Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT03809403 |
| Other study ID # |
APHP180453 |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
February 5, 2019 |
| Est. completion date |
August 2019 |
Study information
| Verified date |
February 2019 |
| Source |
Assistance Publique - Hôpitaux de Paris |
| Contact |
Yann Parc, PU-PH |
| Phone |
0149282540 |
| Email |
yann.parc[@]aphp.fr |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
Colectomy is the most commonly used therapeutic approach for the treatment of non-metastatic
colorectal cancer. This approach is generally very effective however the rate of recurrence
and the appearance of metachronous metastasis remains a major problem in the postoperative
period. One of the hypothesis that can explain this tumor progression is the dissemination of
tumor cells at the time of tumor mobilization. In this work, we wish to verify this
hypothesis by comparing two surgical technics used in our department for left or right
colectomies: respectively either first section of the mesenteric vessels followed by the
mobilization of the tumor or first mobilization of the tumor followed by the section of the
mesenteric vessels. To evaluate the dissemination, we will study two disseminations markers
that have shown their prognostic value: i) circulating tumor cells (which represent a direct
marker of dissemination) and ii) tumor circulating DNA (which is an indirect marker) but has
the advantage of being more representative of all tumor clones and therefore the tumor burden
released into the blood at the time of surgery).
Description:
A / Epidemiology of colorectal cancer
Colorectal cancer remains the third most common cancer in France in 2017 with an incidence of
about 44,000 new cases in 2017 and nearly 18,000 deaths each year. The ColoRectal Cancer
(CRC) ranks second among cancer deaths behind lung cancer and breast cancer. Thanks to
diagnostic and therapeutic progress, the mortality rate decrease of 1.5% in men and 1.1% in
women between 2005 and 2012.
Despite these numbers, colorectal cancer has a good prognosis when diagnosed at an early
stage. In fact, the 5-year relative survival is 91% for localized stages (stage I and II) and
70% for stages with locoregional and lymph nodes invasion (stage III). On the other hand,
about 25% of the patients at the time of diagnosis have metastasis (stage IV), with a
survival at 5 years of 11%. Those data explain that the mortality of this cancer is mainly
due to the spread of the primary tumor to secondary locations, mainly liver or lung.
The unstable microsatellite phenotype (ISM) is an important colonic carcinogenesis pathway,
find in approximately 15% of operable colorectal cancers (CRC) and may be of sporadic or
hereditary origin. Several studies have shown that patients with MSI tumors have a better
prognosis than patients with stable microsatellite tumors (SSM) due to a better response to
platinum-based chemotherapy. The determination of this status therefore determines the
adjuvant treatment of CRC and is determined in anatomopathology on the analysis of the
operative specimen.
B / The place of surgery in the care of the CCR:
Currently, the (tumor node metastasis (TNM) classification is used to establish therapeutic
management. Stages I are treated by surgery alone. Stages II are treated by surgery alone in
the absence of risk factors for recurrence and with adjuvant chemotherapy if not. Stages III
are treated by surgery and adjuvant chemotherapy within 8 weeks and for 6 months. For the
stages IV (metastatic), the management of those patients is discussed on a case-by-case basis
according to the symptomatic or non-symptomatic nature of the primary tumor and the
resectability or non-resectability of the metastasis. If there is no contraindication, most
of these patients are treated with first-line chemotherapy, then reassessed every 2 to 3
months.
C / Surgical technic:
Surgical treatment depends on tumor localisation:
In the case of a tumor located on the right colon, from the caecum to the middle of the
transverse colon, a right colectomy is performed associated with lymph node dissection along
the superior mesenteric vessels.
In the case of a tumor located on the left colon, from the middle of the transverse colon to
the rectum, a left colectomy is performed with a dissection centered on the inferior
mesenteric artery.
In current practice, there are different surgical technics for both types of left and right
colectomies. One of the main differences between those technics is the section of vessels
(superior or inferior mesenteric vessels):
The so-called "no touch" technic involves a ligation of the mesenteric vessels before any
tumor mobilization. This technic is the one used systematically at Saint Antoine Hospital for
left colectomy because of the easy dissection of the inferior mesenteric artery and vein in
the absence of mobilization of the left colon, those vessels being at a distance from other
elements (the left ureter is visualized before the ligation of the inferior mesenteric
artery).
