The+Effect+of+KRAS+Mutations+on+Anti-EGRF+Therapies+in+Colorectal+Cancer

=Introduction to Colorectal Cancer =

 Colorectal cancer is the third most common cancer in the entire world. Behind Lung Cancer, colorectal cancer makes up the second most cancer-related death in the United States. 72% of colorectal cancer is colon caner and 28% is rectal. Like all cancers, colorectal cancer occurance is strongly correlated to environmental stimuli and as a result of increasing age. It is recommended that screening for colorectal cancer start at the age of 50, and continued till the patient is 75. Normally, colorectal cancer is initiated in the lining of the bowel. As the cancer becomes invasive it moves to the muscle layers underneath, and eventually through the bowel wall. If the cancer remains in the bowel wall, it is often curable through surgery. Rectal Bleeding and anemia are the common signs of a colorectal tumor, especially in those over 50. Other symptoms include constipation, bloody stool, loss of appetite and weight, nausea, and vomiting. Diagnostic tests are used for patients that have high-risk symptoms. Regular colorectal cancer screening typically begins at 50 ([|Here] is the CDC page for colorectal cancer screening). Routine Colonoscopies are also used to scan the bowels for tumors and other gastrointestinal tract diseases. Unfortunately, detection of a tumor can be very late, and it may already be beyond stage 1 or 2. And once metastatic a patient is often given chemotherapy intervention to survive as long as possible. The 5-year survival rate of patients with stage 3 colorectal cancer is around 50%, however stage 4 patient's is almost zero. Although surgery is the main and most successful form of treatment, once the cancer is metastatic anti-EGRF antibodies are a main line of treatment.

=Anti-EGFR Treatment =

The epidermal growth factor receptor (EGFR) is often over expressed or mutated in uncontrolled cell proliferation. Growth factors bind to their cell surface receptors that signal for cell division. Some mutated receptors, common in cancers, signal cell proliferation even when receiving no signal from the growth factor leading to uncontrollable cell replication. EGFR, sends its signal down the MAP Kinase pathway, which includes the KRAS protein. KRAS is a major player in the resulting EGFR pathway in lung cancer also.



Anti-EGFR monoclonal antibodies bind to the EGFR receptor to block the signal down the pathway. As seen in Figure 1 b, the anti-EGFR drug Cetuximab prevents ligand binding to the EGFR receptor, thus blocking the RAS protein that leads to cell proliferation. Anti-EGFR also can block the phosphoinositide 3-kinase ( PI3K) pathway, leading to cell apoptosis instead of survival. These anti-EGFR antibodies can then be used to kill malignant tumor cells. However, mutations in RAS gene can signal both pathways to continue even withut ligand binding to the receptor. In figure 1 d, despite cetuximab binding to the receptor instead of the ligand, there is still downstream signaling. There are a variety of mutations that affect the KRAS gene, on exon 2, 3 and that continue the downstream signaling while ignoring the EGFR receptor. Furthermore, there is another mutation that was found after prolonged cetuximab exposure. The tumor cells generated a EGFR mutation (S492R) that created cetuximab-resistant cells. The mutation changes the extracellular domain of the receptor such that cetuximab is blocked but ligands can still bind, as illustrated in figure 1 c. In patients with cetuximab resistance, 2 out of 10 had the S492R mutation.

As expected, those with KRAS mutations faired far worse then their KRAS wildtype counterparts in trails for anti-EGFR drugs, particularly cetuximab and panitumumab. Panitumumab has the same mechanism of action as cetuximab, only being effective with wildtype KRAS patients.In Figure 2, cetuximab and panitumumab alter the same EGFR pathway. The oncogenic pathways that are being changed by the monoclonal antibodies are colored red. Through the RAS or PI3K pathway the MAPK pathway leading to cell proliferation is stopped. However, as previously discussed, KRAS mutations hinder anti-EGFR. Interestingly, when cetuximab resistance is garnered, panitumumab still retains function. The opposite also holds true, panitumumab resistance does not also correlate to instant cetuximab resistance. But they work along the same pathways. The conclusion then, is that the two anti-EGFR therapies must bind to different EGFR epitopes. That is, before they inhibit the pathways they are binding to different parts of the antigen molecule. The antibodies are then binding to different sites, causing the difference in cetuximab and panitumumab treatment. With this information a cocktail of the drugs may be the best option for patients with KRAS mutations, but eventually resistance against all anti-EGFR antibodies is acquired.

