Florence Le Pessot

Tumor-infiltrating lymphocytes in colorectal cancers with microsatellite instability are correlated with the number and spectrum of frameshift mutations

Colorectal cancers with microsatellite instability are characterized by an important density of tumor-infiltrating lymphocytes and a good prognosis. Microsatellite instability results from the inactivation of the DNA mismatch repair system and induces secondary somatic frameshift mutations within target genes harboring repeat sequences in their coding frame. By disrupting the open reading frame, frameshift mutations can result in the appearance of potentially immunogenic neopeptides. To determine the frameshift mutations inducing a T-cell response during the development of a tumor with microsatellite instability, we studied in 61 colorectal cancer patients with microsatellite instability, using a fluorescent multiplex PCR comparative analysis, the relative frequency of frameshift mutations within 19 target genes and analyzed the correlation of these frameshift mutations with the density of CD3+ tumor-infiltrating lymphocytes. The four most frequently mutated genes were ACVR2 (92%), TAF1B (84%), ASTE1/HT001 (80%) and TGFBR2 (77%). The vast majority (95%) of the tumors exhibited at least three frameshift mutations, and the number of frameshift mutations was associated with tumor progression (TNM stage, wall invasion and tumor diameter). Tumor-infiltrating lymphocyte density was associated with the overall number of frameshift mutations and with the presence of frameshift mutations within two target genes, namely ASTE1/HT001 and PTEN. These results strongly argue for the clinical relevance of immunotherapy of colorectal cancers with microsatellite instability.

Metastatic colorectal cancer KRAS genotyping in routine practice: results and pitfalls.

KRAS genotyping is mandatory before anti-epidermal growth factor receptor monoclonal antibody therapy in metastatic colorectal cancer, which is the second leading cause of cancer-related death in the United States and in Europe. Thus, large-scale KRAS mutation screening is needed for efficient patient management and in the future metastatic colorectal cancer genotyping might also include the detection of the BRAF V600E mutation, which is a very strong negative prognostic factor in colorectal cancer. We report our experience of routine KRAS/BRAF mutation screening practice performed on 1130 formalin-fixed paraffin-embedded tumor samples from 992 colorectal cancer patients. DNA was extracted from macrodissected tumor areas highlighted by a pathologist, KRAS codons 12/13 and BRAF V600E mutations were assessed in a single SNaPshot® multiplex assay and each mutation was confirmed by an independent analysis. KRAS and BRAF mutations were, respectively, present in 41.8 and 6.5% of the tumor samples. If KRAS and BRAF mutations were mutually exclusive, four samples presented two concomitant KRAS mutations. Genotyping of paired primary tumors and metastases from 44 patients indicated that 5 patients (11.4%) presented discordant KRAS mutational status. KRAS genotype heterogeneity was also observed within primary tumor sites in seven cases. Non-reproducible KRAS artefactual mutations were detected in 53 samples (4.7%). We found that the prominent mechanism leading to these artefactual mutations was the fragmentation of DNA occurring during tissue processing. Routine KRAS genotyping performed on formalin-fixed paraffin-embedded tissues requires, therefore, the development of quality control scheme for molecular pathology, especially because of DNA damages induced by formalin fixation. The tumor heterogeneity observed in some patients indicates that it should be more appropriate to perform KRAS genotyping on metastases if sample is available.

Overexpression of B-type cyclins alters chromosomal segregation.

To identify genes which overexpression results into chromosomal instability (CIN), we developed a biological approach based on a yeast indicator strain in which CIN can be detected by a sectoring phenotype. Screening in this strain of a yeast genomic library cloned into a high copy vector led us to identify, among the clones generating 100% of sectoring colonies, Clb5, one of the six B-type cyclins present in yeast. Overexpression of cyclin B2 and cyclin B1, the two human homologs of Clb5, in the CIN indicator strain resulted also into a sectoring phenotype and induced, like overexpression of Clb5, an abnormal sensitivity to benomyl, indicating that overexpression of B-type cyclins alters the spindle checkpoint. In a series of 38 primary colorectal cancers, we detected in five tumors (13%) an accumulation of cyclin B1, which was neither related to mRNA overexpression nor to mutation within the coding region, and in five other tumors (13%) a 2–10-fold increase of cyclin B2 mRNA which was not related to gene amplification. These results suggest that overexpression of cyclins B, resulting from different mechanisms, could contribute, through an alteration of the spindle checkpoint, to the chromosomal instability observed in cancer.

