Predictive Biomarkers in Colorectal Cancer

Predictive Biomarkers in Colorectal Cancer: History

Please note this is an old version of this entry, which may differ significantly from the current revision.

Subjects: Oncology

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Colorectal cancer (CRC) is the third most common malignancy worldwide. Surgery remains the most important treatment for non-metastatic CRC, and the administration of adjuvant chemotherapy depends mainly on the disease stage, which is still the strongest prognostic factor. A refined understanding of the genomics of CRC has recently been achieved thanks to the widespread use of next generation sequencing with potential future therapeutic implications. Microsatellite instability (MSI) has been suggested as a predictive marker for response to anti-programmed-cell-death protein 1 (PD-1) therapy in solid tumors, including CRC. It should be noted that not all cancers with MSI phenotype respond to anti-PD-1 immunotherapy, highlighting the urgent need for even better predictive biomarkers. Mitogen-Activated Protein Kinase (MAPK) pathway genes KRAS, NRAS, and BRAF represent important molecular targets and could serve as independent prognostic biomarkers in CRC, and identify those who potentially benefit from anti-epidermal growth factor receptor (EGFR) treatment. Emerging evidence has attributed a significant role to inflammatory markers including blood cell ratios in the prognosis and survival of CRC patients; these biomarkers can be easily assessed in routine blood exams and be used to identify high-risk patients or those more likely to benefit from chemotherapy, targeted therapies and potentially immunotherapy. Analysis of cell-free DNA (cfDNA), circulating tumor cells (CTC) and/or micro RNAs (miRNAs) could provide useful information for the early diagnosis of CRC, the identification of minimal residual disease and, the evaluation of the risk of recurrence in early CRC patients. Even the selection of patients suitable for the new targeted therapy is becoming possible with the use of predictive miRNA biomarkers. Finally, the development of treatment resistance with the emergence of chemo-resistance clones after treatment remains the most important challenge in the clinical practice. In this context it is crucial to identify potential biomarkers and therapeutic targets which could lead to development of new and more effective treatments.

colorectal cancer
biomarkers
prognostic and predictive markers
treatment resistance

DNA Mismatch Repair Genes and Microsatellite Instability

Microsatellite instability is caused by mutations in the mismatch repair gene (MMR) with the consequent inability to correct DNA errors that take place during cell replication. Mismatch repair genes are inactivated either as a result of sporadic MLH1 promoter hypermethylation, or germline mutations in MLH1, MSH2, MSH6 and PMS2 genes. It is now recommended that MSI status should be evaluated in all newly diagnosed CRC cases. This important clinical information with prognostic value for stage II CRC can be used as a screening marker to identify Lynch syndrome patients, and may predict response to immunotherapy in patients with stage IV disease.

The high lymphocyte infiltration and the increased expression of neoantigens in MSI-H CRC, and other tumors, consequently to their high genomic instability can explain this observation . Of note, pembrolizumab is now approved in patients with advanced cancer and MSI-H status in a tissue agnostic fashion. It has been showed that neoantigens induce an active immune microenvironment featuring two opposing forces; an immune stimulatory force represented by increased cytotoxic effector T lymphocytes and an immune inhibitory force including upregulated PD-1/PD-L1 checkpoints. Some MSI-L tumors harbored paradoxically high tumor-infiltrating lymphocytes, resulting in high immune cell PD-L1 expression as well; however, this correlation is not as direct as in the case of MMR-deficient (MSI-H) tumors. Another novel monoclonal antibody that targets PD-1 checkpoints and boosts the immune response against cancer cells is nivolumab. The findings of the CheckMate 142 study suggest that nivolumab is a promising therapeutic option for patients with previously treated MSI-H metastatic CRC.

The use of adjuvant chemotherapy in patients with stage II CRC remains controversial, in terms of the potential benefits versus risks of treatment. Current evidence supports the use of MMR testing for the implication of adjuvant therapy in this subset of patients. The favorable prognosis of patients with stage II MSI-H CRC and the lack of benefit from adjuvant 5-FU-based therapy, indicate that these patients should avoid adjuvant chemotherapy. Therefore, testing for MMR status by MSI analysis or immunohistochemistry should be recommended in stage II CRC in patients where adjuvant treatment is a consideration. On the other hand, the investigation of potential benefits of the current standard FOLFOX regimen (5-fluorouracil, leucovorin and oxaliplatin) in stage III MSI-H CRC subgroup has not been completed. Currently, available data do not justify the exclusion of patients with stage III MSI-H CRC from adjuvant treatment with FOLFOX, since the responsiveness of these tumors to oxaliplatin or FOLFOX has not been confirmed. As such, MMR status in patients with stage III disease is of research interest. However, the use of MMR status represents a further step toward personalized cancer care.

