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.

KRAS status assessment is mandatory in patients with metastatic colorectal cancer before therapy with antiepidermal growth factor receptor monoclonal antibodies, as KRAS mutations are associated with resistance to this treatment. However, KRAS genotyping may be very challenging in case of poor tumor cellularity, particularly when major tumor regression is achieved in locally advanced rectal adenocarcinomas after radiochemotherapy. We aimed at identifying the most reliable strategy to detect KRAS mutations in such samples. DNA was extracted from 31 surgical specimens with major tumor regression, following manual dissection, and from paired pre-treatment biopsies and analyzed by high-resolution melting. DNA samples displaying altered melting curve shapes were then sequenced. Samples with unmodified melting curves or wild-type sequence were further investigated by using an allele-specific PCR assay (TheraScreen) and laser microdissection (followed by high-resolution melting and sequencing analyses). In the 31 post-radiochemotherapy surgical specimens, seven KRAS mutations were identified by high-resolution melting analysis/sequencing. One additional mutation was detected by the TheraScreen assay and two mutations, including the one identified by the TheraScreen assay, were detected following laser microdissection. Altogether, 9/31 surgical specimens (29%) presented KRAS mutations. In the manually dissected pre-treatment biopsies, 12 mutations (39%) were identified by high-resolution melting analysis and sequencing. No additional mutations were found by using the TheraScreen assay or laser microdissection. These results indicate that, in the case of post-radiochemotherapy surgical specimens of colorectal cancer with low tumor cellularity, pre-treatment biopsies might represent the most cost-effective option for reliable KRAS genotyping. The use of more sensitive assays, such as allelespecific PCR or laser microdissection, can be envisaged but with higher costs and longer delays.

DNA mismatch repair (MMR) protein analysis by immunohistochemistry (IHC) can identify colorectal cancer (CRC) with microsatellite instability (MSI). As MLH1-deficient CRC can be hereditary or sporadic, markers to distinguish between them are needed. MLH1 promoter methylation assay is the reference method; however, sometimes, it is challenging on formalin-fixed paraffin-embedded tissue samples. We assessed by IHC the expression of BRAFV600E, p16, MGMT, and CDX2 in 55 MLH1-deficient MSI CRC samples (of which 8 had a germline MLH1 mutation) to determine whether this panel differentiates between sporadic and hereditary CRCs. We also analyzed MLH1 promoter methylation by methylation-specific PCR and pyrosequencing and BRAF status by genotyping. None of the hereditary CRCs showed MLH1 methylation, BRAF mutation, BRAFV600E-positive immunostaining, or loss of p16 expression. We detected MLH1 promoter methylation in 67 % and a BRAF mutation in 42 % of CRC, all showing MLH1 promoter methylation. BRAFV600E IHC and BRAF genotyping gave concordant results in all but two samples. Loss of expression of p16 was found in 30 % of CRC with methylation of the MLH1 promoter, but its expression was retained in all non-methylated and part of MLH1-methylated tumors (100 % specificity, 30 % sensitivity). CDX2 and MGMT expression was not associated with MLH1 status. Thus, BRAFV600E and p16 IHC may help in differentiating sporadic from hereditary MLH1-deficient CRC with MSI. Specifically, p16 IHC might be used as a surrogate marker for MLH1 promoter methylation, because all p16-negative CRCs displayed MLH1 methylation, whereas hereditary CRCs were all p16-positive.

No abstract

No abstract