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Background: In patients with high risk stage II and stage III colon cancer (CC), curative surgery followed by adjuvant FOLFOX-4 chemotherapy has become the standard of care. However, for 20 to 30% of these patients, the current curative treatment strategy of surgical excision followed by adjuvant chemotherapy fails either to clear locoregional spread or to eradicate distant micrometastases, leading to disease recurrence. Preoperative chemotherapy is an attractive concept for these CCs and has the potential to impact upon both of these causes of failure. Optimum systemic therapy at the earliest possible opportunity may be more effective at eradicating distant metastases than the same treatment given after the delay and immunological stress of surgery. Added to this, shrinking the primary tumor before surgery may reduce the risk of incomplete surgical excision, and the risk of tumor cell shedding during surgery., Methods/design: PRODIGE 22--ECKINOXE is a multicenter randomized phase II trial designed to evaluate efficacy and feasibility of two chemotherapy regimens (FOLFOX-4 alone and FOLFOX-4 + Cetuximab) in a peri-operative strategy in patients with bulky CCs. Patients with CC deemed as high risk T3, T4 and/or N2 on initial abdominopelvic CT scan are randomized to either colectomy and adjuvant chemotherapy (control arm), or 4 cycles of neoadjuvant chemotherapy with FOLFOX-4 (for RAS mutated patients). In RAS wild-type patients a third arm testing FOLFOX+ cetuximab has been added prior to colectomy. Patients in the neoadjuvant chemotherapy arms will receive postoperative treatment for 4 months (8 cycles) to complete their therapeutic schedule. The primary endpoint of the study is the histological Tumor Regression Grade (TRG) as defined by Ryan. The secondary endpoints are: treatment strategy safety (toxicity, primary tumor related complications under chemotherapy, peri-operative morbidity), disease-free and recurrence free survivals at 3 years, quality of life, carcinologic quality and completeness of the surgery, initial radiological staging and radiological response to neoadjuvant chemotherapy, and the correlation between histopathological and radiological response. Taking into account a 50% prevalence of CC without RAS mutation, accrual of 165 patients is needed for this Phase II trial., Trial Registration: NCT01675999 (ClinicalTrials.gov).

Colorectal cancer is one of the most common cancers in the world. Histological staging is efficient, but combination with molecular markers may improve tumor classification. Gene expression profiles have been defined as prognosis predictors among stage II and III tumors, but their implementation in medical practice remains controversial. Stage II tumors have been recognized as a heterogeneous group, and high-risk morphologic features have been used to justify adjuvant chemotherapy. We propose here the investigation of clinical features and expression profiles from stage II and stage III colon carcinomas without DNA mismatch repair defects. Two series of 130 and 66 colon cancer samples were obtained. Expression profiles were established on oligonucleotide microarrays and processed in the R/Bioconductor environment. Hierarchical, then supervised, analyses were successively performed by applying a data-sampling approach. A molecular signature of seven genes was found to cluster stage III tumors with adjusted p values lower than 10(-10). A subgroup of stage II tumors aggregated this cluster in both series. No correlation was found with disease severity, but the function of the discriminating genes suggests that tumors have been classified according to their putative response to adjuvant targeted or classic therapies. Further pharmacogenetic studies might verify this observation.

