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Microsatellite Instability and Metastatic Colorectal Cancer – A Clinical Perspective

Approximately 4-5% of patients with metastatic colorectal cancer (mCRC) have mismatch repair deficient (dMMR)/microsatellite instability-high (MSI-H) tumours. These tumours present challenges in the clinical practice due to variant response to fluoropyrimidine-based chemotherapy and, perhaps, also non-immunologic targeted therapies. Recently, a breakthrough in the treatment of dMMR/MSI-H mCRC has been achieved with several clinical trials showing dramatic long-term benefit of immunotherapy using checkpoint inhibitors. Nevertheless, several questions remain regarding the optimisation of immunotherapy regimens and the use of biomarkers to identify populations set to derive the greatest benefit from immunotherapy. Combination regimens and/or the use of immunotherapy as a maintenance after induction non-immunologic systemic therapy may be the way forward to improve outcomes.


INTRODUCTION
Colorectal cancer (CRC) is one of the main causes of morbidity and mortality, representing approximately 10% of all cancers diagnosed worldwide (1). Mismatch repair deficient (dMMR)/ microsatellite instability-high (MSI-H) colorectal tumours are identified in approximately 15% of CRC patients. Due to relatively favourable prognosis in comparison to the mismatch repair proficient (pMMR) CRC, the proportion of these tumours decreases to approximately 4-5% in patients with metastatic CRC (mCRC) (2).
Deficiency in protein products of MSH2, MLH1 and MSH6 genes results in impaired detection of mismatched and unpaired bases, leading to the expression of abnormal proteins that may be recognized as neoantigens by the immune system. Additionally, most likely due to Darwinian selection pressure, the deficiency causes failure of apoptosis upon detection of critical DNA damage (2,3).

NON-IMMUNOLOGIC THERAPIES FOR dMMR/MSI-H CRC
dMMR/MSI-H status has been associated with the lack of benefit from fluoropyrimidines in stage II and III CRC (4)(5)(6). Nevertheless, the benefit from oxaliplatin-containing regimens remains unchanged making them the standard of care in patients with CRC requiring adjuvant chemotherapy (6)(7)(8). In mCRC the prognosis and response to chemotherapy in patients with dMMR/MSI-H tumours is significantly influenced by the presence of somatic BRAF mutations which are associated with poor prognosis (9,10). BRAF V600E mutations are causally linked to MLH1 promoter hypermethylation leading to deficiency in MLH1 and PMS2 proteins, the most common cause of dMMR/MSI-H phenotype in patients without Lynch syndrome (11)(12)(13).
Monoclonal antibodies against the vascular endothelial growth factor (VEGF) or, in patients with KRAS/NRAS wildtype tumours, against the epidermal growth factor receptor (EGFR) are commonly added to chemotherapy in mCRC. dMMR/MSI-H patients receiving the VEGF antagonist bevacizumab achieved longer overall survival (OS) compared to patients treated with the EGFR inhibitor cetuximab in the CALGB/SWOG 80405 clinical trial (14). Similar findings were recently reported in a retrospective study (15).

Pembrolizumab: A Phase 2 Study
Due to the abundance and diversity of neoantigens generated in the absence of functional mismatch repair, immunotherapy using antagonists of the programmed death-1 (PD-1) receptor or its ligand PD-L1), with or without antagonists of the CTLA4 receptor, is an attractive option for patients with dMMR/MSI-H tumours. The pivotal phase 2 study establishing the efficacy of immunotherapy for dMMR/MSI-H tumours was published in 2015 by Le and collaborators and included 10 evaluable patients with colorectal cancer (16). Pembrolizumab was given at a dose of 10 mg/kg biweekly. At 20 weeks, the progression-free survival (PFS) rate in these patients was 78%. The overall response rate (ORR) reached 40% while no responses were seen in patients with pMMR mCRC. In an expanded cohort of 40 patients with mCRC, 2-year PFS and OS were 59 months (95% CI 44 -78 months) and 72 months respectively (95% confidence interval [CI] 58 -89 months). Five patients (12%) achieved complete responses (17).
In another cohort of pre-treated patients, patients received nivolumab 3 mg/kg and ipilimumab 1 mg/kg every 3 weeks for four doses followed by biweekly nivolumab 3 mg/kg (18,19). The majority of patients (76%) had received two or more previous systemic therapies. BRAF mutation was present in 25% and KRAS mutation in 37% of patients' tumours. According to 4-year followup data, the overall response rate (ORR) reached 65%, with 13% of patients achieving complete response. The ORR was similar in patients with BRAFwt/KRASwt tumours and patients with tumours harbouring either mutation. As many as 53% of patients (95% CI 43-62%) were free of progression at 48 months. The OS rate at 48 months was excellent reaching 70.5% (95% CI 61.4-77.9%), far surpassing the expected results for conventional therapy where median OS would fluctuate around 19 months (10).
Finally, results of the cohort receiving nivolumab with ipilimumab as the first-line therapy were published recently (21). Here, 45 patients without previous systemic therapy for mCRC were treated with another combination regimen consisting of nivolumab 3 mg/kg once every 2 weeks and ipilimumab 1 mg/kg once every six weeks until progression or unacceptable toxicity. Interestingly, the 24-month PFS rate was higher for patients with KRAS mutated tumours compared to BRAFwt/KRASwt tumours, reaching 87.5% (95% CI, 38.7 to 98.1) and 68.4% (95% CI, 35.9 to 86.8), respectively. The 24month OS rate was 79.4% (95% CI, 64.1 to 88.7).

