The identification of patients likely to be carrying a germline mutation in a DNA MMR gene (Lynch syndrome) is important; however, universal molecular testing of all colorectal cancers would be expensive and time consuming. Use of family history of cancer as a predictive marker is insensitive and can be difficult to assess accurately in a clinical setting. Particular pathology features of colorectal cancers have previously been associated with MSI-H status in the setting of Lynch syndrome10,26
and in series unselected for family history and likely to comprise mainly MSI-H colorectal cancers not caused by germline MMR mutations (often referred to as “sporadic”).12–16,18,19
We have developed the MsPath score that uses easily assessable clinicopathologic characteristics to capture all MSI-H colorectal cancers presenting in patients younger than 60 years, the age group most likely to be associated with Lynch syndrome, while ruling out colorectal cancers that are highly unlikely to be MSI-H. We show that the probability of a tumor being MSI-H can be estimated at pathology review at no additional cost and indicate how a simple scoring system can then be used to triage tumors for MSI or IHC testing.
For example, for cases diagnosed in patients younger than 50 years (all of which should be tested for MSI or by IHC according to the Bethesda guidelines), we found, in the independent validation data, that only approximately 60% equaled or exceeded our recommended MsPath score of 1.0 (), leaving 40% that we suggest would not require MSI testing. For cases diagnosed in patients between 50 and 59 years, we estimate approximately 60% would not need to be tested. Importantly, the few MSI-H colorectal cancers missed by a cutoff MsPath score of 1.0 were unlikely to be due to MMR deficiency. They showed instability in only 30%–40% of markers, and none of the 8 markers mutated in the 3 tumors were a highly specific mononucleotide marker. Furthermore, none of these tumors showed loss of expression of a DNA MMR protein. These findings validate the inclusion of pathology features in the Bethesda guidelines.9
We have recommended a cutoff MsPath score of 1.0 to maximize the specificity while maintaining a high sensitivity, because it is important not to miss MSI-H cases.
The principal advantage of our multivariate predictive model is its ability to exceed the sensitivity and specificity of models based on individual items taken alone. Some of the features in the MsPath model, such as mucinous differentiation, are more specific for MSI-H status, while others, such as presence of tumor-infiltrating lymphocytes and anatomic subsite, are more sensitive.
The biological explanations for the distinctive morphology of MSI-H colorectal cancer are not well understood. Colorectal cancers secreting abundant mucin have been associated with precursor lesions that also show abundant mucin secretion,27
such as villous adenomas in the case of Lynch syndrome28
and serrated polyps in the case of MSI-H colorectal cancers outside Lynch syndrome. 29
The increased lymphocytic reaction in MSI-H colorectal cancer may be caused by the enhanced immunogenicity associated with the generation of mutant proteins transcribed from genes with frameshift mutations. 30,31
Alternatively, MSI-H colorectal cancers may fail to elaborate Fas ligand implicated in the counterattack by malignant cells on contact with lymphoid cells.32
Loss of CDX2 expression has been linked with poor differentiation and particularly with the undifferentiated variant described as medullary carcinoma.33
Only one medullary carcinoma was diagnosed in this study, but it is likely that examples with focal glandular differentiation would have been included in the poorly differentiated subset.
Our study was based on colorectal cancers selected in a population-based manner at locations in 3 countries. Multiple pathologists using a combination of data from pathology reports and pathology reviews reported different distributions for the pathology features used in the final model. Such differences are expected, given the spectrum of pathologist training and experience. Despite such differences, the model we have developed appears robust, even when comparing across continents. Therefore, our results are likely to be generalizable and indicate that the MsPath scoring should be widely reproducible as a triage procedure for prioritizing colorectal cancers for MSI testing. Our sample size was large, and we used a relatively limited set of variables in constructing our statistical model. We found strong evidence for validity by generating the model on the North American data and validating it against the Australian data. Nonetheless, independent validation of the model in other samples is warranted.
Of patients younger than 60 years diagnosed with an MSI-H colorectal cancer, up to 50% may be due to Lynch syndrome.20,34
In a recent population-based study, 16 colorectal cancer cases diagnosed before the age of 60 years were shown to have an MSI-H tumor; of these, 10 (63%) had a deleterious germline mutation in a DNA MMR gene, while 6 (37%) had methylation of the MLH1
methylation is a highly age-related process,36
and most MSI-H colorectal cancers outside Lynch syndrome are diagnosed after the age of 60 years. Therefore, the use of the MsPath score for directing subsequent MSI analysis should provide a reasonably specific, as well as highly sensitive, screening test for Lynch syndrome.
