tumor progression often occurs according to the scheme of the gradual accumulation of genetic abnormalities that leads to intertumor and intratumor heterogeneity according to the spatial and temporal differences . gerlinger et al .   revealed a classic example of intratumor heterogeneity with a renal cell carcinoma and its implications on tumor adaptation and therapeutic failure through darwinian selection . these results have challenged the clinical practice of performing a single biopsy and genetic analysis of a patient , particularly in cases where the genetic profiles influence the therapeutic decisions . therefore , the need for a clinical rebiopsy at relapse and metastasis after a resected cancer is being evaluated continuously . colorectal cancer ( crc ) have been investigated extensively to evaluate the role of a secondary biopsy due to two factors . first , tissues from the primary and metastatic lesions have been assessable in many cases because a metastasectomy has a proven role in the care of metastatic crc . this has made it feasible to perform extensive molecular pathologic evaluations to evaluate the tumor heterogeneity within a patient . the other important factor is that ras mutations have been important in predicting the responses to anti therefore , there would be a necessity to perform a second biopsy if ras mutation status changes according to time or location with a patient . in addition , other genes that interact with ras and affect the response to therapy can also be present . this is the first study designed to evaluate the mutational profile of crc lesions according to the location ( primary vs. metastatic lesion ) and time ( synchronous vs. metachronous ) in individual patients with paired specimens . this study examined whether the crc spatial and temporal progression could be related to a difference in somatic mutations and correlate them with the clinicopathologic features . retrospective analysis was performed on the database of bio - resource center , a de - identified tissue bank linked with the asan biomedical research environment ( able ) , an anonymized electronic medical record of the asan medical center , in which patients were informed before surgery that their surgical specimens might be used for research purposes and provided consent for using their archival tissues for genetic testing . between june 2004 and june 2012 , patients with histologically confirmed crc , who underwent surgical resection of the primary tumor with an adequate amount of tissue for genomic profiling , were selected . in addition , patients with a surgical resection of a corresponding liver metastasis were included . all tumor samples were obtained from the formalin - fixed , paraffin - embedded ( ffpe ) tumor specimens based on an 80% cutoff for tumor sample purity . synchronous was defined as metastatic disease at the time or within 6 months of the original diagnosis of crc . metachronous was defined as the absence of metastatic disease at the time of the initial diagnosis with metastatic disease developing later than 6 months of the original diagnosis . the genomic dna was extracted from 6-m - thick slides per ffpe block and purification of genomic dna was performed using a qiaamp dna ffpe tissue kit ( # 56404 , qiagen , hilden , germany ) . each genomic dna sample was eluted in 50 l of dnase- and rnase - free water , quantified using the quant - it picogreen dsdna assay kit ( invitrogen / life technologies , grand island , ny ) , and normalized to a 5 ng/l concentration . high - throughput profiling of somatic mutations that span 471 unique mutation sites in 41 oncogenes and tumor suppressor genes was performed using oncomap version 4.4 core ( oncomap_v4.4c ) under the sequenom massarray technology platform ( sequenom , san diego , ca ) . the microsatellite instability ( msi ) status of the tumors was determined based on the bethesda panel ( bat25 , bat26 , d5s346 , d2s123 , and d17s250 ) . the polymerase chain reaction products were run on an abi prism 3130xl dna sequencer ( applied biosystems , foster city , ca ) , and analyzed using genescan 3.1 software ( applied biosystems ) according to the manufacturer 's instructions . tumors with two or more unstable markers were classified as msi - high , whereas those with none or one unstable marker were classified as microsatellite stable or low level msi . additional loss of heterozygosity ( loh ) or msi were assayed for d18s57 , d18s58 , and