Correlation between Density of CD8+ T-cell Infiltrate in Microsatellite Unstable Colorectal Cancers and Frameshift Mutations: A Rationale for Personalized Immunotherapy

Colorectal cancers with microsatellite instability (MSI) represent 15% of all colorectal cancers, including Lynch syndrome as the most frequent hereditary form of this disease. Notably, MSI colorectal cancers have a higher density of tumor-infiltrating lymphocytes (TIL) than other colorectal cancers. This feature is thought to reflect the accumulation of frameshift mutations in sequences that are repeated within gene coding regions, thereby leading to the synthesis of neoantigens recognized by CD8(+) T cells. However, there has yet to be a clear link established between CD8(+) TIL density and frameshift mutations in colorectal cancer. In this study, we examined this link in 103 MSI colorectal cancers from two independent cohorts where frameshift mutations in 19 genes were analyzed and CD3(+), CD8(+), and FOXP3(+) TIL densities were quantitated. We found that CD8(+) TIL density correlated positively with the total number of frameshift mutations. TIL densities increased when frameshift mutations were present within the ASTE1, HNF1A, or TCF7L2 genes, increasing even further when at least one of these frameshift mutations was present in all tumor cells. Through in vitro assays using engineered antigen-presenting cells, we were able to stimulate peripheral cytotoxic T cells obtained from colorectal cancer patients with peptides derived from frameshift mutations found in their tumors. Taken together, our results highlight the importance of a CD8(+) T cell immune response against MSI colorectal cancer-specific neoantigens, establishing a preclinical rationale to target them as a personalized cellular immunotherapy strategy, an especially appealing goal for patients with Lynch syndrome.

Prognostic value of circulating mutant DNA in unresectable metastatic colorectal cancer.

Objective:No validated biologic prognostic marker is presently available in metastatic colorectal cancer (MCRC). We prospectively evaluated the prognostic value of circulating mutant DNA in 31 patients presenting an unresectable MCRC treated by chemotherapy, and we used, as tumor markers, KRAS mutations and methylation of the RASSF2A promoter. Methods:Detection in the serum of KRAS mutation and RASSF2A methylation were performed using sensitive methods, respectively, real-time polymerase chain reaction (PCR) performed in the presence of a peptide nucleic acid specific of the wild-type sequence and methyl-specific PCR after bisulfite treatment. Results:Among 29 MCRC patients for whom DNA from the primary tumor was available, 23 (79%) presented at least one of the markers in their primary tumor, and 12 of them presented the same alteration in serum. For the 2 remaining patients, RASSF2A methylation was detected in serum indicating that this alteration was present in the primary tumor. These 14 patients with a detectable tumor marker in their serum were designed sDNA+ patients. After 6 months of follow-up, 11/14 (79%) sDNA+ and 1/11 (9%) sDNA− patients presented a progressive disease (P = 0.001). The median progression free survival was 5 months in sDNA+ patients versus 14 months in sDNA− patients (P = 0.004). After 1 year of follow-up, 2 of 14 (14%) sDNA+ and 8 of 11 (73%) sDNA− patients presented no signs of disease progression (P = 0.005). Conclusions:This study suggests that the presence of circulating mutant DNA in unresectable MCRC patients, which can be detected using simple methods such as methylation-specific PCR or real-time PCR, is highly predictive of clinical outcome.

Contribution of the BOP1 gene, located on 8q24, to colorectal tumorigenesis.

The most common form of genomic instability observed in colorectal cancer is chromosomal instability (CIN), whose molecular bases remain to be determined. We have previously demonstrated that inactivation in human cells of several components of the Pes1‐Bop1 complex (BOP1, GRWD1, PES1, ORC6L, and RPL3), involved in ribosome biogenesis, altered chromosome segregation. To determine the contribution to colorectal tumorigenesis of somatic alterations of genes involved in ribosome biogenesis, we screened 56 primary colorectal cancers, using quantitative multiplex PCR of short fluorescent fragments, a sensitive method for the detection of gene dosage alterations. We found that dosage increase of the BOP1 gene was a frequent event, being detected in 39% of the tumors, and we show that it is associated with an increase of BOP1 mRNA. Scanning of 8q24, on which BOP1 is located, revealed that in colorectal cancers, gene dosage increase of BOP1 can be independent from that of MYC and was more frequent than that affecting MYC. Finally, transient overexpression of BOP1 in human cells increased the percentage of multipolar spindles. Together with our previous results, the present study strongly suggests that deregulation of the BOP1 pathway contributes to colorectal tumorigenesis.

