WO2017077499A1 - Biomarkers of squamous cell carcinoma of head and neck, prognostic markers of recurrence in squamous cell carcinoma of head and neck, and methods thereof - Google Patents
Biomarkers of squamous cell carcinoma of head and neck, prognostic markers of recurrence in squamous cell carcinoma of head and neck, and methods thereof Download PDFInfo
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Definitions
- the present disclosure relates to the field of Oncology, Molecular Biology, Genomics and Bioinformatics.
- the present disclosure relates to biomarkers of cancer and prognostic markers of recurrence in cancer, particularly head and neck squamous cell carcinomas (HNSCC), method of analysing role of said biomarkers/prognostic markers, corresponding methods of detecting cancer and determining/predicting recurrence, and kits thereof.
- the present disclosure relates to analyzing aberrations and providing biomarkers of identification of HNSCC and prognostic markers of recurrence in HNSCC and associated methods/applications.
- said prognostic markers differentiate non-recurring, loco- regionally recurring and distant metastatic tumors.
- HNSCC head and neck
- Tumors of head and neck region are heterogeneous in nature with different incidences, mortalities and prognosis for different subsites and accounts for almost 30% of all cancer cases in India.
- Oral cancer is the most common subtype of head and neck cancers in humans, with a worldwide incidence in >300,000 cases. The disease is an important cause of death and morbidity, with a 5-year survival of less than 50%.
- Recent studies have identified various genetic changes in many subsites of head and neck using high-throughput sequencing assays and computational methods.
- the TCGA study identified loss of TRAF3 gene, amplification of E2F1 in human papilloma vims (HPV)-positive oropharyngeal tumors, along with mutations in PIK3CA, CASP8 and HRAS, and co- amplifications of the regions l lql3 (harboring CCND1, FADD and CTTN) and l lq22 (harboring BIRC2 and YAP1), in HPV-negative tumors, described to play an important role in pathogenesis and tumor development. Chromosomal losses at 3p and 8p, and gains at 3q, 5p and 8q were also observed in HNSCC.
- oral tongue squamous cell carcinoma tend to be different from those at other subsites as oral tongue tumors are associated more with younger patients and spread early to lymph nodes. Additionally, oral tongue tumors have a higher regional failure compared to gingivo-buccal cases in oral cavity. Tobacco (both chewing and smoking) and alcohol are common risk factors for this group of tumors among older patients.
- HNSCC head and neck
- OTSCC oral tongue squamous cell carcinoma
- predictors/prognostic markers of recurrence and distant metastasis in HNSCC including but not limiting to OTSCC
- present disclosure tries to address the above mentioned drawbacks of prior art.
- the present disclosure relates to a method of predicting recurrence of malignancy in a subject having or suspected of having head and neck squamous cell carcinoma (HNSCC), said method comprising step of detecting aberration in at least one gene selected from a group comprising WASH4P, SLC01A2, LILRB3, RPS9, ADM2, AKR1C2, ARHGEF4, CBR4, HOXB2, KDM4C, KNDC1, LILRA6, MARCH3, NANOG, POLRIC, PPFIAl, RP3-323P13.2, STCl, TPCN2, TTC39A, BVES, EVI5, COL4A4, COL5A3, DNM1P47, DUX4L9, HNFIA, MED24, MGME1, MUC3A, OVOL2, TAP2, AQR, FBN1, MST1, NOTCH3, TATDN2, TRBVl l-1, ABCD1P2, ETV7, HDC1, RP
- the disclosure further relates to aberration of WASH4P, SLCOl A2, LILRB3, RPS9, ADM2, AKR1C2, ARHGEF4, CBR4, HOXB2, KDM4C, KNDC1, LILRA6, MARCH3, NANOG, POLRIC, PPFIAl, RP3-323P13.2, STCl, TPCN2, TTC39A, BVES, EVI5, COL4A4, COL5A3, DNM1P47, DUX4L9, HNFIA, MED24, MGME1, MUC3A, OVOL2, TAP2, AQR, FBN1, MST1, NOTCH3, TATDN2, TRBVl l-1, ABCD1P2, ETV7, HDC1, RP11- 438J1.1, SLC9B1P1 or ZNF618, or any combination thereof for predicting recurrence of malignancy and distant metastasis in a subject having or suspected of having HNSCC.
- the disclosure further relates to use of aberration of WASH4P, SLCOl A2, LILRB3, RPS9, ADM2, AKR1C2, ARHGEF4, CBR4, HOXB2, KDM4C, KNDC1, LILRA6, MARCH3, NANOG, POLRIC, PPFIAl, RP3-323P13.2, STCl, TPCN2, TTC39A, BVES, EVI5, COL4A4, COL5A3, DNM1P47, DUX4L9, HNFIA, MED24, MGME1, MUC3A, OVOL2, TAP2, AQR, FBN1, MST1, NOTCH3, TATDN2, TRBVl l-1, ABCD1P2, ETV7, HDC1, RP11-438J1.1, SLC9B1P1 or ZNF618, or any combination thereof for predicting recurrence and distant metastasis of malignancy in a subject having or suspected of having HNSCC.
- the disclosure further relates to a kit for predicting recurrence of malignancy in a subject having or suspected of having HNSCC, said kit comprising agent for detecting aberration of WASH4P, SLCOl A2, LILRB3, RPS9, ADM2, AKR1C2, ARHGEF4, CBR4, HOXB2, KDM4C, KNDC1, LILRA6, MARCH3, NANOG, POLRIC, PPFIAl, RP3-323P13.2, STC1, TPCN2, TTC39A, BVES, EVI5, COL4A4, COL5A3, D M1P47, DUX4L9, HNF1A, MED24, MGME1, MUC3A, OVOL2, TAP2, AQR, FBN1, MST1, NOTCH3, TATDN2, TRBVl l-1, ABCD1P2, ETV7, HDC1, RP11-438J1.1, SLC9B1P1 or Z F618, or any combination thereof, for
- Figure 1 shows key variants in OTSCC and their relationship with habits, clinical and epidemiological parameters.
- OTSCC samples are represented in color-codes with their corresponding status on; node (Y: positive, N: negative); stage (E: early, L: late), recurrence (Y: loco-regionally recurrent, N: non-recurrent and M: distant metastatic); grade (WD: well-differentiated, MD: moderately-differentiated and PD: poorly-differentiated); disease-free survival or DFS (L: low, M: mid and H: high); HPV DNA (Y: positive and N: negative); and habits (chewing, alcohol and smoking, Y: positive and N: negative).
- node Y: positive, N: negative
- stage E: early, L: late
- recurrence Y: loco-regionally recurrent, N: non-recurrent and M: distant metastatic
- grade WD: well-differentiated
- MD moderately-differentiated
- PD poorly-differentiated
- disease-free survival or DFS L: low, M: mid and H: high
- Somatic mutation frequency per megabase is represented as scatterplot with the median point as a fine dotted line (only somatic non-synonymous mutations/mb is around 1.75/mb).
- Figure 2 shows relationship between genes harboring somatic variants with clinical, epidemiological parameters and signaling pathways.
- A Histograms showing relationship between genes with significant somatic variants (>10% frequency) and various clinical and epidemiological parameters.
- B Stack net charts of relative patient fraction (%) for each of the eight cancer-associated signaling pathways and their relationship with various clinical and epidemiological parameters.
- Figure 3 shows differentially expressed genes, affected pathways and their relationship with clinical and epidemiological parameters.