d18s474 using the same instrument . tumors with a 50% or more signal intensity difference between the two alleles were defined as loh positive cases . the categorical variables are presented using contingency tables and were compared using a chi - square test or fisher exact test . the overall survival ( os ) was the primary endpoint for this study and was calculated from the date of surgery until the date of death . the relapse - free survival ( rfs ) was calculated from the date of surgery until the date of relapse . the patients lost to follow - up were censored at the time of the last visit . retrospective analysis was performed on the database of bio - resource center , a de - identified tissue bank linked with the asan biomedical research environment ( able ) , an anonymized electronic medical record of the asan medical center , in which patients were informed before surgery that their surgical specimens might be used for research purposes and provided consent for using their archival tissues for genetic testing . between june 2004 and june 2012 , patients with histologically confirmed crc , who underwent surgical resection of the primary tumor with an adequate amount of tissue for genomic profiling , were selected . in addition , patients with a surgical resection of a corresponding liver metastasis were included . all tumor samples were obtained from the formalin - fixed , paraffin - embedded ( ffpe ) tumor specimens based on an 80% cutoff for tumor sample purity . synchronous was defined as metastatic disease at the time or within 6 months of the original diagnosis of crc . metachronous was defined as the absence of metastatic disease at the time of the initial diagnosis with metastatic disease developing later than 6 months of the original diagnosis . the genomic dna was extracted from 6-m - thick slides per ffpe block and purification of genomic dna was performed using a qiaamp dna ffpe tissue kit ( # 56404 , qiagen , hilden , germany ) . each genomic dna sample was eluted in 50 l of dnase- and rnase - free water , quantified using the quant - it picogreen dsdna assay kit ( invitrogen / life technologies , grand island , ny ) , and normalized to a 5 ng/l concentration . high - throughput profiling of somatic mutations that span 471 unique mutation sites in 41 oncogenes and tumor suppressor genes was performed using oncomap version 4.4 core ( oncomap_v4.4c ) under the sequenom massarray technology platform ( sequenom , san diego , ca ) . the microsatellite instability ( msi ) status of the tumors was determined based on the bethesda panel ( bat25 , bat26 , d5s346 , d2s123 , and d17s250 ) . the polymerase chain reaction products were run on an abi prism 3130xl dna sequencer ( applied biosystems , foster city , ca ) , and analyzed using genescan 3.1 software ( applied biosystems ) according to the manufacturer 's instructions . tumors with two or more unstable markers were classified as msi - high , whereas those with none or one unstable marker were classified as microsatellite stable or low level msi . additional loss of heterozygosity ( loh ) or msi were assayed for d18s57 , d18s58 , and d18s474 using the same instrument . tumors with a 50% or more signal intensity difference between the two alleles were defined as loh positive cases . the categorical variables are presented using contingency tables and were compared using a chi - square test or fisher exact test . the overall survival ( os ) was the primary endpoint for this study and was calculated from the date of surgery until the date of death . the relapse - free survival ( rfs ) was calculated from the date of surgery until the date of relapse . the patients lost to follow - up were censored at the time of the last visit . this study reviewed 105 patients with various stages of crc , who all received a surgical resection and had sufficient tissue of genomic profiling ( supplementary table 1 ) . of those patients , this study selected a subset of 46 patients ( 43.8% , 46/105 ) , who had paired samples from the primary and metastatic tumor in synchronous ( n=27 ) and metachronous ( n=19 ) lesions as a result of metastasectomy ( table 1 ) . the median follow - up of these patients was 117.1 weeks ( range , 39.7 to 393.6 weeks ) . most ( 16/19 ) of the metachronous patients had adjuvant chemotherapy following the resection of the primary lesion . the median interval between the primary surgery and initial recurrence for the metachronous group