Tumor Mutational Burden and Mismatch Repair Deficiency Discordance as a Mechanism of Immunotherapy Resistance

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  • 1 Department of Medicine, Memorial Sloan Kettering Cancer Center;
  • 2 Tri-Institutional Program in Computational Biology and Medicine, Weill Cornell Medical College; and
  • 3 Marie-Josée and Henry R. Kravis Center for Molecular Oncology,
  • 4 Department of Epidemiology and Biostatistics,
  • 5 Human Oncology and Pathogenesis Program, and
  • 6 Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York.

Lynch syndrome is a heritable cancer syndrome caused by a heterozygous germline mutation in DNA mismatch repair (MMR) genes. MMR-deficient (dMMR) tumors are particularly sensitive to immune checkpoint inhibitors, an effect attributed to the higher mutation rate in these cancers. However, approximately 15% to 30% of patients with dMMR cancers do not respond to immunotherapy. This report describes 3 patients with Lynch syndrome who each had 2 primary malignancies: 1 with dMMR and a high tumor mutational burden (TMB), and 1 with dMMR but, unexpectedly, a low TMB. Two of these patients received immunotherapy for their TMB-low tumors but experienced no response. We have found that not all Lynch-associated dMMR tumors have a high TMB and propose that tumors with dMMR and TMB discordance may be resistant to immunotherapy. The possibility of dMMR/TMB discordance should be considered, particularly in less-typical Lynch cancers, in which TMB evaluation could guide the use of immune checkpoint inhibitors.

Background

Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer, is a heritable cancer syndrome predisposing to colorectal and other cancers, including endometrial, gastric, and ovarian cancer.13 Lynch syndrome is caused by an autosomal dominant heterozygous germline mutation in the DNA mismatch repair (MMR) genes. MMR deficiency (dMMR) increases the likelihood of acquiring somatic genetic mutations, particularly in short repetitive sequences, leading to varying lengths of these regions, termed microsatellite instability.1,4 However, dMMR is a double-edged sword, because although it predisposes to malignancy, it leads to cancers with more mutations, particularly frameshift alterations, which are associated with “non-self” immunogenic antigens, high lymphocyte infiltration, and immune response.5,6 Because of the high immunogenicity of these tumors, microsatellite instability–high (MSI-H) or dMMR cancers have been shown to respond well to immunotherapy, in contrast to their microsatellite-stable (MSS) counterparts.710 However, despite the promising response of immunotherapy in dMMR cancers, approximately 15% to 30% of patients do not derive significant benefit with single or combination immunotherapy.7,8

This report describes 3 patients with Lynch syndrome who developed a dMMR MSI-H cancer with a high tumor mutational burden (TMB) and a second primary malignancy that was dMMR but with a low TMB. All tumors were assessed using a next-generation sequencing (NGS) panel comprising >300 cancer-associated genes.11 MSI status was evaluated with the MSIsensor algorithm, with MSI-H status defined by MSIsensor scores ≥10.12 This cutoff for MSI status has been validated against MSI PCR testing and/or MMR immunohistochemistry performed on 138 colorectal cancers (114 MSS and 24 MSI-H) and 40 uterine endometrioid cancers (25 MSS, 15 MSI-H), with a concordance of 99.4%.12

