Ignite Creation Date:
2024-10-26 @ 3:37 PM
Last Modification Date:
2024-10-26 @ 3:37 PM
Study NCT ID:
NCT06544005
Status:
ACTIVE_NOT_RECRUITING
Last Update Posted:
None
First Post:
2024-08-01
Brief Title:
Implication of Long Non-coding RNA HOTTIP Haplotype on Liver Cancer Metastasis
Sponsor:
None
Organization:
None
Study Overview
Official Title:
Implication of Long Non-coding RNA HOTTIP Haplotype
Status:
ACTIVE_NOT_RECRUITING
Status Verified Date:
2024-08
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
No
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
Hepatocellular carcinoma HCC represents the fourth common cancer and the most common cause of mortality-caused and morbidity-related cancer Different reports implied several lncRNAs role in the progression and metastasis of HCC such as Homeobox A HOXA transcript at the distal tip HOTTIP Dysregulation of HOTTIP is associated with various malignancies including HCC affecting survival and prognosis of cancer patients HOTTIP promoted HCC cell proliferationmetastasis and might act as an oncogenic-lncRNA in HCC
Genetic variations such as single-nucleotide polymorphisms SNPs when inherited together as a group known as haplotypes Haplotypes can alter the expression of coding genes and the protein non-coding genes like lncRNAs therefore affecting the disease course including liver a hypothesis to be addressed
Only few studies have focused on the polymorphisms of the onco-lncRNA HOTTIP gene A study found that specific HOTTIP SNPs have the potential to be biomarkers for HCC risk and prognosis where one haplotype of HOTTIP rs17501292-rs2067087-rs17427960 showed a 191-fold increased risk of HCC
Genetic variations such as single-nucleotide polymorphisms SNPs when inherited together as a group known as haplotypes Haplotypes can alter the expression of coding genes and the protein non-coding genes like lncRNAs therefore affecting the disease course including liver a hypothesis to be addressed
Only few studies have focused on the polymorphisms of the onco-lncRNA HOTTIP gene A study found that specific HOTTIP SNPs have the potential to be biomarkers for HCC risk and prognosis where one haplotype of HOTTIP rs17501292-rs2067087-rs17427960 showed a 191-fold increased risk of HCC
Detailed Description:
1 Introduction Hepatocellular carcinoma HCC is a complex disease which could be caused by viruses as hepatitis B and C infection drug abuse and chemicals such as aflatoxin However treatments for HCC are limited and most of them are only effective at an early stage At an advanced stage this cancer is associated with a poor prognosis due to frequent cancer metastasis tumor recurrence and a lack of curative treatment Different reports implied several lncRNAs role in the progression and metastasis of HCC such as HOXA transcript at the distal tip HOTTIP HOTTIP might act as an oncogenic-lncRNA in HCC
Genetic variations such as single-nucleotide polymorphisms SNPs when inherited together as a group because of high Linkage Disequilibrium LD there tends to be redundant information The regions of the genome with high LD that harbor a specific set of SNPs are inherited together known as haplotypes Haplotypes can alter the expression of coding genes and the protein non-coding genes like lncRNAs
Only few studies have focused on the polymorphisms of the onco-lncRNA HOTTIP gene A recent study found that specific HOTTIP SNPs have the potential to be biomarkers for HCC risk and prognosis where one haplotype of HOTTIP gene rs17501292-rs2067087-rs17427960 showed a 191-fold increased risk of HCC P 0006 in Chinese patients samples
2 Objectives 21 Genotype different haplotype SNPs of lncRNA HOTTIP in whole blood samples Liquid biopsy from metastatic HCC patients and to be compared with sex and age-matched non-metastatic patients
22 Correlate HCC clinicopathological characteristics tumor stage and grade tumor progression TNM and different clinical presentations as presenceabsence of metastasis and other classical clinico-pathological prognostic biomarkers such as α-fetoprotein AFP carcinoembryonic antigen CEA alkaline phosphatase ALP alanine transaminase ALT aspartate transaminase AST International normalized ratio INR prothrombin concentration PC total bilirubin Gamma-glutamyltransferase GGT and complete blood count CBC blood pressure blood glucose level serum insulin and body mass index BMI c-reactive protein CRP albumin blood urea nitrogen BUN and prognostic markersoutcome to HOTTIP genotypes or different haplotype SNPs