was 83.9 weeks ( range , 27.3 to 344.3 weeks ) . msi was confirmed in only one patient with a synchronous metastasis ( 1/44 ) and loh in 24.0% ( 6/25 ) and 42.1% ( 8/19 ) for synchronous and metachronous patients , respectively . mutations were identified in 80.4% ( 35/46 ) of the patients ( table 2 ) , which was similar to the other patients within the bio - resource center ( mutation rate , 70.5% , 74/105 ) ( supplementary table 2 ) . the degree of concordance of the gene mutations between the primary and metastatic tumors were examined in both synchronous and metachronous metastatic lesions . the genomic profile of synchronous and metachronous diseases did not show a significant difference ( table 2 ) . in addition , most of the paired samples between the primary and metastatic tumors were concordant and differed in 7.4% ( 2/27 ) and 10.5% ( 2/19 ) for synchronous and metachronous , respectively . in the synchronous patients , one patient had an apc mutation in the metastatic liver alone , while the other patient had a kras mutation only in the primary lesion . on the other hand , one patient had a tp53 mutation in the metastatic liver alone , whereas the other patient had a braf mutation only in the primary lesion in the metachronous group . although there was one patient with msi in the paired analysis , a total of four patients in the 105 patients were evaluated by oncomap analysis ( supplementary table 2 ) . all four patients had at least one mutation : kras , 1/4 ; apc , 2/4 ; stk11 , 0/4 ; tp53 , 2/4 ; braf , 1/4 ; mlh1 , 0/4 ; akt1 , 1/4 ; pik3ca , 1/4 ; nras , 0/4 ; and ctnnb1 , 0/4 . all the metastatic diseases were resected in the 46 patients with synchronous and metachronous disease ( table 1 ) . the median rfs was 43.0 weeks ( 95% confidence interval , 27.7 to 58.3 ) and there was no significant difference between the synchronous and metachronous disease ( p=0.870 ) ( fig . 1 ) . owing to the limited follow - up , only 19.6% ( 9/46 ) this study reviewed 105 patients with various stages of crc , who all received a surgical resection and had sufficient tissue of genomic profiling ( supplementary table 1 ) . of those patients , this study selected a subset of 46 patients ( 43.8% , 46/105 ) , who had paired samples from the primary and metastatic tumor in synchronous ( n=27 ) and metachronous ( n=19 ) lesions as a result of metastasectomy ( table 1 ) . the median follow - up of these patients was 117.1 weeks ( range , 39.7 to 393.6 weeks ) . most ( 16/19 ) of the metachronous patients had adjuvant chemotherapy following the resection of the primary lesion . the median interval between the primary surgery and initial recurrence for the metachronous group was 83.9 weeks ( range , 27.3 to 344.3 weeks ) . msi was confirmed in only one patient with a synchronous metastasis ( 1/44 ) and loh in 24.0% ( 6/25 ) and 42.1% ( 8/19 ) for synchronous and metachronous patients , respectively . mutations were identified in 80.4% ( 35/46 ) of the patients ( table 2 ) , which was similar to the other patients within the bio - resource center ( mutation rate , 70.5% , 74/105 ) ( supplementary table 2 ) . the degree of concordance of the gene mutations between the primary and metastatic tumors were examined in both synchronous and metachronous metastatic lesions . the genomic profile of synchronous and metachronous diseases did not show a significant difference ( table 2 ) . in addition , most of the paired samples between the primary and metastatic tumors were concordant and differed in 7.4% ( 2/27 ) and 10.5% ( 2/19 ) for synchronous and metachronous , respectively . in the synchronous patients , one patient had an apc mutation in the metastatic liver alone , while the other patient had a kras mutation only in the primary lesion . on the other hand , one patient had a tp53 mutation in the metastatic liver alone , whereas the other patient had a braf mutation only in the primary lesion in the metachronous group . although there was one patient with msi in the paired analysis , a total of four patients in the 105 patients were evaluated by oncomap analysis ( supplementary table 2 ) . all four patients had at least one mutation : kras , 1/4 ; apc , 2/4 ; stk11 , 0/4 ; tp53 , 2/4 ; braf , 1/4 ; mlh1 , 0/4 ; akt1 , 1/4 ; pik3ca , 1/4 ; nras , 0/4 ; and ctnnb1 , 0/4 . all the metastatic diseases were resected in the 46 patients with synchronous and metachronous disease ( table 1 ) . the median rfs was 43.0 weeks ( 95% confidence interval , 27.7 to 58.3 ) and there was no significant difference between the synchronous and metachronous disease ( p=0.870 ) ( fig . 