RAS+Affect+On+Anti-EGRF+Antibodies+in+Colorectal+Cancer

=Introduction to Colorectal Cancer =

=Anti-EGFR Treatment =

=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 cetuxtimab. 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 1. Weeeeeeeee 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 2. Weeeeeeeeeeeeee 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?

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.

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.

The methods to back up the data in parts (d) and (e) are 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.

From the results of these two cellular models, the authors then needed to determine if KRAS mutation and amplification are clinically relevant.

=Clinical Trials =

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

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