The Role of KRAS Mutation Testing in the

The Role of KRAS Mutation Testing in the Management of Colorectal Cancer Mark D. Pool, M. D.

Copyright © 2009 Mark D. Pool All Rights Reserved

Epidermal Growth Factor Receptor (EGFR) Transmembrane growth factor receptor with tyrosine kinase activity HER 1 (ERBB 1), belongs to HER/Erb. B family Selectively binds 10 different ligands After binding, forms dimers that causes autotransphorylation through intrinsic tyrosine kinase on cytoplasmic domain

Activation of EGFR • Triggers signaling cascade via RAS/RAF/MEK/MAPK and PI 3 K/AKT pathways • Via these pathways, critical cell functions affected – – – Survival Proliferation Angiogenesis Cytoskeleton organization and motility Vesicle trafficking Calcium signaling

EGFR Two classes of EGFR inhibitors Anti-EGFR m. Abs: cetuximab, panitumumab Small molecule inhibitors of tyrosine kinase activity (EGFR-TKIs): erlotinib, gefitinib

EGFR EGFR is overexpressed in more than 85% of tumors from patients with metastatic CRC. Only a subset of patients with m. CRC achieve a clinical benefit from treatment with EGFR inhibitors. Why?

KRAS Mutation and CRC Poor correlation between EGFR expression by IHC and treatment response Downstream “effectors” may be as important as receptor expression

RAS • RAS genes are the most common targets for somatic gain-of-function mutations in human cancers • Activating RAS mutations occur in 30% of human cancers – Specific RAS genes are mutated in different cancers • KRAS prevalent in pancreatic, colorectal, endometrial, lung, and cervical cancers

RAS Proteins Small GTPases that act as molecular switches by coupling cell membrane growth factor receptors to intracellular signaling pathways to transcription factors that control various cellular processes 3 genes, 4 proteins HRAS, NRAS, KRAS (4 A and 4 B) Localized to the inner surface of cell membrane

RAS Interacts with more than 20 effector proteins Specific mutations within KRAS can result in the KRAS protein being inherently activated independent of upstream growth factor receptor activation

KRAS Mutations and Pathogenesis • KRAS mutations occur early in colorectal carcinogenesis • Occur in 35% to 40% of CRC • 95% concordance between paired primary cancers and metastases

KRAS Mutation and CRC Only certain mutations lead to constitutive, growth-factor-receptor-independent activation of KRAS Mostly occur at codons 12 and 13 Impair intrinsic GTPase activity of KRAS and prevent GTPase activating proteins from promoting conversion of GTP (active) to GDP (inactive)

Frequency of Significant KRAS Mutations Codon Amino Acid Substitution Amino Acid Change 12 12 12 13 Gly 12 Asp Gly 12 Val Gly 12 Cys Gly 12 Ser Gly 12 Ala Gly 12 Arg Gly 13 Asp Aspartate Valine Cysteine Serine Alanine Arginine Aspartate Incidence, % 32. 5 22. 5 8. 8 7. 6 6. 4 0. 9 19. 5

KRAS Mutations as Prognostic Marker • RASCAL study (1998) multivariate analysis presence of KRAS mutation associated with increased risk of recurrence and death – Only Gly 12 Val – RASCAL II (2001) confirmed but only in Dukes C (Stage 3) tumors • Others studies have not confirmed

KRAS Mutations as Prognostic Marker • Role of KRAS must be interpreted in context of other molecular and signaling abnormalities – Any mutation in KRAS, BRAF, or PI 3 KCA associated with shorter 3 -year survival – BRAF mutation and MSS-tumors: negative prognostic effect? – KRAS mutation status of lymph nodes in stage 2 patients may identify increased risk of recurrence

KRAS Mutation as Predictive Marker Recent studies have examined the correlation between KRAS mutations and response to anti. EGFR m. Abs inhibitors in patients with m. CRC. Efficacy limited to patients whose tumors with wild-type KRAS gene.

Results of the NCIC CTG CO. 17 Trial Karapetis, CS et al. KRAS mutations and benefit from cetuximab in advanced colorectal cancer. New Engl J Med 2008; 359: 1757 -1765.

Results of the OPUS Trial Bokemeier C, et al. Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer. J Clin Oncol 2008; 27: 663 -671.

Results of the CRYSTAL Trial Van Cutsem E, et al. Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. New Engl J Med 2009; 360: 1408 -1417.

KRAS Mutation and CRC Improved survival progression-free and overall ~12 -20 weeks PFS, 8 -12 weeks OS Both drugs are ineffective when the patient's tumor has a KRAS mutation. NCCN and ASCO recommend that KRAS mutation testing be part of the evaluation of patients with metastatic CRC.

KRAS Mutation Testing Direct sequencing by PCR (Sanger sequencing) “Gold standard” but requires large amount of tumor DNA to detect mutation

Direct Sequencing of KRAS exon 2

KRAS Mutation Testing • Allele-specific methods: Thera Screen (Dx. S) – Specific primers are used to detect each of the common KRAS mutations – More sensitive than direct sequencing detecting as little as 1% to 10% of mutant DNA from total DNA in sample

Frequency of Significant KRAS Mutations Codon Amino Acid Substitution Amino Acid Change 12 12 12 13 Gly 12 Asp Gly 12 Val Gly 12 Cys Gly 12 Ser Gly 12 Ala Gly 12 Arg Gly 13 Asp Aspartate Valine Cysteine Serine Alanine Arginine Aspartate Incidence, % 32. 5 22. 5 8. 8 7. 6 6. 4 0. 9 19. 5

KRAS Testing Using the Dx. S Kit

KRAS Mutation Testing Role of Pathologists Specimen collection and handling Selection of appropriate tumor tissue for testing Methodology and quality control “In-house” versus reference lab