Initial comparative evaluation between MSI and IHC tests
A comparative analysis between IHC for MMR protein expression and PCR-based MSI testing was performed in 5,676 GC and 2,553 CRC cases.
In the GC cases, 5,082 cases (89.5%) showed MSS/MSI-L and pMMR expression, 503 cases (8.9%) showed MSI-H and dMMR expression. As a result of the initial comparison between MSI testing and IHC, re-evaluation of MSI testing and IHC was necessary in 91 discordant or indeterminate IHC cases (15.3%) among the 594 cases showing MSI-H and loss of MLH1 (Fig. 2A). After re-evaluation, 37 cases of agreement between MSI testing and IHC for MMR proteins were confirmed. Among them, 17 cases resulted from misinterpretation (Fig. 3A and 3B), and 12 cases were impacted by problems in the immunohistochemical process (Fig. 3C and 3D). The remaining eight cases displayed loss of expression in other MMR proteins (MSH2, MSH6, and PMS2), six of which involved loss of MSH2/MSH6 expression.
In the CRC cases, 2,343 cases (91.8%) showed MSS/MSI-L and pMMR expression, and 171 cases (6.7%) showed MSI-H and dMMR expression. As a result of the initial comparison between MSI testing and IHC, re-evaluation of MSI testing and IHC was necessary in 39 discordant or indeterminate IHC cases (18.6%) among the 210 cases showing MSI-H and dMMR expression (Fig. 2B). After re-evaluation, 10 cases of agreement between MSI testing and IHC for MMR were confirmed. Similar to the GC cases, the discordant cases and indeterminate IHC results stemmed from six cases of misinterpretation and four cases with problems during immunohistochemical staining.
MSI testing and IHC for MMR protein expression after re-evaluation
The MSS, MSI-L, and MSI-H statuses of all GC cases were 4,803 (84.6%), 314 (5.5%), and 559 (9.8%), respectively, and those of all CRC cases were 2,149 (84.2%), 208 (8.1%), and 196 (7.7%), respectively (Fig. 4A and 4C). In MSI-L cases, a DT marker in the NCI panel was unstable in most GCs (92.4%) and CRCs (96.2%) (Fig. 5A and 5C). Additionally, the diagnostic sensitivity of MSI-H was higher with the MT markers than that with DT markers (Fig. 5B and 5D).
Among the total of 5,676 GC cases, dMMR protein expression was found in 524 cases (9.2%), and indeterminate IHC was observed in 35 cases (0.6%) (Fig. 4B). In addition, 5,101 cases (89.9%) showed MSS/ MSI-L and pMMR protein expression, and 521 cases (9.2%) showed MSI-H and, dMMR protein expression (Fig. 6A). After re-evaluation, discordant or indeterminate IHC cases were found in 54 out of 5,676 cases (0.9%) and 575 cases (9.4%), excluding unequivocal MSS/MSI-L and pMMR expression cases (Fig. 6C).
Among the total of 2,553 CRC cases, dMMR protein expression was found in 177 cases (6.9%), and indeterminate IHC was observed in 21 cases (0.8%) (Fig. 4D). In addition, 2,348 cases (92.0%) showed MSS/ MSI-L and pMMR protein expression, and 176 cases (6.9%) showed MSI-H and, dMMR protein expression (Fig. 6B). After re-evaluation, discordant or indeterminate results were found in 29 out of 2,553 cases (1.1%), and 205 cases (14.1%), excluding unequivocal MSS/MSI-L and pMMR expression cases (Fig. 6D).
Discordant and indeterminate IHC cases
In regard to the nineteen discordant GC cases, sixteen cases displayed MSI-H/pMMR protein expression. Seven of the MSI-H/pMMR expression cases were composed of only unstable DT markers, with no unstable MT markers (36.8%), six cases contained only one unstable MT marker (31.6%), and the remaining three cases were MSI-H GCs with two unstable MT markers (15.8%) (Fig. 7A). As for the eight discordant CRC cases, seven cases displayed MSI-H/pMMR protein expression, five of which were MSI-H composed of no unstable MT markers and only unstable DT markers (62.5%), one of which contained only one unstable MT marker (12.5%), and the remaining one was a MSI-H CRC with two unstable MT markers (12.5%) (Fig. 7B).
In cases of GC and CRC—except for indeterminate IHC—when there were only one or no unstable MT markers, MSI-H/pMMR expression cases comprised 13 out of 20 GC cases (65.0%) and six out of 11 CRC cases (54.5%), respectively (Fig. 7C and 7D). MSI-H and pMMR GC and CRC cases with only unstable DT markers were found in seven out of eight GC cases (87.5%) and five out of six CRC cases (83.3%)—except for indeterminate IHC. The BAT-25 MT marker was always unstable in MSI-H/pMMR GC and CRC cases with only one unstable MT marker. In the NCI panel of five markers, the D2S123 DT marker showed the most unstable cases (91.3%) in MSI-H/pMMR GC and CRC cases (Fig. 7E).
In the GC cases with indeterminate IHC results, 14 MSI-H and 10 MSS/MSI-L cases showed focal loss. All focal loss IHC results in GC cases were observed in MLH1 or PMS2 (Fig. 8A). In the indeterminate IHC CRC cases, five MSI-H cases and two MSS/MSI-L cases showed focal loss, and all focal loss IHC results observed in MSI-H cases involved MSH6 (Fig. 8B). Abnormal MMR IHC patterns were observed in only two MSI-H CRC cases, one of which lost all MMR protein expression.