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The impact of microsatellite stability status in colorectal cancer
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Contents lists available at ScienceDirect
Curr Probl Cancer journal homepage: www.elsevier.com/locate/cpcancer
The impact of microsatellite stability status in colorectal cancer Ruby Gupta a,∗, Surajit Sinha b, Rabindra N. Paul c a
Keystone Rural Health Center, Chambersburg, PA National Institutes of Health, MD c Atlantic General Hospital, MD b
a r t i c l e
i n f o
a b s t r a c t Several forms of genomic instability are known to drive the development of colorectal cancer (CRC). Chromosomal instability is the most common type found in 85% of the CRC, while 15% patients have microsatellite instability (MSI). MSI tumors are the subset of CRC that are characterized by dysfunction of mismatch repair genes (MMR) causing failure to repair errors in repetitive DNA sequences called microsatellites. Twelve percent of MSI tumors are acquired, caused by methylation-associated silencing of a gene that encodes a DNA MMR protein, while the remaining 3% have germline mutations in one of the MMR genes (Lynch syndrome). The identification of microsatellite stability status is clinically important as studies have revealed that MSI tumors have a better stage-adjusted survival compared with microsatellite stable tumors, and they respond differently to 5FU-based adjuvant chemotherapy depending on this status. There is recent success of immunotherapy (mainly anti-PD1 drugs) in metastatic CRC with MMR dysfunction that has led to the initiation of multiple trials based on immune checkpoint inhibitors. Additionally, it is important to identify patients with Lynch syndrome so that it can guide the frequency of surveillance of CRCs and recommendations of prophylactic surgery. Even though TNM staging remains a key determinant of patient prognosis and guides management in patients with CRC, molecular tumor heterogeneity contributes to significant variability in clinical outcomes despite the same disease
∗
Correspondence to: Ruby Gupta, 830, 5th Avenue, suite 201, Chambersburg, PA 17201. E-mail address: [email protected] (R. Gupta).
https://doi.org/10.1016/j.currproblcancer.2018.06.010 0147-0272/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: R. Gupta et al., The impact of microsatellite stability status in colorectal cancer, Current Problems in Cancer (2018), https://doi.org/10.1016/j.currproblcancer.2018.06.010
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stage; therefore, it is vital to know the type of genomic instability pathway that the tumor harbors. In this article, we discuss the unique genetic, pathologic, and clinical characteristics of microsatellite unstable (MSI) and stable CRC (MSS), their predictive value in directing the management with conventional chemotherapy or novel-targeted agents, and their prognostic significance in patient outcomes. © 2018 Elsevier Inc. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular landscape of colorectal cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunologic landscape of colorectal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . Microsatellite instability—Prognostic value . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagnosis of microsatellite instability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mismatch repair protein by immunohistochemistry . . . . . . . . . . . . . . . . . . . . Microsatellite instability testing by PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microsatellite instability and response to chemotherapy (predictive value) . Microsatellite instability and response to immunotherapy. . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Supplementary materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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2 2 4 4 5 5 6 7 9 11 11 11
Introduction Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females.1 In the United States, approximately 140,250 new cases of large bowel cancer are diagnosed annually, of which 97,220 are colon and the remainder are rectal cancers. CRC contributes to approximately 8% mortality among all cancer deaths in the United States with reported 50,630 deaths in a year.2 In the United States, CRC incidence rates have declined about 2.5%-4% per year over the last 15 years; however, CRC incidence is increasing in the age group under 50, while it is decreasing in older groups.2
Molecular landscape of colorectal cancer The acquisition of genomic instability is a crucial feature in CRC development, and there are at least 2 distinct pathways in CRC pathogenesis: the chromosomal instability (CIN) pathway (85%) and microsatellite instability (MSI) pathway (15%).3 , 4 CIN tumors are caused by mutations in genes leading to chromosomal aneuploidy, loss of heterozygosity, and structural chromosomal rearrangements.3 , 5 Coupled with the karyotypic abnormalities observed in CIN tumors are the accumulation of a characteristic set of mutations in specific tumor suppressor genes and oncogenes that activate pathways critical for CRC initiation and progression.3 , 5 MSI tumors are known to arise from inactivation of 1 of the 4 mismatch repair (MMR) genes: MSH2, MLH1, MSH6, and PMS2.6-9 During normal DNA replication, proficient MMR genes detect the DNA mismatch error and MLH1 and/or PMS2 heterodimer helps in the excision of mismatch errors and the formation of new, corrected DNA strands. The human genome is known to have >10 0,0 0 0 areas of short tandem repeat sequences called microsatellites which are susceptible to replication “slippage” and heavily dependent on the MMR system for repair.10 Deficiency in MMR leads to faster accumulation of genetic errors at these microsatellites leading to formation of subsequent diffuse MSI. The damage to the MMR process leads to additive mutations Please cite this article as: R. Gupta et al., The impact of microsatellite stability status in colorectal cancer, Current Problems in Cancer (2018), https://doi.org/10.1016/j.currproblcancer.2018.06.010
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3
COLORECTAL CANCERS
Microsatellite Unstable pathway (MSI)
Chromosomal Instability pathway (CIN) – No Microsatellite Instability 85%
MLH1 HYPERMETHYLATION SPORADIC
Familial Adenomatous Polyposis <0.5%
Sporadic / CpG Island Methylator Phenotype (12%)
Germline Mutation in Mismatch repair genes
Lynch Syndrome (3-5%)
Second Hit MSI
BRAF mutation
Frameshi mutaons in genes with coding “microsatellite repeats” +
TUMORIGENESIS
Fig. 1. Schematic classification of colorectal cancers and mechanism of tumor development via microsatellite instability pathway.
throughout the genome, leading to a “hypermutator” phenotype or MSI tumors.10,11 In contrast, the tumors with intact MMR are referred as microsatellite stable tumors (MSS).12 In a given tumor, defective MMR (dMMR) often affects only a proportion of the microsatellites due to which it is important to study more than one microsatellite to study instability in microsatellites.4 Epigenetic changes cause sporadic MSI in most of the cases, in particular methylation of the MLH1 promoter, which leads to subsequent silencing of the MLH1 gene. This methylation may be sporadic or associated with a CpG island methylator phenotype. This happens in 12 % of MSI patients, while only about 3% possess a germline MMR mutation (Lynch syndrome [LS]).11 MSI is more common among stage II (20%) than stage III (12%) CRC and less frequent among stage IV CRC (4%).12 CRC developing from each of these pathways have unique characteristics that guide the management and prognosis of the patients with CRC. Schematic classification of CRCs and the mechanism of tumor development via MSI pathway are shown in Figure 1. Patients with MSI status are known to have distinct phenotype including predilection for the right colon, poor differentiation, and/or mucinous histology, abundant tumor infiltrating lymphocytes (TILs), diploid DNA content, and less aggressive clinical behavior,11 while CIN tumors tend to exhibit aneuploidy and have no site predilection.13 Though sporadic MSI CRC shares most clinicopathological characteristics with germline CRC, there are few differences between the two. Sporadic MSI CRC is commonly seen in older Please cite this article as: R. Gupta et al., The impact of microsatellite stability status in colorectal cancer, Current Problems in Cancer (2018), https://doi.org/10.1016/j.currproblcancer.2018.06.010
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patients, predominant in female gender, and patients with increased rate of cigarette smoking and almost 50% harbor BRAF mutations.11,12 Patients with LS undergo germline mutation in DNA MMR genes commonly in MLH1 or MSH2, inherited in autosomal dominant manner, can have KRAS (but never BRAF), are commonly seen in younger patients, and have increased risk of developing cancers of other sites, including endometrium, stomach, ovary, urinary tract, small intestine, and prostate.3,12 For LS patients, the estimated cumulative risk of CRC by age 70 years is approximately 50% in case of MLH1 or MSH2 mutations.14