- FIG. 4 shows role of CASP8 in HPV-positive and HPV-negative OTSCC cell lines. Results from the A. Matrigel cell invasion assay (plotted with respect to the control cells),
- Figure 5 shows a minimal gene signature for tumor recurrence (loco-regional recurrence) and distant metastasis.
- Figure 6 shows cytoband-wise representation of CNVs found in all 48 samples along with clinical parameters and patient epidemiology.
- Figure 7 shows circular genomic representation using Circos (v0.66) of LOHs with > 10% frequency of patients bearing them, somatic indels- and mutations and genes with significant expression changes (
- Figure 8 shows gene signature for locoregional recurrence of tumor/malignancy in HNSCC
- Figure 9 shows gene signature for distance metastasis of tumor/malignancy in HNSCC DETIALED DESCRIPTION OF THE DISCLOSURE
- the present disclosure relates to indicators/biomarkers of squamous cell carcinoma of head and neck (HNSCC) and predictors/prognostic markers of recurrence in squamous cell carcinoma of head and neck (HNSCC).
- HNSCC head and neck
- the present disclosure relates to a method of predicting recurrence of malignancy in a subject having or suspected of having head and neck squamous cell carcinoma (HNSCC), said method comprising step of detecting aberration in at least one gene selected from a group comprising WASH4P, SLC01A2, LILRB3, RPS9, ADM2, AKR1C2, ARHGEF4, CBR4, HOXB2, KDM4C, KNDCl, LILRA6, MARCH3, NANOG, POLRIC, PPFIAl, RP3- 323P13.2, STCl, TPCN2, TTC39A, BVES, EVI5, COL4A4, COL5A3, DNM1P47
- the disclosure relates to a method for detecting aberration in the gene, said method comprises steps of:
- WGDASL aasay Illumina Total Prep RNA Amplification kit (Ambion), Illumina Hi Scan, Genome Studio, VST (Variance stabilizing transformation), LOESS and R package Lumi, ComBat and any other tool used for gene expression profiling, or any combination thereof; predicting recurrence using random forest analysis, error correction, recomputing, or any combination thereof using tools selected from a group comprising varSelRF package, leave-one-out bootstrap method, .632+ method, Benjamin- Hochberg test and any other tool used for predicting recurrence, error correction, re- computing, or any combination thereof; analyzing pathways using tools selected from a group comprising Graphite Web, KEGG, Reactome databases, CytoScape and any other pathway analysis tool, or any combination thereof; visualizing data using tools selected from a group comprising Circos, Mutation Mapper, GIMP, IGV and any other data visualizing tool, or any combination thereof; validating somatic variants using Sanger sequencing
- the disclosure further relates to aberration of WASH4P, SLCOl A2, LILRB3, RPS9, ADM2, AKR1C2, ARHGEF4, CBR4, HOXB2, KDM4C, KNDCl, LILRA6, MARCH3, NANOG, POLRIC, PPFIAl, RP3-323P13.2, STCl, TPCN2, TTC39A, BVES, EVI5, COL4A4, COL5A3, DNM1P47, DUX4L9, HNFIA, MED24, MGMEl, MUC3A, OVOL2, TAP2, AQR, FBN1, MST1, NOTCH3, TATDN2, TRBVl l-1, ABCD1P2, ETV7, HDCl, RP11- 438J1.1, SLC9B1P1 or ZNF618, or any combination thereof for predicting recurrence and distant metastasis of malignancy in a subject having or suspected of having HNSCC.
- the disclosure furthermore relates to use of aberration of WASH4P, SLCOl A2, LILRB3, RPS9, ADM2, AKR1C2, ARHGEF4, CBR4, HOXB2, KDM4C, KNDCl, LILRA6, MARCH3, NANOG, POLRIC, PPFIAl, RP3-323P13.2, STCl, TPCN2, TTC39A, BVES, EVI5, COL4A4, COL5A3, DNM1P47, DUX4L9, HNFIA, MED24, MGMEl, MUC3A, OVOL2, TAP2, AQR, FBN1, MST1, NOTCH3, TATDN2, TRBVl l-1, ABCD1P2, ETV7, HDCl, RP11-438J1.1, SLC9B 1P1 or ZNF618, or any combination thereof for predicting recurrence and distant metastasis of malignancy in a subject having or suspected of having HNSCC.
- the disclosure furthermore relates to a kit for predicting recurrence of malignancy in a subject having or suspected of having HNSCC, said kit comprising agent for detecting aberration of WASH4P, SLCOl A2, LILRB3, RPS9, ADM2, AKR1C2, ARHGEF4, CBR4, HOXB2, KDM4C, KNDCl, LILRA6, MARCH3, NANOG, POLRIC, PPFIAl, RP3- 323P13.2, STCl, TPCN2, TTC39A, BVES, EVI5, COL4A4, COL5A3, DNM1P47, DUX4L9, HNFIA, MED24, MGMEl, MUC3A, OVOL2, TAP2, AQR, FBN1, MST1, NOTCH3, TATDN2, TRBVl l-1, ABCD1P2, ETV7, HDCl, RPl 1-438J1.1, SLC9B 1P1 or ZNF61
- the aberration in gene selected from a group comprising RPS9, MARCH3, LILRB3, LILRA6, ADM2, TTC39A, TPCN2, STC1, RP3- 323P13.2, PPFIA1, POLR1C, NANOG, K DC1, KDM4C, HOXB2, CBR4, ARHGEF4 and AKR1C2, or any combination thereof, predicts locoregional recurrence of the malignancy in the subject.
- the aberration in gene selected from a group comprising AQR, FBN1, MST1, NOTCH3, ATDN2, TRBVl l-1, ABCD1P2, ETV7, FHDCl, RP11-438J1.1, SLC9B 1P1 and ZNF618, or any combination thereof predicts distant metastasis of the malignancy in the subject.
- the aberration is selected from a group comprising up-regulation, down-regulation, amplification, mutation, loss of heterozygosity, copy number variations, structural variations, somatic mutations, gene fusion events, allelic expression, chromosomal aberrations, epigenetic changes, DNA methylation, histone modification and non-coding RNA (ncRNA)-associated gene silencing, or any combination thereof.
- ncRNA non-coding RNA
- the HNSCC is selected from a group comprising cancer of hypopharynx, laryngeal cancer, cancer of oral cavity, nasopharyngeal cancer, oropharyngeal squamous cell carcinomas and cancer of trachea.
- the present disclosure furthermore relates to a method of detecting head and neck squamous cell carcinoma (HNSCC) in a sample having or suspected of having the HNSCC, said method comprising a step of detecting aberration in TP53, CASP8, OBSCN, ING1, TTK, U2AF1, RASA1, CDKN2A, NOTCH1, NOTCH2, DMD, PIK3CA, AJUBA, ANK3, FAT1, HLAA, KEAP1, KMT2D and NFE212, in the sample to detect the HNSCC.
- HNSCC head and neck squamous cell carcinoma
- the aberration is selected from a group comprising up-regulation, down-regulation, amplification, mutation, loss of heterozygosity, copy number variations, structural variations, somatic mutations, gene fusion events, allelic expression, chromosomal aberrations, epigenetic changes, DNA methylation, histone modification and non-coding RNA (ncRNA)-associated gene silencing, or any combination thereof and wherein the HNSCC is selected from a group comprising cancer of hypopharynx, laryngeal cancer, cancer of oral cavity, nasopharyngeal cancer, oropharyngeal squamous cell carcinomas and cancer of trachea.
