Ignite Creation Date:
2024-05-06 @ 8:11 PM
Last Modification Date:
2024-10-26 @ 3:22 PM
Study NCT ID:
NCT06289270
Status:
RECRUITING
Last Update Posted:
2024-03-01
First Post:
2024-02-25
Brief Title:
Microbiota in Urine and Urothelium Can be a Factor for Induction of Urinary Bladder Cancer The Study Will Examine Urine and Bladder Tissues From Egyptian Male Patients and Controls Using Whole Genomic Sequencing Techniques and 16S rRNA The Aim is to Elucidate Role of Microbiota in Bladder Cancer
Sponsor:
Theodor Bilharz Research Institute
Organization:
Theodor Bilharz Research Institute
Study Overview
Official Title:
Profiling Urinary Microbiota in Male Patients With Bladder Cancer- An Exploratory Study
Status:
RECRUITING
Status Verified Date:
2024-02
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
Cancer bladder
Brief Summary:
Microbes inhabiting the urinary tract that are playing an important role in maintenance of health and the development of diseases and bladder cancer There is correlation between initiation of bladder cancer and microbes Urine and bladder tissues from healthy individuals and patients with bladder cancer were analyzed using 16S rRNA sequencing results show abundant phylum Next-generation sequencing technologies will be applied with whole genomic and 16S ribosomal RNA gene sequencing for microbiota profiling in urine and bladder tissue of male healthy individuals and patients with bladder cancer Results will lead to prevention diagnosis and new treatment strategies for bladder cancer
Detailed Description:
Objectives The aim of this research study is to characterize the urinary microbiota of adult male patients with bladder cancer and to be correlated with microbiota in healthy adult males controls
2-Methodology 2-1 Study type Prospective case control study 2-2 Inclusion exclusion criteria recruitment process included patients in the study will be asked to offer an informed consent Dr Loay Mostafa and Dr Mohamed Sahab from the Urology Department - TBRI will be responsible for this item
Inclusion Criteria
Adult men above 18 years
Diagnosed of carcinoma of the bladder with imaging ultrasonography and or computerized tomography and urine cytology
Confirmed bladder cancer either during diagnostic cystoscopy previous pathology or pathological examination of the biopsy specimens
Exclusion criteria
Previous radiotherapy to the bladder or to adjacent organ
Previous intravesical instillation immunotherapy with bacillus Calmette-Guérin BCG or intravesical instillation of chemotherapeutics
Previous neoadjuvant chemotherapy Recruitment process Patients fulfilling the exclusion and inclusion criteria 2-3 Sample size Thirty eight Egyptian men age 18 will be recruited at the Department of Urology TBRI according to sample size calculation using online software Sample Size Calculator Ten urine samples will be collected from normal individuals
httpswwwcalculatornetsample-size-calculatorhtml Sample size will be 16 for non-muscle invasive bladder cancer NMIBC This means 16 or more measurementssurveys are needed to have a confidence level of 95 Sample size will be 11 for muscle invasive bladder cancer MIBC This means 40 or more measurementssurveys are needed to have a confidence level of 95 Sample size will be 11 for control group This means 10 or more measurementssurveys are needed to have a confidence level of 95 A 38 men will be recruited at the Department of Urology TBRI According to sample size calculation using online software Sample Size Calculator Eleven urine samples will be collected from Sample size will be 16 for non-muscle invasive bladder cancer NMIBC This means 16 or more measurementssurveys are needed to have a confidence level of 95 that the real value is within 1753 of the measuredsurveyed value
Sample size will be 11 for muscle invasive bladder cancer MIBC This means 11 or more measurementssurveys are needed to have a confidence level of 95 that the real value is within 3036 of the measuredsurveyed value
Sample size will be 11 for control group This means 11 or more measurementssurveys are needed to have a confidence level of 95 that the real value is within 3036 of the measuredsurveyed value
normal individuals This sample represents the research on microbiome in bladder cancer
httpswwwcalculatornetsample-size-calculatorhtml 2-4 Sample collection
Bladder cancer