Background: Approximately 15% of colorectal cancers develop because of defective function of the DNA mismatch repair (MMR) system. We determined the association of MMR status with colon cancer recurrence and examined the impact of 5-fluorouracil (FU)-based adjuvant therapy on recurrence variables., Methods: We included stage II and III colon carcinoma patients (n = 2141) who were treated in randomized trials of 5-FU-based adjuvant therapy. Tumors were analyzed for microsatellite instability by polymerase chain reaction and/or for MMR protein expression by immunohistochemistry to determine deficient MMR (dMMR) or proficient MMR (pMMR) status. Associations of MMR status and/or 5-FU-based treatment with clinicopathologic and recurrence covariates were determined using χ(2) or Fisher Exact or Wilcoxon rank-sum tests. Time to recurrence (TTR), disease-free survival (DFS), and overall survival (OS) were analyzed using univariate and multivariable Cox models, with the latter adjusted for covariates. Tumors showing dMMR were categorized by presumed germline vs sporadic origin and were assessed for their prognostic and predictive impact. All statistical tests were two-sided., Results: In this study population, dMMR was detected in 344 of 2141 (16.1%) tumors. Compared with pMMR tumors, dMMR was associated with reduced 5-year recurrence rates (33% vs 22%; P < .001), delayed TTR (P < .001), and fewer distant recurrences (22% vs 12%; P < .001). In multivariable models, dMMR was independently associated with delayed TTR (hazard ratio = 0.72, 95% confidence interval = 0.56 to 0.91, P = .005) and improved DFS (P = .035) and OS (P = .031). In stage III cancers, 5-FU-based treatment vs surgery alone or no 5-FU was associated with reduced distant recurrence for dMMR tumors (11% vs 29%; P = .011) and reduced recurrence to all sites for pMMR tumors (P < .001). The dMMR tumors with suspected germline mutations were associated with improved DFS after 5-FU-based treatment compared with sporadic tumors where no benefit was observed (P = .006)., Conclusions: Patients with dMMR colon cancers have reduced rates of tumor recurrence, delayed TTR, and improved survival rates, compared with pMMR colon cancers. Distant recurrences were reduced by 5-FU-based adjuvant treatment in dMMR stage III tumors, and a subset analysis suggested that any treatment benefit was restricted to suspected germline vs sporadic tumors.

Purpose: Prior reports have indicated that patients with colon cancer who demonstrate high-level microsatellite instability (MSI-H) or defective DNA mismatch repair (dMMR) have improved survival and receive no benefit from fluorouracil (FU) -based adjuvant therapy compared with patients who have microsatellite-stable or proficient mismatch repair (pMMR) tumors. We examined MMR status as a predictor of adjuvant therapy benefit in patients with stages II and III colon cancer., Methods: MSI assay or immunohistochemistry for MMR proteins were performed on 457 patients who were previously randomly assigned to FU-based therapy (either FU + levamisole or FU + leucovorin; n = 229) versus no postsurgical treatment (n = 228). Data were subsequently pooled with data from a previous analysis. The primary end point was disease-free survival (DFS)., Results: Overall, 70 (15%) of 457 patients exhibited dMMR. Adjuvant therapy significantly improved DFS (hazard ratio [HR], 0.67; 95% CI, 0.48 to 0.93; P = .02) in patients with pMMR tumors. Patients with dMMR tumors receiving FU had no improvement in DFS (HR, 1.10; 95% CI, 0.42 to 2.91; P = .85) compared with those randomly assigned to surgery alone. In the pooled data set of 1,027 patients (n = 165 with dMMR), these findings were maintained; in patients with stage II disease and with dMMR tumors, treatment was associated with reduced overall survival (HR, 2.95; 95% CI, 1.02 to 8.54; P = .04)., Conclusion: Patient stratification by MMR status may provide a more tailored approach to colon cancer adjuvant therapy. These data support MMR status assessment for patients being considered for FU therapy alone and consideration of MMR status in treatment decision making.

Aims: To evaluate the prognostic value of immunohistochemical expression of Bcl-2 in colon cancers., Patients and Methods: Two hundred and twenty-six resected and paraffin-embedded colon carcinomas were analysed by immunostaining using monoclonal antibodies for Bcl-2. We evaluated whether the Bcl-2 staining patterns, semi-quantitatively assessed, could be correlated with the pTNM stage, size and tumour circumference, differentiation, appearance, vascular invasion, perineural invasion, colloid component, margins, involvement of adjacent structures, stromal appearance, flow cytometry and the S-phase., Results: Eighty patients (36%) were considered Bcl-2 positive. The extent of Bcl-2 expression by tumour cells decreased significantly with respect to increasing tumour size (P=0.042), the extension of parietal invasion pT (P=0.007), the invasive nature of the tumour (P=0.024), and extent of the circumference (P=0.024). In a multivariate analysis, Bcl-2 expression does not appear as an independent prognosis factor in the overall population as in the 166 patients with optimal resection. Of the 59 stage II patients, using univariate analysis, Bcl-2 appears to be predictive of relapse-free survival (P=0.025) but not of overall survival (P=0.09)., Conclusion: The loss of Bcl-2 expression appears to be correlated with increase in number of relapses in the stage II colon cancers and could be a potential useful additional histo-prognostic marker in therapy decision making. Bcl-2 immunodetection seems to be associated with slower local tumour growth.