The extent to which pathology features may identify examples of Lynch syndrome that are overlooked by other Bethesda guideline criteria cannot be determined in this study. However, when germline status of this study sample is known, it will be possible to compare the performance of the pathology component of the Bethesda guidelines with the other Bethesda guideline components for identifying MMR gene mutation carriers. It should be noted that the MsPath model cannot be used to detect the rare occurrence of a pathogenic MMR gene mutation that does not result in MMR deficiency.37
The distinction between early-onset MSI-H colorectal cancers not due to Lynch syndrome and those that are due to Lynch syndrome remains a problem for both clinicians and laboratory scientists. Although initially combined as an otherwise indistinguishable entity, these subsets of MSI-H colorectal cancer differ with respect to both pathogenesis and genotype.38–41
The subset of MSI-H colorectal cancers outside Lynch syndrome is characterized by frequent mutation of BRAF
and wide-spread DNA methylation.39
Although the pathology features may be shared across the 2 subtypes, certain features such as lymphocytic infiltration may be more frequent in Lynch syndrome, while others, such as mucinous differentiation, may be more common in the subset outside Lynch syndrome.38,41
When information with respect to germline mutations, MLH1
methylation, and BRAF
mutation status becomes available, it may be possible to identify subtle differences in pathology and modify the MsPath model accordingly. It may then function as a much more specific indicator of Lynch syndrome.
This study did not consider colorectal cancers diagnosed after the age of 60 years. To identify late-onset cases of Lynch syndrome, the Bethesda guidelines rely exclusively on personal or family history. However, just as the MsPath model may identify colorectal cancers diagnosed in patients before age 60 years that do not require MSI testing, it is possible that the MsPath model could also be applied to later-onset colorectal cancers but meeting other Bethesda guideline criteria. However, it would be inappropriate to exclude tumors from MSI testing on the basis of the MsPath model when the family history was strongly suggestive of Lynch syndrome. Refinements of the MsPath model that would allow distinction between Lynch syndrome versus “sporadic” MSI-H colorectal cancers might permit the recognition of later-onset Lynch syndrome colorectal cancers. This would be a useful contribution given the later age at onset of Lynch syndrome colorectal cancers in the population setting.42
Loss of MSH2 expression serves as strong evidence of an MSH2
The finding of BRAF
mutation or methylation of MLH1
would argue against a diagnosis of Lynch syndrome40,43,44
but may occur in the context of the recently described “serrated pathway syndrome.” 45
Rarer mechanisms for MSI-H status not caused by germline MMR mutations should also be considered, for example, somatic 2-hit inactivation of a DNA MMR gene or germline hemiallelic methylation (epimutation) of MLH1
Although we used MSI testing as the primary measure of DNA MMR proficiency, we would also expect our model to predict IHC, because it is highly correlated with MSI.21
Most hospital-based laboratories would favor IHC as the first line of testing4
because this approach already has very wide diagnostic applications and IHC has the advantage of being routinely available in diagnostic laboratories as well as having the potential for pinpointing the causative gene. If the results are unequivocal, there is no need to undertake confirmatory MSI testing.49
This study has also highlighted a gray zone in the separation of MSI-L and MSI-H as indicated by the 3 MSI-H cases that would have been missed by the cutoff MsPath score of 1.0. The inclusion of nonmononucleotide markers in the MSI panel carries the risk of overdiagnosing bona fide DNA MMR repair deficiency; a fact that is now formally recognized in the revised Bethesda guidelines.9
With the recognition of such likely overdiagnosed MSI-H cases, the MsPath model has a sensitivity approaching 100%.
The Bethesda guidelines were developed specifically for identifying patients with possible Lynch syndrome. Therefore, those guidelines and our MsPath score are not intended for the identification of other forms of hereditary colorectal cancer.
In conclusion, this study adds to the growing literature demonstrating the feasibility of population-based screening for Lynch syndrome.4,50–52
The present study has validated the inclusion of pathology features within the Bethesda guidelines and shown that the new MsPath model can be used to effectively separate colorectal cancer tumors highly unlikely to be MSI-H from those with a possibility of being MSI-H in a community setting. The features utilized in MsPath may be obtained with a minimum of time, training, and expense, and the risk of MMR deficiency can be estimated without using complex calculations or a computer. By means of the MsPath model, expensive and unnecessary special investigations can be avoided. Finally, the MsPath model provides a screening instrument for identifying Lynch syndrome in subjects aged 50 – 60 years who lack a suggestive family history.