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INTRODUCTION

In 2020, an estimated 147,950 men and women in the United States will be diagnosed with colorectal cancer (CRC) and approximately 53,200 cancer deaths will be attributed to this malignancy.¹ The overall CRC incidence and mortality rates have declined over about the past 4 decades, largely due to improved clinical outcomes associated with screening and early detection, as well as new and more effective treatment options. Significant improvements in survival, particularly in patients with metastatic CRC (mCRC), have resulted from important gains in knowledge regarding oncogenic mutations and molecular drivers for the disease, testing strategies to characterize tumor molecular subtypes and profiling, and advances in available treatments that target these biomarkers and related pathways. Pharmacists provide essential services to optimize patient outcomes by facilitating patients’ access to these therapies, identifying and mitigating treatment-related adverse effects, and educating patients about these potential adverse effects and the importance of prompt reporting of associated signs or symptoms.

COMMON GENETIC ALTERATIONS IN ADVANCED CRC

CRC is a molecularly heterogeneous disease composed of different tumor phenotypes that arise from genetic and epigenetic alterations in tumor suppressor genes, oncogenes, and genes associated with DNA repair. Two distinct molecular carcinogenesis pathways, chromosomal instability (CIN) and microsatellite instability (MSI), lead to genomic and epigenomic instability, a hallmark of colorectal carcinogenesis. Most (~ 85%) CRC develop through the CIN pathway, accumulating mutations in oncogenes and tumor suppressor genes such as APC, TP53, KRAS, and BRAF,SMAD4, and PIK3CA.^2,3 CRC also arise through the MSI pathway, with features characteristic of deficient DNA mismatch repair.

DNA mismatch repair deficiency and MSI

Up to 15% of CRCs possess a hypermutated phenotype, MSI, characterized by sequence alterations in microsatellites, sequences of DNA that are particularly prone to replication errors, caused by inactivation or deletion of one or more DNA mismatch repair (MMR) genes, MLH1, MSH2, MSH6, or PMS2.³ An absent or inactivated DNA MMR gene results in a loss of MMR function, referred to as deficient MMR (dMMR). With the loss of MMR protein activity, DNA replication errors accumulate, causing nucleotide base mismatches, indels and frameshift mutations in DNA microsatellites. These errors – and there can be thousands – accumulate, resulting in MSI. Therefore, tumor cells with dMMR accumulate a large burden of somatic mutations, which accounts for a hypermutated phenotype and high MSI (MSI-H). dMMR can be inherited or develop by somatic causes. Individuals with Lynch syndrome, an inherited cancer syndrome, develop MSI-H CRCs due to germline mutations in one of the MMR genes. The vast majority of MSI-H/dMMR CRC, however, are sporadic cases that result from epigenetic silencing of the MLH1 gene promoter due to hypermethylation.²

MSI-H tumors tend to arise in the proximal, or right colon, and are characterized by a prominent lymphocytic infiltration.² Activated T lymphocytes play a critical role in the immune response to cancer cells and their elimination.⁴ However, as tumor antigens and other factors recruit immune cells to the tumor milieu, inflammatory cytokines and growth factors are released into the tumor microenvironment (TME) and promote immunosuppression. Activated T lymphocytes express programmed death-1 (PD-1), a co-inhibitory checkpoint receptor that provides a physiological feedback mechanism to prevent auto-immunity, which further promotes the immunosuppressed TME. Programmed death ligand-1 (PD-L1), expressed on tumor cells and tumor-infiltrating immune cells, also promotes tumor progression through binding to PD-1 and down-regulating T- cell activity. Positive tumor PD-L1 expression is indicative of a weakened host immune response and may serve as a prognostic marker for poorer overall survival.⁵

BIOMARKER AND CANCER MUTATION TESTING

Treatment decisions for individuals diagnosed with mCRC are driven by the molecular biology of the tumor.⁶ Cancer mutation testing informs these decisions through biomarkers that predict tumor response (or lack of response) to targeted therapies, thereby avoiding unnecessary toxicities due to patient exposure to agents least likely to provide a therapeutic benefit. All patients with CRC should undergo DNA MMR testing for the purposes of identifying potential Lynch syndrome and as a predictive biomarker for immune checkpoint therapy.⁷

Biomarkers for immune checkpoint inhibitors

Established biomarkers to identify patients with mCRC who are appropriate candidates for immune checkpoint inhibitors include determinants of MSI-H status, dMMR, and high tumor mutational burden (TMB). Tumors with dMMR are characterized by MSI-H tumors. Tumor MSI-H/dMMR is most commonly identified using immunohistochemistry (IHC) staining or DNA molecular sequencing.⁸ The dMMR genes encode for 4 proteins, MLH1, MSH2, MSH6, and PMS2, and commercially available antibodies are used to detect the tumor expression of these proteins using IHC. An absence or decreased intensity of staining for one or more proteins will suggest the presence of dMMR. BRAF mutation testing should be performed when IHC shows absent MLH1 tumor expression, since a BRAF mutation in the setting of absent MLH1 indicates a somatic rather than germline mutation, thus excluding Lynch syndrome as a cause of dMMR. Although IHC is widely available and routinely used, false-positive and -negative results can occur due to technical reasons or biological factors. Therefore, molecular testing is required to formally prove MSI-H/dMMR, which involves comparing a reference panel of 5 microsatellites derived from both normal and tumor tissue. The microsatellite areas are amplified using polymerase chain reaction (PCR) and a change in the size of at least 2 microsatellites in the tumor identifies MSI-H.