Different diagnostic and prognostic groups of colorectal carcinoma (CRC) have been defined. However, accurate diagnosis and prediction of survival are sometimes difficult. Gene expression profiling might improve these classifications and bring new insights into underlying molecular mechanisms. We profiled 50 cancerous and noncancerous colon tissues using DNA microarrrays consisting of approximately 8000 spotted human cDNA. Global hierarchical clustering was to some extent able to distinguish clinically relevant subgroups, normal versus cancer tissues and metastatic versus nonmetastatic tumours. Supervised analyses improved these segregations by identifying sets of genes that discriminated between normal and tumour tissues, tumours associated or not with lymph node invasion or genetic instability, and tumours from the right or left colon. A similar approach identified a gene set that divided patients with significantly different 5-year survival (100% in one group and 40% in the other group; P=0.005). Discriminator genes were associated with various cellular processes. An immunohistochemical study on 382 tumour and normal samples deposited onto a tissue microarray subsequently validated the upregulation of NM23 in CRC and a downregulation in poor prognosis tumours. These results suggest that microarrays may provide means to improve the classification of CRC, provide new potential targets against carcinogenesis and new diagnostic and/or prognostic markers and therapeutic targets.

MSH2 and MLH1 are proteins involved in DNA reparation. They are mutated in some forms of colon cancer, i.e. hereditary non-polyposis carcinomas and a subset of sporadic carcinomas. We have studied the expression of MSH2 and MLH1 in a retrospective series of 225 colorectal carcinomas by immunohistochemistry. The results were compared to molecular tests of microsatellite instability using amplification by PCR of BAT-25 and BAT-26 repeated sequences and to histoclinical data. Positivity of both proteins was never associated with MSI phenotype. MSH2 and/or MLH1 negative tumors were frequently tumors of the proximal colon; in this subpopulation or proximal tumors, MSH2 and/or MLH1 negativity was associated with a longer disease-free survival.

Objectives: The objective of this work was to study the reporting of resected colonic carcinomas and to compare them with Consensus Guidelines published in January 1998., Methods: The study included 535 colonic carcinomas collected in 8 French registries in 1995, and chosen by drawing lots. For each report, the presence of the information requested by the Guidelines was looked for. Three synthetic variables were built: 2 scores and one qualitative at 2 classes. The influence of patients, tumour and health care system's characteristics was analysed on the 3 dependent variables., Results: Some important variations were observed for information's percents in reporting. In monovariate analysis, scores were significantly influenced by cancer's sub-location, area of patient's residence, surgical center, type of laboratories and pathologist case volume. In multivariate analysis, significant heterogeneity in practices remained between geographical areas. Types of laboratories and pathologist case volume affected differently dependent variables., Conclusion: This study shows the necessity to assess the practices before consensus because of the impact of pathological forms in therapeutic decisions and variations observed.

The CCK-type B receptors are recognized by gastrin, which is known to be possibly involved in the development of gastro-intestinal cancers; alternate splicing of exon 4 of the human CCK-B receptor gene gives 2 different mRNA isoforms, the exact significance of which still remains to be elucidated. The recently described CCK-type C receptors recognize gastrin but do not discriminate between mature and immature forms of the hormone. A series of healthy and tumoral colon samples, the associated hepatic metastases and four colonic cell lines were examined for gene expression of the 2 isoforms of the CCK-B receptor and the CCK-C receptor using reverse transcription-polymerase chain reaction. Gastrin mRNA expression was also investigated. The short isoform of the CCK-B was detected in 80% of the normal colon tissues, 76.5% of the colon tumors, 100% of the metastasis samples and 75% of the colonic cell lines; whereas the long isoform, which is presumably more strongly activated by gastrin, was expressed in 50% of the normal colon samples, 23% of the colon tumors, 43% of the hepatic metastases and 1 cell line (Sk-Co15). However, although CCK-C transcript was detected in 100% of the tumors tested and gastrin mRNA in 86.5%, only 16.5% also expressed the long isoform of the CCK-B receptor. The gastrin/CCK-B receptor might therefore be involved in an hypothetic autocrine proliferative loop only in some colonic tumors, and the receptor mainly involved in this loop may well be the CCK-C receptor, since its mRNA is expressed as often as gastrin mRNA in tumors and cell lines.