BRAF Mutations

In the RAS signaling pathway, BRAF is the direct downstream target of KRAS. BRAF V600E mutation is present in approximately 10% of CRC patients, which is mutually exclusive of the KRAS mutations found in 35–45% of cases. The majority of these consist of a substitution of glutamic acid for valine at the V600 hotspot in exon. BRAF mutations are more frequent in female sex, older age, right colon and proximal tumors with poor differentiation, mucinous histology and infiltrating lymphocytes, which are usually MSI-H. The presence of BRAF mutation is associated with a shorter DFS and OS in MSI-L tumors. Interestingly, this mutation results in an improved and longer DFS in MSI-H tumors; however, OS has been shown to be not significantly affected. BRAF mutation can also represent a therapeutic target in metastatic CRC but targeting BRAF will also require the blockade of other pathways including, EGFR, MEK and PI3K differently from melanoma and non-small cell lung cancer where BRAF inhibition produces therapeutic benefit. However, it has also been reported that EGFR inhibitors provide limited benefit in patients with BRAF V600E metastatic CRC. SWOG 1406 assessed irinotecan-cetuximab combination as a control arm in the second or third line setting in BRAF V600E metastatic CRC, and demonstrated an RR of 4% and median DFS of two months, which confirm the limited activity of cetuximab in this population. It remains controversial whether patients harboring BRAF V600E mutation should not receive an anti-EGFR treatment in the first line setting. In this context, a molecular subgroup analysis of the TRIBE trial suggests that fit patients can achieve better OS with the combination of 5-fluorouracil, oxaliplatin, and irinotecan (FOLFOXIRI)-bevacizumab compared to irinotecan, 5-fluorouracil (FOLFIRI)-bevacizumab but prospective studies are needed to address this issue.

Despite the fact that BRAF inhibitors have a high response rate in BRAF V600 mutant melanoma, and non-small cell lung cancer, their efficacy as monotherapy in BRAF V600E CRC is limited. It has been supported that high levels of basal receptor tyrosine kinase signaling in CRC underlie rapid adaptive resistance taken that extracellular signal-regulated kinase (ERK) inhibition releases EGFR from negative feedback suppression. Despite the fact that overall prognosis for BRAFV600E mutant metastatic CRC is worse as compared to BRAF-wild-type CRC, the assessment of the BRAF V600E status should be mandatory in future adjuvant trials with the prospect to be incorporated in the clinical practice. Even though there is evidence of response to combined BRAF/EGFR and BRAF/MEK inhibition, the reported overall RR and median DFS should be further improved and there is still need for optimization of this therapeutic strategy. Access to combinations of BRAF, MEK, and other pathway inhibitors is currently not Food and Drug Administration (FDA) approved for the treatment of metastatic BRAF V600E CRC; nevertheless, this off-label strategic implementation deserves consideration based on the growing body of literature. Overall, taken the poor prognosis and distinct clinical features of metastatic BRAFV600E CRC, earlier identification of the mutation may expand therapeutic, including clinical trials, options prior to patients’ deterioration.

Mutations in the RAS Gene

KRAS proto-oncogene encodes the GTPase protein KRAS, which has a pivotal role in several molecular pathways, lies downstream of EGFR and potentially engages effectors that control proliferation, differentiation, and survival. Fifteen percent of CRC carry mutations in exons 2, 3 and 4 of the KRAS gene. KRAS mutations are associated with a shorter DFS and OS in patients with MSI-L tumors, which represent approximately 90% of all stage III colon cancer but have no prognostic implication in patients with MSI-H tumors. Additionally, MSI-L tumors are located in the distal colon while the majority of cases with MSI-H tumors develop in the proximal colon. It has been demonstrated that, among CRC patients with point mutations in exon 2 (codons 12 and 13) or exon 3 of KRAS, prognosis is worse only in those having a distal tumor.

