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. [1] 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 [2]. 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–epidermal growth factor receptor (EGFR) therapy [3]. 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.

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 synchronous and metachronous lesions showed no difference in the mutational profiles. 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.

The clinical impact of a rebiopsy at relapse has been controversial. 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 [3]. Whether the RAS mutation status was stable throughout disease progression and treatment is unclear. A recent meta-analysis by Mao et al. [6], 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 [7]. On the other hand, Misale et al. [8] 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) [8]. Cytotoxic agents alone, however, have not shown changes in the mutational profiles in studies including Misale et al. [8] 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 [9].

Another important issue is the platform for sequencing. Lee et al. [10] 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. [11] 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 detection sensitivity of Sequenom MassARRAY technology is approximately 5%. 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.

Hypermutation and MSI should also be considered when interpreting the heterogeneity of mutations. Two out of three metastatic cases with discordant KRAS mutations showed hypermutation in one study [10]. 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). On the other hand, four patients were identified in the expanded analysis. 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).

The biology of the metachronous and synchronous metastatic tumors in CRC is controversial. While some have observed no prognostic difference between the two entities, others have suggested metachronous disease to have a favorable prognosis [12,13]. Some authors have also suggested that the two entities have a different biology [14]. Rose et al. [15] evaluated KRAS and showed no difference between the two entities. 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.

This study had several limitations. Patients who have received prior anti-EGFR therapy were not included. 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 [16]. 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. Second, metastatic lesions were limited to the liver. Metastatic lesions of the lymph node and lung may have different characteristics to the liver-limited lesions [17,18]. 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.