The total number of studied samples The study will explore the microbiota in urine and in tissue samples so every patient with bladder cancer will give 2 samples The control group will give urine sample one samble Recent research showed that microbiota is detecred in the urothelium of normal undividuals and patients with bladder cancer this microbiota would differ from urine liquid microbiota
References
1-Mansour B Monyók Á Makra N Gajdács M Vadnay I Ligeti B Juhász J Szabó D Ostorházi E Bladder cancer-related microbiota examining differences in urine and tissue samples Sci Rep 2020 Jul 610111042 doi 101038s41598-020-67443-2
2- Parra-Grande M Oré-Arce M Martínez-Priego L DAuria G Rosselló-Mora R Lillo M Sempere A Lumbreras B Sánchez-Hellín V Profiling the Bladder Microbiota in Patients With Bladder Cancer Front Microbiol 2022 Feb 712718776 doi 103389fmicb2021718776
Group I Non-muscle invasive bladder cancer16 patients 16 for bladder tissue and 16 for urine samples Total 32 samples The study will explore the microbiota in urine and in tissue samples so every patient with bladder cancer will give 2 samples The control group will give urine sample one samble Recent research showed that microbiota is detecred in the urothelium of normal undividuals and patients with bladder cancer this microbiota would differ from urine liquid microbiotaReferences Group II Muscle invasive bladder cancer 11 patients 22 samples 11 for bladder tissue and 11 for urine samples obtained from the same patients Group III 11 Normal adults urine samples from serves as control group of the study
Total number of participant is 38 individual
Samples for microbiome analysis
Samples will be collected in clean leak-proof containers without disinfectant or detergent residue and with tight-fitting leak-proof lids and preserved in a refrigerator until be analyzed at the microbiology laboratory of TBRI
Urine samples 50 ml for microbiome analysis will be collected during cystoscopy examination Samples will be stored at -80 C until processing
Tissue and urine samples from patients with bladder cancer Under standard procedure of diagnostic cystoscopy urine sample will be collected under complete aseptic conditions patients who has bladder tumor will undergo transurethral resection of bladder tumor a tissue sample 05x05 cm will be investigated for microbiome analysis Samples will be stored at -80 C until processing Histopathological diagnosis with Hematoxylin and eosin stain will be done by an expert pathologist for tumor grade and stage according to WHO 22 and EAU guidelines 2023 This procedure will be applied for both non-muscle invasive and muscle invasive urothelial carcinoma Patients who will undergo radical cystectomy for bladder cancer will be included in the sample size
Samples for pathological studies
Tissue samples will be fixed with formalin and examined with hematoxylin and eosin stain
Clinical Information The urologists will provide adequate tissue samples which is a small part of the whole resected tumor or a part of cystectomy specimens for pathological evaluation with giving useful clinical information to the pathologist to decide the best approach in handling and processing the surgical specimens and draw up an accurate pathology report
The urologist will specify
Demographic data and clinical history of the patient history of urinary tract infection and if it was recurrent or sporadic bladder cytology if present whether it is the first presentation of the tumor and if not details of previous management
The cystoscopic appearance of bladder mucosa and indicate number size location of the tumors the morphological features of the lesion papillary solid or ulcerate
The state of remaining mucosa if further biopsies were performed
Theses information is necessary for a correct evaluation of urothelium and search in microbiome Because the treatments can have an impact on tumor morphology and on normal looking urothelium
Bladder Cancer Samples Reporting
Pathological assessment The pathologist will diagnose if a bladder cancer is present and the type of bladder cancer by examining cells from the bladder tissue biopsy Different histological tumor types of bladder cancer will be reported according to the 2016 WHO classification of urinary bladder tumors Mazzucchelli et al 2021 modified in 2022
Microbiological assessment Microbial identification methods from Urine and tissue
Sample collection and culture conditions of clinical bacterial isolates
Detection of a limited number of microorganisms mainly aerobic and fast-growing bacteria
Identification of bacterial isolates The isolates will be identified by culturing on blood MacConkey agar and sabaruds agar then will be identified by performing biochemical tests oxidase test mannitol bile esculin and catalase test All isolates will be identified to species level by using Vitek 2 compact system