1 ) . owing to the limited follow - up , only 19.6% ( 9/46 ) had expired and the median os could not be evaluated . in this study with oncomap , mutations were identified in 80.4% of the patients with the following frequency : kras ( 39.1% ) , tp53 ( 28.3% ) , apc ( 28.3% ) , pik3ca ( 6.5% ) , braf ( 6.5% ) , and nras ( 4.3% ) . although 19.6% ( 9/46 ) of the patients showed no gene mutations , 43.5% ( 20/46 ) and 37.0% ( 17/46 ) had mutations in one and two or more genes . the paired samples between the primary and metastatic tumors differed in 8.3% ( 2/24 ) and 9.1% ( 2/22 ) for the synchronous and metachronous lesions , respectively . one important aspect is whether the molecular information changes from the primary biopsy / resection until relapse . this may be related to the temporal and spatial differences between the primary and relapsed lesions . in addition , the therapeutic intervention between the primary and relapsed lesions , such as adjuvant chemotherapy , may also induce changes at the molecular level . ras mutations have been important for predicting the response to anti - egfr therapy . whether the ras mutation status was stable throughout disease progression and treatment is unclear . a recent meta - analysis by mao et al . , who pooled studies on the concordance rates of kras , braf , and pik3ca mutation between primary and metastatic lesion , showed that the pooled concordance rate was 92.0% for kras in 43 studies including 2,774 pairs of primary and metastatic lesions . in that analysis , 9% of patients with wild - type kras in the primary tumors who received anti - egfr treatment had mutant kras in the metastases , whereas 11.3% patients with the mutant kras primary tumors had wild - type kras in the metastases . the concordance for other mutations was also high : 96.8% for braf , 93.9% for pik3ca , and 71.7% for pten . in addition , one study evaluated the impact of the discordant cases on clinical care and found that the information obtained by additional mutation analysis changed the medical decision for subsequent therapy in 2% ( 6/305 ) of the cases . on the other hand , misale et al .   showed that the emergence of ras mutations can be observed both in tissue and plasma dna after anti - egfr therapy . in their study , the acquisition of secondary kras mutations in 60% ( 6/10 ) of the cases were observed by deep sequencing ( high coverage 454 sequence analysis or beaming ) . cytotoxic agents alone , however , have not shown changes in the mutational profiles in studies including misale et al .   and the present study . because anti - egfr therapy has no role in an adjuvant setting , the role of a rebiopsy at relapse after the standard operation and adjuvant cytotoxic chemotherapy may be limited . assessed the polyclonality and genetic heterogeneity in crc by performing targeted exome sequencing and high resolution copy number variation analysis of 15 triplets of normal colorectal tissue , primary , and matched synchronous liver metastatic lesions . in that study , the discordance rate of the kras mutation between the primary - metastatic pairs was 50% ( 3/6 ) . on the other hand , brannon et al .   used impact targeted sequencing , a next generation sequencing method , for 230 key cancer - associated genes for 69 matched primary and metastatic tumors , and reported 100% concordance for the kras , nras , and braf mutational profiles . the present study had a discordance rate of less than 10% for the 41 genes evaluated by oncomap in 46 matched tumors . the identification of somatic mutations using an oncomap system was performed with two different sequential reactions , called iplex and homogenous mass extension ( hme ) chemistries . screening of the mutation candidates was done with sequenom massarray iplex , followed by a specific hme reaction for further validation for each detected mutation . the genotypes were named using the typer analyzer module in the massarray typer 4 ( agena bioscience ) software , and further reviewed manually by two independent researchers to clarify uncertain calls due to clustering artifacts . the results obtained from the iplex and hme assays were compared ; only the concordant calls are regarded as validated mutations . as the samples had a tumor percentage of at least 80% , the discordancy was attributed