Pembrolizumab: A Phase 3 Study (KEYNOTE-177)
KEYNOTE-177 is an ongoing open-label randomised multicentric phase 3 trial for patients without previous systemic treatment for mCRC (22,23). So far, 307 patients with dMMR/MSI-H mCRC have been randomised to either pembrolizumab (200mg in 3-weekly cycle for a maximum of 35 cycles) or fluoropyrimidine-based chemotherapy with or without non-immunologic targeted agents including bevacizumab or cetuximab. Crossover was allowed for patients in the chemotherapy arm following disease progression. Treatment in the control arm consisted mostly of mFOLFOX6 with bevacizumab (44.8%) or FOLFIRI with bevacizumab (25.2%). Only 11.2% of control arm patients received first-line cetuximab with backbone chemotherapy.
Two-thirds of patients enrolled in the KEYNOTE -177 study had right-sided tumours. As expected, the proportion of patients with BRAF V600E mutation in the dMMR/MSI-H population was high (28.1% in the pembrolizumab arm). Unfortunately, molecular analysis data for KRAS/NRAS and BRAF V600E were not available for 23.8% patients in the study. The median PFS reached 16 months for patients allocated to pembrolizumab (95% CI 5.4-38.1 months) compared to 8.2 months in patients on chemotherapy (95% CI 6.1-10.2 months). The PFS curve seemed to plateau approximately 6 months after the treatment start and about 40% of patients experienced long-term responses. According to a subgroup analysis, patients over 70 years of age did not seem to derive as large benefit from immunotherapy as did younger patients. Of note, patients with KRAS/NRAS mutated tumours had numerically superior survival if allocated to the control arm. On the other hand, PFS benefit of immunotherapy was nearly identical for patients with BRAF wild-type tumours and those with BRAF V600E tumours, although arguably, the control arm regimens were inadequate for this subpopulation and a triplet chemotherapy with bevacizumab should be more effective (24).
The response rate was higher in the pembrolizumab arm with 13.1% patients reaching complete response. Furthermore, responses were long-lasting in the immunotherapy arm with 83.5% of responses ongoing at 24 months.
The final survival analysis was presented at the 2021 American Society of Clinical Oncology (23). OS at 36 months was 61% in the pembrolizumab arm compared to 50% in the control arm (95% hazard ratio [HR] 0.74, 95% CI 0.53-1.03). There was, however, a massive crossover with 60.4% of patients from the control arm eventually receiving anti-PD-1/PD-L1 therapies, and some additional individuals receiving other checkpoint inhibitors (23).

