Clinical Trial Details
— Status: Active, not recruiting
Administrative data
NCT number |
NCT03885648 |
Other study ID # |
PI-0538-2017 |
Secondary ID |
|
Status |
Active, not recruiting |
Phase |
|
First received |
|
Last updated |
|
Start date |
January 1, 2018 |
Est. completion date |
December 31, 2024 |
Study information
Verified date |
December 2023 |
Source |
Universidad de Granada |
Contact |
n/a |
Is FDA regulated |
No |
Health authority |
|
Study type |
Observational
|
Clinical Trial Summary
Breast cancer ranks first in women, and is the second cause of death in this gender. In
addition to genetics, the environment contributes to the development of the disease, although
the factors involved are not well known. Among the latter is the influence of microorganisms
and, therefore, attention is recently being paid to the mammary microbiota. The hypothesis of
this study is that the risk of breast cancer could be associated with the composition and
functionality of the mammary/gut microbiota, and that exposure to environmental contaminants
(endocrine disruptors, EDCs) might contribute to alter the microbiota.
This is a case-control clinical study that will be performed in women between 25 and 70 years
of age. Cases will be women diagnosed and surgically intervened of breast cancer (stages I
and II). Women with antecedents of cancer or advanced tumor stage (metastasis), or who have
received antibiotic treatment within 3 months prior to recruitment, or any neoadjuvant
therapy, will be excluded. Controls will be women surgically intervened of breast
augmentation or reduction. Women with oncological, gynecological or endocrine history, and
those who have received antibiotic treatment within 3 months prior to recruitment will also
be excluded. Blood, urine, breast tissue and stool samples will be collected. Data regarding
anthropometric, sociodemographic, reproductive history, tumor features and dietary habits
will be gathered.
Metabolomic studies will be carried out in stool and breast tissue samples. Metagenomic
studies will also be performed in stool and breast tissue samples to ascertain the viral,
fungal, bacterial and archaea populations of the microbiota. Quantitation of estrogens,
estrogen metabolites and EDCs in samples of serum, urine and breast tissue will also be
performed.
This is the first time that the contribution of bacteria, archaea, viruses and fungi together
with their alteration by environmental contaminants to the risk of breast cancer will be
evaluated in the same study. Results obtained could contribute to elucidate risk factors,
improve the prognosis, as well as to propose novel intervention studies in this disease.
Description:
The study will follow the Declaration of Helsinki and the Spaniard legislation regarding
clinical research. Data obtained will be confidential and only the researchers and
participants, upon request, will have access to them. The study will also follow the data
protection legislation of Spain to guarantee data confidentiality, treatment and
availability.
Participation is volunteer. Participants will be informed about the nature of the research
and the usage of biological samples. In addition to verbal information, participants will be
presented and read an informed consent. Permission has been granted by the Ethics Committee
of Andalusia (Spain).
Inclusion and exclusion criteria. Women's age will range 25-70 years. Cases will be women
diagnosed and surgically intervened of incident breast cancer, stages I and II. Controls will
be women surgically intervened of breast augmentation or reduction. Women with antecedents of
cancer or advanced tumor stage (metastasis), or who have received antibiotic treatment 3
months prior to recruitment, or any neoadjuvant therapy, will be excluded of the study. Women
with oncological, gynecological or endocrine antecedents and those who have received of
antibiotic treatment 3 months prior to recruitment will be also excluded. Controls will be
matched to cases by age (± 2 years), category of body mass index and hospital of recruitment.
Sample size. The number of breast cancer cases will include at least 100 women, matched with
100 control women. Three hospitals will participate in the recruitment: The University
Hospital of Jaén, Spain; the University Hospital Campus de la Salud of Granada, Spain; and
Ana Moreno Clinic (Granada, Spain).
Biological samples. Blood, breast tissue, stool and urine samples from cases and controls
will be collected. Serum will be separated from blood by centrifugation. Breast tissue
samples will be taken during surgery, from a marginal area 3-5 cm apart from the tumor. For
controls, samples will be taken from any available breast tissue during breast reduction or
augmentation surgery.
Breast and intestinal microbiota. Bacteria, archaea, fungi and viruses will be investigated
in feces and breast tissue samples. Samples will be pre-treated with pathogen lysis tubes L
and S (QIAGEN, Barcelona, Spain). DNA will be extracted with the QIAamp cador Pathogen Mini
kit (QIAGEN, Barcelona, Spain).
DNA quantification. The DNA concentration of the samples will be evaluated in a NanoDrop2000c
(Thermo Fisher Scientific, Waltham, MA, USA).