2-5 Biochemestry and molecular biology assessment 16S Metagenomic Sequencing analysis Whole genomic sequencing WGS will be applied to all cases methodology and quality control are according to the genomic and metagenomic center in the children cancer hospital 57357 who are going to perform the WGS and 16S sequencing on urine and tissue samples
1 DNA isolation from urine and tissue Urine specimens 50ml will be thawed and centrifuged at 7500g 4C for 10minutes The pellet will be used for DNA extraction using Invitrogen PureLink Microbiome DNA Purification Kit performed according to manufacturers protocol To avoid environmental contamination all isolations from urine samples and from the reagent-only extraction control will be carried out within a PCR hood Isolated DNA samples will be placed at -20 C until PCR amplifcation DNA will be quantifed using DeNovix dsDNA High Sensitivity Assay Kit Illumina 2013
2 PCR based KABA KAPA Library Quantification Kits contain all the reagents needed for the accurate reliable and reproducible qPCR-based quantification of next-generation sequencing NGS libraries prepared for sequencing on Illumina platforms Kits include KAPA SYBR FAST qPCR Master Mix formulated with different passive reference dyes for different qPCR instruments a platform- specific library quantification primer premix and a pre-diluted set of DNA standards
16S rRNA gene V3-V4 region will be amplifed with fusion primers that incorporate Illumina adapters and indexing barcodes The PCR step adds Index 1 i7 and Index 2 i5 sequencing and common adapters P5 and P7 required for cluster generation and sequencing High Sensitivity DNA electrophoresis with the Bioanalyzer system will be done to improve quality control analysis of NGS library 3- 16S rRNA gene library preparation and MiSeq sequencing PCR amplification of 16S rDNA sequencing and analyses will be performed by NGS Illumina MiSeq targeting V3V4 region of bacteria MiSeq Reagent Kit v3 600-cycle 16S rRNA gene amplicon sequencing 16S analysis is widely used to analyze microbiota with next-generation sequencing technologies 16S analysis data from the standard V3-V4 primer V34 sets to optimize the bladder microbiota analysis protocol 4- Bioinformatics and statistical analyses The annotated genome is further analyzed using bioinformatic pipelines to gain insights into the pathogen39s biology and potential for causing disease It includes
- Quality control and improvement of the draft genome by removing errors and filling in gaps
Annotation of the final genome including identifying and describing the functions of genes and other features
Comparative genomics which involves comparing the newly sequenced genome to those of related pathogens to identify differences and similarities
Functional analysis this involves identifying the functions of the annotated genes and understanding the pathways and processes they participate in This can reveal key virulence factors and help to understand the pathogen39s pathogenesis mechanism
Phylogenetic analysis This involves comparing the newly sequenced genome to those of
related pathogens to determine evolutionary relationships and track the spread of pathogens in different populations
Differences in the overall microbial composition between bladder cancer and healthy
samples will be assessed NGS is not available in TBRI the 16S sequencing and bioinformatics will be done paid in Children Hospital 57357 they are welcomed to perform the sequencing on aPaid principle
16S ribosomal RNA rRNASequencing analysis
1 DNA isolation from urine and tissue Urine specimens 50ml will be thawed and centrifuged at 7500g 4C for 10minutes The pellet will be used for DNA extraction using microbiome DNA Purification Kit To avoid environmental contamination all isolations from urine samples and from the reagent-only extraction control will be carried out within a PCR hood Isolated DNA samples will be placed at -20 C until PCR amplification DNA will be quantified using an assay Kit Illumina 2013
2 PCR based KABA KAPA Library Quantification Kits contain all the reagents needed for the accurate reliable and reproducible qPCR-based quantification of next-generation sequencing NGS libraries prepared for sequencing on Illumina platforms A platform-specific library quantification primer premix and a pre-diluted set of DNA standards
