Objective Review the targeted therapies that are currently standard of practice in colorectal and gastric cancers, requiring specific molecular testing for selection of candidate patients for therapy.
Introduction Clinical applications of genomic medicine and molecular diagnostics based on testing of tumor tissues are becoming a reality in clinical practice, with significant impact on personalized therapies for cancer patients. Advances in targeted therapies for cancers of the gastrointestinal tract have recently emerged and are rapidly moving targets.
Gastric and Gastroesophageal Cancers Targeting HER2 Receptors The human epidermal growth factor receptor 2 (Her2 or ErbB- 2) was first described in gastric cancer in 1986. HER2 has no known ligand (orphan receptor), and preferentially heterodimerizes with HER3, which lacks intrinsic tyrosine kinase activity. The HER2 and the HER2/HER3 heterodimers are likely to be the most effective complex for activating downstream pathways.
Targeting Her 2 Overexpression and amplification of HER2 have been described in 6–35% of gastric and gastroesophageal junction (GEJ) adenocarcinomas. Criteria for interpretation of HER2 modified for gastric and GEJ adenocarcinoma have been recently reviewed.
Targeting Her 2 Heterogeneity of HER2 expression occurs frequently in gastric and GEJ adenocarcinoma; however, testing is often done in biopsies when no resection specimen is available. The College of American Pathologists (CAP) reviewed current guidelines for interpretation of HER2 expression
Targeting Her 2 Criteria for interpreting HER2 IHC on gastric and GEJ carcinomas differ significantly from the criteria used in breast cancer. First, gastric carcinoma interpretation criteria use 10% tumor cell staining as a cutoff to distinguish negative from 1+. In gastric carcinoma, the distinction between 1+, 2+, and 3+ depends on the intensity of staining presuming that more than 10% of tumor cells show HER2 expression.
Targeting Her 2 Second, gastric cancers only show expression along the basolateral or lateral cell membranes, while apical membranes are negative. Therefore, the criteria for 2+ and 3+ staining in gastric cancer require only lateral or basolateral staining, in contrast to breast cancer criteria criteria which require complete, circumferential staining. Third, the criteria for HER2 over expression differ when interpreting biopsy and resection specimens due to heterogeneity of HER2 expression in gastric and gastro- esophageal junction carcinomas.
Morphological Features Her 2+ Proximal gastric carcinomas with intestinal phenotype are generally found to have a higher prevalence of HER2-positivity (range 8-34%) than distal diffuse gastric carcinomas. The rate of HER2-positivity in esophageal adenocarcinoma is also variable, with one meta- analysis showing approximately 25% of esophageal adenocarcinomas over-expressing HER2.
Conclusion HER2 expression has become an important biomarker for identifying patients who could respond to HER2 targeting therapy using the fully humanized monoclonal antibody trastuzumab. It is now recommended that all patients with gastric cancer should have their tumors tested for HER2 status at the time of initial diagnosis.
Introduction Colorectal cancer has been shown to arise through at least two distinct genetic pathways: One involves chromosomal instability (demonstrating high frequency of structural chromosomal changes) and The other involves microsatellite instability (MSI) (demonstrating DNA microsatellites changes).
Colon Cancer Genetic Pathways CK7+/p53−/MLH-1−/BRAF- mutated/K-ras-wt/M SI CK7+/CK20+/p53−/MLH- 1+/BRAF wt/K-ras- mutated/MSS
Molecular classification of CRC as described by Jass
Microsatellite Instability (MSI) Microsatellites are normal segments of DNA with repeat sequences of nucleotides of set length. Change in the number of repeats or its length (as a result of nucleotide insertions or deletions) is called microsatellite instability (MSI which is caused by defective DNA mismatch repair genes (dMMR).
MSI Microsatellite instability in tumors has been classified into three categories: 1- High (tumors showing instability in 30% or more of the microsatellites). 2- Intermediate (instability in 10%–30%). 3- Stable tumors (less than 10%).
MSI Patients with MSI-H tumors, have a relatively stable diploid karyotype. They typically have right sided or proximal tumors. They usually exhibit improved overall survival.
MSI Morphological Features Mucinous adenocarcinoma, poor differentiation, medullary carcinoma, tumor infiltrating lymphocytes TILs (five or more in at least one of 10 HPF), Crohn‟s-like reaction and serrated adenocarcinoma heterogeneity Some of the most recent researchers showed that more than 15% of CRCs express CK7 and/or are CK20-negative. This aberrant immunophenotype seems to be related to microsatellite instability being more frequent in BRAF- mutated MSI cases localized in the proximal colon.
MSI Morphological Features The morphology of SAC. A showing epithelial serrations, abundant and eosinophilic cytoplasm, and vesicular nuclei. cell balls floating in the extracellular mucinSAC papillary rods in the extracellular mucin. HE.
Immunoexpression of Cytokeratin 7 in case of possible serrated pathway colorectal adenocarcinomas. Most of mucinous carcinomas present diffuse positivity (A) but the moderately-differentiated ones show focal positivity (B).
MSI Morphological Features Secretory mucin associated with (sporadic MSI-H) CRC comprises both intestinal (MUC2) and gastric (MUC5AC) mucin. A mixed gastric and small intestinal mucinous phenotype.
MSI Morphological Features Medullary carcinomas are usually located in the proximal colon and have female predominance. Is formed of solid sheets of undifferentiated cells with round nuclei, prominent nucleoli and is associated with a Crohn‟s like reaction. They show reduced expression of CK20 and lack of expression of CDX2. Are diploid, MSI high and have a favorable prognosis with reduced incidence of lymph node involvement.