- ncRNA non-coding RNA
- the aberration in CASP8 serve as indicator in HPV negative HNSCC subject.
- the method of detecting aberration in the gene comprises steps of:
- recurrence or “cancer recurrence” includes 'loco-regional recurrence' and 'distant metastasis' or 'distant metastatic tumor' .
- HNSCC head and neck squamous cell carcinoma
- HNSCC head and neck squamous cell carcinoma
- the present disclosure studies the role of genetic aberrations including somatic variations/mutations in genes from exome sequencing, immediate upstream and downstream flanking nucleotides of the somatic mutations, DNA methylation, loss of heterozygosity (LOH), copy number variations (CNVs), SNVs and gene expression changes, along with status of HPV infection, tumor nodal status and altered cellular pathways, in HNSCC.
- the disclosure also identifies the correlation of said genetic aberrations with alteration in cellular pathways of cancer and linking of habits, HPV infection, nodal status, tumor grade and recurrence.
- the present disclosure exploits said aspects to analyze the role of genetic changes in head and neck squamous cell carcinoma (HNSCC) and arrive at biomarkers of HNSCC and predictors/prognostic markers of recurrence in HNSCC patients. Accordingly, the present disclosure relates to a method of analysing the role of genetic aberration(s) in HNSCC, said method comprising steps of:
- CNVs Copy number variations
- LH Loss of Heterozygosity
- the analysing of the role of genetic aberrations in HNSCC involves both qualitative and quantitative analysis.
- the above method determines significantly reliable or improved indicators/biomarkers of HNSCC and predictors/prognostic markers of recurrence and distant metastasis in HNSCC, more particularly, oral tongue squamous cell carcinoma (OTSCC).
- OTSCC oral tongue squamous cell carcinoma
- the above method analyses aberration/alteration in genes to determine their role and arrive at indicators/biomarkers of HNSCC and predictors/prognostic markers of recurrence and distant metastasis in HNSCC.
- the above method of analysing genetic aberrations to arrive at indicators/biomarkers of HNSCC comprises act of performing steps (a) to (1).
- the above method of analysing genetic aberrations to arrive at predictors/prognostic markers of recurrence and distant metastasis in HNSCC comprises act of performing steps (a) to (m).
- the above method links habits, clinical, pathological and epidemiological parameters including but not limiting to chewing tobacco, smoking and alcohol consumption, HPV infection, nodal status and tumor grade, with genetic aberrations including but not limiting to somatic variants and the associated pathways affected in HNSCC, more particularly, OTSCC.
- the correlation/linkage of aforesaid habits and/or parameters provide for improved indicators/biomarkers of HNSCC as described in the present disclosure.
- the above method provides for a holistic analysis of genetic aberrations in HNSCC and identifies a group of genes, which are significantly altered/bear aberrations and serve as indicators/biomarkers of HNSCC and predictors/prognostic markers of recurrence in HNSCC. More particularly, the present disclosure provides a set of 19 genes to be aberrated in HNSCC, particularly OTSCC wherein one or more genes of said set serve as indicators/biomarkers in HNSCC.
- the above method of the present disclosure provides genetic aberrations in one or more genes from a group comprising 19 genes as indicators/biomarkers of HNSCC.
- the said 19 genes of the group are TP53, CASP8, OBSCN, ING1, TTK, U2AF1, RASA1, CDKN2A, NOTCH1, NOTCH2, DMD, PIK3CA, AJUBA, ANK3, FAT1, HLAA, KEAP1, KMT2D and NFE212.
- one or more of said 19 genes harbor genetic aberrations, more particularly somatic variations and act as indicators/biomarkers of HNSCC. Stringent filtering steps are applied and multiple annotation tools are used to come up with the said list of 19 cancer-associated genes that harbor significant somatic variations in HNSCC, more particularly OTSCC.
- genetic variants are cataloged and transcriptomic (significantly up- and down-regulated genes) changes in oral tongue squamous cell carcinoma (OTSCC) are observed and these are used in an integrated approach linking genetic aberrations, more particularly genes harboring somatic variants with common risk factors like tobacco and alcohol, clinical, epidemiological factors like tumor grade and HPV; and gene expression changes with tumor recurrence.
- one of the most important genes harboring somatic mutations identified in the study is CASP8, the product for which is derived from the precursor Procaspase-8.
- Caspase-8 is an important protein implicated in both apoptotic and non-apoptotic pathways. Recent analysis from the TCGA study suggests that mutations in CASP8 co-occur with mutations in HRAS, and are mutually exclusive with amplifications in FADD gene. In the functional study/method of the present disclosure, it is concluded that caspase-8 shows different effects in HPV-positive and HPV-negative cells, the effect being more pronounced in HPV-negative cells ( Figure 4).
- HPV- negative tumors activate a completely different set(s) of pathways and/or may have different chemo sensitivity towards drugs than the HPV-positive tumors.
- HPV-positive HNSCC cell lines are resistant to TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) and treatment of cells with the proteasome inhibitor bortezomib sensitizes HPV-positive cells towards TRAIL-induced cell death mediated by caspase-8.
- TRAIL tumor necrosis factor-related apoptosis-inducing ligand
- the E6 protein of HPV interacts with the DED domain of caspase-8 and induces its activation by recruiting it to the nucleus.
- the present disclosure also provides CASP8 gene to be significantly altered and play an important role in apoptosis-mediated cell death in an HPV-negative OTSCC cell line.
- CASP8 gene serves as indicator/biomarker in HPV negative HNSCC subject, particularly subject having HPV negative OTSCC.
- the above method of present disclosure also provides a set of 38 genes to bear aberrations in HNSCC, particularly OTSCC wherein any combination of genes of said set serve as predictors/prognostic markers of recurrence in HNSCC.
- aberration in any combination of said 38 genes serve as predictors/prognostic markers of recurrence in HNSCC, wherein said combinations distinguish non-recurrence, loco-regional recurrence and distant metastasis in HNSCC, particularly OTSCC.
- aberrations in plurality of genes of the 38-gene set serve as minimal signature for predicting recurrence and distant metastasis in HNSCC.
- any combination of 38 genes serve as minimal signature for predicting recurrence in HNSCC wherein said 38 genes are WASH4P, SLC01A2, LILRB3, RPS9, ADM2, AKR1C2, ARHGEF4, CBR4, HOXB2, KDM4C, KNDCl, LILRA6, MARCH3, NANOG, POLRIC, PPFIA1, RP3-323P13.2, STC1, TPCN2, TTC39A, BVES, EVI5, COL4A4, COL5A3, DNM1P47, DUX4L9, HNFIA, MED24, MGME1, MUC3A, OVOL2, TAP2, AQR, FBN1, MST1, NOTCH3, TATDN2 and TRBVl l-1.
- aberration in all 38 genes serve as a signature for predicting recurrence in HNSCC.
- the recurrence is loco-regional recurrence
- gene signature such as RPS9, MARCH3, LILRB3, LILRA6, ADM2, TTC39A, TPCN2, STC1, RP3-323P13.2, PPFIA1, POLRIC, NANOG, KNDCl, KDM4C, HOXB2, CBR4, ARHGEF4 or AKR1C2, or any combination thereof selected from the said 38 gene signature, predicts loco-regional recurrence of malignancy in a subject having or suspected of having HNSCC with highest sensitivity and specificity (illustrated in Figure 8).