23 Correlate HOTTIP lncRNA influence on some HCC hallmarks tumor growth proliferation andor metastasis
24 Molecular Docking for exploring potential drugs that could be re-purposed to inhibit our target oncogenic lncRNA HOTTIP
3 Subjects - 198 HCC patients attending the Faculty of Medicine Ain Shams University Hospital with 11 or 12 ratio for the HCC groups metastatic vs non metastatic HCC groups according to clinical evidencerelevance andor availability
Group 1 non-metastatic HCC patients Group 2 metastatic HCC patients diagnosed with primary HCC receiving any type of therapy neoadjuvant or radiotherapy
Eligibility criterion are adult age and malefemale 11 Criteria for HCC diagnosis following the Ain Shams University ASU hospital role relying on AFP level and CT scan or the fine needle biopsy
Groups to be matched socioeconomically in age range sex MaleFemale 11 residence case-controlled study
- Exclusion criteria HCC patients who have history of liver transplantation have other cancer types at the time of selection presented by renal insufficiency and thyroid dysfunction will be excluded from the study Additionally patients with incomplete data or histopathology diagnosis
Data to be collected HCC patients Clinico-pathological Criteria Clinical data will be obtained from medical records and the original pathology reports These data to be compiled in an Excel sheet
Clinical data to be recorded and assessed
Cancer family history
Individual cancer history and the tumor clinical assessment done using the tumor-node-metastasis TNM classification of the American Joint Committee on Cancer AJCC
HCC histological grading made in accordance to Edmondson-Steiner ES
The characteristics of the HCC patients with regards to Ultrasound US findings cirrhosis ascites and splenomegaly Total and direct bilirubin ALP Prothrombin time PT PC INR hemoglobin HB CBC White Blood Cells WBCs Platelets and ALT to Platelet Ratio index APRI scoreThe formula for the APRI score is ASTupper limit of the normal AST range X 100Platelet Count serum insulin blood glucose level BMI BP albumin BUN
Tumor size as well as clinico-pathological biomarkers AFP CEA GGT data will be collected from patient files for further correlations and statistical analysis
For both groups treatment type and for group 2 the site of metastatic destination disease-free survival DFS overall survival OS the duration of patient survival from the time of treatment initiation will be considered as a universally accepted direct measure of clinical benefit
Methodology 41 Bioinformatic Analysis Selected Polymorphic Sites The investigators selected polymorphisms using 1000 Genome data httpwwwinternationalgenomeorghome as reported previously The tag SNPs were selected separately using the following criteria 1 Haploview with the Tagger function was used 2 the population of the HapMap selected Yoruba in Ibadan Nigeria YRI population 3 those for which pairwise tagging had r2 of 08 and 4 those with a minor allele frequency of 5 The selection area was enlarged by 10 kb both upstream and downstream for HOTTIP lncRNA gene Fast SNP and f SNP searches were used to predict the potential SNP function httpcompbiocsqueensucaF-SNP
After block identification many SNPs were detected among them 2 SNPs with high LD 08 were selected rs17501292-rs2067087
42 Blood Samples Genomic DNA will be extracted from whole ethylenediaminetetraacetic acid EDTA blood samples from all subjects using the DNA Mini Kit Qiagen Valencia CA according to the manufacturers instructions The yield will be measured by Nano Drop 2000 Thermo Fisher Scientific UK and will be aliquoted into 5 clean Eppendorf tubes and stored at -80C until biochemical assessment at the Faculty of Pharmacy Ain-Shams University Advanced Biochemistry Research Lab ABRL
43 SNPs Genotyping rs17501292 NC_0000071427201853 TC NC_0000071427201853 T G T C G rs2067087 NC_0000071427202040GCNC_0000071427202040GT G CTwill be genotyped using real-time polymerase chain reaction RT-PCR with the TaqMan allelic discrimination assay on a 7900 system using predesigned primerprobe Applied Biosystems Inc
STATISTICAL ANALYSIS 51 SPSS v25 USA SPSS Chicago IL or Stat or Graph Pad will be used for statistical analysis