An other technic is to first mobilize the tumor to have an optimal exposure of the vessels
before the ligation. This technic is the one routinely performed at Saint Antoine Hospital
for the right colectomies because of the anatomy of the ileocolic artery and the right colic
artery at the origin of which is performed the lymphadenectomy. Indeed the complete
mobilization of the right colon allows a section of the adhesions between the right mesocolon
(which contains the colonic vessels) and the duodenum and the right ureter. In this way, the
section of the right colic vessels can be made at the origin with good control of adjacent
structures. Moreover, the lymph node dissection is systematically performed by laparotomy:
indeed even for colectomies performed under laparoscopy it is necessary to make a
mini-laparotomy incision in order to extract the operative piece and to perform the
anastomosis.
Currently, no difference has been demonstrated in terms of survival without recurrence and
overall survival between those different technics.
Moreover, the choice of the approach, laparoscopic or by laparotomy, depends on the
comorbidities of the patient, severe respiratory failure may be a contraindication to
laparotomy and its history: one or more previous surgeries by laparotomy is considered as a
contraindication to laparoscopy.
D / Circulating tumor DNA (ctDNA) and Circulating Tumor Cells (CTC) in colorectal cancer:
One of the metastatic extension factors of colorectal tumors treated at a localized stage
(stage II and III) is the dissemination of circulating tumor DNA and circulating tumor cells
in the peripheral blood.
Tumor cells witch migrate into the blood and lymphatic vessels from a tumor (primary or
metastasis) are called circulating tumor cells (CTCs). Some of these have a tumor inducing
potential (responsible for relapses) or the ability to form metastasis with time.
Advances in molecular biology technologies (high-throughput sequencing and digital polymerase
chain reaction (PCR), for example) have made possible to consider the exploration of
circulating tumor DNA, which is present in small quantities in plasma. The amount of ctDNA is
proportional to the tumor burden. Thus the mutations of the tumor's DNA (or somatic
mutations) can be identified from the ctDNA, the mutations having a theranostic impact on the
KRAS, NRAS and BRAF genes could be identified in the ctDNA of patients presenting colorectal
cancers. These mutations can thus serve as tracers, the amount of tumor DNA can also be
monitored and DNA methylation tests can also be specific for the tumor DNA. The presence of
these molecular abnormalities can therefore be used to detect CTCs and ctDNA in these
patients. Several studies, initially conducted in the context of breast cancer,have shown the
interest of these two parameters in the monitoring and follow-up of patients with CRC: indeed
their short half-life of a few minutes to a few hours allows a follow-up in real time their
kinetics in the peripheral blood, making it a predictor of earlier relapse than commonly
measured tumor markers (ACE and CA 19-9). In addition, their presence is associated with a
significant decrease in recurrence-free survival and overall survival. Studies in colorectal
cancer show similar results to those shown for breast cancer: In the retrospective study of
Linuma et al, multivariate analysis for relapse-free survival (SSR) and overall survival (SG)
found that patients with CTC expression and positive ctDNA had a worse prognosis than the
others: SG (hazard ratio [HR], 3.84, 95% CI, 2.41 to 6.22, P .001) and SSR ( HR, 3.02, 95%
CI, 1.83 to 5.00, P .001). These results are found in the prospective study by Roder et al,
where the detection of CTC in operated patients with colon cancer is associated with 5-year
Overall survival (OS)of 68% against 85% in patients without CTC detectable. In multivariate
analysis, the results are similar with a significant difference on SG (HR1.94, 95% CI
1.0-3.7, p = 0.04) and SSR (HR 1.94, 95% CI 1.1-3.7, p = 0.044).
E / Determination of microsatellite instability (MSI) status on ctDNA:
Patients with MSI tumors have a better prognosis than microsatellite stability (MSS) patients
and have different sensitivities to chemotherapy. The MSI status is currently determined on
the operative specimen in pathology by determining the expression of the MLH1-MSH2-MSH6 and
PMS2 proteins by immunohistochemistry and by a molecular test determining the instability of
microsatellite markers. The determination of the MSI status on the ctDNA is not carried out
in current practice but raises several questions: is it systematically detectable on the
ctDNA found on peripheral blood when this phenotype is present? Are the ctDNA levels
comparable according to the MSI and MSS status?
