An unusual phenotype occurs in 15% of mismatch repair-deficient tumors and is associated with non-colorectal cancers and genetic syndromes

Abstract

Immunohistochemistry (IHC) and/or MSI-PCR (microsatellite instability-polymerase chain reaction) tests are performed routinely to detect mismatch repair deficiency (MMR-D). Classical MMR-D tumors present a loss of MLH1/PMS2 or MSH2/MSH6 with MSI-High. Other profiles of MMR-D tumors have been described but have been rarely studied. In this study, we established a classification of unusual MMR-D tumors and determined their frequency and clinical impact. All MMR-D tumors identified between 2007 and 2017 were selected. Any profile besides the classical MMR-D phenotype was defined as unusual. For patients with unusual MMR-D tumors, IHC, and PCR data were reviewed, the tumor mutation burden (TMB) was evaluated and clinical and genetic features were collected. Of the 4948 cases of MMR testing, 3800 had both the available IHC and MSI-PCR results and 585 of these had MMR-D. After reviewing the IHC and PCR, 21% of the cases initially identified as unusual MMR-D were reclassified, which resulted in a final identification of 89 unusual MMR-D tumors (15%). Unusual MMR-D tumors were more often associated with non-CRC than classical MMR-D tumors. Unusual MMR-D tumors were classified into four sub-groups: i) isolated loss of PMS2 or MSH6, ii) classical loss of MLH1/PMS2 or MSH2/MSH6 without MSI, iii) four MMR proteins retained with MSI and, iv) complex loss of MMR proteins, with clinical characteristics for each sub-group. TMB-high or -intermediate was shown in 96% of the cancers studied (24/25), which confirmed MMR deficiency. Genetic syndromes were identified in 44.9% (40/89) and 21.4% (106/496) of patients with unusual and classical MMR-D tumors, respectively (P < 0.001). Five patients treated with an immune checkpoint inhibitor (ICI) had a prolonged clinical benefit. Our classification of unusual MMR-D phenotype helps to identify MMR deficiency. Unusual MMR-D phenotype occurs in 15% of MMR-D tumors. A high frequency of genetic syndromes was noted in these patients who could benefit from ICI.

Fig. 1. Flow chart.

MMR: mismatch repair, IHC: immunohistochemistry, MSI: microsatellite instability, MMR-P: MMR proficient, MMR-D: MMR deficient.

Fig. 2. Identification of unusual MMR-D tumors after IHC and MSI review: *11/13 cases were clearly assigned to one category of loss of MMR proteins and 2/11 cases remained indeterminate for at least one MMR protein (PMS2).

**: for one case, initial IHC was performed on biopsy with normal expression of MMR proteins but with an isolated loss of PMS2 on the surgical sample, and for the other case, initial IHC was performed on a polyp with normal expression of MMR protein but with PMS2/MLH1 loss on invasive carcinoma.

Fig. 3. Representative images of MMR immunohistochemistry and microsatellite status assessed by MSI-PCR of each unusual MMR-D subgroup.

Line 1 illustrates a group 1 unusual MMR-D: endometrial carcinoma with retained expression of MLH1, PMS2, and MSH2 (A, B, C,) isolated loss of MSH6 (D) and MSI-High (instability at the five markers of Pentaplex-PCR) (E). Line 2 shows an endometrial carcinoma in subgroup 2 with retained expression of MLH1 and PMS2 (F, G), loss of both MSH2 and MSH6 proteins (H, I), and MSI-Low in tumor DNA (J) at Bat 26 microsatellite only (asterisk) (comparison with normal DNA not shown). Line 3 shows a group 3 unusual MMR-D: a colon cancer with retained expression of the four MMR proteins (K, L, M, N) but MSI-High (instability at three of five markers of Pentaplex-PCR) (O). Line 4 illustrates a group 4 unusual MMR-D: colon cancer in a patient with LS with MSH6 germline mutation, retained expression of MLH1 and PMS2 (O, P) but a clonal loss of expression of MSH2 and MSH6 (Q, R) and MSI-High (instability at the five markers of Pentaplex-PCR) (S).

Fig. 4. Distribution of the histological types among the different phenotypes of MMR-D.

(A) Distribution of the histological types among the four sub-groups of unusual MMR-D tumors, and (B) proportion of classical and unusual MMR-D in the different histological types of the entire MMR-D cohort comprising 491 CRC, 42 endometrial carcinomas, 26 non-colorectal GI cancers, and 26 “other types of tumors” (results are in percentages). MMR-D: MMR deficiency, Non:colorectal GI carcinoma: noncolorectal gastrointestinal carcinoma.

Fig. 5. Distribution of each group of unusual MMR-D tumors among the histological types.

Non-colorectal GI carcinoma: non-colorectal gastrointestinal carcinoma, MSI: microsatellite instability, MSS: microsatellite stable.

Fig. 6. NGS analysis.

Tumor mutational burden (TMB) is reported as mutations per megabase (mut/Mb) in the y-axis. Tumor types are indicated on the x-axis. The TMB value is mentioned above each graph bar. Tumors were divided into three categories according to TMB value: TMB-high: ≥20 mut/Mb, TMB-intermediate: between 6 and 19 mut/Mb and TMB-low: ≤5 mut/Mb. Even after excluding the only ultra-mutated tumor (501 mut/Mb) corresponding to LS associated with a somatic PolD1 mutation, the range of TMB values was highly variable, ranging from 4 to 145 mut/Mb. All cases, except for four cases, were MSI by NGS analysis. Stars above the graph bars indicate MSS (microsatellite stable) tumors; yellow stars represent MSS tumors both with NGS and MSI-PCR and brown stars indicate MSS tumors with NGS but MSI-H with MSI-PCR.