- At least 4 gene from the gene signature such as RPS9, MARCH3, LILRB3, LILRA6, ADM2, TTC39A, TPCN2, STC1, RP3-323P13.2, PPFIA1, POLRIC, NANOG, KNDCl, KDM4C, HOXB2, CBR4, ARHGEF4 or AKR1C2, predicts loco-regional recurrence of malignancy in a subject having or suspected of having HNSCC, wherein in the said at least 4 gene mandatorily there should be at least one gene selected from RPS9, LILRA6, LILRB3, MARCH3 or ADM2, to predict the loco-regional recurrence of malignancy in a subject having or suspected of having HNSCC.
- gene signature such as AQR, FBN1, MST1, NOTCH3, TATDN2 or TRBVl l-1, or any combination thereof selected from the said 38 gene signature, predicts distant metastasis of malignancy in a subject having or suspected of having HNSCC.
- gene combinations from the said 38 gene signature that predicts distant metastasis of malignancy with highest sensitivity and specificity (illustrated in Figure 9) in a subject having or suspected of having HNSCC are:
- the gene signature ABCD1P2, ETV7, FHDC1, RP11-438J1.1, SLC9B1P1 or ZNF618, or any combination thereof predicts distant metastasis of malignancy in a subject having or suspected of having HNSCC with highest sensitivity and specificity (illustrated in Figure 9).
- Identifying signature for loco-regional tumor recurrence and distant metastasis prospectively in primary tumors adds significant advantage to disease management.
- a machine learning method is employed in the present disclosure using the molecular changes identified in the study, in three batches of primary tumors; non-recurring, loco-regionally recurring and tumors with distant metastasis.
- a 38-gene signature is identified to be significantly distinguishing the three groups.
- the present method helps in finding novel drug candidates for OTSCC based on the genes that are altered in harboring genetic variants and/or having altered expression.
- the genome-wide study of genetic aberrations in HNSCC, particularly somatic variant identification and gene expression changes in tumors in the present method give rise to possibilities of finding novel drug targets/candidates and/or lead to using existing drugs prescribed/under trial for other indications.
- the present disclosure also identifies the role of genetic aberrations as potential drug targets. In a preferred embodiment, the same is carried out by identifying the significantly altered genes, preferably somatic variant identification and/or gene expression changes and screening for available drugs/drugs under trial against them.
- head and neck squamous cell carcinomas refers to cancers including but not limiting to cancers of oral cavity including the inner lip, tongue, floor of mouth, gingivae, and hard palate, nasopharyngeal cancer, oropharyngeal squamous cell carcinomas (OSCC), cancer of hypopharynx, laryngeal cancer and cancer of trachea.
- OSCC oropharyngeal squamous cell carcinomas
- OTSCC oral tongue squamous cell carcinoma
- the expressions related to "prediction/determination of recurrence” or “predicting/determining recurrence” may be used interchangeably with conventional terms such as prognosis which refers to predicting the likely outcome of recurrence in subject(s) having HNSCC.
- the aforementioned method of analysing the role of genetic aberration(s) to arrive at indicators/biomarkers of HNSCC and predictors/prognostic markers of recurrence in HNSCC specifically involves the following steps:
- the habit is selected from a group comprising smoking, alcohol consumption and chewing tobacco, or any combination thereof
- the clinical and epidemiological parameter is selected from a group comprising detection of HPV status in the tumors and staging, or a combination thereof
- MutSigCV MutSiC2 and any other tool used to detect cross-contamination in the samples, identify significant somatic variants, annotate and analyze the variants, or any combination thereof;
- CNVs Copy number variations
- LH Loss of Heterozygosity
- h carrying out gene expression profiling using tools selected from a group comprising Illumina HumanHT-12-v4 expression Bead chip, PureLink RNA kit, RNeasy (Qiagen) Mini kit, Agilent Bioanalyzer, RNA Nano6000 chip, Illumina WGDASL aasay, Illumina Total Prep RNA Amplification kit (Ambion), Illumina Hi Scan,
- Genome Studio VST (Variance stabilizing transformation), LOESS and R package Lumi, ComBat and any other tool used for gene expression profiling, or any combination thereof;
- the present disclosure specifically relates to a group of 19 genes as indicators/biomarkers of HNSCC, particularly OTSCC wherein one or more genes of said set serve as indicators/biomarkers in HNSCC.
- genetic aberrations in one or more genes from a group comprising 19 genes act as indicators/biomarkers of HNSCC.
- the said 19 genes of the group are TP53, CASP8, OBSCN, ING1, TTK, U2AF1, RASA1, CDKN2A, NOTCH1, NOTCH2, DMD, PIK3CA, AJUBA, ANK3, FAT1, HLAA, KEAP1, KMT2D and NFE212.
- one or more of said 19 genes harbor genetic aberrations, more particularly somatic variations and serve as indicators/biomarkers of HNSCC.
- the present disclosure also provides a set of 38 genes as predictors/prognostic markers of recurrence in HNSCC, particularly OTSCC wherein, any combination of said 38 genes can be employed as predictors/prognostic markers of recurrence in HNSCC.
- aberration in any combination of said 38 genes serve as predictors/prognostic markers of recurrence in HNSCC, wherein said combinations distinguish non-recurrence, loco-regional recurrence and distant metastasis in HNSCC, particularly OTSCC.
- the said 38 genes are WASH4P, SLC01A2, LILRB3, RPS9, ADM2, AKR1C2, ARHGEF4, CBR4, HOXB2, KDM4C, KNDC1, LILRA6, MARCH3, NANOG, POLRIC, PPFIA1, RP3- 323P13.2, STC1, TPCN2, TTC39A, BVES, EVI5, COL4A4, COL5A3, DNM1P47, DUX4L9, HNFIA, MED24, MGME1, MUC3A, OVOL2, TAP2, AQR, FBN1, MST1, NOTCH3, TATDN2 and TRBVl l-1.
- aberrations in all 38 genes serve as a signature for predicting recurrence and distant metastasis in HNSCC.
- the term "aberration” includes but is not limiting to alteration in expression including up-regulation/over expression or down-regulation/under expression, amplification, mutation, loss of heterozygosity, copy number variations, structural variations, somatic mutations, gene fusion events, allelic expression, chromosomal abberations epigenetic changes including DNA methylation, histone modification and non- coding RNA (ncRNA)-associated gene silencing or any combination of aberrations thereof.
- ncRNA non- coding RNA
- mutants include but are not limiting to epigenetic mutation, transgenetic mutation, deletion, substitution and insertion or any combination thereof.
- abbrerations include up-regulation & down-regulation of plurality of the genes from the set of 38-genes and/or somatic variation or mutation in one or more genes from the group of 19 genes.
- the 38 genes are WASH4P, SLC01A2, LILRB3, RPS9, ADM2, AKR1C2, ARHGEF4, CBR4, HOXB2, KDM4C, K DC1, LILRA6, MARCH3, NANOG, POLRIC, PPFIA1, RP3- 323P13.2, STC1, TPCN2, TTC39A, BVES, EVI5, COL4A4, COL5A3, D M1P47, DUX4L9, HNF1A, MED24, MGME1, MUC3A, OVOL2, TAP2, AQR, FBN1, MST1, NOTCH3, TATDN2 and TRBV11-1, and the 19 genes are TP53, CASP8, OBSCN, ING1, TTK, U2AF1, RASAl, CDKN2A, NOTCHl, NOTCH2, DMD, PIK3CA, AJUBA, ANK3, FATl, HLAA, KEAPl, KMT2D and NFE212.