Immunologic landscape of colorectal cancer The immune system plays an important role in the development and progression of CRC. MSI tumors have dense infiltration of lymphocytes called TILs and Th1-associated cytokine-rich environment.15 The abundance of CD3+ and CD8+ cytotoxic TILs triggers an immune response in the host that results in a process called “immunoediting,” leading to immune tolerance where tumor antigen escapes the appropriate immune response.15 A series of checkpoint inhibitors regulate the process of immunoediting, including PD-1, PDL-1, CTLA-4, and LAG-3.16 Studies have shown that MSI tumors have >20 times higher mutational burden or neoantigens that leads to enhanced immunotherapy responsiveness in these “hypermutator” phenotypes in comparison to MSS tumors.15 Both PD-1 expression in TILs and PD-L1 expression on tumor cells differ in MSIH compared with MSS tumors. TILs from MSI-H tumors have 77% PD-1 expression compared with 39% in MSS tumors. MSI-H tumors have 32% PD-L1 expression compared with 13% in MSS tumors.10 Furthermore, 4 consensus molecular subtypes (CMS) of CRC have been categorized.16,17 CMS1 (immune) tumors, with MSI phenotype, constitute 14% of them, are characterized by hypermutation, strong immune activation, and are associated with overexpression of genes specific to cytotoxic lymphocytes. CMS2 (canonical) constitute 37% of mutations, are epithelial, display CIN phenotype and prominent WNT and MYC signaling activation. CMS3 (metabolic) constitute 13% of CRC tumors, are epithelial, and are characterized by metabolic dysregulation.16,17 CMS2 and CMS3 tumors display low inflammatory and immune signatures. In the end, CMS4 (Mesenchymal) accounts for 23% of CMS and expresses lymphocytic and monocytic markers, and is characterized by an angiogenic, inflammatory, and immunosuppressive signature, with a high concentration of fibroblasts and the remaining 13% display mixed features. Developing effective immunotherapy against a particular subtype depends on understanding the molecular and immunological signature displayed by the specific subtype of CRC.17 CMS1 tumors might respond well to immune checkpoint activation and reactivation of arrested immune response.17 CMS2 and CMS3 might respond to immunogenic stimuli like a vaccine or a co-stimulatory compound.17 CMS4 tumors would more likely respond to an approach which targets the suppressive monocytoid cells and related cytokines, alone or in combination with immune checkpoint inhibition.17 Studies have consistently shown that there is better stage-adjusted survival in MSI tumors compared with MSS tumors.18-22
Microsatellite instability—Prognostic value Microsatellite stability status can be used as a prognostic marker in patients with CRC particularly in cases of locally advanced stage II and stage III CRC.18,23 A number of studies have investigated the relationship between MSI status and survival in CRC patients and have shown that sporadic CRCs with a MMR deficiency generally have a favorable prognosis, particularly in early stage tumors and is more prominent in stage II than in stage III disease.22,24 Please cite this article as: R. Gupta et al., The impact of microsatellite stability status in colorectal cancer, Current Problems in Cancer (2018), https://doi.org/10.1016/j.currproblcancer.2018.06.010
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In 2005, a systematic review was performed to study prognosis of patients with MSI and CRC. Data were pooled from 32 studies that included 7642 patients with stage I-IV CRC,22 of which 1277 patients had MSI. This meta-analysis concluded that MSI was associated with better prognosis when compared with those with intact MMR among patients that were untreated or treated with 5-fluorouracil (5FU)-based adjuvant chemotherapy. The hazard ratio (HR) for overall survival (OS) was 0.65 (95% confidence interval (CI), 0.59-0.71) in favor of MSI CRC patients. This study also evaluated progression-free survival from 8 published studies and found that pooled HR was 0.67 (97% CI, 0.53-0.83).22 Gryfe and colleagues reported that 17% of 607 CRC patients who were younger than 50 years had MSI; the HR for patients with MSI-associated tumors was 0.42 compared with patients in the same cohort with non-MSI tumors. Patients with tumors with MSI had lower mortality rates when patients were stratified by tumor stage, including patients with stage IV cancer.25 Among patients with CRC who have MSI, patients with LS seem to have better prognosis than patients with sporadic CRC with MSI.26,27 The most likely explanation is that patients with LS have lower stage disease when they are diagnosed compared with patients with other types of CRC, and it is less common for patients with LS to present with metastatic disease. Germline mutations in tumor-suppressor genes, such as APC, MLH1, and MSH2, indicate a very high risk of CRC and guide the frequency of CRC surveillance and recommendations for prophylactic surgery. Yearly colonoscopy is recommended for carriers of LS, and prophylactic colectomy is considered for patients with high-grade lesions.28,29 The precise explanation of how MSI status offers a better prognosis is not very obvious. Possible explanations of mechanisms by which MSI status affords a better prognosis is the robust lymphocytic response that has been postulated to be correlated with an immunogenic, high neoantigen burden microenvironment due to elevated mutational rate in MSI tumors.30 Second, allele loss at or mutation of DCC, TP53, and KRAS are all associated with a poorer prognosis, and these abnormalities are not common in tumors with MSI.31 However, despite the enormous number of correlative studies exploring the prognostic significance of various molecular features, the microsatellite status is one of the crucial factors that are used for clinical decision-making other than BRAF and RAS mutations. These factors are both prognostic and predictive of chemotherapy efficacy.
Diagnosis of microsatellite instability The identification of a robust molecular prognostic marker to supplement conventional pathologic staging system is highly desirable. Microsatellite status is one of the strong prognostic markers in patients with CRC23 , 32 and is frequently analyzed by testing for loss of an MMR protein by immunohistochemistry (IHC) or testing for MSI using a PCR-based assay. MMR protein by IHC and MSI testing by PCR are complementary but each has its own limitation.33,34 An algorithm for evaluation of newly diagnosed CRC for microsatellite stability status including screening for LS is shown in Figure 2.
Mismatch repair protein by immunohistochemistry • Identifies affected gene by detecting loss of its protein product.33 • Highly concordant with DNA-based MSI testing with good sensitivity (>90%) and excellent specificity (100%).34 • Specific mismatch gene product (MLH1, MSH2, MSH6, and PMS2) can direct germline testing to that specific gene and assists in the identification of patients with LS.34 • In CRCs with loss of MLH1 protein expression, testing for a mutation in the BRAF oncogene is used to confirm a sporadic case and exclude LS. Patients with nonmutated BRAF would then have germline testing for a mutation in the presumed altered MLH1 gene.35 Please cite this article as: R. Gupta et al., The impact of microsatellite stability status in colorectal cancer, Current Problems in Cancer (2018), https://doi.org/10.1016/j.currproblcancer.2018.06.010
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Immunohistochemistry (IHC) for Mismatch Repair (MMR) proteins (MLH1, PMS2, MSH6, MSH2)
All Present
Loss of MSH2 and/or MSH6 or PMS2
Loss of MLH1 and PMS2
Analysis of BRAF V600E mutation by Polymerase chain reaction (PCR) or IHC
Presence of BRAF mutation
Microsatellite stable (MSS)
Sporadic Microsatellite Instable (MSI)
Absence of BRAF mutation
Suspicious Lynch syndrome (LS) age or history
Gene testing for MLH1/PMS2
Not Present
Not suspicious LS age or history
MLH1 methylation
present
Sporadic MSI
If germline mutation present, then LS- refer to genetic counselling
Fig. 2. Algorithm for evaluation of newly diagnosed colorectal cancer for microsatellite stability status including screening for Lynch syndrome.
Microsatellite instability testing by PCR • Evaluates tumors by amplifying microsatellite repeats.34 • In 1997, a National Cancer Institute workshop recommended using a reference panel of microsatellite markers for defining MSI status.36 This panel had 3 dinucleotide markers (D5S346, D2S123, and D17S250), and 2 mononucleotide markers (BAT25 and BAT26). MSI-H was defined as instability at 2 or more markers; MSS as tumors with no evidence of instability at any of the markers, with MSI-L used for the intermediate phenotype.36 • Studies have shown lack of a conclusive cutoff between MSI-L and MSS; therefore, in most of the cases, MSI mainly refers to MSI-H.36 • If MSI analysis is used as the initial screening test rather than IHC, BRAF mutational testing or MLH1 promoter methylation can be used as the second step prior to genetic sequencing in patients found to have MSI.33