16S rRNA hypervariable regionsV1V2 and V3-V4 region will be amplifed with fusion primers that incorporate Illumina adapters and indexing barcodes
High Sensitivity DNA electrophoresis with the Bioanalyzer system will be done to improve quality control analysis of NGS library
3- 16S rRNA gene library preparation and MiSeq sequencing PCR amplification of 16S rRNA sequencing and analyses will be performed by NGS Illumina targeting V1-V2 and V3-V4 region 16S rRNA gene amplicon sequencing 16S analysis will be used to analyze microbiota with next-generation sequencing technologies 16S analysis data from the standard V1-V2 V3-V4 primer sets to optimize the bladder microbiota analysis protocol
4- Bioinformatics and statistical analyses The annotated genome is further analyzed using bioinformatic pipelines to gain insights into the pathogens biology and potential for causing disease
2-Methodology 2-1 Study type Prospective case control study 2-2 Inclusion exclusion criteria recruitment process included patients in the study will be asked to offer an informed consent Dr Loay Mostafa and Dr Mohamed Sahab from the Urology Department - TBRI will be responsible for this item
Inclusion Criteria
Adult men above 18 years
Diagnosed of carcinoma of the bladder with imaging ultrasonography and or computerized tomography and urine cytology
Confirmed bladder cancer either during diagnostic cystoscopy previous pathology or pathological examination of the biopsy specimens
Exclusion criteria
Previous radiotherapy to the bladder or to adjacent organ
Previous intravesical instillation immunotherapy with bacillus Calmette-Guérin BCG or intravesical instillation of chemotherapeutics
Previous neoadjuvant chemotherapy Recruitment process Patients fulfilling the exclusion and inclusion criteria 2-3 Sample size Thirty eight Egyptian men age 18 will be recruited at the Department of Urology TBRI according to sample size calculation using online software Sample Size Calculator Ten urine samples will be collected from normal individuals
httpswwwcalculatornetsample-size-calculatorhtml Sample size will be 16 for non-muscle invasive bladder cancer NMIBC This means 16 or more measurementssurveys are needed to have a confidence level of 95 Sample size will be 11 for muscle invasive bladder cancer MIBC This means 40 or more measurementssurveys are needed to have a confidence level of 95 Sample size will be 11 for control group This means 10 or more measurementssurveys are needed to have a confidence level of 95 A 38 men will be recruited at the Department of Urology TBRI According to sample size calculation using online software Sample Size Calculator Eleven urine samples will be collected from Sample size will be 16 for non-muscle invasive bladder cancer NMIBC This means 16 or more measurementssurveys are needed to have a confidence level of 95 that the real value is within 1753 of the measuredsurveyed value
Sample size will be 11 for muscle invasive bladder cancer MIBC This means 11 or more measurementssurveys are needed to have a confidence level of 95 that the real value is within 3036 of the measuredsurveyed value
Sample size will be 11 for control group This means 11 or more measurementssurveys are needed to have a confidence level of 95 that the real value is within 3036 of the measuredsurveyed value
normal individuals This sample represents the research on microbiome in bladder cancer
httpswwwcalculatornetsample-size-calculatorhtml 2-4 Sample collection
Bladder cancer
The total number of studied samples The study will explore the microbiota in urine and in tissue samples so every patient with bladder cancer will give 2 samples The control group will give urine sample one samble Recent research showed that microbiota is detecred in the urothelium of normal undividuals and patients with bladder cancer this microbiota would differ from urine liquid microbiota
References
1-Mansour B Monyók Á Makra N Gajdács M Vadnay I Ligeti B Juhász J Szabó D Ostorházi E Bladder cancer-related microbiota examining differences in urine and tissue samples Sci Rep 2020 Jul 610111042 doi 101038s41598-020-67443-2
2- Parra-Grande M Oré-Arce M Martínez-Priego L DAuria G Rosselló-Mora R Lillo M Sempere A Lumbreras B Sánchez-Hellín V Profiling the Bladder Microbiota in Patients With Bladder Cancer Front Microbiol 2022 Feb 712718776 doi 103389fmicb2021718776
