Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT04425122 |
| Other study ID # |
CRE-2020.186 |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
July 1, 2020 |
| Est. completion date |
December 31, 2023 |
Study information
| Verified date |
February 2023 |
| Source |
Chinese University of Hong Kong |
| Contact |
Hon Chi Yip, FRCSEd |
| Phone |
35052627 |
| Email |
hcyip[@]surgery.cuhk.edu.hk |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
This is a prospective study on the microbiota associated with oesophageal squamous cell
carcinoma (SCC). Patients with newly diagnosed SCC of oesophagus would be recruited for
taking samples of oral and oesophageal rinse, tissue biopsies for investigation of the
microbiota of oesophageal cancer. Control patients would be identified from patients
scheduled for routine endoscopy.
Description:
Oesophageal carcinoma significantly contributes to the global cancer burden ranking as the
sixth leading cause of global cancer-related death. Oesophageal squamous cell carcinoma
(OESCC) is one of the most common type of upper gastrointestinal (UGI) carcinoma in some
regions globally, particularly China and Japan in Asia. The disease has an extremely poor
prognosis, with overall 5-year survival rates of less than 30%, mainly due to late stage at
diagnosis and high likelihood of distant metastases. Although OESCC is associated with risk
factors including smoking and alcohol, the aetiology of OESCC is still poorly understood.
Increasing evidence indicates a key role for bacterial microbiota in carcinogenesis. Emerging
data implicates the human microbiome in a variety of cancers, most notably Fusobacterium in
colorectal cancer and Helicobacter in gastric cancer. The consortium of bacterial microbiome
colonizing the gastrointestinal tract is extensive and interact in a complex manner. The
microbiome may interact with genetic and environmental factors to metabolize dietary
constituents and xenobiotics, among other functions. When the microbial balance is disturbed,
the microbiota could alter host cell proliferation and death, manipulate the immune system,
and influence host metabolism, giving rise to carcinoma. Several studies have reported an
important role of the human microbiota in upper gastrointestinal carcinoma and found
associations between the microbiota and some diseases of the UGI tract, such as esophagitis
and Barrett esophagus, and with squamous dysplasia and squamous carcinoma of the oesophagus.
Another even less well known is the role of the fungal microbiome in OESCC.
Here the investigators will seek to evaluate the role of the bacterial and fungal microbiome
in OESCC and the host microbe interactions that may play a role in the understanding and
management of OESCC.
Study questions:
1. Does the microbiome differ between OESCC and normal patients?
2. Does the microbiome change from normal through premalignancy to malignancy?
3. What are the mechanisms involved in the microbiome and the development or progression of
OESCC?
Study design:
This is a prospective study. The study will be carried out in accordance with the Declaration
of Helsinki.
Patients would be recruited from the Prince of Wales Hospital from Jun 2020 to May 2025. 100
patients with newly diagnosed OESCC or oesophageal squamous dysplasia will be recruited to
the test group. 100 patients undergoing an oesophagogastroduodenoscopy (OGD) without a
history of malignancy will be recruited to the control group. The expected duration of
subject participation is less than two months.
The study has two visits, as follows:
1. Visit 1:
i. To discuss about the project and to sign the informed consent form; ii. To collect
oral rinse 20ml; iii. To complete questionnaire: Gut Microbe - Medical and health
questionnaire.
2. Visit 2:
To have OGD. Visit 2 will be performed within two months post Visit 1 and must before
treatment.
i. Test group: With examination of the lesion with biopsy of tumor, biopsy of adjacent normal
tissue, flush with normal saline.
ii. Control group: Will have a biopsy of normal tissue and flush with normal saline.
Sample size justification:
This is a prospective study with no previous data based on OESCC. But based on data from head
and neck squamous cell carcinoma (HNSCC) with some overlap in traditional risk factors the
investigators target to recruit at least 100 OESCC patients and 100 healthy subjects. In
HNSCC study using 54 HNSCC tumour tissues and the adjacent normal tissues has generated 16S
rRNA short reads able to discriminate cases from controls. A total of 77 bacterial genera
with >0.1% mean relative abundance was observed in tumour tissues. A rarefaction analysis
indicated that at least 73 genera can be detected from any of 35 samples, suggesting that
recruitment of 100 patients and 100 controls meets the minimized sample size to contain
nearly full spectrum of oral bacteria and is expected to have a power of 0.8 and confidence
level of 95% to characterize oral microbiota dysbiosis between cases and controls.
Independent variables
The following independent variables will be examined to evaluate the association of the
methylation panel and OESCC:
1. Clinical factors: TNM staging of disease, comorbidities;
2. Cancer risk factors: smoking history, alcohol consumption;
3. Patient factors: sex, age;
4. Histologic characteristics: tumour depth, extracapsular spread, lymphovascular invasion,
perineural invasion.