=Cellular Models =

The study was conducted with two colorectal cancer cellular models. One is called DiFi, the other Lim1215. Both lines were generated to be similar on a molecular level to the colorectal cancer patients who most likely would respond to cetuximab. DiFi cells overexpress EGFR due to the amplification of the EGFR gene. On the other hand, Lim1215 cells express normal levels of EGFR, while still being similarly sensitive to cetuximab as DiFi cells. From these two models, the researchers produced the cetuximab-resistant variants (DiFi-R, Lim1215-R), which are extremely sensitive to EGFR inhibition. One concern is that the methods section of the paper did not delve much into the specifics of how the cells were generated, so I am unsure how the cells function and how they are equally resistant to cetuximab.

DiFi cells




Figure 3. Cetuximab resistance in DiFi cells by increasing concentration, genetic composition, FISH analysis, Western blot analysis, and ectopic expression. Graph (a) depicts the resistance of DiFi R1 and DiFi R2 to increasing concentrations of cetuximab over the course of a week. The significant difference between the wild type DiFi and the R lines shows that there is a resistance. The figure does not distinguish the difference between R1 and R2, but it is later clarified that DiFi R1 was generated by exposure to a constant dose of cetuximab for a year, while R2 was exposed to stepwise increasing cetuximab dosage over a year. In addition, graph (a) shows that the cell viability of R1 and R2 are extremely similar and almost equally resistant. The inclusion of these two different drug treatment protocols reduces possibility of the variable that the method of treatment could affect resistance. Parts (b) and (c) display the amplification of the KRAS gene in DiFi R. Part (b) compares the exome gene copy number between the control DiFi and resistant DiFi R, clearly showing the difference and transition from EGFR expression to KRAS expression. Part (c) provides the reader with a visual representation confirming the amplification of KRAS in DiFi R. However, the study consisted of two R lines, so which one is actually shown?

Western blot analysis of the different cells in parts (d) and (e) gives us a better look at the protein levels. The parental DiFi line clearly has a lot of EGFR present and very little KRAS. DiFi R1 and R2 are similar in that both have low levels of EGFR but high levels of KRAS. However, a slight difference between R1 and R2 after exposed to cetuximab is that R2 has more pMEK and pERK, possibly as a result of method of treatment. Despite these minor differences, the resistant lines clearly depict the decrease of EGFR but increase of KRAS from the control, continuing to support the hypothesis. Part (e) was obtained by infecting DiFi cells with a KRAS lentivirus, which is used for gene manipulation because it specializes in infecting non-diving cells. Unfortunately, the function of the lentivirus was not expanded upon neither in the article nor the methods section, but I assumed that it silenced the KRAS gene according to outside sources. The similar presence of KRAS in “KRAS over” (which I assumed to mean “overexpressed”), R1, and R2 connects the resistant lines to having an overexpression of KRAS as well. The details of this Western blot analysis are lacking, which could undermine its significance as a part of the study.

Graph (f) looks again at cell viability in relation to cetuximab concentration but this time in DiFi cells overexpressing KRAS, wildtype cells, and empty vectors. The significant difference between the cell viability of DiFi “KRAS over” and DiFi wildtype and DiFi empty show a resistance in DiFi “KRAS over”. However, as higher concentrations of cetuximab, the cell viability of DiFi “KRAS over” is very close to DiFi wildtype and DiFi empty. In addition, the cell viability of DiFi wildtype in graph (f) is extremely different from graph (a), where graph (a) seemed to show that cetuximab was more effective, while graph (f) showed more resistance in the wildtype. The authors use graph (f) to connect overexpression of KRAS to cetuximab resistance, but with these two possible points, the conclusion may have been weakened.

From this data, the authors conclude that in DiFi cells, KRAS amplification mediates the acquired resistance to cetuximab.

Lim1215 cells




Figure 4. Cetuximab resistance in Lim1215 cells by increasing concentration, genetic composition, FISH analysis, Western blot analysis, and ectopic expression

Graph (a) depicts the relationship between cetuximab concentration and cell viability, ultimately exhibiting the successful resistance created in the R lines. The overlap of error bars of the R lines show that the resistance is similar between the two, despite method of treatment. Once again, the difference between the R1 and R2 lines isn’t explained. Just like the DiFi R lines, the Lim1215 R1 line was exposed to a constant concentration of cetuximab, while R2 was exposed to a concentration that increased stepwise over time for a year. However, there is a striking difference between the Lim1215 R lines and the DiFi R lines. The Lim1215 R lines were exposed to an overall concentration of 1400 nM over a course of 3 months, while DiFi R lines were exposed to an overall concentration of 350 nM over the course of a year. The specifics and reasons behind this procedure are unclear, making the comparison between the two cell lines questionable. How else are the two cellular models different to require different protocols?