There are still no available drugs currently targeting the activating mutations of KRAS. This represents a major therapeutic problem, as KRAS mutations are associated with dismal prognosis among CRC patients. It seems that there will be a long and winding way until the first approved KRAS inhibitor, mainly due to the genetic heterogeneity of the KRAS-mutant disease. Novel and effective approaches are currently under investigation, for targeting KRAS. Identification of molecules that could either bind the mutated sites of KRAS, or inhibit the synthesis at the DNA level of the mutated protein, is in progress. There is an effort for developing small molecules binding to KRAS G12D mutant, and preventing the formation of active KRAS-GTP. miRNAs could be a further therapeutic potential to explore effective targeting of KRAS-mutant CRC. miRNAs regulate critical pathways involved in the CRC pathogenesis, including the p53, PI3K, RAS, MAPK, EMT transcription factors, and Wnt/β-catenin pathways. As such, the development of effective drugs for targeting complicated pathways in CRC remains challenging.

The Epidermal Growth Factor Receptor Family

The crucial role of EGFR signaling in the survival of several tumors, including CRC, is supported by the approval of anti-EGFR targeted therapies; unfortunately, resistance to target therapy inevitably emerges; HER2 has recently been shown to represent one potential resistance mechanism leading to anti-EGFR antibody therapy resistance in KRAS wild type tumors.

HER2 amplification has been detected in approximately 5% of KRAS wild type cancers. Therapeutically, combination treatment with trastuzumab and lapatinib has resulted in a 35% overall RR, and a median DFS of approximately 5.5 months in heavily pretreated patients harboring HER2-amplified CRC. These findings support the role of HER2 expression as a predictive biomarker of anti-HER2 treatment response in this subset of metastatic CRC . Furthermore, in a cohort of KRAS wild type metastatic CRC patients treated with anti-EGFR treatment, those with higher expression and activation of EGFR and HER3 membrane receptors had a better OS, independently of the line of treatment. EGFR pathway activation is associated with a better OS in KRAS wild type metastatic CRC patients receiving anti-EGFR treatment. This could be explained by the fact that these tumors are more dependent on EGFR signaling and thus more sensitive to its inhibition.

In KRAS wild type patients, the PI3K pathway can explain the variability of response to anti-EGFR therapies; in this subset of tumors, several potential mechanisms could be at the origin of the EGFR activation, including the overexpression of EGFR ligands and specifically epiregulin (EREG) and amphiregulin (AREG). This has been associated with a better response to anti-EGFR therapy in KRAS wild type CRC.

Apart from KRAS status, and resistance to anti-EGFR therapy, mutations in PIK3CA are associated with proximal colonic tumors, and adverse outcomes for patients with BRAF wild-type tumors.

The lack of association between EGFR protein expression by immunohistochemistry and response to EGFR-targeted agents is likely due to several technical reasons. Immunohistochemistry is not a strictly quantitative method, which has a substantial impact on the determination of EGFR immunoreactivity. Furthermore, EGFR expression might differ between primary tumors and metastatic sites. As such, the evaluation of EGFR expression in the primary tumor may not be suitable for predicting the treatment response of metastases. In addition, there is no correlation between EGFR protein expression and EGFR gene amplification. Based on this evidence, cetuximab and panitumumab have been approved by the FDA without the need for EGFR testing, as a second and third line therapy for advanced CRC.

Several clinical trials with a second or first generation of tyrosine kinase inhibitors are ongoing. In metastatic settings, tyrosine kinase inhibitors could have a role as maintenance treatment. The combination of different targeted therapies in order to overcome tumor resistance is reasonable. There is evidence that involves different molecular networks as far as resistance to targeted therapies against one pathway is concerned. Several approaches targeting the EGFR and its downstream pathways exist. There is urgent need for further biomarkers in both clinical practice and the process of drug development to make prediction of responses to different targeted therapies feasible.

Blood Biomarkers

Circulating tumor cells (CTCs) are identified in the peripheral blood based either on the level of epithelial surface marker expression or on physical features of cancer cells. Their presence indicates active disease, proliferation and metastatic potency, and is followed by genomic analyses that provide data in terms of the tumor biology and real-time monitoring of the therapeutic efficacy. The prognostic value of CTC in early stages of the disease has been established by several studies. However, comparison of their findings seems difficult due to the variety of patients’ population, analytical techniques and timing of specimen collection.

