Is There a Role for Programmed Death Ligand-1 Testing and Immunotherapy in Colorectal Cancer With Microsatellite Instability? Part I—Colorectal Cancer: Microsatellite Instability, Testing, and Clinical Implications

At the molecular level, CRC is not a homogenous disease but can be classified into different subtypes, which are characterized by specific molecular, pathologic, and clinical characteristics. Recently, 2 molecular pathologic classification systems for CRC were proposed, one by The Cancer Genome Atlas (TCGA) project (US National Institutes of Health, Bethesda, Maryland), and a second by the Consensus Molecular Subtypes (CMS) Consortium.¹ 

The integrated molecular analysis by TCGA project integrated multiple platforms, including exome sequencing, DNA copy number, promoter methylation, and messenger RNA and microRNA expression, to develop comprehensive CRC profiles. This approach classified CRCs into 2 major groups consistent with previous classification systems.⁴ 

Tumors associated with chromosomal instability represent approximately 84% of all CRCs. The tumors are characterized by gross changes in chromosome number and structure, including deletions, gains, translocations, and other chromosomal rearrangements. Most of these tumors arise by the adenoma-carcinoma sequence with inactivating mutations or losses in the APC tumor-suppressor gene. Other common mutations include TP53, KRAS, SMAD4, PIK3CA, ARID1A, SOX9, and AMER1 (previously called FAM123B). In addition, amplifications of ERBB2 and IGF2 have been found in a subset of tumors and are potentially drug targetable. These tumors are microsatellite stable (MSS) and tend to be left sided.

The microsatellite instability–high (MSI-H) category represents approximately 15% to 16% of all CRCs. These tumors are hypermutated cancers either because of defective MMR (13%) or because of DNA polymerase-ɛ proofreading mutations (3%).⁴ 

The recent CMS Consortium (Colorectal Cancer Subtyping Consortium [CRCSC]) analyzing CRC expression profiling data from multiple studies described a novel classification by aggregated gene expression. They proposed 4 robust CMS groups⁵ :

1. CMS1 (MSI-H, 14%).—The CMS1 tumors include almost all hypermutated, microsatellite-unstable CRC cancers with strong immune activation that are found frequently in women with right-sided tumors.

2. CMS2 (Canonical, 37%).—The CMS2 tumors display epithelial differentiation, are chromosomally unstable, and show marked WNT and MYC signaling activation. They are mainly left-sided tumors.

3. CMS3 (Metabolic, 13%).—The CMS3 tumors are epithelial, show evident metabolic dysregulation, and possess KRAS-activating mutations.

4. CMS4 (Mesenchymal, 23%).—The CMS4 tumors have prominent transforming growth-factor β activation, stromal invasion, and angiogenesis and are frequently found at an advanced stage, with worse overall survival and disease-free survival.

Tumors with mixed features (13%) represent a transition phenotype or intratumoral heterogeneity.⁵ 

Although further research is required to validate these 2 systems, they may be useful for clinical trial designs and future postsurgical adjuvant-treatment decisions, particularly for tumors with aggressive features or predicted responsiveness to immune checkpoint blockade.⁵ 

Errors of DNA replication are inevitable, but specialized MMR systems have evolved that identify and correct mismatched pairs of DNA, acting like “proofreaders” or “spell checkers.” Microsatellite instability is the molecular fingerprint of a deficient MMR system.

Microsatellites are tandem repeats of 1 to 6 nucleotides scattered throughout the genome and are particularly prone to DNA replication errors, mainly because the DNA polymerases cannot bind efficiently during DNA synthesis.⁶  Microsatellite sequences of an individual are fixed for life and are the same in every tissue. The most frequent errors associated with microsatellites are base-base mismatches that escape the intrinsic proofreading activity of DNA polymerases, and insertion-deletion loops. These partnerless nucleotides occur when the first nucleotide and template strand dissociate and incorrectly reanneal in a microsatellite. Insertions or deletions in microsatellites located in the DNA coding regions generate frameshift mutations leading to protein truncations.