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Microsatellite instability and response to chemotherapy (predictive value) Currently, all patients with stage III CRC and some patients with high-risk stage II CRC are recommended to receive 5FU-based chemotherapy, but studies have shown no benefit of 5FUbased chemotherapy among patients with CRC with MSI.26,37 , 38 Thus, MSI status is considered a strong predictive factor for nonresponse to 5FU-based chemotherapy in CRC patients. Several biological mechanisms have been proposed to explain why 5FU-based adjuvant chemotherapy fails to improve outcomes in patients with MSI. This includes the immunosuppressive effects of chemotherapy diminishing the antitumor immune response involving the lymphocytic infiltration characteristic of MSI tumors.11 Second, the intact MMR system ensures that 5FU is efficiently removed from DNA before it can interfere with essential DNA metabolic processes, such as transcription. This also increases the intracellular concentration of the drug and thus exacerbates its cytotoxicity in MSS patients, whereas the absence of MMR system may reduce DNA repair and, thus, attenuate the effect of 5FU in MSI patients.39 In vitro studies have shown that resistance to 5FU in MSI tumors with hypermethylated defective MMR CRC can be reversed after treatment with demethylation agents.39 Randomized trials evaluating outcomes of chemotherapy in CRC with MSI have been summarized in Table 1. The first major study indicating that patients with stage II or stage III MSI CRC did not benefit from FU-based chemotherapy was done by Ribic and colleagues in 2003.26 This study clearly showed that among patients with MSI tumors, treatment was associated with a worse outcome for both stage II and stage III cancer. Among stage II MSI CRC, HR for death was 3.28 (95 % CI, 0.86-12.48) and among patients with stage III MSI CRC, HR was 1.42 (95 % CI, 0.36-5.56).26 Another meta-analysis to study role of MSI status in predicting the efficacy of adjuvant chemotherapy in CRC was done in 2009.37 This meta-analysis involved 7 studies and included 3690 patients with CRC. Eight hundred and ten patients were stage II, and 2444 were stage III patients. Four hundred and fifty four patients were found to have MSI. A total of 1444 patients received 5FU-based chemotherapy, whereas 1518 patients did not. No statistically significant difference was found in MSI patients for recurrence-free survival (RFS) whether or not they received chemotherapy. In addition, the study showed that there was lesser benefit for MSI than for MSS patients, HR interaction RFS 0.77 (95% CI: 0.67-0.87).37 Later in 2010, study done by Sargent and colleagues evaluated the predictive and prognostic values of MSI in 457 stage II and stage III CRC patients from 5 randomized trials.38 These patients were randomly assigned to 5FU-based therapy (either FU + levamisole or FU + leucovorin; n = 229) versus no postsurgical treatment (n = 228). The primary end point was disease-free survival (DFS). Overall, 70 (15%) of 457 patients exhibited MSI. Patients with MSI tumors receiving 5FU had no improvement in DFS (HR, 1.10; 95% CI, 0.42-2.91; P = 0.85) compared with those randomly assigned to surgery alone. In the pooled data set of 1027 patients (165 patients exhibited MSI), these findings were reproducible. In patients with stage II disease and with MSI CRC, treatment was associated with reduced OS (HR, 2.95; 95% CI, 1.02-8.54; P = 0.04).38 The role of oxaliplatin in addition to 5FU and leucovorin has been studied in the adjuvant setting for treatment of MSI stage II and stage III CRC. In an analysis of the Multicenter International Study of Oxaliplatin, 5FU, and Leucovorin (FOLFOX) in adjuvant treatment of colon cancer (MOSAIC) study,40 microsatellite status was evaluated in 986 patients out of 2246 patients, and 90 patients were found to be MSI. Of the patients with MSI CRC, 45 patients had stage II and 45 stage III. In this modest number of patients with MSI colon cancers, a DFS benefit from FOLFOX compared with 5FU alone was observed. HRs for stages II and III MSI were 0.52 (0.21-1.28) for RFS, 0.52 (0.24-1.14) for DFS, and 0.45 (0.19-1.05) for OS, respectively; therefore, this study supported the use of FOLFOX in pts with MSI stage III cancer.41 Another drug that has shown increased sensitivity to MSI cancer cell lines is irinotecan (IFL) though studies have shown conflicting results. Study performed by Bertagnolli and colleagues showed that patients with stage III CRC who had MSI tumors and treated with 5FU and IFL had improved 5-year DFS when compared with MSS tumors with HR of 0.76 (95% CI, 0.64-0.88) versus 0.59 (95% CI, 0.53-0.64), respectively (P = 0.03).42 In contrast, another study that included Please cite this article as: R. Gupta et al., The impact of microsatellite stability status in colorectal cancer, Current Problems in Cancer (2018), https://doi.org/10.1016/j.currproblcancer.2018.06.010
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Study design
Number of patients Stage of CRC
Adjuvant chemotherapy regimen
Ribic et al.26
RCT
570
II/III
5FU or 5FU/levamisole
Des Guetz et al.37
Meta-analysis
(MSI = 95) 3690 (MSI = 454)
II/III
5FU
Sargent et al.38
RCT
457 (MSI = 70)
II/III
Flejou et al.41
Pooled analysis RCT
1027 (MSI = 165) 986 (MSIH = 90)
II II/III
5FU/levamisole Or 5FU/leucovorin 5FU or 5FU + oxaliplatin
RCT
1264 (MSI = 96) 1254
III
Bertagnolli et al.42 Klingbiel et al.
43
RCT
II/III
5FU or 5FU + Irinotecan 5FU/leucovorin Or FOLFIRI
FOLFIRI = Folinic
Worse [stage II- OS: HR 3.28, 95% CI, 0.86-12.48] Stage III- OS: HR 1.42, 95%CI, 1.02-8.54 No benefit [RFS: HR 0.96, 95% CI, 0.62-1.49; OS: HR 0.70, 0.44-1.09] No benefit [DFS: HR 1.10, 95% CI, 0.42-2.91] Worse [OS: HR 2.95, 95% CI, 1.02-8.54] Added benefit with oxaliplatin [OS: HR 0.45,95% CI, 0.19-1.05]; DFS: HR 0.52, 95% CI, 0.24-1.14] Added benefit with Irinotecan [DFS: HR 0.76, 95% CI 0.42-0.71] No added benefit with Irinotecan [Stage II- OS, HR 0.16, 95% CI, 0.04-0.64; RFS, HR 0.26, 95% CI, 0.10-0.65; stage IIIOS, HR 0.70, 95% CI, 0.44-1.09; RFS, HR 0.67, 95% CI, 0.46-0.99] acid + Fluorouracil + Irinotecan;
OS = Overall
survival;
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MSI = Microsatellite instability; RCT = Randomized controlled trial; 5FU = 5Fluorouracil; DFS = Disease-free survival; RFS = Recurrence-free survival; CI = Confidence Interval.
Patient outcomes with chemotherapy in MSI
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Table 1 Randomized trials evaluating outcomes of chemotherapy in CRC with MSI.
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1254 patients with stages II and III CRC showed no benefit in OS or RFS on addition of IFL to 5FU compared with 5FU alone for MSS or MSI tumors warranting further studies.43 Thus, the lack of evidence of benefit from 5FU-based adjuvant chemotherapy in stage II MSI CRC with their favorable prognosis suggests that these patients should not receive adjuvant chemotherapy. In patients with MSS tumors, decisions regarding adjuvant therapy for stage II CRC should be based on other factors that indicate a high-risk patient, such as a T4 tumor, tumor perforation, bowel obstruction, poor differentiation, venous invasion, or fewer than 12 lymph nodes examined. Among patients with stage III disease, the predictive impact of MMR status for adjuvant chemotherapy remains controversial. Multiple prior studies have demonstrating a lack of benefit for 5FU as adjuvant chemotherapy, although only limited data exist for patients with stage III MSI CRCs treated with standard adjuvant FOLFOX regimen. Therefore, as of now, FOLFOX has become current standard of care for adjuvant chemotherapy for stage III colon cancer irrespective of MSI status.
Microsatellite instability and response to immunotherapy The role of immunotherapy in CRC has recently become area of significant interest due to unprecedented successes in advanced solid tumors such as lung cancer and melanoma.16 Based upon knowledge of the immunogenic tumor microenvironment in MSI-H tumors, a trial of anti-PD1 therapy, pembrolizumab was undertaken, enrolling 3 cohorts of patients: MSI-H CRCs, MSI-H non-CRCs, and MSS CRCs. In the initial report, 40% of MSI-H CRC patients achieved an immune-related objective response and 78% achieved 20-week immune-related progression-free survival rate, respectively.15 Updated results continue to show progress wherein 16 (57%) of 25 patients achieved objective responses and additional 9 (32%) with stable disease.15 In another study, 70 patients with MSI-H tumors were enrolled and treated with a different PD-1 inhibitor, nivolumab 3 mg/kg q2week.44 At the initial presentation, 12 (26%) of the 47 patients achieved an objective response with at least 12 weeks of follow-up, with a median time to response of 2.12 months (range 1.3-13.6). An additional 14 (30%) of patients achieved stable disease, for a disease control rate of 55%. In a follow-up presentation at the 2017 American Society of Clinical Oncologists GI cancers symposium, the objective response rate (ORR) was 31% with 69% of patients achieving stable disease. 45 On May 23, 2017, pembrolizumab was granted approval by the U.S. Food and Drug Administration for patients with unresectable or metastatic, MSI or dMMR solid tumors that have progressed following prior treatment and who have no satisfactory alternative treatment options or with MSI or dMMR CRC that has progressed following treatment with a fluoropyrimidine, oxaliplatin, and irinotecan. On July 31, 2017, the Food and Drug Administration also granted accelerated approval to nivolumab for patients metastatic CRC who have MSI-H or dMMR solid tumors whose disease has progressed after chemotherapy. While immunotherapy has shown significant therapeutic response in patients with MSI CRC, its activity against MSS tumors has not been established. Various strategies and clinical trials are being done to evaluate response of immunotherapy to MSS CRC. A phase 1b trial done by Bendall and colleagues46 has used combination therapy involving PDL-1and/or PD-1 inhibitor atezolizumab and cobimetinib (MEK pathway inhibitor) in metastatic MSS CRC showing promising results. The cobimetinib and/or atezolizumab combination showed an investigator-assessed objective response rate (ORR) of 17% in 23 patients overall. An ORR of 20% was observed in 22 patients with KRAS-mutant tumors. The 6-month OS rate was 72%.46 A phase III study is ongoing, where patients with refractory metastatic disease are randomized to cobimetinib and atezolizumab, atezolizumab alone or regorafenib (NCT02788279). Cobimetinib is also being combined with nivolumab and ipilimumab (NCT02060188) as well as being utilized in a trial with atezolizumab and bevacizumab (NCT02876224). Major ongoing trials of immunotherapy in the setting of stage IV CRC have been summarized in Table 2. Please cite this article as: R. Gupta et al., The impact of microsatellite stability status in colorectal cancer, Current Problems in Cancer (2018), https://doi.org/10.1016/j.currproblcancer.2018.06.010