Next generation sequencing (NGS) is another method that can be used to determine tumor MSI status and TMB, as well as other potential biomarkers such as PD-L1 expression and specific mutations arising from single-nucleotide variants (SNV), insertions or deletions (indels) and copy-number variants (CNVs). Traditionally, PD-L1 expression in tumors has been assessed using IHC; several commercially available assays are used to determine PD-L1 expression where FDA-approved indications for immune checkpoint inhibitors are based on this biomarker. Depending on the malignancy, PD-L1 expression is quantified by the degree of positive staining in tissue tumor cells, as a combined positive score (CPS) that includes PD-L1 expression in tumor and immune cells, or as a tumor proportion score (TPS), which is based on the proportion of viable tumor cells that show partial or complete membrane staining at any intensity.⁹ Unlike its role in other malignancies, however, PD-L1 expression has not proven useful to date as a predictive marker for clinical outcomes in mCRC.¹⁰

Tumor mutational burden (TMB)

Tumors with a high degree of MSI and an associated high number of mutations, referred to as TMB, are more immunogenic. High tumor mutational burden can result from endogenous impaired DNA repair or endogenous factors such as carcinogen exposure-induced DNA damage.¹¹ As such, they are more likely to be recognized by the immune system and thus more sensitive to immune checkpoint inhibitors. The general correlations between responses to immune checkpoint inhibitors and among these factors – mismatch repair deficiency, high MSI, and high TMB – have led to the agnostic approvals of anti-PD-1 therapy on this basis. However, even among patients with both MSI-H and dMMR tumors, responses to anti-PD-1 therapy vary considerably, and almost half of patients experience limited clinical benefit from treatment. The reason for this variable response is largely unknown, and of great interest to define. Since not all MSI-H/dMMR CRC respond to immune checkpoint inhibitors, TMB may help further predict which of these patients may benefit from these therapies.¹² TMB is a measure of the number of mutations carried by the tumor cells and, although MSI-H tumors are associated with a higher TMB, tumors with a high TMB may not be MSI-H. Quantification methods and thresholds for establishing high tumor TMB are not standardized among tumor types; however, ³ 10 mutations per megabase (Mb) was established for the initial FDA-approved use indication for pembrolizumab for TMB-H tumors.¹³

In the KEYNOTE-158 study, treatment outcomes for patients with advanced solid tumors treated with pembrolizumab were analyzed with respect to tissue TMB (tTMB), which was determined using the FoundationOne CDx assay.¹⁴ Among all participants evaluable for TMB and response to therapy, 102 of 790 (13%) patients had tTMB-high status and the remaining 688 (87%) of patients had non-tTMB-high status. Thirty patients in the tTMB-high group (29%; 95% CI 21-39) experienced an objective response compared to 43 (6%; 95% CI 5-8) in the non-tTMB-high group. In a separate study that examined the association between TMB and survival in multiple cancer types, patients with a higher somatic TMB (the highest 20% in each histology) experienced better overall survival.¹² The TMB cut point was 52.2/Mb for the top 20% of CRC patients. Although neither of these trials focused specifically on patients with advanced CRC, high TMB may be useful in identifying patients with microsatellite stable (MSS)/proficient MMR (pMMR) who might benefit from immune checkpoint inhibitors. Alternatively, it may be reasonable to consider chemotherapy as first line treatment for patients with MSI-H/dMMR mCRC but low TMB. TMB is elevated in approximately 8% to 25% of CRC, among which about 75% are considered as MSI-H.³ Ongoing studies are evaluating these strategies, as much additional information will be helpful to guide treatment decisions.

Emerging biomarkers for checkpoint inhibitor immunotherapy

Other potential biomarkers to predict response to immunotherapy are promising. Specific mutations, characteristics of immune cell infiltration, and biological features may be associated with tumor responsiveness to immunotherapy. As mentioned, certain tumor mutations may be associated with differentially improved outcomes to immune checkpoint inhibition. One group of investigators utilized mouse models and interrogated the genome wide MSI intensity and mutational load of MSI-H tumors from patients treated with immune checkpoint inhibitors using clinical data and tumor sequencing data from the Cancer Genome Atlas project.¹⁵ These investigators determined that the intensity of MSI using MSI scores (to indicate greater genomic instability) and the nature of tumor mutation – indel – were associated with a greater response to anti-PD-1 therapy. These findings require validation but suggest the potential to improve upon methods using MSI and tumor mutational burden.