MSI MSI is a molecular marker that can provide valuable prognostic and predictive information in colon cancer patients. MSI data can be used in clinical decision-making. Specifically, the favorable outcome of stage II colon cancers with MSI indicates that such patients should not receive adjuvant chemotherapy. Stage III colon cancers with MSI suggest a lack of benefit from 5- fluorouracil alone, the benefit of the current standard treatment, 5-fluorouracil, leucovorin, and oxaliplatin, in this subgroup remains unknown and awaits further study.
Target Therapy CRC The targeted biologic therapies available for CRC fall into three groups: (1) Inhibitors of the vascular endothelial growth factor (VEGF) proangiogenic system (the monoclonal VEGF-A antibody bevacizumab, and aflibercept, a unique fusion protein derived from extracellular receptor components of the VEGF system). (2) Monoclonal antibodies against the epidermal growth factor receptor (EGFR) on the surface of tumor cells (cetuximab and panitumumab); and (3) Regorafenib, an oral small-molecule inhibitor of intracellular kinases involved in various signaling cascades.
Targeting EGFR in CRC EGFR (also known as ErB1 or HER1) is a member of the ERbB trans membrane tyrosine kinase receptor family. EGFR is known to be over- expressed in tumors of epithelial origin, including CRC. EGFR alterations have prompted the development of two classes of anti-EGFR agents: Monoclonal anti-EGFR antibodies (such as cetuximab, panitumumab, etc) and small molecule TKIs directed against EGFR tyrosine kinase (such as gefitinib, erlotinib, etc).
There has been no FDA approval granted to TKIs as new targeted agents for the treatment of mCRC. As a consequence, we will confine our discussions of anti-EGFR therapies to monoclonal antibodies.
Biomarkers and Response to Anti-EGFR Therapy. Analysis of EGFR expression via IHC is not reliable in predicting the efficacy of EGFR therapy. Investigations have lead to focus on downstream effectors of EGFR signaling, including RAS family members, BRAF and the PI3K pathway.
KRAS Mutations KRAS is a member of the RAS proto-oncogene family which is transiently activated by the action of ligand binding of EGFR. The majority (85%) of these mutations occur in codons 12 and 13, with a smaller number occurring in codons 61, 117 and 146.
Testing KRAS Mutations The American Society of Clinical Oncology recommended that patients who are candidates for anti-EGFR therapy should have their tumor tested for KRAS mutation.
K RAS Mutated CRC Enteric Phenotype. Non Mucinous. Usually positive in CDX2-, CK20- colorectal cancer.
Approach used for KRAS mutation testing The analysis of tumor samples for KRAS mutations is undertaken using samples of fresh-frozen or paraffin- embedded tumor material. A pathology review of the material is essential to establish that the sample contains sufficient tumor cells for analysis. DNA is then extracted from the sample and used for molecular testing using sequencing or pyro sequencing- based approaches, or an allele-specific PCR-based approach.
Table 1. Characteristics of the specimens for KRAS mutational analysis. Normanno N, Pinto C, Castiglione F, Bardelli A, et al. (2011) KRAS Mutations Testing in Colorectal Carcinoma Patients in Italy: From Guidelines to External Quality Assessment. PLoS ONE 6(12): e29146. doi:10.1371/journal.pone.0029146 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029146
Figure 1. Methods used for KRAS genotyping by the centers participating to the Italian Quality assessment scheme. Normanno N, Pinto C, Castiglione F, Bardelli A, et al. (2011) KRAS Mutations Testing in Colorectal Carcinoma Patients in Italy: From Guidelines to External Quality Assessment. PLoS ONE 6(12): e29146. doi:10.1371/journal.pone.0029146 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029146
Take-home messages: KRAS mutation status has emerged as an important predictive marker for anti-EGFR therapy in patients with mCRC. A pathologist is involved in KRAS testing in 10 stages of the procedure. KRAS mutation testing should be performed in pathology departments, only with CE- IVD/FDA- approved KRAS tests.
Testing BRAF Mut BRAF is a serine-threonine kinase, which is downstream of RAS. BRAF is an important oncogene and is found to be mutated in 15% of CRC. BRAF mutation has been associated with the serrated pathway of tumor development. The vast majority of BRAF mutations in CRC are the V600E mutation, which leads to constitutive activation of the kinase
BRAF mutant The BRAF mutant genotype impacts the molecular and phenotypic characteristics of colorectal cancer. The mutation occurs fairly early in the progression of colorectal cancer and is associated with CpG island methylation. In the end, you get a distinct type of tumor that is diploid and microsatellite-unstable.
Testing BRAF Mut Testing for BRAF is becoming part of the clinical routine for the management of mCRC patients in some centers. BRAF mutation testing is being incorporated into commercially available test kits for this purpose. It has also been shown that KRAS and BRAF analyses together have a favorable cost- benefit profile
Testing BRAF Mut BRAF oncogene by polymerase chain reaction amplification followed by direct sequencing Or RT PCR Allele Specific.
Summary BRAF-mutated colorectal cancer is a distinct molecular, phenotypic, and clinical subset that is in dire need of new treatment strategies.
Summary The role of the histopathologist is no longer limited to issuing an accurate tissue diagnosis but is increasingly directed towards the provision of prognostic information and additional findings directly relevant to patient management. This ongoing refinement of reporting practice should not obscure the more fundamental role of the pathologist in the classification of disease.
In summary, molecular pathology is a new and rapidly evolving field challenging pathologists involved in cancer diagnosis. It is crucial to make sure that the testing would be carried out in specialized centers that can address the molecular, as well as the histopathological aspects of the disease, to allow accurate diagnoses of high quality.