Indeed, both timing and the site of sampling are factors that influence the outcome of CTC analysis. Regarding this, a meta-analysis of 14 studies revealed that there is a role of CTCs as predictors of recurrence in six out of nine studies in which blood samples were collected at least 24 h postoperatively. In contrast, the prognosis was not associated with perioperative CTC levels.

The cfDNA releases by cancer cells, and refers to degraded DNA fragments released to the blood plasma or other biological fluids. Analysis of cfDNA, for mutations and genetic aberrations provides real-time monitoring of tumor progression, particularly in stage II resected colon cancer. Currently, improved PCR-based methods were implemented to identify genomic alterations in cfDNA.

Different studies demonstrated that higher levels of total cfDNA or mutant ctDNA correlate with a shorter OS in patients with metastatic CRC. The RAS tumor mutational status can be evaluated through analysis of the cfDNA, alternatively to tissue testing. In addition, cfDNA analysis provides information on the mechanisms of acquired resistance to anti-EGFR drugs in metastatic CRC patients. This is likely to be multi-clonal rather than monoclonal. Taking the heterogeneity of metastatic CRC, cfDNA analysis is a strategy that can depict the complex landscape of genetic alterations that lead to resistance to targeted agents. There is still not established evidence for the implementation of cfDNA analysis with regard to treatment decision in clinical practice. Due to a lack of prospective clinical trials, the potential detection of somatic mutations in cfDNA should not modify the treatment.

Liquid biopsy may be a novel approach toward identification of the minimal residual disease in patients with resected CRC. However, the obtained prognostic information through detection of clonal mutations should be translated into sophisticated therapeutic options for the improvement of survival. At the present time, plasma testing has a limited use for RAS mutation in CRC. The possibility of false negative result of a RAS test on plasma is not rare. However, it may provide complementary information to histology, particularly in patients with recurrence disease following resection of the primary tumor.

The use of blood biomarkers in CRC is still extremely limited in clinical practice. Several reasons, including low level of standardization of the tests used, limited number of patients enrolled, and the lack of clear clinical benefit, explain this statement. The importance of liquid biopsy to the current screening policy will be upgraded with the design of prospective studies.

MiRNAs

MiRNAs represent extremely stable, single stranded molecules with hairpin-loop shape and small size found in exosomes. They play a vital role in the genetic control of CRC development, progression and metastatic potential. Several studies highlight the potential utility of miRNAs as biomarkers in either tissues or blood for the assessment of response to the agents implemented in CRC, including the 5-fluorouracil based therapies, and EGFR inhibitors. Development of miRNA signature for predicting treatment response designates a personalized therapeutic approach of CRC.

At the same time, miRNAs have been found to induce chemoresistance. Indeed, FOLFOX-resistance in advanced CRC is significantly associated with upregulation and downregulation of several serum miRNAs. In terms of treatment response to anti-VEGF or anti-EGFR inhibitors in metastatic CRC, upregulation of miR-126 was correlated with bevacizumab resistance, whereas overexpression of miR-31, miR-100, miR-125b, and downregulation of miR-7, with resistance to cetuximab, respectively [145,146,147].

Conclusions

Colorectal cancer is a highly heterogeneous clinical entity, and pathological evaluation is a suboptimal method for the consideration of disease prognosis. Multiple time point sequencing for the evaluation of the clonal development process, treatment response, and resistance throughout the course of CRC are hugely important. In this era, recent advances in the identification of molecular signatures contribute to the development of novel therapeutic strategies. MSI-H tumors are potentially responsive to immunotherapy, particularly the PD-1 blockades, as they are found to be infiltrated by lymphocytes. The utilization of the KRAS, NRAS, and BRAF genes as prognostic and predictive biomarkers is an important step toward a personalized therapeutic approach of CRC patients. Aberrant miRNA expression may serve as a biomarker as well, despite the fact that an optimal strategy for their detection and validation is still required.

Adaptive clinical trials should be designed for the determination of real-time pharmacodynamic markers. They could be integrated into a prediction model for the evaluation of the benefit from either adjuvant chemotherapy, or targeted therapies for metastatic CRC

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