Description
Treatment
Primary end point
Identifier
Phase II—Pretreated stage IV CRC with MSI or dMMR
Pembrolizumab monotherapy
ORR
Phase III—1st line- stage IV CRC with MSI or dMMR
Pembrolizumab monotherapy vs standard of care chemotherapy
PFS
NCT02460198 Keynote 164 NCT02563002
Atezolizumab vs atezolizumab + FOLFOX + bevacizumab vs FOLFOX + bevacizumab Cobimetinib plus atezolizumab vs atezolizumab monotherapy vs regorafenib Nivolumab vs nivolumab combinations Pembrolizumab and cetuximab Pembrolizumab and nintedanib Pembrolizumab plus napabucasin
PFS
NCT02997228
OS
NCT02788279
ORR Objective tumor response, OS, PFS MTD of nintedanib Immune-related ORR determined by their response evaluation criteria in solid tumors Degree of change in TILs Frequency, duration, and severity of adverse events, ORR MTD of poly-ICLC, response rate Percentage of participants with adverse events PFS
NCT02060188 NCT02713373 NCT02856425 NCT02851004
NCT02834052 NCT02876224 NCT02873195
Determination of the recommended phase II dose safety, ORR Safety and tolerability, ORR, PFS, OS
NCT02327078
JID: YMCN
10
Keynote 177 Phase III—1st line Stage IV CRC with MSI or dMMR
Phase III—Pretreated metastatic CRC II—Recurrent and metastatic CRC 1b/II—Pretreated recurrent or metastatic CRC Ib—stage IV CRC Ib/II—refractory CRC
Pembrolizumab plus oral azacitidine and romidepsin Pembrolizumab plus azacitidine plus epacadostat
Phase I/II—Stage IV MSS CRC Phase Ib—stage IV metastatic CRC Phase II—Stage IV CRC
Phase I/II—Advanced solid tumors including CRC
Pembrolizumab plus poly-ICLC Atezolizumab plus cobimetinib plus bevacizumab capecitabine/bevacizumab + atezolizumab vs capecitabine/bevacizumab + placebo Durvalumab in combination with olaparib and/or cediranib Nivolumab plus epacadostat vs nivolumab plus epacadostat + chemotherapy Nivolumab plus varlilumab
Phase I—Stage IV CRC and pancreatic cancer Phase I/Ib—Incurable MSI CRC, solid cancers
Durvalumab and pexidartinib Atezolizumab and CPI-444
Phase 1b/II—Locally advanced rectal cancer Phase II study—Stage IV CRC, NSCLC
Nivulomab following chemoradiotherapy Durvalumab and tremelimumab alone or in combination with high or low-dose radiation
Phase I/II—Refractory CRC Phase I/II—Advanced solid tumors including CRC
Number of participants with treatment-related adverse events, ORR DLT, ORR Incidence DLTs, ORR, Incidence of treatment-emergent adverse events, mean and median area under the curve, mean and median maximum concentration, MDL (maximum dose level) of single agent Pathological complete response ORR
NCT02512172 NCT02959437
NCT02484404
NCT02335918 NCT02777710 NCT02655822
NCT02948348 NCT02888743
ORR = Objective response rate; PFS = Progression-free survival; OS = Overall survival; MTD = Maximum tolerated dose; DLT = Dose-limiting toxicity; NSCLC = Non-small cell lung cancer.
[mUS1Ga;August 14, 2018;12:5]
Phase I—Pretreated MSS advanced CRC Phase I/II—stage IV MSS CRC and NSCLC
ARTICLE IN PRESS
Phase Phase Phase Phase
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Table 2 Key immunotherapy trials in progress for stage IV CRC.
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Conclusion All patients with resected CRC are recommended to undergo testing to determine their microsatellite status directly through MSI testing or indirectly through IHC of the 4 MMR proteins. Microsatellite status serves not only as a predictive marker for response to chemotherapy and immunotherapy but also as a prognostic marker for patient outcome and a screening tool for detecting patients with LS. Overall, the prognosis for individuals with metastatic CRC remains poor. While the advent of immunotherapies has shown significant promises in improving survival in minority of patients with advanced CRC, additional strategies are required to sensitize patients with MSS CRC.
Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.currproblcancer.2018.06.010.
References 1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65:87–108. 2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. CA Cancer J Clin. 2017;67:7–30. 3. Markowitz SD, Bertagnolli MM. Molecular origins of cancer: molecular basis of colorectal cancer. N Engl J Med. 2009;361:2449–2460. 4. de la Chapelle A, Hampel H. Clinical relevance of microsatellite instability in colorectal cancer. J Clin Oncol. 2010;28:3380–3387. 5. Pino MS, Chung DC. The chromosomal instability pathway in colon cancer. Gastroenterology. 2010;138:2059–2072. 6. Ionov Y, Peinado MA, Malkhosyan S, Shibata D, Perucho M. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature. 1993;363:558–561. 7. Aaltonen LA, Peltomäki P, Leach FS, Sistonen P, Pylkkänen L, Mecklin JP. Clues to the pathogenesis of familial colorectal cancer. Science. 1993;260:812–816. 8. Jiricny J. The multifaceted mismatch-repair system. Nat Rev Mol Cell Biol. 2006;7:335–346. 9. Strand M, Prolla TA, Liskay RM, Petes TD. Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair. Nature. 1993;365:274–276. 10. Lee V, Murphy A, Le DT, Diaz Jr LA. Mismatch repair deficiency and response to immune checkpoint blockade. Oncologist. 2016;21:1200–1211. 11. Boland CR, Goel A. Microsatellite instability in colorectal cancer. Gastroenterology. 2010;138 2073–2087 e3. 12. Kawakami H, Zaanan A, Sinicrope FA. Microsatellite instability testing and its role in the management of colorectal cancer. Curr Treat Options Oncol. 2015;16:30. 13. Ilyas M, Straub J, Tomlinson IP, Bodmer WF. Genetic pathways in colorectal and other cancers. Eur J Cancer. 1999;35:335–351. 14. Bonadona V, Bonaïti B, Olschwang S, Grandjouan S, Huiart L, Longy M, et al. Cancer risks associated with germline mutations in MLH1, MSH2, and MSH6 genes in Lynch syndrome. JAMA. 2011;305:2304–2310. 15. Le DT, Durham JN, Smith KN, Wang H, Bartlett BR, Aulakh LK, et al. PD-1 blockade in tumors with mismatch-repair deficiency. N Engl J Med. 2015;372:2509–2520. 16. Boland PM, Ma WW. Immunotherapy for colorectal cancer. Cancers. 2017;9(5):50. 17. Thanki K, Nicholls ME, Gajjar A, Senagore AJ, Qiu S, Szabo C, et al. Consensus molecular subtypes of colorectal cancer and their clinical implications. Int Biol Biomed J. 2017;3:105–111. 18. Sinicrope FA, Rego RL, Halling KC, Foster N, Sargent DJ, La Plant B, et al. Prognostic impact of microsatellite instability and DNA ploidy in human colon carcinoma patients. Gastroenterology. 2006;131:729–737. 19. Gafà R, Maestri I, Matteuzzi M, Santini A, Ferretti S, Cavazzini L, et al. Sporadic colorectal adenocarcinomas with high-frequency microsatellite instability. Cancer. 20 0 0;89:2025–2037. 20. Halling KC, French AJ, McDonnell SK, Burgart LJ, Schaid DJ, Peterson BJ, et al. Microsatellite instability and 8p allelic imbalance in stage B2 and C colorectal cancers. J Natl Cancer Inst. 1999;91:1295–1303. 21. Lanza G, Gafà R, Santini A, Maestri I, Guerzoni L, Cavazzini L. Immunohistochemical test for MLH1 and MSH2 expression predicts clinical outcome in stage II and III colorectal cancer patients. J Clin Oncol. 2006;24:2359–2367. 22. Popat S, Hubner R, Houlston RS. Systematic review of microsatellite instability and colorectal cancer prognosis. J Clin Oncol. 2005;23:609–618. 23. Samowitz WS, Curtin K, Ma KN, Schaffer D, Coleman LW, Leppert M, et al. Microsatellite instability in sporadic colon cancer is associated with an improved prognosis at the population level. Cancer Epidemiol Biomarkers Prev. 2001;10:917–923. 24. Vilar E, Gruber SB. Microsatellite instability due to hMLH1 deficiency is associated with increased cytotoxicity to irinotecan in human colorectal cancer cell lines. Br J Cancer. 2008;99:1607–1612. Please cite this article as: R. Gupta et al., The impact of microsatellite stability status in colorectal cancer, Current Problems in Cancer (2018), https://doi.org/10.1016/j.currproblcancer.2018.06.010
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25. Gryfe R, Kim H, Hsieh ET, Aronson MD, Holowaty EJ, Bull SB. Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer. N Engl J Med. 20 0 0;342:69–77. 26. Ribic CM, Sargent DJ, Moore MJ, Thibodeau SN, French AJ, Goldberg RM, et al. Tumor microsatellite-instability status as a predictor of benefit from fluorouracil-based adjuvant chemotherapy for colon cancer. N Engl J Med. 2003;349:247–257. 27. Maccaroni E, Bracci R, Giampieri R, Bianchi F, Belvederesi L, Brugiati C, et al. Prognostic impact of mismatch repair genes germline defects in colorectal cancer patients: are all mutations equal? Oncotarget. 2015;6:38737–38748. 28. Lynch HT, de la Chapelle A. Hereditary colorectal cancer syndromes: molecular genetics, genetic counseling, diagnosis and management. Fam Cancer. 2008;7:27–39. 29. Järvinen HJ, Aarnio M, Mustonen H, Aktan-Collan K, Aaltonen LA, Peltomäki P, et al. Controlled 15-year trial on screening for colorectal cancer in families with hereditary nonpolyposis colorectal cancer. Gastroenterology. 20 0 0;118:829–834. 30. Linnebacher M, Gebert J, Rudy W, Woerner S, Yuan YP, Bork P, et al. Frameshift peptide-derived T-cell epitopes: a source of novel tumor-specific antigens. Int J Cancer. 2001;93:6–11. 31. Klump B, Nehls O, Okech T, Hsieh CJ, Gaco V, Gittinger FS, et al. Molecular lesions in colorectal cancer: impact on prognosis? Original data and review of the literature. Int J Colorectal Dis. 2004;19:23–42. 