Another example of potentially predictive mutations in patients with MSS/pMMR CRC are in DNA polymerase epsilon (POLE) and DNA polymerase delta (POLD1), two enzymes involved in DNA synthesis and repair.¹⁶ Mutations in the genes for these enzymes are associated with very high numbers of accumulated tumor mutations, often even higher than seen with MSI-H/dMMR CRC. They are found in only about 1% to 2% of pMMR CRCs but are more frequent in patients under 50 years of age.¹⁶ Tumors that carry POLE/POLD1 mutations share clinical-pathological features with dMMR CRC and are believed to have enhanced immunogenicity, thus rendering them more likely to respond to immunotherapy.

MSI-H tumors tend to be diagnosed more frequently in the proximal (right-sided) colon and contain higher numbers of tumor-infiltrating lymphocytes (TILs), along with a high expression of PD-L1. Since tumors that contain a higher number of TILs are associated with high clinical response rates and survival benefit, methods to quantify tumor lymphocyte invasion have been developed.¹⁷ The distribution, density, type, and functionality of TILs has been incorporated into a scoring system designated “Immunoscore” and validated to predict risk of recurrence in operable CRC, but its value in advanced CRC has been less studied. Additional studies are warranted to determine the true utilization of Immunoscore to help define optimal therapy for patients with metastatic CRC.

Although most CRC tissue used for biomarker testing is derived from formalin-fixed paraffin-embedded (FFPE) tissue, use of plasma cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA) as “liquid biopsies” is gaining wider acceptance, particularly as a noninvasive biomarker to monitor tumors longitudinally.² Tumor tissue profiling remains the gold standard upon which to base initial treatment decisions for targeted therapies, but ctDNA offers potential advantages, particularly when tumor tissue is not easily available.¹⁸

OVERVIEW OF CURRENT TREATMENT OF ADVANCED CRC

Initial treatment for unresectable metastatic disease

For patients with metastatic disease that is not deemed completely resectable, systemic therapy can be initiated with consideration of patient characteristics and comorbidities, goals of therapy, timing and type of prior therapies, tumor histopathological and molecular characteristics, and toxicity profiles of the individual drugs.⁷ The selection of the specific initial treatment regimen will be determined by the tumor KRAS, NRAS, and BRAF gene mutation, MMR/MSI status, and HER2 amplification as well as whether the patient is a candidate for intensive therapy. In medically fit patients with proficient MMR (pMMR) and MSS tumors, intensive chemotherapy regimens include a fluoropyrimidine (infusional fluorouracil or capecitabine) doublet (FOLFOX, CapeOX, FOLFIRI) or triplet regimen (FOLFOXIRI) as initial therapy. Patients with MSI-H/dMMR tumors are candidates for first-line treatment with pembrolizumab.

Patients who are not considered appropriate to receive intensive therapy should be considered for treatment with a fluoropyrimidine, with or without bevacizumab, or an EGFR inhibitor alone (cetuximab or panitumumab) for RAS/BRAF wild-type and left-sided tumors.⁷ Dual HER2 blockade (eg, trastuzumab plus pertuzumab or lapatinib) can be used to treat patients with HER2 amplified, RAS/BRAF wild-type tumors.¹⁹ Patients with MSI-H/dMMR tumors can be considered for initial treatment with single-agent pembrolizumab or nivolumab, or nivolumab plus ipilimumab, although only pembrolizumab is currently approved for use in this setting.⁷ A strategy for incorporated targeted therapies for initial and subsequent treatment regimens for mCRC is depicted in Figure 1.²⁰

Second-line and subsequent treatment for metastatic CRC

Second-line chemotherapy regimens and subsequent treatment decisions are based on prior treatment regimens administered.⁷ Subsequent treatment regimens for prior oxaliplatin-containing therapy without irinotecan can include FOLFIRI or irinotecan, whereas patients who received prior irinotecan-containing therapy without oxaliplatin can be considered for FOLFOX, CapeOX, or irinotecan plus cetuximab or panitumumab. Bevacizumab or a different anti-vascular endothelial growth factor (VEGF) agent can be used with second-line and subsequent therapies, but not concurrently with an EGFR inhibitor. Regardless of the first-line chemotherapy regimen used, pembrolizumab, nivolumab, or nivolumab plus ipilimumab can be used for MSI-H/dMMR tumors. Targeted agents can be administered with chemotherapy as determined by the presence or absence of a tumor gene mutation.

Treatment options for individuals who have received both oxaliplatin- and irinotecan-containing regimens include regorafenib, trifluridine plus tipiracil, and targeted therapies based on tumor markers (eg, RAS/BRAF gene status, HER2 amplification, MMR/MSI status).^6,7 Participation in a clinical trial is also encouraged for all patients with mCRC.

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