1. Simoneaux, Richard

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Colorectal cancer death rates for men and women have been steadily decreasing for the last few decades. This fact may be due to a number of reasons, including enhanced screening and removal of precancerous colorectal polyps, improved dietary and medical prevention, and the development of specifically targeted systemic therapies.

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Despite the advances that have been made in the prevention and treatment of CRC, it is still the second-most lethal cancer, thus highlighting the need for the ongoing development of not only treatment options, but also of predictive and diagnostic markers for better disease targeting. Further complicating the treatment of this condition is the fact that CRC is not one homogenous disease. Comprehensive genetic testing has shown that individual CRC disease states are distinct, each having a median of 76 non-silent mutations (Science 2007;318:1108-1113).


In a mini-review published last year, a number of authors, including Celia Chao, MD, FACS, Associate Professor in the Department of Surgery, University of Texas Medical Branch at Galveston, discussed the differentiation of CRC into four different consensus molecular subtypes (CMS)(Int Biol Biomed J 2017;3(3);105-111).


"In an effort to correlate cancer cell phenotype with clinical behavior and guide rational treatment with specific targeted therapies, six independent molecular classification systems were unified by the CRC Subtyping Consortium (CRCSC), based on gene expression data, into a single consensus system with four distinct groups known as the CMS," Chao stated.


The CRC Subtyping Consortium

With the advances made in genomic testing, much research has been done by a number of groups in order to better classify CRC. With the data being generated by more than 15 separate institutes, significant overlap was noted between the different reported subtypes. Consequently, collaboration among these 15 institutes was initiated as the CRCSC with the ultimate goal of establishing a consensus-based subtyping model.


The 15 institutions that comprise the CRCSC analyzed more than 30 different gene expression sets across multiple platforms and sample preparation methods. These researchers applied six previously published classification systems to data from a set of more than 4,000 predominantly stage II or III CRC samples (Nat Med 2013;19;614-618). Analysis concordance of subtype calls, as well as clinical, molecular or pathology annotation, were provided by a central independent team using consortium consensus-derived methodology. The four subtypes identified (CMS1-CMS4) cover approximately 87 percent of all CRC cases, thus leaving roughly 13 percent molecularly uncharacterized (Nat Med 2015;21;1350-1356).



Approximately 14 percent of all CRC are considered CMS1, of which approximately 12 percent are sporadic (non-inherited), while the remaining patients have inherited disease (Lynch syndrome).


Precursor lesions for the CMS1 subtype are also known as serrated polyps. The serrated pathway to CRC has the following characteristics: located in the proximal colon; has a high BRAF V600E mutation rate; associated with impaired DNA mismatch repair (MMR); has tumor microenvironment (TME) with immunogenic lymphocyte infiltration; and has CpG island hypermethylation (CpG island methylator phenotype), resulting in loss of tumor suppressor function. In CMS1 disease, the serrated polyps are accompanied by a low transforming growth factor beta (TGF[beta]) microenvironment.


When hypermethylation or mutation to the promoter regions of the MMR genes occurs, microsatellite instability (MSI) arises. "Cancers displaying MSI have approximately 47 mutations per million bases and are thus considered hypermutated. In contrast, microsatellite stable (MSS) disease is characterized by an average of 2.8 mutations per million bases," Chao explained. Although CMS1 disease is characterized by a high mutation rate, this subtype is also associated with low copy numbers for the affected genes.


Patients presenting with early stage MSI cancer (the majority of CMS1 tumors) tend to have a better prognosis compared to those with MSS disease. Stage II cancers are associated with a low recurrence rate and, consequently, these patients are generally not considered candidates for adjuvant chemotherapy. "Patients with stage III MSI tumors do not benefit from fluorouracil monotherapy; however, they are responsive to adjuvant FOLFOX therapy (fluorouracil, leucovorin, and oxaliplatin combination)," Chao elaborated.


Generally, CMS1 tumors are treatable if they are detected before metastasis has occurred. These favorable outcomes may, in part, be due to the nature of the TME. "For this subtype, the TME is characterized as having a relative lack of proliferation-supporting cancer-associated fibroblasts (CAFs) as well as the presence of immunity-promoting cells, such as natural killer cells, CD8+ cytotoxic T lymphocytes, and CD4+ activated type 1 T helper cells (Th1)," Chao explained.


As a result of the immunogenic nature associated with this CRC subtype, the use of checkpoint inhibitors, such as pembrolizumab or nivolumab, for patients having advanced CRC is being explored in early phase clinical trials. This class of inhibitors acts by disrupting the immunosuppressive PD-1/PD-L1 interaction, thus enhancing the ability for the patient's T cells to recognize and destroy tumor cells.


Although CMS1 tumors do tend to have a lower rate of relapse, if relapse does occur, they are associated with worse survival (9 months). The 5-year survival rate for those with CMS1 disease is 73 percent.



The largest number of CRC patients, approximately 39 percent, has the CMS2 (canonical) subtype. This subtype is thought to arise from the canonical adenoma-to-carcinoma pathway. The gene expression profile in CMS2 is consistent with that of a differentiated epithelial cell phenotype, often characterized by the initial loss of APC, a tumor suppressor gene, followed by an activating mutation in KRAS and loss of TP53.


A high degree of chromosomal instability was noted in CMS2 disease, often with aneuploidy, gains and/or losses of large portions of chromosomes, and loss of heterozygosity. CMS2 CRC was found to exhibit high somatic copy number alterations and have more frequent copy number losses in tumor suppressor genes and copy number gains in oncogenes.


Although CMS2 cancers had high copy number alterations, the mutation rate was low (fewer than eight mutations per million bases), and thus was termed non-hypermutated. "One interesting finding revealed by the Cancer Genome Atlas network is that APC and TP53 were relatively less-mutated in the hypermutated CMS1 disease than was noted in CMS2 cancers," Chao said. In addition, CMS2 tumors typically have activated Myc and Wnt/[beta]-catenin signal transduction pathways.