to actual sample characteristic rather than the low mutant allele frequency . future studies and practice may differ according to platforms ; therefore , the issues among platforms must be validated in precedent to make further conclusions . two out of three metastatic cases with discordant kras mutations showed hypermutation in one study . the discordance may be the result of acquired mutations in the dna mismatch repair genes after the carcinogenesis of primary tumors , which leads to dynamic polyclonal evolution . on the other hand , in the present study , all four cases with discordant mutations were microsatellite stable . as msi is not often checked in a metastatic setting , more data will be needed to fully evaluate this issue . the clinicopathologic factors associated with msi were difficult to characterize due to the limited number of patients ( one in the synchronous group ) . all patients had right side colon cancer , but there was no specific pattern in the comutations or number of mutations ( kras , 1/4 ; apc , 2/4 ; tp53 , 2/4 ; braf , 1/4 ; akt1 , 1/4 ; and pik3ca , 1/4 ) . while some have observed no prognostic difference between the two entities , others have suggested metachronous disease to have a favorable prognosis . this finding was expanded to 41 genes that cover the major signal pathways including the ras  raf mitogen - activated protein kinase ( mapk ) pathway for both primary and metastatic lesions . although limited in size , this study showed a similar clinical outcome and mutational profiles of metachronous and synchronous metastatic tumors . the use of anti - egfr therapy and other molecular targeted agents is increasing prior to surgery in borderline resectable or conversion cases of metastatic crc . these agents may have an impact on the mutational profile , particularly the ras - raf - mapk pathway . in a relapsed setting , however , most of the patients will be exposed only to cytotoxic agents due to the limited role of anti - egfr therapy in an adjuvant setting . metastatic lesions of the lymph node and lung may have different characteristics to the liver - limited lesions . in the present clinical practice , however , the majority of metastasectomy cases will be related to liver - limited disease because its role has been established in other settings . these results suggest that a conventional second biopsy or mutational profiling may not be helpful in managing crc patients who have relapsed after adjuvant chemotherapy . in addition , metachronous and synchronous crc has concordant mutational profiles in both primary and metastatic tumors . on the other hand , as highly reliable and sensitive mutation profiling methods , such as beaming , emerge and more patients are exposed to preoperative anti - egfr therapy , this may need to be reevaluated in both tissue and liquid biopsy specimens .
purposealthough the mutation status of kras is highly concordant in primary and metastatic lesions , it has not been generalized to other major pathway genes.materials and methodsin this study , 41 genes were evaluated and the mutational profiles were compared in 46 colorectal cancer patients with paired surgical specimens of primary and metastatic lesions : synchronous ( n=27 ) and metachronous ( n=19 ) lesions . a high - throughput mass spectrometry - based genotyping platform validated by orthogonal chemistry , oncomap v.4.4 , was used to evaluate the formalin - fixed , paraffin - embedded surgical specimens . 
 the patients demographics , tumor characteristics , and microsatellite instability status were analyzed by a retrospective chart review.resultsin this study , with oncomap , mutationswere identified in 80.4% of patientswith the following frequency : kras ( 39.1% ) , tp53 ( 28.3% ) , apc ( 28.3% ) , pik3ca ( 6.5% ) , braf ( 6.5% ) , and nras ( 4.3% ) . 
 although 19.6% ( 9/46 ) of the patients showed no gene mutations , 43.5% ( 20/46 ) and 37.0% ( 17/46 ) had mutations in one and two or more genes , respectively . 
 the synchronous and metachronous lesions showed similar mutational profiles . 
 paired samples between primary and metastatic tumors differed in 7.4% ( 2/27 ) and 10.5% ( 2/19 ) for synchronous and metachronous according to oncomap.conclusionthese findings indicate the major pathway genes , including kras , tp53 , apc , pik3ca , braf , and nras , are often concordant between the primary and metastatic lesions regardless of the temporal relationship of metastasis .