- the present disclosure relates to a method of detecting HNSCC in a subject having or suspected of having HNSCC, wherein said method comprises determining aberration(s) in one of more genes from the group comprising 19 genes, which are identified by the present disclosure to be critical biomarkers of HNSCC.
- determination of aberration(s) in one or more genes from the group of 19 genes includes analysing somatic variation/somatic mutations in said genes of the present disclosure.
- somatic variations in one or more genes of the group comprising 19 genes is determined to detect HNSCC in a subject.
- the 19 genes are TP53, CASP8, OBSCN, ING1, TTK, U2AF1, RASAl, CDKN2A, NOTCHl, NOTCH2, DMD, PIK3CA, AJUBA, ANK3, FATl, HLAA, KEAPl, KMT2D and NFE212.
- the HNSCC is OTSCC.
- the method detecting HNSCC in a subject having or suspected of having HNSCC comprises acts of:
- aberration in one or more genes from the group comprising 19 genes is determined with the help of an agent selected from a group comprising primer, probe, antibody, nanoparticles and a suitable interacting protein/biological agent capable of interacting with one or more genes of the group, in order to detect presence or absence of aberrations.
- said agent is employed for determining aberration(s) in one or more genes selected from the group TP53, CASP8, OBSCN, ING1, TTK, U2AF1, RASA1, CDKN2A, NOTCH1, NOTCH2, DMD, PIK3CA, AJUBA, ANK3, FAT1, HLAA, KEAP1, KMT2D and NFE212.
- aberration(s) in one or more genes from the group of 19-genes is identified by employing techniques selected from a group comprising but not limiting to solution- based assays and solid-support based assays, or a combination thereof.
- gene aberration is determined by employing techniques selected from a group comprising but not limiting to Sequencing, Reporter gene technique, PCR, Northern Blotting, Western blotting, ELISA, fluorescence- based assays, luminescence/chemiluminescence-based assays, in-situ hybridization, Serial analysis of gene expression (SAGE), microarrays, tiling array, RNA Sequencing/Whole Transcriptome Shotgun Sequencing (WTSS) and electrochemical assays, or any combination of techniques thereof.
- techniques selected from a group comprising but not limiting to Sequencing, Reporter gene technique, PCR, Northern Blotting, Western blotting, ELISA, fluorescence- based assays, luminescence/chemiluminescence-based assays, in-situ hybridization, Serial analysis of gene expression (SAGE), microarrays, tiling array, RNA Sequencing/Whole Transcriptome Shotgun Sequencing (WTSS) and electrochemical assays
- the solution-based assays to detect aberration(s) in one or more genes from the group of 19 genes is selected from a group comprising but not limiting to Solution hybridization, PCR and luminescence- based assay, or any combination thereof.
- the solid support based assays employed to detect aberration(s) in one or more genes from the group of 19 genes is selected from a group comprising but not limiting to Northern Blot, fluorescence- based assays, ELISA and Microarray, or any combination thereof.
- sample' used in the method of detecting HNSCC refers to any biological material/fluid/cell having or suspected of having tumor/cancer. Further, the sample may be derived from subject including humans and/or mammals, or the sample may be any biological fluid prepared/obtained in a laboratory.
- kits for detecting HNSCC in a subject having or suspected of having HNSCC comprises suitable agent(s) to determine aberration in one or more genes of the group comprising 19 genes, wherein said 19 genes are TP53, CASP8, OBSCN, ING1, TTK, U2AF1, RASA1, CDKN2A, NOTCH1, NOTCH2, DMD, PIK3CA, AJUBA, ANK3, FATl, HLAA, KEAPl, KMT2D and NFE212; and an instruction manual thereof which provides step-wise protocol of determining said aberration and correlating the same with HNSCC detection.
- 19 genes are TP53, CASP8, OBSCN, ING1, TTK, U2AF1, RASA1, CDKN2A, NOTCH1, NOTCH2, DMD, PIK3CA, AJUBA, ANK3, FATl, HLAA, KEAPl, KMT2D and NFE212; and an instruction manual thereof which provides step-wise protocol of determining said aberration and correlating the same with
- the agent is selected from a group comprising primer, probe, antibody, nanoparticle, suitable interacting protein/biological agent capable of interacting with one of more genes of the group comprising 19 genes.
- the present disclosure also relates to a method of predicting or prognosing recurrence of HNSCC in a sample having HNSCC, wherein said method comprises determining aberration(s) in the set consisting of 38 genes, which are identified by the present disclosure to be critical in HNSCC. In an exemplary embodiment, aberration in any combinations of 38 genes can predict recurrence of HNSCC in a sample.
- determination of aberration(s) in the set consisting of 38 genes includes analysing expression levels of said genes.
- up-regulation and down-regulation of said 38 genes is determined to predict recurrence and distant metastasis of HNSCC in a sample.
- the 38 genes are WASH4P, SLC01A2, LILRB3, RPS9, ADM2, AKR1C2, ARHGEF4, CBR4, HOXB2, KDM4C, KNDCl, LILRA6, MARCH3, NANOG, POLRIC, PPFIA1, RP3-323P13.2, STC1, TPCN2, TTC39A, BVES, EVI5, COL4A4, COL5A3, DNM1P47, DUX4L9, HNF1A, MED24, MGME1, MUC3A, OVOL2, TAP2, AQR, FBN1, MST1, NOTCH3, TATDN2 and TRBV11-1.
- determination of aberration(s) in the set of 38 genes is performed wherein aberration in any combinations of 38 genes can predict recurrence.
- aberration in any combinations of 38 genes distinguish non- recurrant, locoregional recurrant and distant metastatic tumors.
- the method of predicting the recurrence in a subject having HNSCC comprises acts of:
- RPS9 RPS9, ADM2, AKR1C2, ARHGEF4, CBR4, HOXB2, KDM4C, KNDC1, LILRA6, MARCH3, NANOG, POLRIC, PPFIA1, RP3-323P13.2, STC1, TPCN2, TTC39A, BVES, EVI5, COL4A4, COL5A3, DNM1P47, DUX4L9, HNF1A, MED24, MGME1, MUC3A, OVOL2, TAP2, AQR, FBN1, MST1, NOTCH3, TATDN2 and TRBVl l-l; and
- step (b) predicting recurrence and distant metastasis in HNSCC based on step (a) wherein aberration(s) in any combination of genes within the set of 38 genes correlates to the recurrence of HNSCC in said sample or vice-versa.
- the above method of predicting recurrence distinguishes non- recurrence, loco-regional recurrence and distant metastasis in HNSCC subjects.
- aberration in the 38 gene set is determined with the help of an agent selected from a group comprising primer, probe, antibody, nanoparticles and a suitable interacting protein/biological agent capable of interacting with said genes, in order to detect presence or absence of aberrations.
- said agent is employed for determining aberration(s) in the set of 38 genes, wherein genes are WASH4P, SLC01A2, LILRB3, RPS9, ADM2, AKR1C2, ARHGEF4, CBR4, HOXB2, KDM4C, KNDC1, LILRA6, MARCH3, NANOG, POLRIC, PPFIA1, RP3-323P13.2, STC1, TPCN2, TTC39A, BVES, EVI5, COL4A4, COL5A3, DNM1P47, DUX4L9, HNF1A, MED24, MGME1, MUC3A, OVOL2, TAP2, AQR, FBN1, MST1, NOTCH3, TATDN2 and TRBV11-1.