Case Reports

The first patient was a man aged 43 years who initially presented with multiple primary colon masses. At total colectomy, the most advanced lesion was stage IIB. Pathology was notable for tumor-infiltrating lymphocytes (TILs) and the absence of MLH1 and PMS2 staining on immunohistochemistry (Figure 1A). Germline genetic testing revealed a deleterious MLH1 likely pathogenic variant confirming Lynch syndrome, and tumor genomic analysis showed an MSI-H phenotype with an MSIsensor score of 40.67 and a TMB of 54.4 mutations per megabase (mt/Mb). This tumor had driver mutations in APC and TP53, a genomic profile common for colorectal tumors (Figure 1B). Three years later, imaging showed a pancreatic tail mass consistent with a new primary, which was completely resected. Surgical pathology on the resected mass showed a poorly differentiated neuroendocrine carcinoma (60% of tumor cells staining for the proliferation marker Ki-67). Immunohistochemistry confirmed the loss of MLH1 and PMS2 in the neuroendocrine carcinoma (Figure 1A). Surprisingly, despite the patient’s Lynch syndrome status, NGS revealed an MSS tumor with an MSIsensor score of 1.8 and a low TMB (9.7 mt/Mb; Figure 1B). Unlike in the patient’s colon tumor, increased TILs were not seen in the neuroendocrine carcinoma. This second tumor also had a completely different set of somatic mutations, with a truncating RB1 driver mutation consistent with neuroendocrine carcinoma. Unfortunately, within 6 months after resection, the patient was found to have multiple hypervascular metastatic lesions in the liver, consistent with his pancreatic primary. He was started on pembrolizumab given the dMMR status of his primary tumor. His initial scan after 3 cycles showed a mixed response; however, a subsequent scan showed definitive disease progression with the development of new lesions.

Figure 1.
Figure 1.

Comparison of histopathologic and genomic features across paired primary dMMR tumors. (A) H&E and IHC stains of each tumor pair. In patients 1 and 2, IHC showed normal staining for MSH2 and MSH6 and loss of staining for MLH1 and PMS2 in both tumors. In patient 3, IHC showed normal staining for MLH1 and PMS2 and loss of staining for MSH2 and MSH6 in both tumors. (B) Spectrum of oncogenic mutations and differences in MSIsensor scores, TMB, and FGA, along with depiction of MMR protein status from IHC. The paired tumors from the 3 patients appear genomically distinct, harboring few shared oncogenic mutations. The patients’ secondary primary malignancies all have lower TMBs and MSIsensor scores.

Abbreviations: CHOL, cholangiocarcinoma; COAD, colorectal adenocarcinoma; dMMR, deficient mismatch repair; FGA, fraction of genome altered; H&E, hematoxylin-eosin; IHC, immunohistochemistry; MMR, mismatch repair; MSIsensor, microsatellite instability sensor; OCCC, ovarian clear cell carcinoma; PNEC, pancreatic neuroendocrine carcinoma; TMB, tumor mutational burden; UCEC, uterine corpus endometrial carcinoma.

Citation: Journal of the National Comprehensive Cancer Network J Natl Compr Canc Netw 19, 2; 10.6004/jnccn.2020.7680

The second patient was a man aged 54 years with 2 resected colon primaries that were dMMR with loss of MLH1/PMS2 on immunohistochemistry (Figure 1A) and germline NGS confirming an MLH1 likely pathogenic variant. He later presented with a new liver lesion on surveillance imaging. Before it was clear that the patient had a second primary, he received ipilimumab and nivolumab for presumed liver metastasis from colon cancer, with clear progression (the mass grew from 5.7 × 5.4 cm to 9.0 × 8.4 cm) after 3 cycles of therapy. Biopsy of the mass showed a morphologically different tumor from the prior colon cancer that was poorly differentiated, suggestive of a new gastrointestinal primary. Subsequent resection of the mass showed a cholangiocarcinoma with positive staining for albumin in situ hybridization. The cholangiocarcinoma was MMR-deficient (absent MLH1/PMS2) and MSI-H, with an MSIsensor score of 13.51 but a low TMB of 7 mt/Mb (Figure 1B). The colorectal tumor had driver mutations in APC, SOX9, and NF1. No somatic mutations were shared between the 2 tumors from this patient. Despite treatment using ipilimumab and nivolumab, no lymphocytic infiltrate was seen in the resected tumor.