The MMR system, comprising 6 proteins (MutL protein homolog, MLH1; MutS protein homologs, MSH2, MSH3, and MSH6; PMS1 protein homolog, PMS1; and MMR endonuclease, PMS2) and the corresponding genes, recognizes and repairs errors that occur during DNA replication. When a mismatch is detected, 3 steps take place: MSH2 associates with either MSH6 or MSH3 (forming MutSα and MutSβ complexes, respectively) and MLH1 couples with PMS2, PMS1, or MSH3 (forming MutLα, MutLβ or MutLγ complexes, respectively). The recognition of mismatches and insertion-deletion loops is performed by a sliding clamp formed by the combination of a MutS and a MutL complex, which interacts with replication factor C. Excision of human exonuclease 1 (hExo1) is performed by proteins such as exonuclease-1 and proliferating cell nuclear antigen. Resynthesis and relegation is carried out by DNA polymerase δ and DNA ligase.^7,8  Mutations in the genes responsible for the recognition step lead to an accumulation of errors in DNA, which results in MSI-H.

The unrepaired microsatellite alterations contribute to carcinogenesis along a distinct pathway (the MSI pathway) that differs from the chromosomal instability pathway.^9–13 

There are 2 general mechanisms for MSI occurrence.

1. Acquired (Somatic) MSI.—Acquired (somatic) MSI is seen in 10% to 13% of all CRCs. These tumors can be broadly defined as sporadic CRC without germline MMR mutations. This type of CRC exhibits MSI-H and loss of function of the MLH1 protein because of CpG island hypermethylation in the promoter of MLH1 gene.¹⁴  More than 95% of sporadic MSI-H CRCs are associated with MLH1 promoter hypermethylation.¹⁵  These tumors exhibit in 60% of cases with a missense mutation in the BRAF gene, which leads to an amino acid substitution in codon 600 (p.V600E), with constitutive signaling of the BRAF protein. BRAF V600E mutations are mutually exclusive with KRAS mutations, the latter being more commonly associated with MSI tumors. The presence of a BRAF mutation indicates a sporadic MSI-H CRC and essentially excludes a diagnosis of an inherited Lynch syndrome.¹³  Most sporadic MSI-H CRCs are thought to arise via the sessile-serrated adenoma-carcinoma pathway.¹⁶ 

2. Inherited (Germline) MSI.—Inherited MSI occurs in 2% to 3% of all CRCs. These tumors occur because of germline mutations in 1 of the MMR genes (most commonly MLH1, MSH2, MSH6, and PMS2), germline deletions of EPCAM, a gene upstream from MSH2 or germline MLH1 promoter hypermethylation.^11,14,17  Deletions affecting the 3′ exons of the EPCAM gene lead to a transcriptional read through and mediate epigenetic silencing of the MSH2 allele in a mosaic pattern. Therefore, CRCs in individuals with heterozygous germline EPCAM deletions will be MSH2⁻ MSI-H cancers.¹⁸  Inherited CRC, referred to as hereditary nonpolyposis CRC, is a highly penetrant, autosomal-dominant cancer syndrome. Hereditary nonpolyposis CRC comprises at least 2 distinct clinical entities: Lynch syndrome from MMR repair gene mutations and familial CRC type X, which develops in families with similar patterns of heredity but without disease-predisposing, germline MMR mutations.¹⁹ 

Lynch syndrome is further categorized as (1) Lynch syndrome type 1, with an early onset of colon cancers only, which may be multiple (either synchronous or metachronous), and has 50% risk of colon cancers in first-degree relatives; (2) Lynch syndrome type 2, which, in addition to colon cancers, has a high risk for the development of endometrial, ovarian, renal, ureteral, and gastric carcinomas and cholangiocarcinoma; and (3) the rare Muir-Torre syndrome, presenting with sebaceous neoplasms, keratoacanthomas, and colorectal and/or urogenital malignancies.^11,17 

Clinically, patients with Lynch syndrome are identified by Amsterdam I and II screening criteria and, recently, by modified Bethesda guidelines, which also incorporate pathologic features of MSIH CRC.^11,17,20,21 

Lynch syndrome is due to heterozygous germline mutations in 1 of the MMR genes MLH1 (weighted proportion 32%), MSH2 (weighted proportion 39%), MSH6 (weighted proportion 10%), and PMS2 (weighted proportion 15%).²²  The BRAF V600E mutation is not associated with Lynch syndrome–related CRCs.