Samples:
Oral rinse: Before the procedure, an oral rinse would be performed by asking all patients to
rinse the oral cavity with 20ml normal saline. The resultant solution would be kept for
subsequent analysis.
Endoscopic procedures Endoscopic procedures would be performed by doctors with expertise on
performing upper endoscopy. It would be conducted at the Combined Endoscopy Center, Prince of
Wales Hospital. Topical local anaesthetic will be applied to the oropharynx. Endoscopes with
magnification /dual focus and NBI function would be used. A soft black hood would be attached
to the tip of the endoscope for better focal length adjustment.
Patients in the test group would then undergo endoscopy with one biopsy of the tumor and one
biopsy of the adjacent normal esophageal mucosa. In addition, oesophageal rinse would also be
performed by flushing the esophagus gently with 20ml normal saline and the fluid suctioned
and collected for analysis.
Patients in the control group would undergo endoscopy as per usual clinical practice. One
biopsy would be taken from normal esophageal mucosa for analysis. Similarly, oesophageal
rinse would be performed.
Experiments DNA extraction and bisulphite modification: DNA from microdissected fresh tissues
and saliva will be extracted with phenol-chloroform, precipitated in 100% ethanol,
centrifuged at 5100 rpm for 45 minutes, washed in 70% ethanol twice, dissolved in LoTE buffer
(10mM TRIS hydrochloride, 1mM EDTA buffer, pH 8), and stored at -80°C.
RNA extraction: Fresh tissue will be stored in RNAlater for extraction of RNA and stored in
-80°C.
Genomic analysis: Utilizing next generation sequencing analysis for copy number aberrations
and single nucleotide variations to analyse the samples collected using shotgun massive
parallel sequencing as previously described. We will also use sequencing to analyse the
microbiota of the cohort. Bacterial 16S rRNA gene sequencing: An aliquot of DNA will be used
to screen oral microbial community using 16S rRNA gene V3- V4 region amplicon sequencing 24.
QIIME2 with the latest Silva ribosomal RNA database will be used to classify amplicon
sequence variant, with operational taxonomic table showing the proportion of bacterial reads
per sample. Mycobiota ITS gene sequencing: we will target mycobiota internal transcribed
spacer gene (ITS) to characterize oral fungal community. A short region of ITS1 (~270 bp)
will be PCR amplified and sequenced using MiSeq. The reads will be processed against a fungal
UNITE database, and the classification tables will be generated at the species-, genera- and
OTU-level for statistical analyses.
Expression analysis: The investigators will determine if these genomically altered tumors
express specific changes in signaling proteins using immunohistochemical staining (IHC) and
protein profiling by protein arrays. FFPE-sections or frozen tissues will be harvested for
alongside with genomic characterization study for IHC analyses, or protein profiling. For IHC
analysis or protein profiling, a total of 5 FFPE-slides will be employed for IHC or protein
extraction for protein profiling as previously published. RNA will be extracted and subjected
to RNA analyses, including RNA-seq, expression array and RT-PCR analyses so as to determine
specific gene expression patterns of oesophageal tumors, as well as expression of potential
biomarkers.
Bacterial Culture: To further understand the role of the microbiome as a biomarker in OESCC
tissue specimens will be cultured for bacteria identified in sequencing to be significantly
enriched in tumours. Tissue specimens were aseptically macerated with disposable scalpels and
vortexed for 30 seconds in PBS (500 l), and the neat suspensions were used to make 10-fold 2
) dilutions. Neat suspensions (50 l) were each spread onto blood agar (BA), fastidious
anaerobe agar (FAA) (BA and FAA supplemented with 5% defibrinated sheep blood; TCS
Biosciences Ltd.), and Sabouraud's agar (Lab MTM; International Diagnostics Group plc). The
dilutions and final PBS washes of the specimens (see above) were spread onto BA and FAA. BA
and Sabouraud's agar plates were incubated aerobically at 37°C for 48 h. FAA plates were
incubated in an anaerobic cabinet at 36°C for 96 h.
Outcomes measures
1. Loco-regional recurrence free survival will be recorded from time of diagnosis to time
of recurrence locally, regionally or local and regionally.
2. Disease specific survival will be recorded from time of diagnosis to time of death from
OESCC.
3. Overall survival will be recorded from time of diagnosis to time of death from all
causes.
Statistical Analysis Association between microbes, surgery treatment, and individuals'
metadata (e.g., gender, age, smoking, drinking, T/N stages, survival, recurrence) will be
analysed using a mix of ordination, non-parametric multivariate analysis of variance, matrix
correlation, chi-square test, generalized linear model, or conditional logistic regression
models. The performance of the final biomarkers will be assessed by cross-validation and
summarized by c-index score to assess the concordance between model and comparison groups
using ROC curves. All multiple testing corrections will be performed by computing FDRs using
the BenjaminiHochenberg method, and adjusted p values < 0.05 will be considered statistically
significant.