The third patient was a woman aged 40 years with an MSH2 pathologic germline mutation who had synchronous primary endometrial and ovarian cancers found on screening. Both tumors showed a loss of MSH2/MSH6 by immunohistochemistry (Figure 1A). The endometrial adenocarcinoma was evaluated as MSI-intermediate (MSIsensor score, 7.16) with a high TMB of 35.1 mt/Mb. However, sequencing of the patient’s ovarian clear cell carcinoma showed an MSS phenotype (MSIsensor score, 0.12) and a low TMB (7.9 mt/Mb; Figure 1B). Hematoxylin-eosin stains identified increased TILs in the primary endometrial adenocarcinoma, compared with the ovarian clear cell carcinoma without TILs. Consistent with what is expected in these cancer types, both tumors from this patient harbored an ARID1A mutation; however, these mutations were in different positions and private to each of their respective tumors.

Discussion

We have reported 3 patients with Lynch syndrome who each developed 2 primary tumors with confirmed dMMR status. Unexpectedly, despite confirmed dMMR within each tumor pair, one tumor had an expected high TMB, but the other had a lower TMB. In agreement with their TMB status, the 2 patients treated with immunotherapy for their TMB-low tumors did not experience a response to the therapy. Using FACETs, an allele-specific copy number algorithm, we assessed ploidy and loss of heterozygosity of MLH1 in the paired cancers from the first 2 patients discussed herein.13 Both paired cancers showed a loss of the normal MMR allele. However, the germline MLH1 mutant allele was enriched in the first TMB-high cancer compared with the second cancer.

Although research has shown that patients with Lynch syndrome can develop sporadic cancers with retained MMR machinery, Lynch syndrome–associated tumors with confirmed dMMR but with a discordantly low TMB have not been well described. It is known that some MMR variants are associated with dMMR on immunohistochemistry but not MSI-H—but in this case, the same germline MMR variant in each tumor pair led to an MSI-H and TMB-high phenotype in one tumor and a TMB-low phenotype in the other tumor.14 Consistent with our data, Georgiadis et al15 reported that although TMB and MSI status were highly correlated, TMB and MSI discordance did occur, and 6 of 7 MSI-H tumors with progressive disease on immunotherapy had low TMB. Similarly, Schrock et al16 found that within MSI-H colorectal cancer, TMB was predictive of immunotherapy response.

We acknowledge that the immunogenic response in Lynch syndrome tumors is complicated because Lynch syndrome polyps with a low mutational rate still elicit an immune response.17 In the 3 patients analyzed herein, high levels of TILs were not detected in the TMB-low tumors, in contrast to the TMB-high tumors. In addition, in patient 2, no immune activation was seen after the dual immune checkpoint blockade.

Conclusions

These case reports reveal an important practical clinical point: patients with Lynch syndrome can develop dMMR tumors with a low TMB. Although we cannot draw general conclusions from a 3-person case series, these results, in combination with previous evidence showing that TMB is associated with a response to immunotherapy within MSI-H tumors, suggest that dMMR tumors with a low TMB would not respond to immunotherapy.15,16 We propose that dMMR/TMB discordance should be considered in patients with Lynch syndrome, particularly those who develop secondary, less-typical Lynch syndrome cancers, and that TMB should be evaluated in these tumors to inform the use of immune checkpoint inhibitors.

References

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    Sinicrope FA. Lynch syndrome–associated colorectal cancer. N Engl J Med 2018;379:764773.

  • 2.

    Watson P, Vasen HFA, Mecklin JP, . The risk of extra-colonic, extra-endometrial cancer in the Lynch syndrome. Int J Cancer 2008;123:444449.

  • 3.

    Latham A, Srinivasan P, Kemel Y, . Microsatellite instability is associated with the presence of Lynch syndrome pan-cancer. J Clin Oncol 2019;37:286295.

  • 4.

    Lynch HT, Snyder CL, Shaw TG, . Milestones of Lynch syndrome: 1895-2015. Nat Rev Cancer 2015;15:181194.

  • 5.

    Buckowitz A, Knaebel HP, Benner A, . Microsatellite instability in colorectal cancer is associated with local lymphocyte infiltration and low frequency of distant metastases. Br J Cancer 2005;92:17461753.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 6.

