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 [1] , 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 [6], 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.

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