Metagenomic library construction and sequencing. The Nextera XT DNA Library Preparation Kit
(Illumina, San Diego, CA, USA) will be used for metagenomic library construction. The
amplicon tagment mix (ATM) in Nextera XT, which includes the enzyme used for tagmentation,
will be diluted 1:10 in nuclease-free water for library construction using 1 to 100 pg input
DNA. Each 20 μL of the tagmentation reaction mixture consists of 10 μL TD buffer, 5 μL of
input DNA, and 5 μL of diluted ATM. PCR cycles for library construction will be 12, 14, 17,
and 20 cycles for 1,000, 100, 10, and 1 pg DNA, respectively, following the manufacturer's
protocol. The manufacturer recommends 12 cycles of the PCR reaction for no less than 1 ng
input DNA. Amplified libraries will be purified using AMPure XP (Agencourt, Brea, CA, USA).
The quality of the purified libraries will be assessed using an Agilent High Sensitivity DNA
Kit on an Agilent 2100 Bioanalyzer (Santa Clara, CA, USA). The sequencing libraries will be
further quantified using the KAPA Library Quantification Kit. Metagenomic libraries will be
mixed with PhiX Control v3 (Illumina) at a ratio of 9:1 and sequenced with an Illumina MiSeq
Reagent Kit v3 (600 cycles). All samples to be analyzed will be combined in a pool before
starting the massive sequencing. The latter will be done with the MiSeq apparatus (Illumina).
Data processing for metagenomic libraries. Metagenomic reads will be subjected to adaptor
clipping and quality trimming using Trimmomatic v0.3. The first three nucleotides with
quality scores less than 20 will be cut from the 3' and 5' read ends. Reads will be processed
using a sliding window method, cutting once the average quality within the window (4 base)
fell below the threshold (Q20). Reads with a length of fewer than 100 nucleotides will be
then removed. Low-complexity reads will be filtered out using PRINSEQ version 0.20.4. PCR
duplicates will be removed with Picard version 2.8.0
(http://broadinstitute.github.io/picard). The processed high-quality and clean reads in each
library will be used in subsequent analyses. In the community analysis based on metagenomic
sequences, small subunit (SSU) rRNA gene sequences will be identified using Metaxa2 software.
Taxonomy assignments will be performed based on the results of a BLAST search against the
SILVA123 database, using the MEGAN program [23] with the following settings: Min Support 1,
Min Score 50, Max Expected 1xe-5, Top Percent 10.0. SSU rRNA gene community compositions were
compared among the metagenomic libraries.
Metabolomic study. Sample preparation. The samples will be prepared using the automated
MicroLab STARR (Hamilton Company, Salt Lake City, UT, USA) robot system. For quality control
(QC) purposes, a recovery standard will be added to 100 L of the serum samples before the
first step in the extraction process. The proteins will be precipitated with methanol by
vigorously shaking for 2 min. Next, the samples will be centrifuged to remove the proteins,
dissociate the small molecules bound to the proteins or trapped in the precipitated protein
matrix, and to recover chemically diverse metabolites (Glen Mills GenoGrinder 2000, Lebanon,
USA). Then, the samples will be placed on a TurboVapR (Zymark, California, USA) to remove the
organic solvent. For liquid chromatography (LC) analysis, the samples will be stored
overnight in nitrogen before preparation. For gas chromatography (GC) analysis, each sample
will be dried overnight under vacuum before preparation.
Quality controls. Several types of control experiments will be performed in parallel with the
experimental samples. A pooled matrix sample, generated by taking a small volume of each
experimental sample, will serve as a technical replicate throughout the extracted data set.
Water aliquots will serve as process blanks. Moreover, a cocktail of QC standards carefully
chosen not to interfere with the measurement of endogenous compounds will be spiked into
every analyzed sample. This will allow instrument performance monitoring and aid in the
chromatographic alignment. The instrument variability will be determined by calculating the
median relative standard deviation (RSD) for the standards that will be added to each sample
before injection into the mass spectrometers (MS). The entire process variability will be
determined by calculating the median RSD for all the endogenous metabolites (i.e.,
non-instrument standards) present in 100% of the pooled matrix samples. The experimental
samples will be randomized across the platform run with the QC samples spaced every 5 or 10
injections to verify overall assay performance. The internal standards will be used to
measure the instrument variability and will have a median RSD of 3%. Additionally, the total
process variability will be 8%.