52 Data will be collected excel tabulated and tested for normality by Kolmogorov-Smirnov test
53 Between-group differences in sex variability will be compared by the χ2 test and by analysis of variance for age variability Multivariate logistic regression with adjustments for age and sex will be used to show the association between selected lncRNA polymorphisms and HCC risk
54 Normally distributed variables will be expressed as mean SEM and analyzed using two samples independent t-test Median interquartile range will be used to express nonparametric data and subsequently analyzed using Mann Whitney U test
55 The two-way pairwise interactions of lncRNA SNP-SNP will be calculated using multivariate logistic regression Univariate then multivariate survival analyses will be carried out by the log-rank test and the Cox proportional hazards model
56 Pearsons Chi-square analysis or Fishers exact test will be employed to compare the difference of categorical variables
57 Receiver Operating Characteristics ROC curves will be drawn using Medcalc to get the sensitivity and specificity of some markers together with the SNPs
58 Survival curves will be drawn using Kaplan-meier curve 59 Hardy-Weinberg test will be used to assure equilibrium of our study participants with the population or not
510 For all analyses a two-tailed P value of 005 or less will be considered as statistically significant
Genetic variations such as single-nucleotide polymorphisms SNPs when inherited together as a group because of high Linkage Disequilibrium LD there tends to be redundant information The regions of the genome with high LD that harbor a specific set of SNPs are inherited together known as haplotypes Haplotypes can alter the expression of coding genes and the protein non-coding genes like lncRNAs
Only few studies have focused on the polymorphisms of the onco-lncRNA HOTTIP gene A recent study found that specific HOTTIP SNPs have the potential to be biomarkers for HCC risk and prognosis where one haplotype of HOTTIP gene rs17501292-rs2067087-rs17427960 showed a 191-fold increased risk of HCC P 0006 in Chinese patients samples
2 Objectives 21 Genotype different haplotype SNPs of lncRNA HOTTIP in whole blood samples Liquid biopsy from metastatic HCC patients and to be compared with sex and age-matched non-metastatic patients
22 Correlate HCC clinicopathological characteristics tumor stage and grade tumor progression TNM and different clinical presentations as presenceabsence of metastasis and other classical clinico-pathological prognostic biomarkers such as α-fetoprotein AFP carcinoembryonic antigen CEA alkaline phosphatase ALP alanine transaminase ALT aspartate transaminase AST International normalized ratio INR prothrombin concentration PC total bilirubin Gamma-glutamyltransferase GGT and complete blood count CBC blood pressure blood glucose level serum insulin and body mass index BMI c-reactive protein CRP albumin blood urea nitrogen BUN and prognostic markersoutcome to HOTTIP genotypes or different haplotype SNPs
23 Correlate HOTTIP lncRNA influence on some HCC hallmarks tumor growth proliferation andor metastasis
24 Molecular Docking for exploring potential drugs that could be re-purposed to inhibit our target oncogenic lncRNA HOTTIP
3 Subjects - 198 HCC patients attending the Faculty of Medicine Ain Shams University Hospital with 11 or 12 ratio for the HCC groups metastatic vs non metastatic HCC groups according to clinical evidencerelevance andor availability
Group 1 non-metastatic HCC patients Group 2 metastatic HCC patients diagnosed with primary HCC receiving any type of therapy neoadjuvant or radiotherapy
Eligibility criterion are adult age and malefemale 11 Criteria for HCC diagnosis following the Ain Shams University ASU hospital role relying on AFP level and CT scan or the fine needle biopsy
Groups to be matched socioeconomically in age range sex MaleFemale 11 residence case-controlled study
- Exclusion criteria HCC patients who have history of liver transplantation have other cancer types at the time of selection presented by renal insufficiency and thyroid dysfunction will be excluded from the study Additionally patients with incomplete data or histopathology diagnosis
Data to be collected HCC patients Clinico-pathological Criteria Clinical data will be obtained from medical records and the original pathology reports These data to be compiled in an Excel sheet