BIOMARKERS OF IMMUNOTHERAPY EFFICACY IN dMMR/MSI-H mCRC
The marked difference in the proportion of dMMR/MSI-H tumours between early and advanced CRC stages points to a strong role of immunoediting in the progression of these tumours. The activation of immunosuppressive pathways linked to PD-1 and CTLA4 protects these tumours from destruction by the immune system and could explain the high efficacy of checkpoint inhibitors in this setting (25). However, only 40-50% of patients with dMMR/MSI-H mCRC derive long-term benefit from the treatment. Possible mechanisms of intrinsic and acquired resistance have been comprehensively reviewed by Sahin et al. (26).
PD-L1 expression was not correlated with benefit from immunotherapy in patients with dMMR/MSI-H mCRC or in the tumour-agnostic trial by Le et al. (16,18,19).
The efficacy of checkpoint inhibitors in dMMR/MSI-H patients was not associated with BRAF V600E mutation which is present in approximately 30-40% of dMMR/MSI-H mCRC (19,23). On the other hand, the apparent lower efficacy of immunotherapy in patients with KRAS/NRAS mutated tumours treated with anti-PD-1 monotherapy in the KEYNOTE -177 and CheckMate-142 studies, contrasted with a preserved benefit of combined immunotherapy in CheckMate-142, deserved further study as the subgroups were too small for a valid conclusion (19)(20)(21)23).
Tumour mutation burden (TMB) was a better biomarker for the efficacy of immunotherapy than the absence of an MMRrelated protein. It directly correlated with the number of neoantigens (27). Although the great majority of dMMR/MSI-H tumours had high TMB, some dMMR/MSI-H tumours with low TMB have been identified, and they exhibited resistance to therapy with checkpoint inhibitors (28,29).
Salem et al. compared the TMB according to the type of MMR deficiency. In a heterogeneous cohort of cancers, the largest subgroups comprised endometrial and colorectal tumours. The loss of MSH2/MSH6 was associated with approximately double rate of mutations compared to loss of MLH1/PMS2 (30).
Approximately 3% of colorectal tumours classified as microsatellite stable (MSS) have high TMB, mostly associated with the mutation of polymerase epsilon (POLE) or variant MSH2 (28,31,32). These mutations resulted in a high sensitivity to checkpoint inhibitors. Clinical trials enrolling patients with POLEmutated mCRC along those with dMMR/MSI-H mCRC are ongoing (NCT03150706 and NCT03435107, Table 1).
There is growing evidence that the genomic instability in tumours with mismatch-repair deficiency may result in the formation of gene fusions. Cocco and collaborators found that potentially targetable fusions of oncogenes such as NTRK, BRAF, RET, FGFR, ROS1 and ALK were present in 5% of dMMR/MSI-H CRC as compared to only 0.4% of MSS CRC cases. Particular enrichment was seen in the subgroup of dMMR/MSI-H B R A F w t / R A S w t C R C c a s e s w i t h M L H 1 p r o m o t e r hypermethylation where the occurrence of these aberrations was as high as 42% (13). Vanǩova et al. confirmed these findings identifying oncogenic gene fusions in as many as 9 of 23 colorectal tumours that were dMMR/MSI-H due to MLH1 promoter hypermethylation and simultaneously BRAFwt/ KRASwt. The fusions involved NTRK, ALK and BRAF genes (33). These studies suggest that BRAFwt/KRASwt tumours with MLH1 promoter hypermethylation should also be tested for the presence of oncogene fusions.
It has been shown that dMMR-MSI-H colorectal cancer was associated with specific changes in gut microbiome. Fusobacterium nucleatum was enriched in dMMR/MSI-H CRC and associated with lower number of tumour-infiltrating lymphocytes while the opposite has been suggested for MSS colorectal cancer (34). However, somewhat counterintuitively, F. nucleatum has been found to be positively associated with response to checkpoint inhibitors, enhancing the expression of PD-1 and PD-L1 and activating the stimulator of interferon genes (STING) signalling (35). The activity of interferon-g pathway was also associated with response in dMMR/MSI-H CRC in a recent transcriptomics study while VEGF-A expression characterised poor responders (36).
Other putative molecular markers of response to checkpoint inhibitors have been proposed, in particular b2-microglobulin mutations (37). Nevertheless, a recent study failed to corroborate their association with treatment outcomes (38).
Finally, peritoneal involvement manifesting with ascites is associated with resistance to immune checkpoint blockade in dMMR/MSI-H gastrointestinal cancers and markedly inferior patient outcomes, even compared to peritoneal disease without ascites (39).
The search for reliable predictors is ongoing and detailed reviews of potential biomarkers for immunotherapy of colorectal cancer are available (40,41).

CONCLUSIONS
Immunotherapy is the preferred treatment option for dMMR/ MSI-H CRC resulting in long-term treatment responses in approximately 40-50% of patients. As most non-responders will experience progression within 6-8 months of the treatment initiation, the obvious clinical strategies that have been successfully applied in other tumours with similar early treatment characteristics include the combinations of checkpoint inhibitors with chemotherapy and the use of checkpoint inhibitors as a maintenance therapy in non-progressing patients after the induction (42,43) In both situations, inclusion of a VEGF-targeted therapy in the regimen appears beneficial. Multiple clinical trials are ongoing and our knowledge on the use of immunotherapy in this setting will keep expanding in the following years ( Table 1).

AUTHOR CONTRIBUTIONS
TB has designed and written the manuscript. The author confirms being the sole contributor of this work and has approved it for publication.

FUNDING
The work was supported by grant TN-00064190 from the Ministry of Health, Czech Republic to the Thomayer University Hospital. Publication fee was covered by unrestricted grants from Servier, Roche, AstraZeneca, and Bristol Myers Squibb to the First Faculty of Medicine, Charles University, Prague, Czech Republic. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.

ACKNOWLEDGMENTS
The author would like to thank Dr Alena Novakova for critical reading of the manuscript and helpful comments.