"At the time of diagnosis and treatment, 39 percent of CMS2 CRC patients present with stage III disease. For these patients, the recommended treatment is standard adjuvant chemotherapy," Chao explained.


In terms of location, the majority (59%) of CMS2 lesions occur on the left side of the colon. Additionally, for those having this subtype of CRC, higher survival rates (35 months) after relapse were noted. The 5-year survival rate for those with CMS2 CRC is 77 percent, the best for the four different CRC subtypes.



CMS3 CRC, which is known as the metabolic subtype of disease, typically has chromosomal instability but with lower somatic copy number alterations than those found in the CMS2 or CMS4 subtypes. The MSI observed for this subtype was intermediate in nature, being less than that found for CMS1 but greater than the levels found in the CMS2 and CMS4 CRCs. A fair number of CMS3 tumors (30%) are characterized as hypermutated.


Mutations to KRAS are present in every CRC subtype; however, they are the most frequently found in patients having CMS3 disease (68%). Pathway analyses of CMS3 mRNA showed enhancement in nine of the 10 metabolic pathways studied, including glutamine, fatty acid, and lysophospholipid metabolism.


The high percentage of KRAS mutations in CMS3 CRC limits the therapeutic options for this disease subtype, as monoclonal antibodies (mABs) targeting the epidermal growth factor receptor (EGFR), such as cetuximab, have shown limited effectiveness against KRAS-driven cancers. "For the subset of CMS3 CRC patients not having KRAS mutations," Chao observed, "EGFR-targeting therapies may have some utility." For those patients with KRAS-mutant, EGFR-mAb-resistant CRC, a therapy consisting of MEK and pan-RAF inhibitor combinations may be evaluated.


Approximately 3 percent of CMS3 CRC patients have a high copy number for HER2. For this small subset of patients, the use of human epidermal growth factor-targeting tyrosine kinase inhibitors (TKI), such as dacomitinib (HER1, HER2, HER4) or neratinib (HER2), may have some utility. In particular, a phase II study is being performed at Memorial Sloan Kettering Cancer Center, New York, N.Y., which is evaluating the use of neratinib in patients having solid HER2-positive tumors, including but not limited to CRC. In the future, TKI therapy may be utilized alone or in combination with the HER2-targeting mAb trastuzumab.


"One thing hampering the treatment of patients with CMS3 CRC is the fact that many with this disease subtype do not have an identifiable gene target for therapy," Chao noted. "However, this subtype is often characterized by metabolic reprogramming, which may prove useful for targeting in future therapeutic strategies."


The 5-year survival rate for patients having CMS3 CRC is approximately 75 percent, the second-highest of the four subtypes. Approximately 13 percent of CRC patients have CMS3 disease.



Preclinical studies utilizing precancerous serrated adenoma showed that, depending on TGF[beta] concentration, the cells developed into either those with CMS1 (low TGF[beta] levels) or CMS4 (high TGF[beta] levels) phenotype.


Unlike CMS1 CRC, CMS4 tumors (also termed mesenchymal) are characterized by MSS (low methylation), chromosomal instability, very low levels of hypermutation, and very high levels of somatic copy number alterations. The stromal TME in this subtype is considered to be inflammatory, having high numbers of CAFs with characteristic TGF[beta] signaling, displaying angiogenesis, prominent innate immune cells, matrix protein-bound integrins, and an epithelial-mesenchymal transition. "Genes that are frequently mutated in this CRC subtype include APC, KRAS, PIK3CA, and TP53," Chao added.


"CMS4 cancers are often diagnosed at advanced stages, and thus their prognosis is poor, with the worst 5-year overall survival (62%) and relapse-free survival (60%) of any molecular subtype," Chao said. "Although standard FOLFOX adjuvant chemotherapy is recommended for stage III CRC, CMS4 cancers show no benefit from systemic adjuvant treatments." Metastatic CMS4 disease is often EGFR inhibitor resistant, independent of KRAS mutation status.


Future therapies for this very lethal form of CRC may target elements present in the TME. As an example, the mAb abituzumab, which targets the integrin av[beta]6 (fibronectin-binding protein) present on tumor cell surfaces, has shown some promise in the phase I/II POSEIDON clinical trial (NCT01008475).


That trial compared the standard of care (cetuximab plus irinotecan) with or without abituzumab in metastatic CRC patients who had failed first-line oxaliplatin therapy. In the phase I portion of the trial, abituzumab was shown to be safe up to 1,000 mg every 2 weeks (ESMO 2014, Abstract 507PD).


The subset of patients for whom this combined therapy of abituzumab, cetuximab, and irinotecan held the most promise were for those having high plasma levels of the chemokine ligand 23 (CCL23) biomarker. In that group, patients in the abituzumab arm (n=68) had a median OS of 19.4 months, as compared to 7.4 months obtained for the standard of care patients (n=28), affording a hazard ratio of 0.41 (95% CI: 0.23-0.75; p=0.0048).



"Comprehensive transcriptomic analysis has permitted the identification of four consensus molecular subtypes of colorectal carcinoma into which most CRC can be categorized based on their genomic signature," Chao observed. "These subtypes aid not just in prognostication, but also to determine treatment strategies for individual patients, based on the mutations and activated pathways in those tumors as well as the phenotypic characteristics and therapeutic responses of other tumors with similar signatures."


Chao, a surgeon, concluded, "Many surgeons are not aware of these different CRC subtypes, and it is incumbent upon the surgical community to understand where oncology is going; this is why I decided to write this mini-review."


Richard Simoneaux is a contributing writer.


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