Clinical data to be recorded and assessed
Cancer family history
Individual cancer history and the tumor clinical assessment done using the tumor-node-metastasis TNM classification of the American Joint Committee on Cancer AJCC
HCC histological grading made in accordance to Edmondson-Steiner ES
The characteristics of the HCC patients with regards to Ultrasound US findings cirrhosis ascites and splenomegaly Total and direct bilirubin ALP Prothrombin time PT PC INR hemoglobin HB CBC White Blood Cells WBCs Platelets and ALT to Platelet Ratio index APRI scoreThe formula for the APRI score is ASTupper limit of the normal AST range X 100Platelet Count serum insulin blood glucose level BMI BP albumin BUN
Tumor size as well as clinico-pathological biomarkers AFP CEA GGT data will be collected from patient files for further correlations and statistical analysis
For both groups treatment type and for group 2 the site of metastatic destination disease-free survival DFS overall survival OS the duration of patient survival from the time of treatment initiation will be considered as a universally accepted direct measure of clinical benefit
Methodology 41 Bioinformatic Analysis Selected Polymorphic Sites The investigators selected polymorphisms using 1000 Genome data httpwwwinternationalgenomeorghome as reported previously The tag SNPs were selected separately using the following criteria 1 Haploview with the Tagger function was used 2 the population of the HapMap selected Yoruba in Ibadan Nigeria YRI population 3 those for which pairwise tagging had r2 of 08 and 4 those with a minor allele frequency of 5 The selection area was enlarged by 10 kb both upstream and downstream for HOTTIP lncRNA gene Fast SNP and f SNP searches were used to predict the potential SNP function httpcompbiocsqueensucaF-SNP
After block identification many SNPs were detected among them 2 SNPs with high LD 08 were selected rs17501292-rs2067087
42 Blood Samples Genomic DNA will be extracted from whole ethylenediaminetetraacetic acid EDTA blood samples from all subjects using the DNA Mini Kit Qiagen Valencia CA according to the manufacturers instructions The yield will be measured by Nano Drop 2000 Thermo Fisher Scientific UK and will be aliquoted into 5 clean Eppendorf tubes and stored at -80C until biochemical assessment at the Faculty of Pharmacy Ain-Shams University Advanced Biochemistry Research Lab ABRL
43 SNPs Genotyping rs17501292 NC_0000071427201853 TC NC_0000071427201853 T G T C G rs2067087 NC_0000071427202040GCNC_0000071427202040GT G CTwill be genotyped using real-time polymerase chain reaction RT-PCR with the TaqMan allelic discrimination assay on a 7900 system using predesigned primerprobe Applied Biosystems Inc
STATISTICAL ANALYSIS 51 SPSS v25 USA SPSS Chicago IL or Stat or Graph Pad will be used for statistical analysis
52 Data will be collected excel tabulated and tested for normality by Kolmogorov-Smirnov test
53 Between-group differences in sex variability will be compared by the χ2 test and by analysis of variance for age variability Multivariate logistic regression with adjustments for age and sex will be used to show the association between selected lncRNA polymorphisms and HCC risk
54 Normally distributed variables will be expressed as mean SEM and analyzed using two samples independent t-test Median interquartile range will be used to express nonparametric data and subsequently analyzed using Mann Whitney U test
55 The two-way pairwise interactions of lncRNA SNP-SNP will be calculated using multivariate logistic regression Univariate then multivariate survival analyses will be carried out by the log-rank test and the Cox proportional hazards model
56 Pearsons Chi-square analysis or Fishers exact test will be employed to compare the difference of categorical variables
57 Receiver Operating Characteristics ROC curves will be drawn using Medcalc to get the sensitivity and specificity of some markers together with the SNPs
58 Survival curves will be drawn using Kaplan-meier curve 59 Hardy-Weinberg test will be used to assure equilibrium of our study participants with the population or not
510 For all analyses a two-tailed P value of 005 or less will be considered as statistically significant
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None