- genes are WASH4P, SLC01A2, LILRB3, RPS9, ADM2, AKR1C2, ARHGEF4, CBR4, HOXB2, KDM4C, KNDC1, LILRA6, MARCH3, NANOG
- aberration(s) in 38 gene set is identified by employing techniques selected from a group comprising but not limiting to solution- based assays and solid-support based assays, or a combination thereof.
- gene aberration is determined by employing techniques selected from a group comprising but not limiting to Sequencing, Reporter gene technique, PCR, Northern Blotting, Western blotting, ELISA, fluorescence- based assays, luminescence/chemiluminescence-based assays, in-situ hybridization, Serial analysis of gene expression (SAGE), microarrays, tiling array, RNA Sequencing/Whole Transcriptome Shotgun Sequencing (WTSS) and electrochemical assays, or any combination of techniques thereof.
- the solution-based assays to detect aberration(s) in the 38 gene set is selected from a group comprising but not limiting to Solution hybridization, PCR and luminescence- based assay, or
- the solid support based assays employed to detect aberration(s) in 38 gene set is selected from a group comprising but not limiting to Northern Blot, fluorescence- based assays, ELISA and Microarray, or any combination thereof.
- the HNSCC of the aforementioned methods is selected from group comprising cancers of oral cavity including the inner lip, tongue, floor of mouth, gingivae, and hard palate, nasopharyngeal cancer, oropharyngeal squamous cell carcinomas (OPSCC), cancer of hypopharynx, laryngeal cancer and cancer of trachea.
- the cancer is oral tongue squamous cell carcinoma (OTSCC).
- OTSCC oral tongue squamous cell carcinoma
- the term ' sample' used in the method of predicting or prognosing recurrence refers to any biological material/fluid/cell having tumor/cancer.
- the sample may be derived from subject including humans and/or mammals, or the sample may be any biological fluid prepared/obtained in a laboratory.
- the present disclosure also provides a kit for prognosis or predicting recurrence or distant metastasis in a subject having HNSCC.
- said kit comprises suitable agent(s) to determine aberration in genes comprising the set of 38 genes, wherein said 38 genes are WASH4P, SLC01A2, LILRB3, RPS9, ADM2, AKR1C2, ARHGEF4, CBR4, HOXB2, KDM4C, KNDC1, LILRA6, MARCH3, NANOG, POLRIC, PPFIA1, RP3- 323P13.2, STC1, TPCN2, TTC39A, BVES, EVI5, COL4A4, COL5A3, DNM1P47, DUX4L9, HNFIA, MED24, MGME1, MUC3A, OVOL2, TAP2, AQR, FBN1, MST1, NOTCH3, TATDN2 and TRBVl l-1; and an instruction manual thereof which provides step-wise protocol of determining the aberration and correlating said aberration with recurrence in HNSCC.
- the agent is selected from a group comprising primer, probe,
- the present disclosure also provides a method of detecting HNSCC, particularly OTSCC in a HPV negative subj ect, the method comprising act of determining aberration in CASP8 gene in the sample of said subject.
- HPV is detected by using any of the four different assays, immunohistochemistry (THC) using antibodies against pl6 (AM540-5M, Biogenex, CA, USA), and viral E6 and E7 antigens (sc-460 and sc-58661 respectively, Santa Cruz Biotechnology, TX, USA); HPV DNA PCR using either type-specific (HP VI 6 LI and E6 and HPV18 LI and E7) or consensus primers (PGMY09/11, MY09/11, GP5 + /6 + and CPI/II); q-PCR using HPV16- and HPV18-specific TaqMan probes and primers and digital PCR using TaqMan probes and primers to detect HPV in primary tumor samples.
- THC immunohistochemistry
- pl6 AM540-5M, Biogenex, CA, USA
- viral E6 and E7 antigens sc-460 and sc-58661 respectively, Santa Cruz Biotechnology, TX, USA
- HPV DNA PCR using either type-specific (HP VI 6 LI and E
- Tumor and matched control samples are collected from 50 patients diagnosed with OTSCC, with informed consent. Data from patient habits, epidemiology and clinical parameters are presented in Figure 1A and Table 1.
- Table 1 Patient details used in the study (habits, epidemiology, clinical parameters)
- HPV infection status in the primary tumors is established with at least one of the assay (pi 6 fflC, consensus or type (HPV16/18)-specific PCR, qPCR or digital PCR as described in Palve et al., 2016; http: ⁇ iorxiv.org/content/early/2017/10/24/082651). Thirty- three percentage of the patients deceased at the time of completing the analysis.
- Exome libraries are prepared using Agilent SureSelect, Illumina TruSeq and Nextera exome capture kits following manufacturers' specifications. Paired end sequencing is performed using HiSeq 2500 or GAIIx and raw reads are generated using standard Illumina base caller. Read pairs are filtered using in house and only those reads having >75% bases with > 20 phred score and ⁇ 15 Ns are used for sequence alignment against human hgl9 reference genome using NovoAlign (v3.00.05). The aligned files (*.sam) are processed using Samtools (vO.1.12a) and only uniquely mapped reads from NovoAlign are considered for variant calling.
- the alignments are pre-processed using GATK (vl .2-62) in three steps before variant calling.
- the indels are realigned using the known indels from 1000G (phasel) data.
- duplicates are removed using Picard (vl .39).
- base quality recalibration is done using CountCovariates and Table Recalibration from GATK (vl .2-62), taking into account known SNPs and indels from dbSNP (build 138).
- Unified Genotyper from GATK (v2.5-2) is used for variant calling, using known SNPs and indels from dbSNP (build 138).
- Raw variants from GATK are filtered to only include the PASS variants (standard call confidence > 50) within the merged exomic bait boundaries. Two out of 50 tumor samples did not confirm to the QC standards, therefore excluded from all further analyses. Therefore, all the downstream analyses are restricted to 48 primary tumors. The variants are further flagged as novel or present in either dbS P138 or COSMIC (v67) databases, based on their overlap.
- Dindel is also used to call indels. Both GATK and Dindel calls are filtered for microsatellite repeats (flagged as STR). The raw variant calls are used to estimate frequencies of nucleotide changes and transitiomtransversion (ti/tv) ratios.
- Exome-filtered PASS variants specific to the tumor samples, with respect to both location and actual call, are retained as somatic variants, which are further filtered to exclude variants where the region bearing the variant is not callable in the matched control sample, and those where the matched control sample had even one read covering the variant allele.
- Cross-contamination is estimated using ContEst in the tumor samples. Locus-wise and gene- wise driver scores are estimated by CRAVAT using the head and neck cancer database with the CHASM analysis option. Genes with a CHASM score of at least 0.35 is considered significant for comparison with other functional analyses. Somatic mutations are normalized with respect to the exome bait size (MB) to calculate the somatic mutation frequency per MB. Annotation and functional analyses of variants
- Somatic variants are filtered to contain only those callable in the matched normal but not covered by any read in the control samples (VCF), are used for IntoGen with the 'cohort analyses' option. Also, MutsigCVl .3 is run with these variants using coverage from un-filtered variants of all tumor samples. Pooled alignments for all normal and tumor samples (bam), each, along with pooled variants for all normal samples (MAF) are analyzed using MuSiC2 to calculate the background mutation rates (bmrs) for all genes, and a list of significantly mutated genes are identified (p-value of convolution test ⁇ 0.05). A condensed list of 19 genes, common between at least two analyses is compiled ( Figure ID).
