Trial Information

Colorectal Cancer: a Prospective Multicentric Study of the in Situ Immune Infiltrate for the Identification of the Patients at High Risk of Relapse

The main objective is to assess the impact of the immunoscore in the clinical practice. To
this end, the immune parameter (CD3, CD8, CD45RO) of colorectal cancers (stage I-IV) will be
evaluated prospectively through a large multicenter study. 400 patients with CRC stage I to
IV will be included (6 centers for inclusion; Paris-HEGP, Dijon, Bobigny-Avicenne, Besancon,
Poitiers, Rouen). The number of patients to be included has been calculated on the basis of
an inclusion period of 24 months, a follow-up of 3 years (a collection of data during the
4th and 5th year is planned with a co-financing), an expected event rate of 20%, a
prognostic value of the immune parameter similar to that observed in the retrospective study
of 399 patients with CRC (*1) and a power of the test of 90%. During both initial management
and follow-up, all therapeutic procedures will be registered (surgery, radiotherapy,
chemotherapy…). Data will be collected from clinical examination, radiological exams and
biological tests that are usually performed during a hospitalization for colorectal cancer:
Full clinical examination and questioning about the presence of personal and family history
of CRC, adenomas and other cancers; Abdominal ultrasound, chest X-ray; Computed tomography
(CT) and magnetic resonance imaging (MRI) according to clinician's requirement; Standard
laboratory tests and blood assay for carcinoembryonic antigen (CEA); The Histopathologic
study of the tumor containing the status of the surgical excision margins, the histological
type of cancer, the level of parietal invasion, the number of lymph nodes examined and the
number of metastatic lymph nodes, according to the TNM classification. Patients will be
followed-up every 3 months during the first 2 years, and every 6 months for the rest of the
follow-up period, as recommended. Each patient will be followed up to 5 years. Clinical,
biological and radiological data will be registered into a specific CRF. Verification of
inclusions and monitoring of data will be conducted by the HEGP Clinical Research Unit (URC
HEGP) and the principal investigator. A plan for data-management will be built in line with
the monitoring. A questionnaire will also be sent to the patients every six month for five
years. This questionnaire was built by Pr F Pagès and by psychologists and psychiatrists to
search for information concerning (i) the presence of an immune disorder (such as allergy,
autoimmunity, inflammatory process) and (ii) the psychological status of the patients (eg.
depression). After monitoring, all data will be stored using a double data entry system in a
MS-Access database designed by the URC-HEGP and in the TME.dB database created by the team
of the principal investigator. The 2 databases will be merged, and the final data base will
be frozen for statistical analysis. Sample preparation: The pathologist of each center will
choose a tumor block containing the core of the tumor and the invasive margin. This tumor
block is part of the tumor samples routinely processed by the department of Pathology for
diagnosis. three tissue sections of 4-microns will be made and deposit on a glass slide to
perform the immunohistochemistry. Four tissue sections of 4-microns will be made and deposit
on an Eppendorf safe lock tubes to perform the genetic analyses. Anonymous patient names on
the slides and tubes will be provided by using a code number and initials of the patient. If
biopsies for diagnostic purposes are available (i.e. those performed for the patients during
their initial hospitalization (50% of cases), A tissue sections of each biopsy will be done.
All slides and tubes will be sent by mail to the HEGP platform for analysis of the immune
infiltrate and to the Biology department for the genetic analyses. The immunoscore will be
determine using the following steps: Immunostainings for CD3, CD8, CD45RO on Benchmark XT
automate; Scanning the immunostainings on Hamamatsu scanner: Quantification of the immune
populations in specific tumor areas (the core and the invasive margin) with a dedicated
image analysis module based on the recognition of histological structures adapted to
colorectal cancer. This module has been developed by our group in connection with the
Definiens company. A semi-automatic procedure allows the quantification of stained cells in
each tumor region. Each region is divided into tiles of 800 microns side (500 to 700 tiles
analyzed per tumor). The density of each marker is calculated using the mean density of the
three tiles the most infiltrated in each tumor region. A categorization into high density
(Hi) or low (Lo) for each marker in each tumor region of interest will be performed. This
categorization is done by comparing the observed density with the optimal cut-off density
(min p value approach) we previously determined for the DFS and OS in a retrospective study
of 250 CRC. Genetic analyses: The tubes containing the sections for the genetic analyses of
patients with inform consent signed for genetic investigation will be transfer to the
Department of Biology, Hôpital Européen Georges Pompidou. The % of tumor cells of each
specimen will be determined by a pathologist (based on a H&E evaluation; less than 10%,
10-20%, 20-50%, up to 50% tumor cells) and the DNA extraction will be performed. Genetic
investigation will comprise the MSI status, the presence of a K-Ras and BRAF mutation