Group I Non-muscle invasive bladder cancer16 patients 16 for bladder tissue and 16 for urine samples Total 32 samples The study will explore the microbiota in urine and in tissue samples so every patient with bladder cancer will give 2 samples The control group will give urine sample one samble Recent research showed that microbiota is detecred in the urothelium of normal undividuals and patients with bladder cancer this microbiota would differ from urine liquid microbiotaReferences Group II Muscle invasive bladder cancer 11 patients 22 samples 11 for bladder tissue and 11 for urine samples obtained from the same patients Group III 11 Normal adults urine samples from serves as control group of the study
Total number of participant is 38 individual
Samples for microbiome analysis
Samples will be collected in clean leak-proof containers without disinfectant or detergent residue and with tight-fitting leak-proof lids and preserved in a refrigerator until be analyzed at the microbiology laboratory of TBRI
Urine samples 50 ml for microbiome analysis will be collected during cystoscopy examination Samples will be stored at -80 C until processing
Tissue and urine samples from patients with bladder cancer Under standard procedure of diagnostic cystoscopy urine sample will be collected under complete aseptic conditions patients who has bladder tumor will undergo transurethral resection of bladder tumor a tissue sample 05x05 cm will be investigated for microbiome analysis Samples will be stored at -80 C until processing Histopathological diagnosis with Hematoxylin and eosin stain will be done by an expert pathologist for tumor grade and stage according to WHO 22 and EAU guidelines 2023 This procedure will be applied for both non-muscle invasive and muscle invasive urothelial carcinoma Patients who will undergo radical cystectomy for bladder cancer will be included in the sample size
Samples for pathological studies
Tissue samples will be fixed with formalin and examined with hematoxylin and eosin stain
Clinical Information The urologists will provide adequate tissue samples which is a small part of the whole resected tumor or a part of cystectomy specimens for pathological evaluation with giving useful clinical information to the pathologist to decide the best approach in handling and processing the surgical specimens and draw up an accurate pathology report
The urologist will specify
Demographic data and clinical history of the patient history of urinary tract infection and if it was recurrent or sporadic bladder cytology if present whether it is the first presentation of the tumor and if not details of previous management
The cystoscopic appearance of bladder mucosa and indicate number size location of the tumors the morphological features of the lesion papillary solid or ulcerate
The state of remaining mucosa if further biopsies were performed
Theses information is necessary for a correct evaluation of urothelium and search in microbiome Because the treatments can have an impact on tumor morphology and on normal looking urothelium
Bladder Cancer Samples Reporting
Pathological assessment The pathologist will diagnose if a bladder cancer is present and the type of bladder cancer by examining cells from the bladder tissue biopsy Different histological tumor types of bladder cancer will be reported according to the 2016 WHO classification of urinary bladder tumors Mazzucchelli et al 2021 modified in 2022
Microbiological assessment Microbial identification methods from Urine and tissue
Sample collection and culture conditions of clinical bacterial isolates
Detection of a limited number of microorganisms mainly aerobic and fast-growing bacteria
Identification of bacterial isolates The isolates will be identified by culturing on blood MacConkey agar and sabaruds agar then will be identified by performing biochemical tests oxidase test mannitol bile esculin and catalase test All isolates will be identified to species level by using Vitek 2 compact system
2-5 Biochemestry and molecular biology assessment 16S Metagenomic Sequencing analysis Whole genomic sequencing WGS will be applied to all cases methodology and quality control are according to the genomic and metagenomic center in the children cancer hospital 57357 who are going to perform the WGS and 16S sequencing on urine and tissue samples
1 DNA isolation from urine and tissue Urine specimens 50ml will be thawed and centrifuged at 7500g 4C for 10minutes The pellet will be used for DNA extraction using Invitrogen PureLink Microbiome DNA Purification Kit performed according to manufacturers protocol To avoid environmental contamination all isolations from urine samples and from the reagent-only extraction control will be carried out within a PCR hood Isolated DNA samples will be placed at -20 C until PCR amplifcation DNA will be quantifed using DeNovix dsDNA High Sensitivity Assay Kit Illumina 2013
