in bootstrap models; C — ROC curves for classification of samples using optimized (solid line) and non-optimized (dashed line) models.

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11. Supplement
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