Identification of MSI-H (or MMR-deficient) colorectal tumors is important because MMR deficiency may serve as a prognostic marker of patient outcome: MSI-H CRCs have been shown to have a better overall prognosis compared with MSS tumors^23,24 ; it can be a predictive marker of response to 5-fluorouracil–based chemotherapy²⁵ ; and it can be used as a screening tool for Lynch syndrome.

The gross and microscopic features of MSI-H CRC have been described extensively.^21,26–28 

The MSI-H CRC, regardless of its molecular pathway (sporadic or inherited), may show similar histologic features; morphologically, it may be poorly differentiated and show a medullary pattern (Figure 1, A). They may have a significant mucinous component (>50%), with signet ring cells (Figure 1, B), a lack of the typical dirty necrosis usually seen in MSS CRC, and have increased tumor-infiltrating lymphocytes (Figure 1, C). They frequently have a circumscribed/expansile growth pattern (Figure 1, D), intratumoral heterogeneity (mixed conventional, mucinous, and poorly differentiated carcinoma), and a prominent inflammatory reaction at the advancing edge of the tumor (Crohn-like reaction) (Figure 1, E).²⁷ 

Tumor-infiltrating lymphocytes are mostly cytotoxic T cells, are CD8⁺ (Figure 1, F), and are closely associated with MSI and medullary architecture. They should be distinguished from Crohn-like, peritumoral infiltrates (lymphoid aggregates or follicles at the tumor edge, not associated with a preexisting lymph node tumor). Although absolute cutoff values have not been established, only moderate- and high-density intratumoral lymphocytes (≥3 tumor-infiltrating lymphocytes/high-power field) should be considered significant.^26–28 

There are no distinct pathologic features that can predict sporadic from germline MSI-H CRCs. Both tend to occur in the right colon; however, sporadic tumors tend to occur in older women (older than 70 years), whereas germline MSI-H CRC of Lynch/hereditary nonpolyposis CRC syndrome tends to occur in younger men (younger than 60 years) who may have a history of other cancers. If the tumor is sporadic because of MLH1 hypermethylation, a residual, preexisting, sessile-serrated polyp may be evident, whereas if it is due to germline MMR mutations, a preexistent, classic adenoma may be evident.

The presence or absence of MSI should be confirmed in all patients with CRC to ensure accurate prognosis, appropriate treatment, and risk assessment for the patient and relatives. The Amsterdam criteria (1990), revised Amsterdam criteria (1998), Bethesda guidelines (1997), and revised Bethesda guidelines (2004) are used in clinical practice to identify individuals at risk for Lynch syndrome who require further evaluation by testing the tumor (Table 1).^2,11,17 

There are 5 separate tests used to varying degrees in the workup of MSI-H CRC.

The current gold standard for assessing tumor DNA MMR activity is molecular MSI testing. Frameshift mutations in microsatellites can be identified by extraction of DNA from healthy and tumor tissue from formalin-fixed, paraffin-embedded tissue, amplification of selective microsatellites by polymerase chain reaction (PCR), and analysis of fragment size by gel electrophoresis or an automated sequencer. Criteria have been developed to standardize the molecular classification of MSI, using either a Bethesda consensus-defined panel of microsatellites or a recently revised panel standardized by the National Cancer Institute, which uses 5 mononucleotide markers: 2 mononucleotide repeats (BAT26 and BAT25) and 3 dinucleotide repeats (D5S346, D2S123, and D17S250).²⁰  The sensitivity of this revised panel to detect MSI CRC is 90%. Using this reference panel, tumors can be characterized as MSI-H if 2 or more of the 5 markers show instability in ≥30% of loci tested (ie, have insertion/deletion mutations), and as low-frequency MSI-Low if only 1 of the 5 markers shows instability in 10% to 30% of the loci tested and MSS if no loci show instability. Although MSI-H is characteristic of Lynch syndrome tumors, it may be found in about 15% of unselected groups of CRCs. Of this subset of MSI-H tumors, 20% to 25% represent Lynch syndrome and the other 75% to 80% are sporadic MSI-H tumors caused by methylation-induced silencing of MLH1.¹⁷  A finding of MSI-H in a tumor should prompt further analysis, such as sequencing of DNA MMR genes (germline-mutation testing).

