Tumor Tissue And Plasma Kras Mutations And The Prognosis Of Patients With Colorectal Cancer Study

With the constant emergence of new anti-tumor drugs, the efficiency of thetreatment for advanced colorectal cancer and the whole survival time of the patientsare increasing constantly, especially with the use of targeted drug EGFR (Epidermalgrowth factor receptor) McAb, combined chemotherapy can extend the patients'survival time as long as two years. Besides, the study has discovered the outcomepredictor of the EGFR McAb, which is KRAS genetic mutation. Before takingmedication, susceptive group can be screened through detecting whether the KRASgenes of the patients' tumor tissues have mutated, so EGFR McAb has realizedpreliminary individual application.However, in the process of individual application, there exist some big defects.For the reason that it is relatively difficult to obtain tumor tissues and that it does nothave the real-time, it is an urgent problem to find out the substitute containing thegenetic information of the tumor tissues, which satisfies the need of real-time andeasy access, to replace the tumor tissues for genetic detection.With the development of detecting techniques, the detecting methods of KRASgenetic mutation are also faced with reform. A study shows that the commonlyaccepted sequencing methods for the moment has low sensitivity, which might lead tothe existence of false-negative results, so it is necessary to detect with methods ofhigh sensitivity. We considerd that there is bound to be a kind of patients whendetected with two methods of different sensitivity. When applied to the method of lowsensitivity, the patients belong to the wild type. As for the method of high sensitivity,the patients may belong to the mutant type. That is to say, this kind of patients mayhave relatively low quantity of mutation. Whether it is suitable to apply EGFR McAbto this kind of patients and whether the quantity of the KRAS mutation (mutationabundance), except for whether the KRAS mutation happens, will have influence onthe curative effect of the EGFR McAb and the patients' prognosis? No relative studyhas been done by now. Though KRAS mutation is the outcome predictor for EGFRMcAb, it still has difference with the relativity of the prognosis of the simple chemotherapy patients. The reasons for differences, we believed that one of theimportant point may be the detection methods used by the various studies was notunified, he detection sensitivity of the method was different.So we carry out the study in order to investigate the different detection methodsto KRAS mutation and patients' prognosis; and the relations between the KRASmutation abundance and the curative effects and prognosis of the simplechemotherapy patients, and the feasibility of using plasma to replace tumor tissues inthe process of detecting KRAS genetic mutation.From January,2008to June,2011, we have screened117advanced colorectalcancer patients, who have received at least two schemes of simple chemotherapy(schemes centered on oxaliplatin or on irinotecan). Based on RECIST(ResponseEvaluation Criteria in Solid Tumors),37patients in the first-line chemotherapy havegained partial response (PR)(31.6%). The median PFS(Progression free survival) forall the patients is6.3months and the time of median OS(Overall survival) is19.5months. We have applied methods of direct sequencing and PNA-PCR(PeptideNucleic Acid-mediated PCR Clamping) to detect the KRAS mutation status in thepatients' tumor tissues and in the paired serum. There are46patients of KRASmutation detected in the tumor tissues with the method of direct sequencing and thedetection rate is39.3%. As for the KRAS mutation in the plasma,18patients havebeen detected and the detection rate is15.4%. Its concordance rate with the tumortissues is32.6%. The general concordance rate of the wild type and the mutant type is70.9%and the consistency is relatively bad(Kappa0.318,p<0.001). By using themethod of PNA-PCR to detect KRAS genetic mutation of the tumor tissues and theplasma,56patients of KRAS mutation of tumor tissues have been detected, whosedetection rate is47.9%, and35patients of KRAS mutation in the plasma have beendetected, whose detection rate is29.9%and whose concordance rate with tumortissues is55.4%. The general concordance of the wild type and the mutant type is75.2%and the consistency is ordinary(Kappa0.496,p<0.001). By applying themethod of direct sequencing to detect KRAS mutation in tumor tissues and themethod of PNA-PCR to detect the plasma, we find that the concordance rate of KRAS mutation in the plasma and tumor tissues is63.0%. The general concordance rate is80.3%and the consistency is good(Kappa0.570,p<0.001). We can conclude fromthe above results that because the plasma DNA contains very low amount of tumortissue-specific DNA, it is more suitable to use the detecting methods of highsensitivity to detect KRAS genetic mutation and so as to increase the concordancerate with the KRAS mutation of tumor tissues.Through measurement and analysis of the KRAS mutation conditions of thetumor tissues and the curative effects of the simple chemotherapy, we find that neitherpatients of the wild type nor patients of the mutant type have statistical difference inORR(over response rate) and PFS when referred to both methods of direct sequencingand PNA-PCR. In the aspect of the patients' prognosis, the