32. Sinicrope FA, Sargent DJ. Molecular pathways: microsatellite instability in colorectal cancer: prognostic, predictive, and therapeutic implications. Clin Cancer Res. 2012;18:1506–1512. 33. Chen W, Swanson BJ, Frankel WL. Molecular genetics of microsatellite-unstable colorectal cancer for pathologists. Diagn Pathol. 2017;12:24. 34. Lindor NM, Burgart LJ, Leontovich O, Goldberg RM, Cunningham JM, Sargent DJ, et al. Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors. J Clin Oncol. 2002;20:1043–1048. 35. Ladabaum U, Wang G, Terdiman J, Blanco A, Kuppermann M, Boland CR, et al. Strategies to identify the Lynch syndrome among patients with colorectal cancer: a cost-effectiveness analysis. Ann Intern Med. 2011;155:69–79. 36. Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, Burt RW, et al. A National Cancer Institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res. 1998;58:5248–5257. 37. Des Guetz G, Schischmanoff O, Nicolas P, Perret GY, Morere JF, Uzzan B. Does microsatellite instability predict the efficacy of adjuvant chemotherapy in colorectal cancer? A systematic review with meta-analysis. Eur J Cancer. 2009;45:1890–1896. 38. Sargent DJ, Marsoni S, Monges G, Thibodeau SN, Labianca R, Hamilton SR, et al. Defective mismatch repair as a predictive marker for lack of efficacy of fluorouracil-based adjuvant therapy in colon cancer. J Clin Oncol. 2010;28:3219–3226. 39. Arnold CN, Goel A, Boland CR. Role of hMLH1 promoter hypermethylation in drug resistance to 5-fluorouracil in colorectal cancer cell lines. Int J Cancer. 2003;106:66–73. 40. André T, Boni C, Mounedji-Boudiaf L, Navarro M, Tabernero J, Hickish T, et al. Oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment for colon cancer. N Engl J Med. 2004;350:2343–2351. 41. Flejou J-F, André T, Chibaudel B, Scriva A, Hickish T, Tabernero J, et al. Effect of adding oxaliplatin to adjuvant 5-fluorouracil/leucovorin (5FU/LV) in patients with defective mismatch repair (dMMR) colon cancer stage II and III included in the MOSIAC study. J Clin Oncol. 2013;31(15_suppl):3524. 42. Bertagnolli MM, Niedzwiecki D, Compton CC, Hahn HP, Hall M, Damas B, et al. Microsatellite instability predicts improved response to adjuvant therapy with irinotecan, fluorouracil, and leucovorin in stage III colon cancer: cancer and leukemia group B protocol 89803. J Clin Oncol. 2009;27:1814–1821. 43. Klingbiel D, Saridaki Z, Roth AD, Bosman FT, Delorenzi M, Tejpar S. Prognosis of stages II and III colon cancer treated with adjuvant 5-fluorouracil or FOLFIRI in relation to microsatellite status: results of the PETACC-3 trial. Ann Oncol. 2015;26:126–132. 44. Overman MJ, Kopetz S, McDermott RS, Leach J, Lonardi S, Lenz H-J, et al. Nivolumab ± ipilimumab in treatment (tx) of patients (pts) with metastatic colorectal cancer (mCRC) with and without high microsatellite instability (MSI-H): CheckMate-142 interim results. J Clin Oncol. 2016;34(15_suppl):3501. 45. Overman MJ, McDermott R, Leach JL, Lonardi S, Lenz HJ, Morse MA, et al. Nivolumab in patients with metastatic DNA mismatch repair-deficient or microsatellite instability-high colorectal cancer (CheckMate 142): an open-label, multicentre, phase 2 study. Lancet Oncol. 2017;18:1182–1191. 46. Bendell JC, Kim TW, Goh BC, Wallin J, Oh D-Y, Han S-W. Clinical activity and safety of cobimetinib (cobi) and atezolizumab in colorectal cancer (CRC). J Clin Oncol. 2016;34(15_suppl):3502.
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Contents lists available at ScienceDirect
Curr Probl Cancer journal homepage: www.elsevier.com/locate/cpcancer
The impact of microsatellite stability status in colorectal cancer Ruby Gupta a,∗, Surajit Sinha b, Rabindra N. Paul c a
Keystone Rural Health Center, Chambersburg, PA National Institutes of Health, MD c Atlantic General Hospital, MD b
a r t i c l e
i n f o
a b s t r a c t Several forms of genomic instability are known to drive the development of colorectal cancer (CRC). Chromosomal instability is the most common type found in 85% of the CRC, while 15% patients have microsatellite instability (MSI). MSI tumors are the subset of CRC that are characterized by dysfunction of mismatch repair genes (MMR) causing failure to repair errors in repetitive DNA sequences called microsatellites. Twelve percent of MSI tumors are acquired, caused by methylation-associated silencing of a gene that encodes a DNA MMR protein, while the remaining 3% have germline mutations in one of the MMR genes (Lynch syndrome). The identification of microsatellite stability status is clinically important as studies have revealed that MSI tumors have a better stage-adjusted survival compared with microsatellite stable tumors, and they respond differently to 5FU-based adjuvant chemotherapy depending on this status. There is recent success of immunotherapy (mainly anti-PD1 drugs) in metastatic CRC with MMR dysfunction that has led to the initiation of multiple trials based on immune checkpoint inhibitors. Additionally, it is important to identify patients with Lynch syndrome so that it can guide the frequency of surveillance of CRCs and recommendations of prophylactic surgery. Even though TNM staging remains a key determinant of patient prognosis and guides management in patients with CRC, molecular tumor heterogeneity contributes to significant variability in clinical outcomes despite the same disease
∗
Correspondence to: Ruby Gupta, 830, 5th Avenue, suite 201, Chambersburg, PA 17201. E-mail address: [email protected] (R. Gupta).
https://doi.org/10.1016/j.currproblcancer.2018.06.010 0147-0272/© 2018 Elsevier Inc. All rights reserved.
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stage; therefore, it is vital to know the type of genomic instability pathway that the tumor harbors. In this article, we discuss the unique genetic, pathologic, and clinical characteristics of microsatellite unstable (MSI) and stable CRC (MSS), their predictive value in directing the management with conventional chemotherapy or novel-targeted agents, and their prognostic significance in patient outcomes. © 2018 Elsevier Inc. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular landscape of colorectal cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunologic landscape of colorectal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . Microsatellite instability—Prognostic value . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagnosis of microsatellite instability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mismatch repair protein by immunohistochemistry . . . . . . . . . . . . . . . . . . . . Microsatellite instability testing by PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microsatellite instability and response to chemotherapy (predictive value) . Microsatellite instability and response to immunotherapy. . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Supplementary materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Introduction Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females.1 In the United States, approximately 140,250 new cases of large bowel cancer are diagnosed annually, of which 97,220 are colon and the remainder are rectal cancers. CRC contributes to approximately 8% mortality among all cancer deaths in the United States with reported 50,630 deaths in a year.2 In the United States, CRC incidence rates have declined about 2.5%-4% per year over the last 15 years; however, CRC incidence is increasing in the age group under 50, while it is decreasing in older groups.2
Molecular landscape of colorectal cancer The acquisition of genomic instability is a crucial feature in CRC development, and there are at least 2 distinct pathways in CRC pathogenesis: the chromosomal instability (CIN) pathway (85%) and microsatellite instability (MSI) pathway (15%).3 , 4 CIN tumors are caused by mutations in genes leading to chromosomal aneuploidy, loss of heterozygosity, and structural chromosomal rearrangements.3 , 5 Coupled with the karyotypic abnormalities observed in CIN tumors are the accumulation of a characteristic set of mutations in specific tumor suppressor genes and oncogenes that activate pathways critical for CRC initiation and progression.3 , 5 MSI tumors are known to arise from inactivation of 1 of the 4 mismatch repair (MMR) genes: MSH2, MLH1, MSH6, and PMS2.6-9 During normal DNA replication, proficient MMR genes detect the DNA mismatch error and MLH1 and/or PMS2 heterodimer helps in the excision of mismatch errors and the formation of new, corrected DNA strands. The human genome is known to have >10 0,0 0 0 areas of short tandem repeat sequences called microsatellites which are susceptible to replication “slippage” and heavily dependent on the MMR system for repair.10 Deficiency in MMR leads to faster accumulation of genetic errors at these microsatellites leading to formation of subsequent diffuse MSI. The damage to the MMR process leads to additive mutations Please cite this article as: R. Gupta et al., The impact of microsatellite stability status in colorectal cancer, Current Problems in Cancer (2018), https://doi.org/10.1016/j.currproblcancer.2018.06.010
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3
COLORECTAL CANCERS
Microsatellite Unstable pathway (MSI)
Chromosomal Instability pathway (CIN) – No Microsatellite Instability 85%
MLH1 HYPERMETHYLATION SPORADIC
Familial Adenomatous Polyposis <0.5%
Sporadic / CpG Island Methylator Phenotype (12%)
Germline Mutation in Mismatch repair genes
Lynch Syndrome (3-5%)
Second Hit MSI
BRAF mutation
Frameshi mutaons in genes with coding “microsatellite repeats” +
TUMORIGENESIS
Fig. 1. Schematic classification of colorectal cancers and mechanism of tumor development via microsatellite instability pathway.