An MSI assessment by PCR is sensitive, but not specific, for Lynch syndrome because only 20% to 25% of all CRC tumors are associated with germline mutations in a DNA MMR gene. In addition, PCR will miss about 5% of all Lynch syndrome mutation carriers and is only 86% sensitive in CRCs from families with germline mutations in MSH6.²⁹  For this reason, IHC is of value to supplement molecular MSI testing. Moreover, IHC is a much less labor-intensive and more cost-effective method than MSI testing by PCR. It has a sensitivity of more than 90%, comparable to that of an MSI analysis, a specificity of 100%, with a positive predictive value of MSI-H of 100%, with an abnormal staining pattern and an excellent concordance between IHC and molecular testing. Immunohistochemistry is being performed in many academic and community hospitals, either to complement MSI testing or as a substitute. The IHC analysis can direct genetic testing to the appropriate MMR gene when loss of a MMR protein expression is identified.

Tumor testing with IHC on formalin-fixed, paraffin-embedded tissue uses 4 commercially available antibodies that are specific for MMR proteins: MLH1, MSH2, MSH6 and PMS2.

In their functional state, the MMR proteins form heterodimers. As previously mentioned, during normal DNA MMR activation, MLH1 recruits its binding partner PMS2 to the site of DNA repair. If MLH1 is mutated, as in Lynch syndrome, and is lost from the DNA MMR complex, then PMS2 will also be absent from the repair-protein complex. Therefore, mutation and loss of MLH1 protein expression, demonstrated by IHC, is almost always accompanied by loss of PMS2 expression. The same holds true for MSH2 and its binding partner MSH6. In contrast, germline mutations of either PMS2 or MSH6 are usually associated with the loss of the respective protein alone. This is because the function of the secondary proteins, PMS2 and MSH6, may be compensated for by other proteins, such as MSH3, MLH3, and PMS1.³⁰ 

Normal colonic mucosa, stromal fibroblasts and lymphocytes, and MSS CRC show retained protein nuclear IHC expression for all 4 proteins (Figure 2, A). An abnormal result shows loss of nuclear IHC expression with either protein, resulting in 4 staining patterns (Table 2): (1) loss of MLH1 and PMS2 (Figure 2, B and C), (2) loss of PMS2 alone, (3) loss of MSH2 and MSH6, and (4) loss of MSH6 alone.

Because IHC is so widely used, pathologists must be aware of some of the pitfalls and limitations in stain interpretation:

1. Weak or patchy staining (heterogeneous staining) corresponds to differences in MMR status within the tumor, and is, therefore, important to recognize to prevent false-positive or false-negative evaluations. Three distinct patterns of heterogeneous expression have been described: intraglandular (retained/lost staining within or in between glands), clonal (retained/lost staining in whole glands or groups of glands), and compartmental (retained/lost staining in larger tumor areas/compartments leading to retained/lost staining in between different tumor blocks).³¹  Multiple causes may apply, such as variable epitope expression, expression related to variable differentiation (mucinous areas versus poor differentiation), second-hit mutations or methylation in selected tumor clones, and possibly, influence from factors linked to the tumor microenvironment, such as hypoxia and oxidative stress.³² 

2. Cytoplasmic staining (rather than nuclear) may be interpreted erroneously as normal/intact (Figure 2, B).

3. Neoadjuvant therapy, especially in rectal cancers, may affect IHC staining, especially for MSH6, which may be absent, weak, or stain just the nucleoli.^33,34  In these cases, pretreatment biopsy, if available, may be more suitable for MSI testing.

