Impact of MLH1, PMS2, MSH2 and MSH6 alterations on tumor mutation burden (TMB) and PD-L1 expression in 1,057 microsatellite instability-high (MSI-H) tumors
Mohamed E. Salem, Axel Grothey, Edward Kim, Joanne Xiu, Richard M. Goldberg, W. Michael Korn, Anthony F. Shields, Alberto Puccini, Andreas Seeber, Jimmy J. Hwang, Philip A. Philip, Heinz-Josef Lenz, Derek Raghavan, and John L. Marshall
Immune checkpoint inhibitors (ICI) have revolutionized cancer therapy, resulting in remarkable and long-lasting clinical responses, although in a small subsets of patients.
Response to ICIs has been shown to correlate with MSI-high status  , TMB, and PD-L1 overexpression [2,3].
TMB-high and MSI-high rates, and PD-L1 overexpression vary significantly across solid tumors [4,5].
Although the majority of MSI-H tumors exhibit high TMB and PD-L1 overexpression , the precise relationship between TMB level and individual MMR gene alterations remains to be elucidated.
Herein, we aimed to assess the relationship between the level of TMB and the four MMR genes alterations in different types of cancers among 1057 MSI-H tumors.
Patients: MSI-H tumor samples profiled by Caris Life Sciences between 2015 and January of 2018 were analyzed.
Next generation sequencing was performed using the NextSeq platform (Illumina, Inc., San Diego, CA) o a 592-gen panel.
MMR protein expression was tested by IHC using antibody clones (MLH1, M1 antibody; MSH2, G2191129 antibody; MSH6, 44 antibody; PMS2, EPR3947 antibody [Ventana Medical Systems, Inc., Tucson, AZ, USA]). dMMRP was the complete loss of protein expression (0+ in 100% of cells)
Microsatellite instability (MSI) was examined using over 7,000 target microsatellite loci and compared to the reference genome . The number of altered microsatellite loci was counted for each sample. MSI-NGS results were compared with results from over 2,000 matching clinical cases analyzed with traditional PCR-based methods. (sensitivity of > 95% and specificity of > 99%.
Tumor mutational burden (TMB) was measured by counting all non-synonymous missense mutations found per tumor that had not been previously described as germline alterations (592 genes and 1.4 megabases [MB] sequenced per tumor). The threshold to define TMB-high was greater than or equal to 17 mutations/MB and was established by comparing TMB with MSI by fragment analysis in CRC cases
PD-L1 IHC analysis was performed using the primary antibody SP142 (Spring Biosciences). The staining was regarded as positive if its intensity on the membrane of the tumor cells was ≥ 2+ and the percentage of positively stained cells was 5%.
Statistical Analysis ANOVA and chi-square tests were used for comparisons.
The prevalence of MMR genes alterations differs among different tumor types. For instance, MSH2 and MSH6 are more frequently altered in CRC than endometrial cancers.
MSH2 and/or MSH6 alterations are associated with a significantly higher TMB than MLH1 and/or PMS2 across several cancer types.
TMB varies significantly across MSI-H tumors. MSI-H CRCs carry the highest TMB compared to MSI-H endometrial cancers and others MSI-H solid tumors. Furthermore, left side CRC tumors exhibit higher TMB than right side tumors.
There is a significant association between the altered microsatellite loci and the level of TMB. Furthermore, the association between MSI-H, TMB status and microsatellite loci is tumor type specific.
PD-L1 overexpression was seen at a significantly higher frequency in tumors with MSH2 (23%) compared to MSH6 (16%), MMLH1 (16%), or PMS (14%) alterations in all tumor types.
Our findings suggest that alteration in specific MMR gene may have different impact on MS indels, frameshift mutations or microsatellite loci length and TMB level.
Further investigations into the biology, etiology and optimal treatment of MSI-H tumors are still appropriate.
Additional analysis to assess the correlation between specific MMR gene alterations and response to checkpoint inhibitors is underway.