wild type patients and themutant type patients detected with the method of direct sequencing approachstatistical meaning in the difference of OS (p=0.052), while patients of wild type andmutant type detected with the method of PNA-PCR have obvious statistical differencein OS(p=0.010). According to results of the KRAS detected with the methods ofdirect sequencing and PNA-PCR(abundance of KRAS mutation), we divide thepatients into wild type group(wide type for the two methods), high mutationgroup(mutation for the two methods) and low mutation group(wild type for the directsequencing, while mutation for the PNA-PCR method). The patients of the threegroups have no difference in efficiency and PFS, but the median OS for the wild typegroup is as long as21.3months,17.1months for the low mutation group,and15.6months for the high mutation group, the difference has obvious statistical meaning(p=0.033). Comparing each two of the three groups, we find that the OS differencebetween the wild type group and the high mutation group has obvious statisticalmeaning (p=0.029) and the OS difference between the wild type group and the lowmutation group approaches statistical meaning(p=0.058). Besides, there is nostatistical difference of OS between high mutation group and low mutation group. Aswe can see, the OS of the low mutation group approaches that of the high mutationgroup and the OS of the wild type group is still the longest. The KRAS mutation conditions in the plasma detected with the method of eitherdirect sequencing or PNA-PCR have no influence on the curative effects of the simplechemotherapy, but it has obvious relativity with the patients' OS. The median OSdetected by the method of direct sequencing is five months longer than that of thepatients of mutant type and there exists statistical difference (p=0.009). Similarly, theOS for the patients of the wild type and the mutant type are21.3months and15.1months respectively, and the difference also has obvious statistical meaning (p=0.001).The above results show that the conditions of the KRAS genetic mutation in theplasma have relativity with the OS of patients and that the prognosis for the KRASmutation patients is bad. The KRAS mutation in the plasma is the outcome predictorfor the patients of simple chemotherapy.The results show that the KRAS mutation in the plasma and tumor tissueshas relatively high consistency when detected by PNA-PCR, so detecting the KRASgenetic mutation in the plasma can be considered for guiding individual medicationwhen it is difficult to access the tumor tissues. In whichever way to detect KRASgenetic mutation, there is no relativity between KRAS mutation and the curativeeffects of the simple chemotherapy, so KRAS mutation cannot be regarded as thepredictor of the curative effects. However, as for the relativity between KRASmutation and the OS of patients of simple chemotherapy, different detecting methodsmay lead to different conclusions, which confirmed that one of the reasons fordifferent conclusions of the relationship between KRAS mutation and patients'prognosis. In this study, the overall survival for the patients of wild type and mutanttype detected by direct sequencing has no statistical difference, while the overallsurvival for the patients of the wild type and mutant type detected by PNA-PCR hasobvious difference. Therefore, the method of PNA-PCR with high sensitivity may bemore suitable than the method of direct sequencing for the detection of KRASmutation in the tumor tissues. But through analyzing the OS of the three groups, wefind that the OS of the wild type patients is the longest, and that of the low mutationgroup and high mutation group come as the second and third longest respectively.Therefore, it may have more important predicting meaning if we carry out detailed categorization according to the abundance of the KRAS genetic mutation. Thissuggests that we should combine the two methods to detect KRAS genetic mutation,which means that the patients of the wild type should be applied to the method ofPNA-PCR, which can reduce the false negative results. and that the patients of themutant type should be applied to the traditional method-----direct sequencing,to avoid the exaggeration of KRAS mutation detected by high sensitivity methods,such as the PNA-PCR method. As for the KRAS mutation in the plasma, bothmethods have showed that KRAS mutation has no relativity with the patients'curative effects, but it has obvious relativity with the patients' prognosis, suggestingthat the KRAS mutation in the plasma is the predictor for the prognosis of thepatients.In conclusion, different detecting methods may lead to different conclusions,which confirmed that one of the reasons for different conclusions of therelationship between KRAS mutation and patients' prognosis. it may have betterpredicting effects for the patients' prognosis by detecting the KRAS mutation with thecombination of PNA-PCR of high sensitivity and the traditional direct sequencing,and by categorizing the patients according to the abundance of the KRAS mutation. Itwould be better to use methods of high sensitivity to detect KRAS mutation in theplasma and the prognosis for the mutant patients is relatively bad. When it is difficultto gain the patients' tumor tissues, plasma can be taken as the replacement of thetumor tissues for the detection of the KRAS genetic mutation.