- High quality DNA (200ng), quantified by Qubit (Invitrogen), is used as the starting material for whole-genome genotyping experiments following the manufacturer's specifications. Briefly, the genomic DNA is denatured at room temperature (RT) for 10 mins using 0.1N NaOH, neutralized and used for whole genome amplification (WGA) under isothermal conditions, at 37°C for 20 hrs. Post WGA, the DNA is enzymatically fragmented at 37°C for lhr. The fragmented DNA is precipitated with isopropanol at 4°C and resuspended in hybridization buffer.
- RT room temperature
- WGA whole genome amplification
- the samples are then denatured at 95°C for 20 mins, cooled at RT for 30 mins and 35 ⁇ 1 of each sample is loaded onto the Illumina HumanOmni 2.5-8 beadchip for hybridization (20hrs at 48°C) in a hybridization chamber.
- the unhybridized probes are washed away and the Chips (Human Omni2.5-8 vl .O and vl . l) are prepared for staining, single base extension and scanning using Illumina's HiScan system.
- the SNP locations are filtered to retain only those, called without any error, contain within the exome boundaries as per the sequencing baits, and which are callable (covered by at least five sequencing reads). At these locations, the overlap is estimated for individual SNP calls, i.e., chr/pos/ref/alt and for no calls; i.e., chr/pos/ref/ref; between sequencing and array platforms.
- CNVs Copy number Variations
- LH Loss of Heterozygosity
- CNVs and LOHs are identified using cnvPartition 3.1.6 plugin in Illumina GenomeStudio v2011.1, with default settings except for a minimum coverage of at least 10 probes per CNV7LOH with a confidence score threshhold of at least 100.
- Somatic CNVs and LOHs are extracted by filtering out any region common to CNVs and LOHs detected in its matched control. Somatic CNVs and LOHs are further filtered with respect to common and disease- related CNVs and LOHs using CNV Annotator. Overlaps with common CNVs and LOHs are discarded, reporting only the overlaps with disease-related, and novel CNVs and LOHs.
- the CNVs and LOHs are categorized within each cytoband and those with an occurrence in at least 10% of the patient samples are reported.
- the somatic mutation frequency per MB ranges from 10-45 with a median around 25 (Figure IB).
- the median value for transition to transversion (ti/tv) ratio for both the tumor and its matched control samples is -2.5.
- T->C changes are most frequent, followed by G- >A and then T->G.
- Habits (smoking and alcohol consumption), nodal status, HPV infection, tumor grade and stage has no significant impact on the distribution of these nucleotides.
- the workflow described in the Methods section is used to identify somatic mutations and indels in tumor samples following which three functional tools, IntOGen , MutSigCV and MuSiC2 are used for variant interpretations.
- CDKN2A, HLA-A and TTK form a mutually exclusive set with TP53;
- RASA1, OBSCN, HLA-A, AJUBA and TTK are mutually exclusive with either NOTCH 1 alone, or NOTCH2 and ANK3 together;
- NOTCH 1, NOTCH2, HLA-A, AJUBA, ANK3, TTK, MLL2, INGl or KEAP1 are mutually exclusive with CASP8 alone, or FAT1 and DMD together;
- FAT1, HLA-A, AJUBA, ANK3, TTK, MLL2, INGl or KEAP1 are mutually exclusive with PIK3CA or DMD or NOTCH1 and OBSCN, or CDKN2A and OBSCN, U2AF1, MLL2 and TTK form a small mutually exclusive set.
- CNV Copy Number Variation
- RNA quality profiling is carried out using Illumina HumanHT-12 v4 expression BeadChip (Illumina, San Diego, CA) in tumor and matched normal tissues following manufacturer's specifications.
- Total RNA is extracted from 20mg of tissue using PureLink RNA (Invitrogen) and RNeasy (Qiagen) Mini kits. RNA quality is checked using Agilent Bioanalyzer using RNA Nano6000 chip. Samples with poor RIN numbers, indicating partial degradation of RNA, are processed using Illumina WGDASL assay as per manufacturer's recommendations. The RNA samples with no degradation are labelled using Illumina TotalPrep RNA Amplification kit (Ambion) and processed according to the array manufacturer's recommendations.
- Gene expression data is collected using Illumina' s HiScan and analyzed with the GenomeStudio (v2011.1 Gene Expression module 1.9.0) and all assay controls are checked to ensure quality of the assay and chip scanning.
- Raw signal intensities are exported from GenomeStudio for pre-processing and analyzed using R further.
- Gene-wise expression intensities for tumor and matched control samples from GenomeStudio are transformed and normalized using VST (Variance Stabilizing Transformation) and LOESS methods, respectively, using the R package lumi.
- the data is further batch-corrected using ComBat.
- the pre-processed intensities for tumor and matched control samples are subjected to differential expression analyses using the R package, limma .
- the varSelRFBoot function is used from the varSelRF Bioconductor package to perform bootstrapping.
- the .632+ method is described by the following formula: where Er 632' Er 632 Err ⁇ and err are errors estimated by the .632+ method, the original .632 method, leave-one-out bootstrap method and err represents the error. R ' represents a value between 0 and 1. Another popular error correction method used is leave-one-out bootstrap method.
- the .632+ method is designed to correct the upward bias in the leave-one- out and the downward bias in the original .632 bootstrap methods.
- variable importance remains the same before and after correcting for multiple hypotheses comparisons using pre- and post- Benjamin-Hochberg FDR-corrected P values.
- Consensus list of genes from analysis, filtering and annotation of variant calls and from differential expression analysis using whole genome micro-arrays, are mapped to pathways using the web version of Graphite Web employing KEGG and Reactome databases.
- the network of interactions between genes is drawn originally using CytoScape (v3.1.1) using the .sif file created by Graphite Web.
- Circos (v0.66) is used for multi-dimensional data visualization. Additionally, the cbioportal portal (http://www.cbioportal.org/) is used to visualize variants within the 19 genes harboring significant variants. All of the mandatory fields accepted by Mutation Mapper are provided for select genes from the study to create structural representations for each gene including domains. Such diagrams from the study, the HNSCC study and all cancer studies from TCGA are collated using the image-editing tool, GIMP (www.gimp.org). SNPs and indels are visualized for each individual tumor sample using IGV (vl .5.54), along with the reads supporting variants.
- EXAMPLE 10 EXAMPLE 10:
- the specificity of the designed primers is tested using UCSC's tool, In Silico PCR.
- the variant-bearing region is amplified by using specific primers and used in Sanger sequencing.
- the somatic variants are confirmed by sequencing in the entire tumor and matched control DNA set used for the exome sequencing followed by further validation in 60 additional tumor samples.
- the 19 cancer-associated genes are classified from the previous analyses and those are linked with habits, clinical parameters and HPV infection.
- CDKN2A is found to be mutated patients only in the no-smoking category (never smokers and past smokers), and NOTCH1 only in those that consumed alcohol (Figure 2A).
- HPV-negative patients harbor fewer TP53 somatic variants, while HPV- positive patients alone has somatic variants in the RASA1.
- DMD and PIK3CA are mutated only in the HPV-negative patients.
- DMEM Dulbecco's Modified Eagles' Media
- Caspase-8 is transiently knocked down using ON-TARGETplus Human CASP8 smart pool siRNA (L-003466-00-0010; Dharmacon) along with an ON-TARGET plus Non-targeting siRNA (D-001810-01 -20; Dharmacon).
- the transfection efficiency for the two cell lines are optimized using siGLO Red Transfection Indicator (D-001630; Dharmacon).
- the siRNA duplexes are transfected using Lipofectamine-2000 according to the manufacturer's instructions (Invitrogen).
