Colorectal cancer is the abnormal proliferation of cells, that forms a tumor, located at the distal end of the digestive tract (colon or rectum). Various treatments have been employed to slow the growth progression in hopes to eradicate it. The main cause of metastatic colorectal cancer (mCRC) is still unclear and research persists to better maximize patient benefit from medically prescribed plan of actions. FOLFIRI is a first-line chemotherapy regimen consisting of three drugs: fluorouracil, leucovorin, and irinotecan. Leucovorin decrease the effects of certain toxins. Fluorouracil incorporate a pyrimidine antimetabolite to inhibit DNA replication. Irinotecan is a topoisomerase inhibitor that impedes DNA from uncoiling and replicating. Integrating cetuximab, an epidermal growth factor receptor (EGFR) inhibitor, into FOLFIRI regimen “significantly improved overall survival OS, progression-free survival PFS, and objective response” in the first-line treatment of patients with the wild type KRAS codon 12/13 mCRC. Essentially, this article reassesses this finding by conducting an extended RAS mutation study. The purpose of this particular research article is to investigate RAS mutations – a family of genes involved in cell signaling, cell growth, and apoptosis – in association to treatment effects. In particular, the article examined the treatment effects of FOLFIRI plus cetuximab vs. only FOLFIRI in “patients with tumors carrying predefined mutations at RAS loci other than KRAS codon 12 and 13” (633). The analysis was also applied to patients with neoplasms wild-type at all RAS loci. The study design of this article employs the administration of FOLFIRI in 14-day cycles with weekly cetuximab for those expressing EGFR mCRC – this was then was compared to those only getting FOLFIRI. Unless the disease progressed, or toxicity was evident, or patient withdrew consent the treatment was continued. The RAS mutation testing was done on DNA samples already identified to have the wild type at KRAS codon 12 and 13. Prior to the DNA extraction, a pathologist estimated that the fraction of tumor cells was between 5% and 60%. However, in the application of the 5% analysis cutoff – the estimation of the neoplastic cells were not put into consideration. BEAM technology – beads, emulsion, amplification (through PCR with phusion DNA polymerase), and magnetics – was used for RAS mutation analysis. This technique enables detection and count mutant DNA sequences vs wild type DNA sequences at a ratio of 1:10,000./ The presence of 26 particular mutations in KRAS exon 3 and 4, along with the NRAS exon 2, 3, 4, were analysed. Tumors were classified as a “RAS mutant if the sum of the individual percentages of mutant sequences over total amplified sequences of the individual percentages was 5%, regardless of whether all loci were valuable for mutation status”. Those that were classified as RAS wild type had a summed mutation prevalence of <5%and if all 26 mutations could be evaluated. In terms of statistical analyses, hazard ratios (HR) and odd ratios (OR) were attained for FOLFIRI plus cetuximab and FOLFIRI alone. HR is the ratio of hazard rates calculated by dividing an event occurring in the treatment by an event occurring in the control group. This calculation takes into account the total number of event and the timing of each of the events. OR measures the odds that an outcome happens given an exposure compared to the odds when the exposure is absent. To quantify the OS and PHS for analysis, HRs and 95% CIs were calculated by the COX proportional hazards model to compare the treatments. 95% CI is the range of values that you are 95% certain contains the true mean of the population. The survival times was calculated by the Kaplan-Meier analysis - which measures the fraction of patients living for a given time after exposure to treatment. The p-value was also computed to either support or reject the null hypothesis. The analysis for the objective response between treatment groups was compared using the Cochran-Mantel-Haenszel test. All analyses, asides the cutoff value, were arranged according to the Eastern Oncology Group performance status and region. This is a scale to assess the progression of the patient's disease. The results of the OS, PFS, and objective response for KRAS 12 or 13 wild-type and wild-type RAS (all loci) tumors show that the efficacy outcome favored that addition of cetuximab to FOLFIRI (Fig 3 and 4). While patients that had other RAS mutations, did not show evidence of benefit from the FOLFIRI plus cetuximab treatment. There was also no difference in efficacy outcomes between the two treatment groups for tumors with any RAS mutation. The BEAMing analysis, at 5% cutoff, showed that the HRs and ORs did not imply improvement or worsening of effectiveness in patients with RAS tumor mutations when cetuximab was added to FOLFIRI. Extrapolating from the data, the addition of cetuximab to FOLFIRI can benefit patients with other RAS mutations (KRAS codon 12 or 13 not included) that are less than 5%. This means that integrating an EGFR inhibitor in mCRC treatment may increase the effectiveness of the outcome in those with other RAS mutation signals less than 5%. Patients with KRAS 12 or 13 and those with other RAS mutations, along with patients with any RAS mutations had no clear indication of a positive or negative outcome when cetuximab was added (Fig 3C and 4C). The absence of a negative outcome strongly opposes the prior studies that suggested a worse result in RAS-mutants when cetuximab was included to FOLFOX4 (another chemotherapeutic regimen). The data implying that adding cetuximab to FOLFIRI coincides with another study's data and conclusion that the addition of panitumumab (another EGFR inhibitor) to FOLFIRI with KRAS exon 2 or other RAS mutations exhibited no negative effects,. This is important because the basis of such studies updates and transgresses into current clinical settings to which now suggest the integration of cetuximab and panitumumab into FOLFIRI or FOLFOX chemotherapy for patients with only RAS wild-type neoplasms. Another important finding was that KRAS exon 4 was the other most common location for a mutation outside of KRAS exon 2 in CRYSTAL (Cetuximab Combined With Irinotecan in First-line Therapy) patients., Another area of interest is the NRAS exon 4 mutations - which was in another study showing its significance in mCRC formation. Such findings could be further explored and investigated to progress mCRC research and treatment. Ultimately, this study supports the use of cetuximab in combination with FOLFIRI in patients with wild-type RAS neoplasms. This treatment combination cannot necessarily be prescribed to patients with other RAS mutations because it does not have a clear and beneficial efficacy. To facilitate the selection for the most apt treatment, this article emphasized the importance of molecular testing of all tumors, especially for KRAS and NRAS genes, prior to considering any anti-EGFR chemotherapy. The information in this article does not necessarily differ from what was learned in lecture - especially in regards to gene mutations and gene regulation. Perhaps, one that struck out to me the most was the use of the Phusion DNA polymerase, instead of the usel Taq DNA polymerase. Phusion is a hybrid enzyme that makes PCR products at a higher speed and with higher accuracy. Therefore, this allows the high confidence in the recognition of mutations in the original DNA template.