4. Prolonged ischemic time may impair MMR function in otherwise MMR-proficient tumors.³²  Better tissue preservation could potentially reduce such effects of tissue microenvironment and improve the performance of IHC. In our laboratory, we select a “scout” block at the time of specimen arrival, before exposure to 10% formalin, which includes both tumor and healthy adjacent colonic mucosa; all ancillary testing is performed on the selected block.

5. Intact expression of all 4 proteins does not entirely exclude Lynch syndrome because approximately 5% of families may have missense mutation (especially in MLH1), resulting in a nonfunctional protein with retained antigenicity.

6. The IHC antibodies are not available for other, lesser-known MMR enzymes (rare).

The serine/threonine protein kinase BRAF is important in the epidermal growth factor receptor (EGFR)–mediated, mitogen-activated protein kinase (MAPK) pathway. BRAF activates the MAPK pathway that affects cell growth, proliferation, and differentiation but also affects other key cellular processes, such as cell migration, apoptosis (through the regulation of BCL2), and survival.³⁵  Mutations in the BRAF gene have been identified exclusively in CRC with wild-type KRAS genes (KRAS and BRAF mutations are mutually exclusive) and is usually associated with significant poorer prognosis. Almost all BRAF mutations are V600E point mutations assayed by PCR methodologies. BRAF mutations are present almost exclusively in serrated pathway neoplasms, and if present, it excludes Lynch syndrome. BRAF mutation testing is used in diagnostic-testing algorithms as a means to differentiate sporadic, MLH1-deficient colon cancer (methylated) from hereditary MLH1-deficient cancer with germline mutations (Figure 3).³⁵ 

MSI-H sporadic CRCs arise through a process that involves the CpG island methylator phenotype. About half of the genes in the human genome have promoters that are embedded in clusters of cytosine-guanosine residues called CpG islands. Cytosines in those regions can be methylated by DNA methyltransferases. Methylation is a means by which a cell permanently silences genes.⁹  Methylation increases with advancing age, is accelerated in the colon in response to chronic inflammation, and might be an adaptive response to injury. A subset of CRCs progresses by methylating tumor-suppressor genes, such as CDKN2A (encoding p16 tumor suppressor protein) and insulin-like growth factor 2 (IGF2), and DNA repair genes, such as O-6-methylguanine-DNA methyltransferase gene (MGMT) and MLH1.⁹ 

The sporadic, hypermethylated MLH1-deficient CRC may be differentiated from MLH1-deficient Lynch syndrome by MLH1 methylation testing with molecular PCR assays that determine the presence or absence of promoter hypermethylation. If methylation is present, the tumor is presumed to be sporadic and not due to Lynch syndrome, and no further testing may be required. If no MLH1 promoter hypermethylation is present and no BRAF mutation was identified, then additional testing for germline mutations and referral for genetic counseling is required.

Genetic testing for germline MMR mutations is complicated, time-consuming, and expensive. Germline testing should be undertaken only after informed consent is obtained from the patients and is usually directed by the IHC and/or MSI results. Demonstration of the loss of particular MMR proteins can then direct which genes should be examined by germline mutation analysis. For example, loss of MSH2 and MSH6 expression by IHC generally indicates the presence of a germline MSH2 mutation. Loss of MSH6 or PMS2 alone generally indicates the presence of a germline MSH6 or PMS2 mutation, respectively. For tumors with loss of MLH1 and PMS2 expression, MLH1 promoter hypermethylation must be excluded first before genetic testing for MLH1 is recommended (see above).