CD44 cell-surface receptor expresses multiple isoforms, some of which are believed to play a role in tumor growth and metastasis. The CD44 gene is composed of 19 exons, of which 9 (exons 6 to 14) are alternatively spliced to form inclusions in the intervening membrane proximal region. Sequences present in the shortest metastatic variant cloned from a rat metastatic cell line have been shown to correspond to human exons 10 and 11, also called exons v6 and v7. Using RT-PCR, we have addressed in detail the CD44 isoforms produced in human breast and colon tumors. We analyzed 53 breast-tumor- and 58 colon-tumor-related samples as well as 1 benign mastopathy, 1 normal breast, 4 non-invaded lymph nodes and 8 normal colon tissues. All tumors analyzed expressed the hemopoietic CD44 (CD44H) isoform (no alternatively spliced exons added), whereas 81% expressed the CD44E form (addition exons 12, 13 and 14). Furthermore, 85% of tumors presented complex patterns of expression, with an average number of 5 to 6 bands detected. In view of their implication in the metastatic process, we investigated in greater detail the isoforms containing exons 10 and 11 (v6 and v7). Exon 10 was more frequently expressed than exon 11, 80% and 57% of the samples respectively. The great majority of cases showed ladder-like patterns starting from the shortest forms (exons 5-10 or 5-10-11) and larger-molecular-weight bands corresponding predominantly to sequential inclusions of exons from 3' to 5'. Exon-10 and exon-11 variants were also found in one benign mastopathy. The majority of normal tissues (1 breast and 6/8 colon) expressed only the CD44H isoform. These data indicate that expression of metastatic variants is common in human breast and colon tumors and can occur early during cancer progression, as testified by their presence in a benign breast tumor. While expression of exon-10 variants were correlated with presence of distal metastases in colon tumors, exon-11 variants were not (metastatic events were too rare in our breast-tumor series to reach significance). This suggest that exon 10 may correspond to the minimal sequences required to favor metastatic events.

To investigate the hypothesis that gastrin might be synthesized by tumour tissues in cancer of the colon, samples from six human colon tumours, one hepatic metastasis, four normal colonic mucosal samples and two antral and one fundic gastric mucosal samples from nine patients were analysed to determine whether gastrin mRNA was present. RNA was extracted from surgical specimens by ultracentrifugation on a CsCl cushion, purified using the guanidinium thiocyanate method, reverse-transcribed and amplified by polymerase chain reaction. Gastrin mRNA was detected in each colonic carcinoma sample (including the hepatic metastasis), while no such signal was observed in normal colon biopsies. Positive and negative controls (gastric antrum and fundus respectively) gave the expected results. In each of the positive samples, the chemiluminescent revelation of amplified products after Southern blotting corresponded to gastrin mRNA without the intron. These findings demonstrate the ability of primary and metastatic human colonic tumours to produce gastrin mRNA. Since malignant cell lines have been reported to produce gastrin peptide, and since gastrin receptors were present in some cases, our results support the idea that gastrin may be involved in an autocrine mechanism.

Increased basal serum gastrin level has been described in patients presenting with colorectal cancer. The aim of this work was to study the evolution of serum gastrin levels after cancer treatment. We measured basal serum gastrin levels before and 1 to 2 months after treatment in 15 patients (7 men, 8 women; mean age: 61.6 years). There were 3 malignant polyps, 4 Dukes A, 3 Dukes B, 4 Dukes C and 1 Dukes D colonic cancers. Treatment included 3 endoscopic polypectomies, 2 laser photodestructions, and 10 surgical resections, Mean basal gastrin level after treatment (49.07 +/- 12.65 mIU/l) was significantly lower (P less than 0.002) than before treatment (104.47 +/- 26.98 mIU/l). In the 2 patients treated by laser therapy, recurrences were associated with reincreasing serum gastrin levels. These results suggest an "autocrine" secretion of gastrin.