Accumulation of frameshift mutations at genes containing coding mononucleotide repeats is thought to be the major molecular mechanism by which mismatch repair-deficient cells accumulate functional alterations. These mutations resulting from microsatellite instability (MSI) can affect genes involved in pathways with a putative oncogenic role, but may also arise in genes without any expected role in MSI carcinogenesis because of the high mutation background of these tumours. We here screened 39 MSI colorectal tumours for the presence of mutations in 25 genes involved in DNA damage signalling and repair pathways. Using a maximum likelihood statistical method, these genes were divided into two different groups that differed significantly in their mutation frequencies, and likely represent mutations that do or do not provide selective pressure during MSI tumour progression. Interestingly, the so-called real-target mutational events were found to be distributed among genes involved in different functional pathways of the DNA metabolism, for example, DNA damage signalling (DNA-PKcs, ATR), double-strand break (DSB) repair (DNA-PKcs, RAD50), mismatch repair (MSH3, MSH6, MBD4) and replication (POLD3). In particular, mutations in MRE11 and/or RAD50 were observed in the vast majority of the tumours and resulted in the concomitant loss of immunohistochemical expression of both proteins. These data might explain why MSI colorectal cancers (CRC) behave differently in response to a wide variety of chemotherapeutic agents, notably those targeting DNA. More generally, they give further insights into how MSI leads to functional changes with synergistic effects in oncogenic pathways.

The detection of point mutations correlated with diseases, in enzymatically amplified DNA sequences (Polymerase Chain Reaction), is currently performed by digestion of PCR products when an existing restriction site disappears at least in one allele of the amplified mutated sequence or by allele specific radiolabeled probes in all other cases. These methods are the most sensitive but they cannot detect a mutation if it is present in less than 5% of the studied cells. We describe here a method based on the introduction of an artificial restriction site, using a modified primer during the PCR, which creates a RFLP indicative of the studied mutation. This RFLP is detected by a radiolabeled oligonucleotide probe which is not related to the mutation. Our approach multiplies the sensitivity by a factor of 1000 and it is practical for use in screening purposes and the detection, after treatment, of the residual disease in human malignancies. Using this method we detected 20% more mutations at codon 12 in the Ki ras oncogene in DNA from colorectal cancers that were undetectable with all the previous methods.

The presence of MSI and tolerance to methylation in LCs identified patients with CMMRD with 100% sensitivity and specificity. These features could be used in diagnosis of patients.

Our series of CDH1 mutation carriers is the largest to date and demonstrates that LBC might be the first manifestation of HDGC. A personal or family history of multiple LBCs at a young age, even without DGC, should prompt CDH1 mutation screening. It is paramount to identify mutation carriers early, so that they can benefit from prophylactic gastrectomy before they develop symptomatic, highly lethal DGC. We recommend a revision of the HDGC-defining criteria and propose for consideration the name 'Hereditary Diffuse Gastric and Lobular Breast Cancer' instead of HDGC.