    Schwitalle Y, Kloor M, Eiermann S, . Immune response against frameshift-induced neopeptides in HNPCC patients and healthy HNPCC mutation carriers. Gastroenterology 2008;134:988997.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 7.

    Le DT, Uram JN, Wang H, . PD-1 blockade in tumors with mismatch-repair deficiency. N Engl J Med 2015;372:25092520.

  • 8.

    Overman MJ, Lonardi S, Wong KYM, . Durable clinical benefit with nivolumab plus ipilimumab in DNA mismatch repair-deficient/microsatellite instability-high metastatic colorectal cancer. J Clin Oncol 2018;36:773779.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 9.

    Marabelle A, Le DT, Ascierto PA, . Efficacy of pembrolizumab in patients with noncolorectal high microsatellite instability/mismatch repair–deficient cancer: results from the phase II KEYNOTE-158 study. J Clin Oncol 2020;38:110.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 10.

    Le DT, Durham JN, Smith KN, . Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science 2017;357:409413.

  • 11.

    Cheng DT, Mitchell TN, Zehir A, . Memorial Sloan Kettering-integrated mutation profiling of actionable cancer targets (MSK-IMPACT): a hybridization capture-based next-generation sequencing clinical assay for solid tumor molecular oncology. J Mol Diagn 2015;17:251264.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 12.

    Middha S, Zhang L, Nafa K, . Reliable pan-cancer microsatellite instability assessment by using targeted next-generation sequencing data. JCO Precis Oncol 2017;2017:117.

    • Search Google Scholar
    • Export Citation
  • 13.

    Shen R, Seshan VE. FACETS—allele-specific copy number and clonal heterogeneity analysis tool for high-throughput DNA sequencing. Nucleic Acids Res 2016;44:16.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Cerretelli G, Ager A, Arends MJ, . Molecular pathology of Lynch syndrome. J Pathol 2020;250:518531.

  • 15.

    Georgiadis A, Durham JN, Keefer LA, . Noninvasive detection of microsatellite instability and high tumor mutation burden in cancer patients treated with PD-1 blockade. Clin Cancer Res 2019;25:70247034.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16.

    Schrock AB, Ouyang C, Sandhu J, . Tumor mutational burden is predictive of response to immune checkpoint inhibitors in MSI-high metastatic colorectal cancer. Ann Oncol 2019;30:10961103.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17.

    Chang K, Taggart MW, Reyes-Uribe L, . Immune profiling of premalignant lesions in patients with Lynch syndrome. JAMA Oncol 2018;4:10851092.

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Submitted May 16, 2020; accepted for publication October 27, 2020.

Disclosures: The authors have not received any financial consideration from any person or organization to support the preparation, analysis, results, or discussion of this article.

Funding: Research reported in this article was supported by the NIH under award number P30 CA 008748. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Work for this article was also supported by the American Cancer Society (134065-PF-19-125-01-CSM).

Correspondence: Rona Yaeger, MD, Department of Medicine, Memorial Sloan Kettering Cancer Center, 300 East 66th Street, 10th Floor, New York, NY 10065. Email: yaegerr@mskcc.org
  • View in gallery

    Comparison of histopathologic and genomic features across paired primary dMMR tumors. (A) H&E and IHC stains of each tumor pair. In patients 1 and 2, IHC showed normal staining for MSH2 and MSH6 and loss of staining for MLH1 and PMS2 in both tumors. In patient 3, IHC showed normal staining for MLH1 and PMS2 and loss of staining for MSH2 and MSH6 in both tumors. (B) Spectrum of oncogenic mutations and differences in MSIsensor scores, TMB, and FGA, along with depiction of MMR protein status from IHC. The paired tumors from the 3 patients appear genomically distinct, harboring few shared oncogenic mutations. The patients’ secondary primary malignancies all have lower TMBs and MSIsensor scores.