F / Influence of the surgical technic on the amount of ctDNA:
In order to limit the metastatic spread of CRC, studies have evaluated standard surgical
technics, particularly in order to limit the release of ctDNA or CTC.
Studies have compared laparoscopy versus laparotomy on the level of ctDNA and intraoperative
CTC in portal and peripheral blood. The results showed a significant decrease in the overall
cDNA level in laparoscopic patients. The randomized prospective study of Wind et al reports
CTC levels in the laparotomy and laparoscopic groups, respectively 44% vs. 5%. 9% (p = 0.046)
in portal blood after mobilization of the tumor. And after resection of the tumor
respectively 29% vs. 20% (p = 0.590) in the portal blood. One of the hypothesis of the
authors to explain this difference is that laparoscopic tumor mobilization is significantly
less important than the one performed during laparotomy.
This hypothesis led to the principle of the "no touch" technic: to achieve the minimum of
intraoperative tumor mobilization with an early ligation of mesenteric vessels.
A few low-level studies have compared ADNct and CTC levels in perioperative peripheral blood
comparing the "no touch" technic with those that mobilize the tumor first. The results show a
tendency to decrease the rates of both markers in the no-touch technic. In the prospective
study by Hayashi et al, 8 out of 11 patients (73%) operated according to the "mobilization
first" technique had detectable CTCs in portal and postoperative portal blood samples whereas
only 1 patient (14%) from the "no touch" group was detected.
G / Objective:
The objective of this work is to compare the amount of ctDNA and CTC in the peripheral blood
at D-1, D1 and D3 after colectomy for localized adenocarcinoma using the surgical "no touch"
technic against mobilization first and to determine the MSI status on ctDNA.
The standard procedure for hospitalization for patients requiring colectomy for CCR is as
follows:
- A preoperative consultation with the surgeon and one with the anesthesiologist.
- Hospitalization the day before the intervention with visit of the surgeon and the
anesthesiologist, blood sample of a complete preoperative assessment (D-1).
- Intervention (D0): right or left colectomy depending on tumor localization.
- Systematic blood test after surgery on D1, D3: samples of 4ml tubes for blood count
determination, blood Ionogram and assay of C-reactive protein.
- Release on D4 depending on the clinical condition and the results of the blood tests.
As part of the research:
- An information note is given to the subject the day before the intervention and a
written statement is collected before the J-1 levy
- During the blood samples taken as part of the treatment, an additional volume is
collected 2 samples of 6mL at D-1 then at D1 and D3 to measure the levels of circulating
tumor cells and tumor circulating DNA by PCR and the MSI status. There is no blood
sample added by the research, the tubes are thus collected at the same time as the
standard blood samples of usual perioperative follow-up.
D / ctDNA Extraction, Molecular Tests, and CTC Detection:
The extraction of complementary deoxyribonucleic acid (cDNA) will be performed with an
extraction automaton (MaxwellTM) from 6 ml of plasma obtained from the blood sample. The cDNA
obtained will be quantified by fluorimetry and quantitative polymerase chain reaction (qPCR).
Molecular analyzes will be performed by high-throughput sequencing on targets of interest in
colorectal cancer and supplemented with digital PCR targeting mutations previously identified
by sequencing the primary tumor (from the operative specimen). A set up of DNA methylation
tests can also be done.
Due to the rarity of CTCs, it is necessary to proceed in two steps: i) enrichment followed
ii) ScreenCell® detection is a non-invasive technique that allows to enrich from the
peripheral blood a cell fraction containing circulating cells The ScreenCell® Molecular
Biology (MB) kit then makes it possible to carry out molecular analyzes such as PCRs in order
to sequence the gene of interest. This ScreenCell® MB kit allows DNAse free filtration.
Before filtration, 6 ml of blood are diluted in 1 ml of the buffer provided. Two minutes
after 1.6 ml of the culture medium adapted to the blood cells studied. After filtration, the
kit allows extraction of RNA directly from isolated cells in the filter. An adequate volume
of the lysis buffer is added directly to the filter capsule (closed in an eppendorf) to
extract RNA from the isolated cells in the filter. After centrifugation, the genetic material
will be analyzed. For this, a reverse transcriptase will be performed in order to obtain the
cDNA of interest.
Statistical analyzes will be performed to compare tumor cell and circulating DNA levels
according to non parametric Fisher or chi² tests. A value of p <0.005 is considered
significant.
