Abstract #1417: Identification and biological characterization of exon 4 KRAS mutations by MALDI-TOF Mass Spectrometry based genotyping
Authors: Manickam Janakiraman, Zeng Zhaoshi, Dhananjay Chitale, Ensar Halilovic, Neal Rosen, Evi Vakiani, Adriana Heyguy, Marc Ladanyi, Suresh Jhanwar, Len Saltz, Philip Paty, David Solit
Journal: Cancer Research (2009)

The kinase pathway comprising RAS, RAF, mitogen-activated protein kinase kinase (MEK) and extracellular signal regulated kinase (ERK) is activated in most human tumors, often through gain-of-function mutations of RAS and RAF family members. We have previously reported that mutations of BRAF are associated with enhanced and selective sensitivity to MEK inhibition when compared to either receptor tyrosine kinase-driven cells or cells harboring a RAS mutation. RAS mutant cell lines are variably MEK-dependent with those cell lines expressing concurrent PIK3CA mutations being relatively refractory to MEK inhibition. A small minority of cell lines with wild-type RAS and BRAF are MEK-dependent suggesting that these tumors may harbor occult mutations in the MAPK pathway. To further explore this possibility, we used a MALDI-TOF Mass Spectrometry based genotyping method to genomically characterize 489 colorectal tumors (382 primary tumors, 68 metastasis, 39 polyps) and 66 colorectal cancer cell lines for mutation in the RAS and PI3-kinase pathways. Consistent with prior data, we found exon 2 KRAS mutations in 32.1% and exon 15 BRAF mutations (27 V600E, 1 V600G) in 5.7% of the tumors. Mutations in exons 3 and 4 of KRAS and NRAS (1.6%) mutations were observed in 8.6% of patients. The incidence of Exon 4 KRAS mutations (5.3%) was higher than would have been expected based upon prior studies. A146 KRAS mutations were identified not only in colorectal primaries but in polyps and liver metastases suggesting that these alterations occur early within the natural history of the disease. Four cell lines were found to harbor exon 4 KRAS mutations (all A146T, 1 homozygous and 3 heterozygous). Two of the 4 also had concurrent PIK3CA mutations. Matched tumor tissue was available for 2 of these cell lines and in both cases confirmed the presence of A146T KRAS and PIK3CA alterations in the primary cancers from which the cell lines were derived. Cell lines expressing A146T KRAS had high, constitutive RAS-GTP expression versus those harboring V600E BRAF. To determine the MEK dependence of cell lines expressing A146T KRAS, we used PD0325901, a selective allosteric inhibitor of MEK1/2. All four A146T KRAS cell lines were MEK-dependent for proliferation. However, the two cells with concurrent PIK3CA mutation were 20-fold less sensitive to MEK inhibition than those with A146T KRAS mutation alone and demonstrated a purely cytostatic response to PD0325901. In such models combined inhibition of both MEK and PI3 kinase was superior to either alone. These data suggest that Exon 4 KRAS mutations are common in colorectal cancer, lead to KRAS activation and are predictive of MEK/MAPK-dependence. Based upon these data, we have proposed prospective genotyping of colorectal cancers for A146 KRAS mutation with the goal of using this data to select patients for treatment with MEK pathway inhibitors.   Citation Information:  In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1417.