    Abbreviations: CHOL, cholangiocarcinoma; COAD, colorectal adenocarcinoma; dMMR, deficient mismatch repair; FGA, fraction of genome altered; H&E, hematoxylin-eosin; IHC, immunohistochemistry; MMR, mismatch repair; MSIsensor, microsatellite instability sensor; OCCC, ovarian clear cell carcinoma; PNEC, pancreatic neuroendocrine carcinoma; TMB, tumor mutational burden; UCEC, uterine corpus endometrial carcinoma.

  • 1.

    Sinicrope FA. Lynch syndrome–associated colorectal cancer. N Engl J Med 2018;379:764773.

  • 2.

    Watson P, Vasen HFA, Mecklin JP, . The risk of extra-colonic, extra-endometrial cancer in the Lynch syndrome. Int J Cancer 2008;123:444449.

  • 3.

    Latham A, Srinivasan P, Kemel Y, . Microsatellite instability is associated with the presence of Lynch syndrome pan-cancer. J Clin Oncol 2019;37:286295.

  • 4.

    Lynch HT, Snyder CL, Shaw TG, . Milestones of Lynch syndrome: 1895-2015. Nat Rev Cancer 2015;15:181194.

  • 5.

    Buckowitz A, Knaebel HP, Benner A, . Microsatellite instability in colorectal cancer is associated with local lymphocyte infiltration and low frequency of distant metastases. Br J Cancer 2005;92:17461753.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 6.

    Schwitalle Y, Kloor M, Eiermann S, . Immune response against frameshift-induced neopeptides in HNPCC patients and healthy HNPCC mutation carriers. Gastroenterology 2008;134:988997.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 7.

    Le DT, Uram JN, Wang H, . PD-1 blockade in tumors with mismatch-repair deficiency. N Engl J Med 2015;372:25092520.

  • 8.

    Overman MJ, Lonardi S, Wong KYM, . Durable clinical benefit with nivolumab plus ipilimumab in DNA mismatch repair-deficient/microsatellite instability-high metastatic colorectal cancer. J Clin Oncol 2018;36:773779.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 9.

    Marabelle A, Le DT, Ascierto PA, . Efficacy of pembrolizumab in patients with noncolorectal high microsatellite instability/mismatch repair–deficient cancer: results from the phase II KEYNOTE-158 study. J Clin Oncol 2020;38:110.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 10.

    Le DT, Durham JN, Smith KN, . Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science 2017;357:409413.

  • 11.

    Cheng DT, Mitchell TN, Zehir A, . Memorial Sloan Kettering-integrated mutation profiling of actionable cancer targets (MSK-IMPACT): a hybridization capture-based next-generation sequencing clinical assay for solid tumor molecular oncology. J Mol Diagn 2015;17:251264.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 12.

    Middha S, Zhang L, Nafa K, . Reliable pan-cancer microsatellite instability assessment by using targeted next-generation sequencing data. JCO Precis Oncol 2017;2017:117.

    • Search Google Scholar
    • Export Citation
  • 13.

    Shen R, Seshan VE. FACETS—allele-specific copy number and clonal heterogeneity analysis tool for high-throughput DNA sequencing. Nucleic Acids Res 2016;44:16.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Cerretelli G, Ager A, Arends MJ, . Molecular pathology of Lynch syndrome. J Pathol 2020;250:518531.

  • 15.

    Georgiadis A, Durham JN, Keefer LA, . Noninvasive detection of microsatellite instability and high tumor mutation burden in cancer patients treated with PD-1 blockade. Clin Cancer Res 2019;25:70247034.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16.

    Schrock AB, Ouyang C, Sandhu J, . Tumor mutational burden is predictive of response to immune checkpoint inhibitors in MSI-high metastatic colorectal cancer. Ann Oncol 2019;30:10961103.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17.

    Chang K, Taggart MW, Reyes-Uribe L, . Immune profiling of premalignant lesions in patients with Lynch syndrome. JAMA Oncol 2018;4:10851092.

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