Recently, several groups have evaluated new methods to assess MSI using massively parallel DNA-sequencing technologies from tumor-normal tissue pairs.^36–39  They used tumor exomes from the TCGA Research Network to comprehensively examine MSI across different tumor types, including CRC. Hause et al³⁶  designed a weighted-tree, microsatellite instability classifier to distinguish MSI-H from MSS cancers. Compared with MSI testing by PCR, the microsatellite instability classifier separated MSI-H from MSS cancers with 96.6% cross-validation accuracy (95.8% sensitivity, 97.6% specificity) in a set of 617 specimens. The microsatellite instability classifier was discordant with clinical testing in classifying 11 of 171 MSI-H tumors (6%; 1 rectal and 10 endometrial) as MSS and 7 (1 rectal, 1 colon, and 5 endometrial) of 446 MSS cancers (2%) as MSI-H. Discordant classifications were primarily in endometrial cancers.³⁶  They postulated that, because the microsatellite instability classifier was more comprehensive and less prone to cancer type–specific biases, it may serve as a better clinical strategy for pancancer MSI determination and ascertainment of instability burden.

Several testing algorithms were proposed to assess MSI in CRCs by various organizations, including National Comprehensive Cancer Network (Fort Washington, Pennsylvania), American College of Medical Genetics and Genomics (Bethesda, Maryland), American College of Gastroenterology (Bethesda, Maryland), Evaluation of Genomic Applications in Practice and Prevention (Centers for Disease Control and Prevention, Atlanta, Georgia),^{14,18,22,40,41}  as well as several clinical investigations, beautifully summarized by Funkhouser et al.¹⁴  Most of them include IHC as prescreening, followed by confirmation with MSI by PCR, BRAF mutation analysis, and MLH1 methylation. Concomitant MSI and IHC analyses of all new CRCs (universal testing) effectively identifies CRC patients at increased risk for Lynch syndrome who should be offered germline mutation analysis. The decision to perform MSI analysis, IHC, or both is dependent on institutional preferences and resources. Once a tumor is determined to be MSI-H by PCR and/or demonstrates loss of MMR protein expression by IHC, the individual can, after appropriate genetic counseling, elect to have molecular genetic testing to identify a germline mutation in 1 of the MMR genes. A suggested practical and cost-effective testing algorithm for MSI is depicted in Figure 3.

Recently, there has been an increased recognition of the host immune system importance in controlling tumor progression and new immunologic biomarkers have been included as a tool for the prediction of prognosis and response to therapy. A group of researchers led by Galon et al⁴¹  looked into developing a standardized immune-based assay for classification of cancer named Immunoscore (IS; Laboratory of Integrative Cancer Immunology, INSERM, Paris, France), which may have a more significant prognostic value than the TNM classification of malignant tumor staging system (American Joint Committee on Cancer, Chicago, Illinois and Union for International Cancer Control, Geneva, Switzerland) and MSI status in CRC. The IS may provide better staging of the disease; hence, an improved clinical decision-making process for patients with colon cancer.^42–46  The IS quantifies the number, density, and distribution of CD3⁺ T lymphocytes and CD8⁺ cytotoxic T cells in the tumor core and invasive margins. The IS analysis is performed on IHC slides stained with CD3 and CD8, analyzed by an image-analysis software of the scanned slides, with a score from 0 (low densities of CD3/CD8⁺ T cells) to 4 (high densities of CD3/CD8⁺ T cells) given. In CRC, the IS significantly improved prognostic accuracy independent of clinical variables for all patients with CRC stages I/II/III and had superior prognostic power versus the classic TNM staging system. Recently, IS was shown to be a stronger predictor of survival than MSI status. Patients with high immune infiltrate (IS 3 and IS 4) have significant, prolonged, disease-specific recurrence, regardless of their MSI status.^46,47  The MSI-H tumors often contain numerous, intraepithelial T cells (Figure 1, F) in response to the expression of neoantigens on the cell surface. It is unclear to what extent MMR deficiency per se, versus high numbers of lymphocytes, or both contribute to the better prognosis of patients with MSI-H tumors. Although patients with tumors showing high IS (IS-High) are overrepresented in the MSI-H group, compared with the MSS group, there is a substantial number of IS-High cases in the MSS group, indicating that there is a good number of MSS cases that are immunogenic. These findings indicate that assessment of the immune status via IS provides a potent indicator of tumor recurrence beyond MSI status, which could be an important guide for immunotherapy strategies.