<span style="font-family: Arial,Helvetica,sans-serif;">According to the Sanger sequencing results in part (b), which is just a method of DNA sequencing, the two resistant lines show inconsistencies compared to the control, as pointed out by the arrows. The other peaks not pointed out do not match perfectly with the control results, but the peaks seem to be relatively proportional, so they are deemed as insignificant differences. In Lim1215 R1, the difference turned out to be a G12R mutation, which means that at position 12 in KRAS, there is an amino acid substitution from a glycine to an arginine. On the other hand, the difference in Lim1215 R2 is a G13D mutation, where an aspartic substituted a glycine at position 13 in KRAS. Because the mutation differed in R1 and R2, I would assume that the type of cetuximab treatment could possibly affect the type of mutation, but the researchers do not expound further on this topic.

<span style="font-family: Arial,Helvetica,sans-serif;">The Western blot analysis in part (c) shows that regardless of cetuximab exposure, active GTP-KRAS is present in the resistant lines but not the parent Lim1215 line. All the other protein expression levels seem similar, emphasizing the significance of the active GTP-KRAS. The evidence of active GTP-KRAS paired with the KRAS mutations magnifies the possibility that there is a correlation between KRAS mutation and acquired cetuximab resistance.

<span style="font-family: Arial,Helvetica,sans-serif;">The methods to back up the data in parts (d) and (e) are not focused on much in the paper. Part (d) is a schematic representation of how the G12R and G13D mutations were “knocked-in” to the genome of Lim1215 parental cells. The paper does not explain this procedure or its significance further, but its relationship with the graph in part (e) can help one understand how the authors come to their conclusions. One can assume that the purpose of knocking-in the mutations into the genome of the parent cells is to test if the mutations are the reason why there is resistance, or if they are just side products. Illustrated in graph (e), the control parent line has significantly lower cell viability as the cetuximab concentration increases, consistent with the control in graph (a) as well. The Lim KI G12R, which corresponds to the mutation in Lim1215 R1, has a similar resistance when compared to graph (a). Lim KI G13D, which corresponds to the mutation in Lim1215 R2, is still resistant compared to the control, but does not show the same cell viability compared to R2 in graph (a). Therefore, another variable may play a role in affecting the resistance in R2. However, the mutation in Lim1215 R1 seems to be a very strong candidate.

<span style="font-family: Arial,Helvetica,sans-serif;">From the results of these two cellular models, the authors then needed to determine if KRAS mutation and amplification are clinically relevant.

=<span style="font-family: Arial,Helvetica,sans-serif;">Clinical Trials =

<span style="font-family: Arial,Helvetica,sans-serif;">Through two different models, the authors concluded that KRAS amplification and/or mutation had an effect on resistance to cetuximab. To determine if these conclusions are clinically relevant, the researchers examined tumor biopsies from colorectal cancer patients. The results are presented in this figure.



<span style="display: block; font-family: Arial,Helvetica,sans-serif; text-align: center;">Figure 5. Difference in patient status after developing chemotherapy and anti-EGFR resistant tumors.

<span style="font-family: Arial,Helvetica,sans-serif;">This study compares the KRAS mutational statuses of colorectal cancer patients who had chemotherapy resistant tumors and those with anti-EGFR resistant tumors. The patients with anti-EGFR resistant tumors have either been treated with cetuximab or panitumumab. The other group had been treated by cytotoxic chemotherapy and had not been previously exposed to anti-EGFR therapies. Through multiple methods of sequencing, KRAS mutations in the patients’ tumors were identified. Some patients who had anti-EGFR therapies, shown in table (b), acquired KRAS mutations, while the chemotherapy patients, table (a), were all wild type for KRAS.

<span style="font-family: Arial,Helvetica,sans-serif;">The sample size for the chemotherapy group is sufficient because they all are wild type, but a larger sample size for the anti-EGFR group would be rewarding and valuable to this study. This could show possibly a variety of mutations as well as the frequency of these mutations in colorectal cancer patients. Also, three anti-EGFR patients are KRAS wild type, leading to the question of what caused their acquired resistance? Would it be similar to the chemotherapy patients?

<span style="font-family: Arial,Helvetica,sans-serif;">The molecular experiments focused solely on the drug cetuximab, but this study allows patients who have also used panitumumab. How similar are these drugs to assume that panitumumab could also lead to the same mutations? The authors of this paper made their conclusions in terms of cetuximab, so incorporating panitumumab data may weaken the strength of their claim. The type of sequencing also differed for some patients, which could lead to inconsistencies in the data.