throughout the genome, leading to a “hypermutator” phenotype or MSI tumors.10,11 In contrast, the tumors with intact MMR are referred as microsatellite stable tumors (MSS).12 In a given tumor, defective MMR (dMMR) often affects only a proportion of the microsatellites due to which it is important to study more than one microsatellite to study instability in microsatellites.4 Epigenetic changes cause sporadic MSI in most of the cases, in particular methylation of the MLH1 promoter, which leads to subsequent silencing of the MLH1 gene. This methylation may be sporadic or associated with a CpG island methylator phenotype. This happens in 12 % of MSI patients, while only about 3% possess a germline MMR mutation (Lynch syndrome [LS]).11 MSI is more common among stage II (20%) than stage III (12%) CRC and less frequent among stage IV CRC (4%).12 CRC developing from each of these pathways have unique characteristics that guide the management and prognosis of the patients with CRC. Schematic classification of CRCs and the mechanism of tumor development via MSI pathway are shown in Figure 1. Patients with MSI status are known to have distinct phenotype including predilection for the right colon, poor differentiation, and/or mucinous histology, abundant tumor infiltrating lymphocytes (TILs), diploid DNA content, and less aggressive clinical behavior,11 while CIN tumors tend to exhibit aneuploidy and have no site predilection.13 Though sporadic MSI CRC shares most clinicopathological characteristics with germline CRC, there are few differences between the two. Sporadic MSI CRC is commonly seen in older Please cite this article as: R. Gupta et al., The impact of microsatellite stability status in colorectal cancer, Current Problems in Cancer (2018), https://doi.org/10.1016/j.currproblcancer.2018.06.010
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patients, predominant in female gender, and patients with increased rate of cigarette smoking and almost 50% harbor BRAF mutations.11,12 Patients with LS undergo germline mutation in DNA MMR genes commonly in MLH1 or MSH2, inherited in autosomal dominant manner, can have KRAS (but never BRAF), are commonly seen in younger patients, and have increased risk of developing cancers of other sites, including endometrium, stomach, ovary, urinary tract, small intestine, and prostate.3,12 For LS patients, the estimated cumulative risk of CRC by age 70 years is approximately 50% in case of MLH1 or MSH2 mutations.14
Immunologic landscape of colorectal cancer The immune system plays an important role in the development and progression of CRC. MSI tumors have dense infiltration of lymphocytes called TILs and Th1-associated cytokine-rich environment.15 The abundance of CD3+ and CD8+ cytotoxic TILs triggers an immune response in the host that results in a process called “immunoediting,” leading to immune tolerance where tumor antigen escapes the appropriate immune response.15 A series of checkpoint inhibitors regulate the process of immunoediting, including PD-1, PDL-1, CTLA-4, and LAG-3.16 Studies have shown that MSI tumors have >20 times higher mutational burden or neoantigens that leads to enhanced immunotherapy responsiveness in these “hypermutator” phenotypes in comparison to MSS tumors.15 Both PD-1 expression in TILs and PD-L1 expression on tumor cells differ in MSIH compared with MSS tumors. TILs from MSI-H tumors have 77% PD-1 expression compared with 39% in MSS tumors. MSI-H tumors have 32% PD-L1 expression compared with 13% in MSS tumors.10 Furthermore, 4 consensus molecular subtypes (CMS) of CRC have been categorized.16,17 CMS1 (immune) tumors, with MSI phenotype, constitute 14% of them, are characterized by hypermutation, strong immune activation, and are associated with overexpression of genes specific to cytotoxic lymphocytes. CMS2 (canonical) constitute 37% of mutations, are epithelial, display CIN phenotype and prominent WNT and MYC signaling activation. CMS3 (metabolic) constitute 13% of CRC tumors, are epithelial, and are characterized by metabolic dysregulation.16,17 CMS2 and CMS3 tumors display low inflammatory and immune signatures. In the end, CMS4 (Mesenchymal) accounts for 23% of CMS and expresses lymphocytic and monocytic markers, and is characterized by an angiogenic, inflammatory, and immunosuppressive signature, with a high concentration of fibroblasts and the remaining 13% display mixed features. Developing effective immunotherapy against a particular subtype depends on understanding the molecular and immunological signature displayed by the specific subtype of CRC.17 CMS1 tumors might respond well to immune checkpoint activation and reactivation of arrested immune response.17 CMS2 and CMS3 might respond to immunogenic stimuli like a vaccine or a co-stimulatory compound.17 CMS4 tumors would more likely respond to an approach which targets the suppressive monocytoid cells and related cytokines, alone or in combination with immune checkpoint inhibition.17 Studies have consistently shown that there is better stage-adjusted survival in MSI tumors compared with MSS tumors.18-22
Microsatellite instability—Prognostic value Microsatellite stability status can be used as a prognostic marker in patients with CRC particularly in cases of locally advanced stage II and stage III CRC.18,23 A number of studies have investigated the relationship between MSI status and survival in CRC patients and have shown that sporadic CRCs with a MMR deficiency generally have a favorable prognosis, particularly in early stage tumors and is more prominent in stage II than in stage III disease.22,24 Please cite this article as: R. Gupta et al., The impact of microsatellite stability status in colorectal cancer, Current Problems in Cancer (2018), https://doi.org/10.1016/j.currproblcancer.2018.06.010
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In 2005, a systematic review was performed to study prognosis of patients with MSI and CRC. Data were pooled from 32 studies that included 7642 patients with stage I-IV CRC,22 of which 1277 patients had MSI. This meta-analysis concluded that MSI was associated with better prognosis when compared with those with intact MMR among patients that were untreated or treated with 5-fluorouracil (5FU)-based adjuvant chemotherapy. The hazard ratio (HR) for overall survival (OS) was 0.65 (95% confidence interval (CI), 0.59-0.71) in favor of MSI CRC patients. This study also evaluated progression-free survival from 8 published studies and found that pooled HR was 0.67 (97% CI, 0.53-0.83).22 Gryfe and colleagues reported that 17% of 607 CRC patients who were younger than 50 years had MSI; the HR for patients with MSI-associated tumors was 0.42 compared with patients in the same cohort with non-MSI tumors. Patients with tumors with MSI had lower mortality rates when patients were stratified by tumor stage, including patients with stage IV cancer.25 Among patients with CRC who have MSI, patients with LS seem to have better prognosis than patients with sporadic CRC with MSI.26,27 The most likely explanation is that patients with LS have lower stage disease when they are diagnosed compared with patients with other types of CRC, and it is less common for patients with LS to present with metastatic disease. Germline mutations in tumor-suppressor genes, such as APC, MLH1, and MSH2, indicate a very high risk of CRC and guide the frequency of CRC surveillance and recommendations for prophylactic surgery. Yearly colonoscopy is recommended for carriers of LS, and prophylactic colectomy is considered for patients with high-grade lesions.28,29 The precise explanation of how MSI status offers a better prognosis is not very obvious. Possible explanations of mechanisms by which MSI status affords a better prognosis is the robust lymphocytic response that has been postulated to be correlated with an immunogenic, high neoantigen burden microenvironment due to elevated mutational rate in MSI tumors.30 Second, allele loss at or mutation of DCC, TP53, and KRAS are all associated with a poorer prognosis, and these abnormalities are not common in tumors with MSI.31 However, despite the enormous number of correlative studies exploring the prognostic significance of various molecular features, the microsatellite status is one of the crucial factors that are used for clinical decision-making other than BRAF and RAS mutations. These factors are both prognostic and predictive of chemotherapy efficacy.