2 PCR based KABA KAPA Library Quantification Kits contain all the reagents needed for the accurate reliable and reproducible qPCR-based quantification of next-generation sequencing NGS libraries prepared for sequencing on Illumina platforms Kits include KAPA SYBR FAST qPCR Master Mix formulated with different passive reference dyes for different qPCR instruments a platform- specific library quantification primer premix and a pre-diluted set of DNA standards
16S rRNA gene V3-V4 region will be amplifed with fusion primers that incorporate Illumina adapters and indexing barcodes The PCR step adds Index 1 i7 and Index 2 i5 sequencing and common adapters P5 and P7 required for cluster generation and sequencing High Sensitivity DNA electrophoresis with the Bioanalyzer system will be done to improve quality control analysis of NGS library 3- 16S rRNA gene library preparation and MiSeq sequencing PCR amplification of 16S rDNA sequencing and analyses will be performed by NGS Illumina MiSeq targeting V3V4 region of bacteria MiSeq Reagent Kit v3 600-cycle 16S rRNA gene amplicon sequencing 16S analysis is widely used to analyze microbiota with next-generation sequencing technologies 16S analysis data from the standard V3-V4 primer V34 sets to optimize the bladder microbiota analysis protocol 4- Bioinformatics and statistical analyses The annotated genome is further analyzed using bioinformatic pipelines to gain insights into the pathogen39s biology and potential for causing disease It includes
- Quality control and improvement of the draft genome by removing errors and filling in gaps
Annotation of the final genome including identifying and describing the functions of genes and other features
Comparative genomics which involves comparing the newly sequenced genome to those of related pathogens to identify differences and similarities
Functional analysis this involves identifying the functions of the annotated genes and understanding the pathways and processes they participate in This can reveal key virulence factors and help to understand the pathogen39s pathogenesis mechanism
Phylogenetic analysis This involves comparing the newly sequenced genome to those of
related pathogens to determine evolutionary relationships and track the spread of pathogens in different populations
Differences in the overall microbial composition between bladder cancer and healthy
samples will be assessed NGS is not available in TBRI the 16S sequencing and bioinformatics will be done paid in Children Hospital 57357 they are welcomed to perform the sequencing on aPaid principle
16S ribosomal RNA rRNASequencing analysis
1 DNA isolation from urine and tissue Urine specimens 50ml will be thawed and centrifuged at 7500g 4C for 10minutes The pellet will be used for DNA extraction using microbiome DNA Purification Kit To avoid environmental contamination all isolations from urine samples and from the reagent-only extraction control will be carried out within a PCR hood Isolated DNA samples will be placed at -20 C until PCR amplification DNA will be quantified using an assay Kit Illumina 2013
2 PCR based KABA KAPA Library Quantification Kits contain all the reagents needed for the accurate reliable and reproducible qPCR-based quantification of next-generation sequencing NGS libraries prepared for sequencing on Illumina platforms A platform-specific library quantification primer premix and a pre-diluted set of DNA standards
16S rRNA hypervariable regionsV1V2 and V3-V4 region will be amplifed with fusion primers that incorporate Illumina adapters and indexing barcodes
High Sensitivity DNA electrophoresis with the Bioanalyzer system will be done to improve quality control analysis of NGS library
3- 16S rRNA gene library preparation and MiSeq sequencing PCR amplification of 16S rRNA sequencing and analyses will be performed by NGS Illumina targeting V1-V2 and V3-V4 region 16S rRNA gene amplicon sequencing 16S analysis will be used to analyze microbiota with next-generation sequencing technologies 16S analysis data from the standard V1-V2 V3-V4 primer sets to optimize the bladder microbiota analysis protocol
4- Bioinformatics and statistical analyses The annotated genome is further analyzed using bioinformatic pipelines to gain insights into the pathogens biology and potential for causing disease
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None