Characterization of an adaptive immune response in microsatellite-instable colorectal cancer

Sporadic or hereditary colorectal cancer (CRC) with microsatellite instability (MSI) is frequently characterized by inflammatory lymphocytic infiltration and tends to be associated with a better outcome than microsatellite stable (MSS) CRC, probably reflecting a more effective immune response. We investigated inflammatory mechanisms in 48 MSI CRCs and 62 MSS CRCs by analyzing: (1) the expression of 48 cytokines using Bio-Plex multiplex cytokine assays, and (2) the in situ immune response by immunohistochemical analysis with antibodies against CD3 (T lymphocytes), CD8 (cytotoxic T lymphocytes), CD45RO (memory T lymphocytes), T-bet (Th1 CD4 cells), and FoxP3 (regulatory T cells). MSI CRC exhibited significantly higher expression of CCL5 (RANTES), CXCL8 (IL-8), CXCL9 (MIG), IL-1β, CXCL10 (IP-10), IL-16, CXCL1 (GROα), and IL-1ra, and lower expression of MIF, compared with MSS CRC. Immunohistochemistry combined with image analysis indicated that the density of CD3+, CD8+, CD45RO+, and T-bet+ T lymphocytes was higher in MSI CRC than in MSS CRC, whereas the number of regulatory T cells (FoxP3+) was not statistically different between the groups. These results indicate that MSI CRC is associated with a specific cytokine expression profile that includes CCL5, CXCL10, and CXCL9, which are involved in the T helper type 1 (Th1) response and in the recruitment of memory CD45RO+ T cells. Our findings highlight the major role of adaptive immunity in MSI CRC and provide a possible explanation for the more favorable prognosis of this CRC subtype.

Lugol chromo-endoscopy versus Narrow Band Imaging for endoscopic screening of esophageal squamous-cell carcinoma in patients with a history of cured esophageal cancer: a feasibility study

To date, Lugol chromo-endoscopy is the reference technique to detect an esophageal neoplasia in patients with prior esophageal squamous-cell carcinoma (ESCC), but is not easy to perform without general anesthesia, which can limit its use in routine practice. The objective of this study were to compare the accuracy of white light, narrow band imaging (NBI), and Lugol to detect esophageal neoplasia in patients with a history of cured ESCC, in a prospective study. Thirty patients were prospectively included between June 2006 and June 2009. They all had a history of cured ESCC. Esophageal mucosa was examined first using white light, second NBI, and third after Lugol staining. Histology was obtained in all abnormalities detected by white light, NBI, and/or Lugol. Five neoplastic lesions in five different patients were identified at histology, four cancers, and one high-grade dysplasia. NBI and Lugol both detected all esophageal neoplastic lesions, whereas white light detected the four cancers but missed the high-grade dysplasia. In this feasibility study, NBI and Lugol both detected all identified esophageal neoplasia in very high-risk patients of ESCC. This result suggests that NBI could be used instead of Lugol to detect an esophageal neoplasia in patients with high risk of ESCC, but needs to be confirmed in a larger study.

Regulatory T Lymphocytes Are Associated with Less Aggressive Histologic Features in Microsatellite-Unstable Colorectal Cancers

Background Colorectal cancers (CRCs) with microsatellite instability (MSI) are associated with a good prognosis and a high density of tumor-infiltrating lymphocytes (TILs). We have undertaken to determine the link between TIL densities and MSI CRC histologic features. Patients and Methods Using tissue microarrays, T-cell sub-population infiltration, including T cells (CD3), cytotoxic T cells (CD8) and regulatory T cells (FoxP3) were studied in 86 MSI CRCs. We separately analyzed TILs of the stromal and epithelial compartments in the tumor center, the tumoral invasion margin and associated normal tissue. Results For FoxP3+ TIL density in the tumor center stromal compartment, we found a strong negative correlation with T4 stage (p = 0.01), node invasion (p<0.001) and VELIPI (vascular emboli, lymphatic invasion and perinervous invasion) criteria (p = 0.002). Conclusion The strong correlation between regulatory T cell density and the absence of VELIPI criteria suggests that this sub-group of T cells is preferentially associated with less invasive tumors.

Phlegmonous Gastritis in a 32-Week Pregnant Woman Managed by Conservative Surgical Treatment and Antibiotics

Phlegmonous gastritis is an extremely rare and life-threatening condition. We report the case of a 32-week pregnant women presenting a peritonitis owing to phlegmonous gastritis caused by a group A streptococcus and successfully managed by conservative surgical treatment and antibiotics. Multiple endoscopies with biopsies illustrate progressive and complete gastric recovery.