Diagnosis of microsatellite instability The identification of a robust molecular prognostic marker to supplement conventional pathologic staging system is highly desirable. Microsatellite status is one of the strong prognostic markers in patients with CRC23 , 32 and is frequently analyzed by testing for loss of an MMR protein by immunohistochemistry (IHC) or testing for MSI using a PCR-based assay. MMR protein by IHC and MSI testing by PCR are complementary but each has its own limitation.33,34 An algorithm for evaluation of newly diagnosed CRC for microsatellite stability status including screening for LS is shown in Figure 2.
Mismatch repair protein by immunohistochemistry • Identifies affected gene by detecting loss of its protein product.33 • Highly concordant with DNA-based MSI testing with good sensitivity (>90%) and excellent specificity (100%).34 • Specific mismatch gene product (MLH1, MSH2, MSH6, and PMS2) can direct germline testing to that specific gene and assists in the identification of patients with LS.34 • In CRCs with loss of MLH1 protein expression, testing for a mutation in the BRAF oncogene is used to confirm a sporadic case and exclude LS. Patients with nonmutated BRAF would then have germline testing for a mutation in the presumed altered MLH1 gene.35 Please cite this article as: R. Gupta et al., The impact of microsatellite stability status in colorectal cancer, Current Problems in Cancer (2018), https://doi.org/10.1016/j.currproblcancer.2018.06.010
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Immunohistochemistry (IHC) for Mismatch Repair (MMR) proteins (MLH1, PMS2, MSH6, MSH2)
All Present
Loss of MSH2 and/or MSH6 or PMS2
Loss of MLH1 and PMS2
Analysis of BRAF V600E mutation by Polymerase chain reaction (PCR) or IHC
Presence of BRAF mutation
Microsatellite stable (MSS)
Sporadic Microsatellite Instable (MSI)
Absence of BRAF mutation
Suspicious Lynch syndrome (LS) age or history
Gene testing for MLH1/PMS2
Not Present
Not suspicious LS age or history
MLH1 methylation
present
Sporadic MSI
If germline mutation present, then LS- refer to genetic counselling
Fig. 2. Algorithm for evaluation of newly diagnosed colorectal cancer for microsatellite stability status including screening for Lynch syndrome.
Microsatellite instability testing by PCR • Evaluates tumors by amplifying microsatellite repeats.34 • In 1997, a National Cancer Institute workshop recommended using a reference panel of microsatellite markers for defining MSI status.36 This panel had 3 dinucleotide markers (D5S346, D2S123, and D17S250), and 2 mononucleotide markers (BAT25 and BAT26). MSI-H was defined as instability at 2 or more markers; MSS as tumors with no evidence of instability at any of the markers, with MSI-L used for the intermediate phenotype.36 • Studies have shown lack of a conclusive cutoff between MSI-L and MSS; therefore, in most of the cases, MSI mainly refers to MSI-H.36 • If MSI analysis is used as the initial screening test rather than IHC, BRAF mutational testing or MLH1 promoter methylation can be used as the second step prior to genetic sequencing in patients found to have MSI.33
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Microsatellite instability and response to chemotherapy (predictive value) Currently, all patients with stage III CRC and some patients with high-risk stage II CRC are recommended to receive 5FU-based chemotherapy, but studies have shown no benefit of 5FUbased chemotherapy among patients with CRC with MSI.26,37 , 38 Thus, MSI status is considered a strong predictive factor for nonresponse to 5FU-based chemotherapy in CRC patients. Several biological mechanisms have been proposed to explain why 5FU-based adjuvant chemotherapy fails to improve outcomes in patients with MSI. This includes the immunosuppressive effects of chemotherapy diminishing the antitumor immune response involving the lymphocytic infiltration characteristic of MSI tumors.11 Second, the intact MMR system ensures that 5FU is efficiently removed from DNA before it can interfere with essential DNA metabolic processes, such as transcription. This also increases the intracellular concentration of the drug and thus exacerbates its cytotoxicity in MSS patients, whereas the absence of MMR system may reduce DNA repair and, thus, attenuate the effect of 5FU in MSI patients.39 In vitro studies have shown that resistance to 5FU in MSI tumors with hypermethylated defective MMR CRC can be reversed after treatment with demethylation agents.39 Randomized trials evaluating outcomes of chemotherapy in CRC with MSI have been summarized in Table 1. The first major study indicating that patients with stage II or stage III MSI CRC did not benefit from FU-based chemotherapy was done by Ribic and colleagues in 2003.26 This study clearly showed that among patients with MSI tumors, treatment was associated with a worse outcome for both stage II and stage III cancer. Among stage II MSI CRC, HR for death was 3.28 (95 % CI, 0.86-12.48) and among patients with stage III MSI CRC, HR was 1.42 (95 % CI, 0.36-5.56).26 Another meta-analysis to study role of MSI status in predicting the efficacy of adjuvant chemotherapy in CRC was done in 2009.37 This meta-analysis involved 7 studies and included 3690 patients with CRC. Eight hundred and ten patients were stage II, and 2444 were stage III patients. Four hundred and fifty four patients were found to have MSI. A total of 1444 patients received 5FU-based chemotherapy, whereas 1518 patients did not. No statistically significant difference was found in MSI patients for recurrence-free survival (RFS) whether or not they received chemotherapy. In addition, the study showed that there was lesser benefit for MSI than for MSS patients, HR interaction RFS 0.77 (95% CI: 0.67-0.87).37 Later in 2010, study done by Sargent and colleagues evaluated the predictive and prognostic values of MSI in 457 stage II and stage III CRC patients from 5 randomized trials.38 These patients were randomly assigned to 5FU-based therapy (either FU + levamisole or FU + leucovorin; n = 229) versus no postsurgical treatment (n = 228). The primary end point was disease-free survival (DFS). Overall, 70 (15%) of 457 patients exhibited MSI. Patients with MSI tumors receiving 5FU had no improvement in DFS (HR, 1.10; 95% CI, 0.42-2.91; P = 0.85) compared with those randomly assigned to surgery alone. In the pooled data set of 1027 patients (165 patients exhibited MSI), these findings were reproducible. In patients with stage II disease and with MSI CRC, treatment was associated with reduced OS (HR, 2.95; 95% CI, 1.02-8.54; P = 0.04).38 The role of oxaliplatin in addition to 5FU and leucovorin has been studied in the adjuvant setting for treatment of MSI stage II and stage III CRC. In an analysis of the Multicenter International Study of Oxaliplatin, 5FU, and Leucovorin (FOLFOX) in adjuvant treatment of colon cancer (MOSAIC) study,40 microsatellite status was evaluated in 986 patients out of 2246 patients, and 90 patients were found to be MSI. Of the patients with MSI CRC, 45 patients had stage II and 45 stage III. In this modest number of patients with MSI colon cancers, a DFS benefit from FOLFOX compared with 5FU alone was observed. HRs for stages II and III MSI were 0.52 (0.21-1.28) for RFS, 0.52 (0.24-1.14) for DFS, and 0.45 (0.19-1.05) for OS, respectively; therefore, this study supported the use of FOLFOX in pts with MSI stage III cancer.41 Another drug that has shown increased sensitivity to MSI cancer cell lines is irinotecan (IFL) though studies have shown conflicting results. Study performed by Bertagnolli and colleagues showed that patients with stage III CRC who had MSI tumors and treated with 5FU and IFL had improved 5-year DFS when compared with MSS tumors with HR of 0.76 (95% CI, 0.64-0.88) versus 0.59 (95% CI, 0.53-0.64), respectively (P = 0.03).42 In contrast, another study that included Please cite this article as: R. Gupta et al., The impact of microsatellite stability status in colorectal cancer, Current Problems in Cancer (2018), https://doi.org/10.1016/j.currproblcancer.2018.06.010
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Study design
Number of patients Stage of CRC
Adjuvant chemotherapy regimen
Ribic et al.26
RCT
570
II/III
5FU or 5FU/levamisole
Des Guetz et al.37
Meta-analysis
(MSI = 95) 3690 (MSI = 454)
II/III
5FU
Sargent et al.38
RCT
457 (MSI = 70)
II/III
Flejou et al.41
Pooled analysis RCT
1027 (MSI = 165) 986 (MSIH = 90)
II II/III
5FU/levamisole Or 5FU/leucovorin 5FU or 5FU + oxaliplatin
RCT
1264 (MSI = 96) 1254
III
Bertagnolli et al.42 Klingbiel et al.
43
RCT
II/III
5FU or 5FU + Irinotecan 5FU/leucovorin Or FOLFIRI
FOLFIRI = Folinic
Worse [stage II- OS: HR 3.28, 95% CI, 0.86-12.48] Stage III- OS: HR 1.42, 95%CI, 1.02-8.54 No benefit [RFS: HR 0.96, 95% CI, 0.62-1.49; OS: HR 0.70, 0.44-1.09] No benefit [DFS: HR 1.10, 95% CI, 0.42-2.91] Worse [OS: HR 2.95, 95% CI, 1.02-8.54] Added benefit with oxaliplatin [OS: HR 0.45,95% CI, 0.19-1.05]; DFS: HR 0.52, 95% CI, 0.24-1.14] Added benefit with Irinotecan [DFS: HR 0.76, 95% CI 0.42-0.71] No added benefit with Irinotecan [Stage II- OS, HR 0.16, 95% CI, 0.04-0.64; RFS, HR 0.26, 95% CI, 0.10-0.65; stage IIIOS, HR 0.70, 95% CI, 0.44-1.09; RFS, HR 0.67, 95% CI, 0.46-0.99] acid + Fluorouracil + Irinotecan;
OS = Overall
survival;
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MSI = Microsatellite instability; RCT = Randomized controlled trial; 5FU = 5Fluorouracil; DFS = Disease-free survival; RFS = Recurrence-free survival; CI = Confidence Interval.