- the siRNA-oligo complexes medium is changed 8 hrs post transfection.
- the efficiency of transfection along with the mRNA expression is analyzed at 24 and 48 hrs post transfection by qRT-PCR.
- the specific down-regulation of CASP8 is confirmed by three independent experiments as given below:
- RNA is extracted from cell pellets and tissues using RNeasy Mini kit spin columns (Qiagen) following manufacturer's protocol. Genomic DNA contamination is removed by RNase- Free DNase Set (Qiagen) and the total RNA is eluted in nuclease free water (Ambion). The RNA samples are estimated using Qubit 2.0 fluorometer (Invitrogen) and the integrity is checked by gel electrophoresis. The RNA samples are stored at -80°C until further used. The cDNA is synthesized with 400ng total RNA, using a SuperScript-III first strand cDNA synthesis kit, and following the manufacturer's instructions (Invitrogen).
- the cDNA is then subjected for quantitative real-time PCR (q-RT-PCR) using KAPA SYBR FAST qPCR Master Mix (KK4601, KAPA).
- the primer pairs used for testing the expression of caspase- 8 in q-RT-PCR are, forward 5'-ATGATGACATGAACCTGCTGGA-3' and reverse 5'- C AGGCTCTTGTTGATTTGGGC-3 ' .
- the amplification is done on Stratagene MX300P real time machine. To normalize inter-sample variation in RNA input, the expression values are normalized with GAPDH. All amplification reactions are done in triplicates, using nuclease free water as negative controls. The differential gene expression is calculated by using the comparative CT method of relative quantification. Assessment of cell viability
- MTT cell proliferation assay is performed as per manufacturer's instructions (Sigma) to assess cell viability. Briefly, cells are seeded on 96-well plates containing DMEM with 10% FBS & incubated overnight. After treatment with 0.1% DMSO (vehicle control), or Cisplatin for 48 hrs, medium is changed and 100 ⁇ of MTT solution (lmg/ml) is added to each well. The cells are further incubated for 4hrs at 37°C. The formazan crystals in viable cells are dissolved by adding ⁇ of dimethyl sulfoxide (DMSO) (Merck). The absorbance is recorded at 540 nm using reference wavelength of 690 nm on micro plate reader (Tecan Systems). Data is normalized to vehicle treatment, and the cell viability is calculated using GraphPad Prism software (version 4.03; La Jolla, CA). All the experiments are performed in triplicates.
- DMSO dimethyl sulfoxide
- Cells are cultured up to 80% confluency in 12 well plates; serum-starved for 24 hrs and then wounded using a 200 ⁇ pipette tip. The wound is washed with lx PBS and the cells are grown in DMEM containing 10% FBS. Cells are imaged at lOx magnification at 0 hr, 15 hrs, 23 hrs and 42 hrs. For each well, three wounds are made and the migration distance is photographed and measured using Carl Zeiss software (Zeiss). Each experiment is performed in triplicates.
- the ECM gel (E1270, Sigma) is thawed overnight at 4°C and plated at requisite concentrations (for UPCLSCC040: 1.5mg/ml and UMSCC047: 2mg/ml) onto the transwell inserts and incubated overnight in the CO2 incubator at 37°C with 5% CO2.
- Cells were serum-starved for overnight, harvested, counted and seeded (UPCI: SCC040: 50,000 cells and UMSCC047: 20,000 cells per well) on top of the matrigel transwell-inserts (2 mg/ml) in serum free medium as per manufacturer's specifications (Sigma).
- D-MEM containing 10% FBS and 1% NEAA was added to the lower chamber.
- the 24-well plates containing matrigel inserts with cells were incubated in 37°C incubator for 48 hrs. At the end of incubation time, cells in the upper chamber were removed with cotton swabs and cells that invaded the Matrigel to the lower surface of the insert were fixed with 4% paraformaldehyde (Merk Milipore), permeabilized with 100% methanol, stained with Giemsa (Sigma), mounted on glass slides with DPX mounting agent and counted under a light microscope (Zeiss). Each experiment was performed in triplicates. RESULTS:
- CASP8 is mutated in a significant number of oral tongue tumors.
- Caspase-8 is an important and versatile protein that plays a role in both apoptotic (extrinsic or death receptor-mediated) and non-apoptotic processes.
- the functional consequences of CASP8 knockdown through a siRNA-mediated method in an HPV-positive UM:SCC-47 and an HPV-negative UPCLSCC040 OTSCC cell lines is studied. Prior to the functional assay, the concentration of siRNA required for silencing, extent of CASP8 knockdown and cisplatin sensitivity (ICso) in both these cell lines is tested (Figure 4).
- the present disclosure thus presents a comprehensive study on FtNSCC, particularly oral tongue tumors by molecular characterization of OTSCC and determines variants linked with habits, nodal status, tumor recurrence and FtPV infection.
- Exome sequencing, whole- genome gene expression, and genotyping arrays is performed using fifty primary tumors along with their matched control samples, towards identification of somatic variants (mutations and indels), significantly up- and down-regulated genes, loss of heterozygosity (LOH) and copy number variations (CNVs). All the molecular data along with the clinical parameters and epidemiology such as tumor stage, nodal status, FtPV infection, risk habits and tumor recurrence is integrated to interpret the effect of changes in the process of cancer development in oral tongue.
- TP 53 significant somatic variations are identified in TP 53 (38%>), RAMI (8%), CASP8 (8%), CDKN2A (6%), NOTCH 1 (4%), NOTCH2 (4%), and PIK3CA (4%) from the exome sequencing study in OTSCC.
- the key variants are validated using an additional set of primary tumor samples. Variants in TP53 and NOTCH! are found in mutually exclusive sets of tumors. Additionally, frequent aberrations are found in chromosomes 6-9, and 11 in tumor samples.
- MMPs matrix metalloproteases
- An ensemble machine learning method is employed and a minimal gene signature set that distinguishes a group of tumors with loco-regional recurrence from the non-recurrent set is determined.
- a 38-gene minimal signature is determined that predicts tumor recurrence using an ensemble machine learning method.
- functional analysis of CASP8 gene is performed in HPV-negative and FtPV-positive OTSCC cell lines to establish its role in the process of tumor development.
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Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009039341A2 (en) * | 2007-09-21 | 2009-03-26 | Wayne State University | Neoepitope detection of disease using protein arrays |
-
2016
- 2016-11-04 WO PCT/IB2016/056652 patent/WO2017077499A1/en active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009039341A2 (en) * | 2007-09-21 | 2009-03-26 | Wayne State University | Neoepitope detection of disease using protein arrays |
Non-Patent Citations (2)
Title |
---|
KRISHNAN NM ET AL.: "Integrated analysis of oral tongue squamous cell carcinoma identifies key variants and pathways linked to risk habits", HPV, CLINICAL PARAMETERS AND TUMOR RECURRENCE, 11 October 2015 (2015-10-11), pages 1 - 18, XP055381918, Retrieved from the Internet <URL:http://dx.doi.org/10.1101/028845.> * |
KRISHNAN NM ET AL.: "Integrated analysis of oral tongue squamous cell carcinoma identifies key variants and pathways linked to risk habits", HPV, CLINICAL PARAMETERS AND TUMOR RECURRENCE, vol. 4, no. 1215, 11 October 2015 (2015-10-11), pages 1 - 18, XP055381915, Retrieved from the Internet <URL:http://dx.doi.org/10.1101/028845.> * |
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