A major study has been conducted by the IS worldwide consortium, led by the Society for Immunotherapy of Cancer (Milwaukee, Wisconsin) involving 23 pathology centers from 17 countries and including more than 3800 patients with stage I to III colon cancer, and its results were presented at the American Society of Clinical Oncology annual meeting (June 2–6, 2016; Chicago, Illinois) and European Society for Medical Oncology annual meeting (October 7–11, 2016; Copenhagen, Denmark).⁴²  The study showed that IS predicted time to recurrence, disease-free survival, and overall survival; was statistically significant in multivariate models, including TNM stage; identified a subgroup of patients with high-risk stage II cancer who could potentially benefit from adjuvant chemotherapy; and is a robust and reproducible test across testing centers. The results of this international validation may result in the implementation of the IS as a new component for the classification of cancer, designated TNM-Immune.⁴⁵ 

This is a particularly timely finding in the era of immunotherapy because IS-based assays could be used to predict which patients would be more likely to benefit from treatment modalities, such as checkpoint blockade, or whether strategies such as adjuvant therapy or cancer vaccines to prime immunity might be more appropriate.

The diagnosis of hereditary Lynch syndrome and recognition of sporadic CRCs with MSI-H have important implications regarding cancer prevention, surveillance, and management. Studies have shown that MSI-H CRCs carry a better prognosis, stage for stage, compared with MSS CRCs.⁴⁸  In addition, stage II and stage III MSI-H CRCs achieved similar progression-free survival and overall survival, with or without 5-fluorouracil-based neoadjuvant chemotherapy.^25,49  Therefore, patients with stage II and stage III MSI-H CRC are not recommended to receive single-agent 5-fluorouracil chemotherapy in the adjuvant setting.²⁵  As mentioned earlier, individuals with Lynch syndrome have significantly higher risks of developing extracolonic malignancies in addition to early onset of CRC. Intensive cancer surveillance has been shown to substantially reduce cancer-related death in this group of patients.

A recent small phase 2 clinical trial showed that patients with MSI-H CRCs are good candidates for checkpoint immunotherapy with the anti–programmed death receptor-1 drug pembrolizumab (Merck & Co., Kenilworth, New Jersey). This led the US Food and Drug Administration to grant a breakthrough therapy designation in November 2015 to pembrolizumab for the treatment of metastatic/refractory MSI-H CRC.⁵⁰  The role of immunotherapy in MSI-H CRC is detailed in part II of this article (in this issue).

Microsatellite instability-high is a molecular subtype of CRC that displays a well-defined histopathologic and therapeutic profile that is distinct from other molecular subtypes. Microsatellite instability can be reliably detected by molecular testing, IHC techniques, and genetic testing developed during the past 2 decades. All efforts must be made to identify the presence of MSI in all CRCs, either germline or sporadic, in all patients to ensure appropriate treatment, accurate prognosis, and risk assessment for relatives.

Today, the TNM staging system provides the most reliable reference for the routine prognostications and treatment decisions for CRC. Recently, the IS was validated as a prognostic marker in stage I/II/III colon cancer superior to TNM staging and MSI in predicting patients' disease-specific recurrence and survival. This may result in the implementation of the IS as a new component for the TNM classification of cancer, designated TNM-Immune, although the latest eighth edition of AJCC Cancer Staging Manual,⁵¹  published in November 2016, failed to include it.

The MSI status also has implications in selecting the appropriate chemotherapy. Patients with MSI-H CRC do not show a clinical benefit from single-agent 5-fluorouracil chemotherapy in the adjuvant setting, and very recently pembrolizumab was approved for the treatment of metastatic/refractory MSI-H CRC. Drug-development strategies focused on specific molecular subtypes clearly represent the future of cancer therapeutics. Clinical trials based on molecular classifications are likely to have stronger rationales than previously performed classic studies and will increase the likelihood of obtaining a relevant therapeutic response, thus improving the health and well-being of patients.