Patient outcomes with chemotherapy in MSI
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Table 1 Randomized trials evaluating outcomes of chemotherapy in CRC with MSI.
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1254 patients with stages II and III CRC showed no benefit in OS or RFS on addition of IFL to 5FU compared with 5FU alone for MSS or MSI tumors warranting further studies.43 Thus, the lack of evidence of benefit from 5FU-based adjuvant chemotherapy in stage II MSI CRC with their favorable prognosis suggests that these patients should not receive adjuvant chemotherapy. In patients with MSS tumors, decisions regarding adjuvant therapy for stage II CRC should be based on other factors that indicate a high-risk patient, such as a T4 tumor, tumor perforation, bowel obstruction, poor differentiation, venous invasion, or fewer than 12 lymph nodes examined. Among patients with stage III disease, the predictive impact of MMR status for adjuvant chemotherapy remains controversial. Multiple prior studies have demonstrating a lack of benefit for 5FU as adjuvant chemotherapy, although only limited data exist for patients with stage III MSI CRCs treated with standard adjuvant FOLFOX regimen. Therefore, as of now, FOLFOX has become current standard of care for adjuvant chemotherapy for stage III colon cancer irrespective of MSI status.
Microsatellite instability and response to immunotherapy The role of immunotherapy in CRC has recently become area of significant interest due to unprecedented successes in advanced solid tumors such as lung cancer and melanoma.16 Based upon knowledge of the immunogenic tumor microenvironment in MSI-H tumors, a trial of anti-PD1 therapy, pembrolizumab was undertaken, enrolling 3 cohorts of patients: MSI-H CRCs, MSI-H non-CRCs, and MSS CRCs. In the initial report, 40% of MSI-H CRC patients achieved an immune-related objective response and 78% achieved 20-week immune-related progression-free survival rate, respectively.15 Updated results continue to show progress wherein 16 (57%) of 25 patients achieved objective responses and additional 9 (32%) with stable disease.15 In another study, 70 patients with MSI-H tumors were enrolled and treated with a different PD-1 inhibitor, nivolumab 3 mg/kg q2week.44 At the initial presentation, 12 (26%) of the 47 patients achieved an objective response with at least 12 weeks of follow-up, with a median time to response of 2.12 months (range 1.3-13.6). An additional 14 (30%) of patients achieved stable disease, for a disease control rate of 55%. In a follow-up presentation at the 2017 American Society of Clinical Oncologists GI cancers symposium, the objective response rate (ORR) was 31% with 69% of patients achieving stable disease. 45 On May 23, 2017, pembrolizumab was granted approval by the U.S. Food and Drug Administration for patients with unresectable or metastatic, MSI or dMMR solid tumors that have progressed following prior treatment and who have no satisfactory alternative treatment options or with MSI or dMMR CRC that has progressed following treatment with a fluoropyrimidine, oxaliplatin, and irinotecan. On July 31, 2017, the Food and Drug Administration also granted accelerated approval to nivolumab for patients metastatic CRC who have MSI-H or dMMR solid tumors whose disease has progressed after chemotherapy. While immunotherapy has shown significant therapeutic response in patients with MSI CRC, its activity against MSS tumors has not been established. Various strategies and clinical trials are being done to evaluate response of immunotherapy to MSS CRC. A phase 1b trial done by Bendall and colleagues46 has used combination therapy involving PDL-1and/or PD-1 inhibitor atezolizumab and cobimetinib (MEK pathway inhibitor) in metastatic MSS CRC showing promising results. The cobimetinib and/or atezolizumab combination showed an investigator-assessed objective response rate (ORR) of 17% in 23 patients overall. An ORR of 20% was observed in 22 patients with KRAS-mutant tumors. The 6-month OS rate was 72%.46 A phase III study is ongoing, where patients with refractory metastatic disease are randomized to cobimetinib and atezolizumab, atezolizumab alone or regorafenib (NCT02788279). Cobimetinib is also being combined with nivolumab and ipilimumab (NCT02060188) as well as being utilized in a trial with atezolizumab and bevacizumab (NCT02876224). Major ongoing trials of immunotherapy in the setting of stage IV CRC have been summarized in Table 2. Please cite this article as: R. Gupta et al., The impact of microsatellite stability status in colorectal cancer, Current Problems in Cancer (2018), https://doi.org/10.1016/j.currproblcancer.2018.06.010
Description
Treatment
Primary end point
Identifier
Phase II—Pretreated stage IV CRC with MSI or dMMR
Pembrolizumab monotherapy
ORR
Phase III—1st line- stage IV CRC with MSI or dMMR
Pembrolizumab monotherapy vs standard of care chemotherapy
PFS
NCT02460198 Keynote 164 NCT02563002
Atezolizumab vs atezolizumab + FOLFOX + bevacizumab vs FOLFOX + bevacizumab Cobimetinib plus atezolizumab vs atezolizumab monotherapy vs regorafenib Nivolumab vs nivolumab combinations Pembrolizumab and cetuximab Pembrolizumab and nintedanib Pembrolizumab plus napabucasin
PFS
NCT02997228
OS
NCT02788279
ORR Objective tumor response, OS, PFS MTD of nintedanib Immune-related ORR determined by their response evaluation criteria in solid tumors Degree of change in TILs Frequency, duration, and severity of adverse events, ORR MTD of poly-ICLC, response rate Percentage of participants with adverse events PFS
NCT02060188 NCT02713373 NCT02856425 NCT02851004
NCT02834052 NCT02876224 NCT02873195
Determination of the recommended phase II dose safety, ORR Safety and tolerability, ORR, PFS, OS
NCT02327078
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Keynote 177 Phase III—1st line Stage IV CRC with MSI or dMMR
Phase III—Pretreated metastatic CRC II—Recurrent and metastatic CRC 1b/II—Pretreated recurrent or metastatic CRC Ib—stage IV CRC Ib/II—refractory CRC
Pembrolizumab plus oral azacitidine and romidepsin Pembrolizumab plus azacitidine plus epacadostat
Phase I/II—Stage IV MSS CRC Phase Ib—stage IV metastatic CRC Phase II—Stage IV CRC
Phase I/II—Advanced solid tumors including CRC
Pembrolizumab plus poly-ICLC Atezolizumab plus cobimetinib plus bevacizumab capecitabine/bevacizumab + atezolizumab vs capecitabine/bevacizumab + placebo Durvalumab in combination with olaparib and/or cediranib Nivolumab plus epacadostat vs nivolumab plus epacadostat + chemotherapy Nivolumab plus varlilumab
Phase I—Stage IV CRC and pancreatic cancer Phase I/Ib—Incurable MSI CRC, solid cancers
Durvalumab and pexidartinib Atezolizumab and CPI-444
Phase 1b/II—Locally advanced rectal cancer Phase II study—Stage IV CRC, NSCLC
Nivulomab following chemoradiotherapy Durvalumab and tremelimumab alone or in combination with high or low-dose radiation
Phase I/II—Refractory CRC Phase I/II—Advanced solid tumors including CRC
Number of participants with treatment-related adverse events, ORR DLT, ORR Incidence DLTs, ORR, Incidence of treatment-emergent adverse events, mean and median area under the curve, mean and median maximum concentration, MDL (maximum dose level) of single agent Pathological complete response ORR
NCT02512172 NCT02959437
NCT02484404
NCT02335918 NCT02777710 NCT02655822
NCT02948348 NCT02888743
ORR = Objective response rate; PFS = Progression-free survival; OS = Overall survival; MTD = Maximum tolerated dose; DLT = Dose-limiting toxicity; NSCLC = Non-small cell lung cancer.
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Phase I—Pretreated MSS advanced CRC Phase I/II—stage IV MSS CRC and NSCLC
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Table 2 Key immunotherapy trials in progress for stage IV CRC.
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Conclusion All patients with resected CRC are recommended to undergo testing to determine their microsatellite status directly through MSI testing or indirectly through IHC of the 4 MMR proteins. Microsatellite status serves not only as a predictive marker for response to chemotherapy and immunotherapy but also as a prognostic marker for patient outcome and a screening tool for detecting patients with LS. Overall, the prognosis for individuals with metastatic CRC remains poor. While the advent of immunotherapies has shown significant promises in improving survival in minority of patients with advanced CRC, additional strategies are required to sensitize patients with MSS CRC.
Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.currproblcancer.2018.06.010.
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Please cite this article as: R. Gupta et al., The impact of microsatellite stability status in colorectal cancer, Current Problems in Cancer (2018), https://doi.org/10.1016/j.currproblcancer.2018.06.010