<span style="font-family: Arial,Helvetica,sans-serif;">The number of mutated reads from sequencing quantifies the total number of KRAS mutations for the two groups of patients. Graph (c) illustrates the significant difference between the two groups, including a small p-value, supporting the significance. However, this study focused on sequencing KRAS in patients that have already had a form of treatment, but without the knowledge of their KRAS status before the treatment, one cannot say that anti-EGFR therapy causes KRAS mutations, leading to resistance. We only know from this data that there is a correlation, but in combination with the molecular data, other assumptions can be made.

<span style="font-family: Arial,Helvetica,sans-serif;">Another Trial
We wanted to investigate the relationship of KRAS mutations and anti-EGFR therapies in more than one type of study, so the molecular data and clinical trial data work well together. However, these three data sets are from the same research paper, meaning that they could have been exposed to the same biases. We decided to look at another clinical trial and compare the results.





=<span style="font-family: Arial,Helvetica,sans-serif;">Conclusions =

Estimates are that around 40% of patients with metastatic colorectal cancer have KRAS mutations. For most patients with KRAS mutations, this will mean they forgo the use of anti-EGFR drugs like cetuximab and panitumumab to avoid the high cost and adverse side effects. In 2009 the FDA updated the labels of cetuximab (Erbitux) and panitumumab (Vectibix) to include information about the effects of KRAS mutations, then licensed the drugs for use only for patients with wildtype KRAS. Although the licensing remains (in the USA and Europe), the National Comprehensive Cancer Network revised their guidelines to recommend anti-EGFR therapy to all patients, even those with any KRAS mutation. Clearly, more specific anti-EGFR drugs need to be researched to provide more personalized care. A drug that blocks the ligands and signals from KRAS mutations can help inhibit the MAPK pathway. Otherwise patients with a KRAS mutation and metastatic colorectal cancer will find themselves looking at less effective interventions then their wildtype counterparts taking anti-EGFR monoclonal antibodies.

=Sources=

<span style="background-color: rgba(255,255,255,0.0980392); font-family: 'Times New Roman',Times,serif; font-size: 16px;">"Emergence of KRAS Mutations and Acquired Resistance to Anti EGFR Therapy in Colorectal Cancer." //<span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;">NIH Public Access //<span style="background-color: rgba(255,255,255,0.0980392); font-family: 'Times New Roman',Times,serif; font-size: 16px;">. NCBI, n.d. Web. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3927413/?report=classic#SD1

<span style="background-color: rgba(255,255,255,0.0980392); font-family: 'Times New Roman',Times,serif; font-size: 16px;">"Molecular Mechanisms of Resistance to Cetuximab and Panitumumab in Colorectal Cancer." Journal of Clinical Oncology, n.d. Web. 02 June 2014. http://jco.ascopubs.org/content/28/7/1254.long

<span style="background-color: rgba(255,255,255,0.0980392); font-family: 'Times New Roman',Times,serif; font-size: 16px;">"KRAS Mutation Status Is Predictive of Response to Cetuximab Therapy in Colorectal Cancer." //<span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;">KRAS Mutation Status Is Predictive of Response to Cetuximab Therapy in Colorectal Cancer //<span style="background-color: rgba(255,255,255,0.0980392); font-family: 'Times New Roman',Times,serif; font-size: 16px;">. The Journal of Cancer Research, n.d. Web. 02 June 2014. http://cancerres.aacrjournals.org/content/66/8/3992.long

<span style="background-color: rgba(255,255,255,0.0980392); font-family: 'Times New Roman',Times,serif; font-size: 16px;">"The Road to Resistance: EGFR Mutation and Cetuximab." //<span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;">Nature Medicine //<span style="background-color: rgba(255,255,255,0.0980392); font-family: 'Times New Roman',Times,serif; font-size: 16px;">. Nature Medicine, n.d. Web. http://www.nature.com/nm/journal/v18/n2/full/nm.2646.html

<span style="background-color: rgba(255,255,255,0.0980392); font-family: 'Times New Roman',Times,serif; font-size: 16px;">"FDA Approval for Cetuximab." //<span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;">National Cancer Institute //<span style="background-color: rgba(255,255,255,0.0980392); font-family: 'Times New Roman',Times,serif; font-size: 16px;">. National Cancer Institute, n.d. Web. 02 June 2014. http://www.cancer.gov/cancertopics/druginfo/fda-cetuximabAchor-Kras-7612

<span style="background-color: rgba(255,255,255,0.0980392); font-family: 'Times New Roman',Times,serif; font-size: 16px;">"Colon, Rectal, and Anal Cancers." //<span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;">Oncology and Hematology News and Journal Articles //<span style="background-color: rgba(255,255,255,0.0980392); font-family: 'Times New Roman',Times,serif; font-size: 16px;">. Cancer Management Handbook, n.d. Web. 02 June 2014.