Ultra-performance liquid chromatography-tandem mass spectrometry. The liquid
chromatography/mass spectrometry (LC/MS) portion of the platform to be used will employ a
Waters ACQUITY ultra performance liquid chromatography (UPLC), a Thermo Scientific Q-Exactive
high-resolution/accurate MS interfaced with a heated electrospray ionization (HESI-II)
source, and an Orbitrap mass analyzer operated at a 35,000 mass resolution. The sample
extract will be dried and reconstituted in acidic or basic LC-compatible solvents, each of
which will contain eight or more injected QC standards at fixed concentrations to ensure
injection and chromatographic consistency. One aliquot will be analyzed using acidic,
positive ion-optimized conditions, and another will be performed using basic, negative ion
optimized conditions in two independent injections using separate dedicated columns (Waters
UPLC BEH C18-2.1 × 100 mm, 1.7 m). The extracts reconstituted in acidic conditions will be
gradient-eluted from a C18 column using water and methanol containing 0.1% formic acid. A
second aliquot, from the basic extract, will be similarly eluted from a C18 column using
methanol, water, and 6.5 mM ammonium bicarbonate. A third aliquot will be analyzed via
negative ionization following elution from a hydrophilic interaction chromatography column
(Waters UPLC BEH Amide 2.1 × 150 mm, 1.7 m) using a gradient consisting of water and
acetonitrile with 10 mM ammonium formate.
Gas chromatography-mass spectrometry. The samples destined for analysis by GC-MS will be
dried under a vacuum for a minimum of 18 h before being derivatized under dried nitrogen with
bistrimethylsilyltrifluoroacetamide. The derivatized samples will be separated on a 5%
diphenyl/95% dimethyl polysiloxane fused silica column (20 m × 0.18 mm id; 0.18 m film
thickness) with helium as the carrier gas and a temperature ramp from 60C to 340C over a
17.5-min period. The samples will be analyzed on a Thermo-Finnigan Trace DSQ fast-scanning
single-quadrupole MS using electron impact ionization (EI) operated at unit mass resolving
power. The scan range will be 50-750 m/z.
Data extraction and compound identification. The raw data will be extracted, and the peaks
will be aligned and identified. QCs will be processed using specific hardware and software.
The peaks will be quantified using the area-under-the-curve (AUC) calculation. The compounds
will be identified comparing the data to library entries of purified standards or recurrent
unknown entities. The biochemical identifications will be based on three criteria: the
retention index (RI) within a narrow RI window of the proposed identification, an accurate
mass match to the library ± 0.005 amu, and the MS/MS forward and reverse scores between the
experimental data and authentic standards. The MS/MS scores will be based on a comparison of
the ions present in the experimental spectrum with the ions present in the library spectrum.
Although there may be similarities between these molecules based on one of these factors, the
use of all three data points can be utilized to distinguish and differentiate the
biochemicals with precision.
Quantitation of the exposure to EDCs in urine samples. Analysis of non-persistent EDCs will
be carried out by dispersive liquid-liquid microextraction (DLLME) and ultra-high performance
liquid chromatography with tandem mass spectrometry detection (UHPLC-MS/MS). Briefly, urine
samples are thawed completely at room temperature, centrifuge at 2600 x g for 10 min to
sediment particulate matter and 0.75 mL are taken to carry out the analysis. In order to
determine total EDCs amount (free plus conjugated), each sample is spiked with 50 μL of
enzyme solution (β-glucuronidase/sulfatase) and incubated at 37 °C for 24 h. The treated
urine is placed in a 15 mL screw-cap glass tube and spiked with 30 μL of the surrogate
standard solution (1.25 mg/L of BPA-d16). Urine is diluted to 10.0 mL with 5% NaCl aqueous
solution (w/v) and the pH is adjusted to 2.0. Next, 0.75 mL of acetone and 0.75 mL of
trichloromethane are mixed and injected rapidly into the aqueous sample with a syringe. After
manual shaking, centrifugation and evaporation of the extract, the residue is dissolved with
100 μL of a mixture consisting of water (0.1% ammonia)/acetonitrile (0.1% ammonia), 70:30
(v/v), and finally 10 μL is injected in the LC system. Urinary creatinine concentration is
determined using an automated colorimetric determination in the same urine samples in which
the environmental chemical is assessed.
Quantitation of urinary concentrations of the parent estrogens and estrogen metabolites.
Concentrations of estrone, estradiol and 13 estrogen metabolites will be determined adjusting
for urinary creatinine levels (mg/dl). The quantitation of urinary estrogens will be assessed
by gas chromatography tandem mass spectrometry using a triple quadrupole analyzer. Briefly,
one mL of urine with 25 µL of internal standards solution is extracted using C18 SPE
cartridge (Sep-Pack C18) previously conditioned with 4 mL of methanol and 4 mL of water.
After loading the urine sample, the cartridge is washed with 4 mL of water. The treated urine
is then evaporated. Three mL of acetate buffer 0.1 M (pH 4.5) and 3 µL of β-glucuronidase are
added to the evaporated, and the mixture is incubated for 3 h at 55 °C. After cooling to room
temperature, pH was increased to approximately 9.5. Then, samples are extracted with 6 mL of
tert-butyl methyl ether by shaking and centrifuged. Finally, the organic layer is evaporated
to dryness under a stream of nitrogen.
Other sources of information and covariables. Participants' anthropometric, sociodemographic
and reproductive features will be recorded. They will also grant permission to access their
clinical history. Tumor features and dietary habits will be recorded.