The reported 5-year overall survival of patients with colorectal liver metastases (CLM) undergoing hepatic resection has increased from 20–30% to 40–50% since early publications in the 1970s (1,2). This is likely due to improvements in surgical management (3,4) and the introduction of modern chemotherapy with a better understanding of patient selection (5). Fifty years ago, surgeons experienced discrepancy between clinicopathological features of tumor and long-term outcomes after resection. While aggressive unresectable recurrence was observed in several patients with small solitary metastasis, long-term survival was observed in a patient group presenting with multiple or large metastases. Although prognostic drivers were unknown, these observations opened for a new aggressive approach for advanced colorectal cancer. With the introduction of new effective chemotherapy, even more aggressive surgeries are performed in the responders (6). However, surgeons are still troubled with a black box which determines the fate of patients, regardless of a multidisciplinary approach (7).
The surgical repertoire for patients with CLM has become extensive. Unresectable disease can no longer be defined by absolute anatomical criteria (8,9). Liver growth through portal flow modulation methods allows safer resection in patients with insufficient future liver remnant (FLR) (10,11). Vascular reconstruction can expand surgical indication for a disease previously assessed as unresectable (12). Finally, liver transplantation has been performed in a super selected population with unresectable CLM with reported long-term outcome exceeding that of patients with resectable disease (13). In modern series of patients undergoing resection of CLM, roughly one third of the patients experience recurrence within 1 year (14). Another third achieves a “steady state” with prolonged disease-free survival between recurrences of resectable disease. Patients undergoing the 3th and 4th liver resection for recurring disease is not uncommon in modern series, and the survival after each hepatectomy is similar (6). Finally, the last third of patients achieves long-term survival.
Patient selection is still of major interest and a challenge among surgeons treating CLM. Selection to perform surgery is as important as selection not to perform surgery, and is part of the concept of personalized treatment (15). The timing of surgery is also important as a part of selection and considering the phenotype of the tumor biology. With the use of modern chemotherapy and targeted agents, conventional clinicopathological factors may be less important for the purpose of selection (16). Next generation patient selection is likely to incorporate, or be largely based on, molecular markers that may affect tumor biology. Here, we review clinical and genomic limitations regarding the implementation of current molecular markers for clinical decision making in management of patients with resectable CLM.
Clinical significance of RAS mutations in patients with resectable CLM and its limitations
Recently, mutations in the genes of the rat sarcoma viral oncogene homolog (RAS) subfamily have gained interest as molecular markers in CLM. The genes, in their wild-type form, acts as a molecular on/off switch in the epidermal growth factor receptor (EGFR) pathway, a mitogenic pathway, and predict response to anti-EGFR treatment. The literature also reports a prognostic role of RAS mutations and association between RAS mutational status and survival outcomes after resection of CLM (17). It has also been reported that RAS mutations may predict poorer response to modern preoperative chemotherapy, even in the cases without anti-EGFR antibodies (18).
One important limitation of these studies was the retrospective nature in which the patients were identified and included in analysis (19-21). The early publications reported data from 60 to 193 patients, representing merely 15% to 30% of the total respective institutional volumes (19-22). Known RAS mutation status was a necessity for inclusion, while RAS mutation testing was not performed routinely. Targeted anti-EGFR treatment is seldom used perioperatively, and testing may have been performed more often in patients assessed as borderline resectable or initially unresectable tumors. Patients achieving a resectable state after treatment with modern chemotherapy may have caused imbalance towards patients with worse biology in retrospective analyses of the impact of RAS mutations. Interestingly, in a number of publications, even the survival among RAS wild-type patients was reported inferior to that expected and reported in complete cohorts of resectable patients. As a consequence, it has been questioned whether RAS mutations are truly biomarkers for biology or byproducts of patient selection (23). However, in these retrospective studies, RAS mutations are still significantly associated with worse outcome compared to RAS wild-type. The important point is whether these observations in retrospective studies are generalizable for decision making in actual clinical management of patients with CLM according to RAS mutational status.
Prospective studies are now confirming the association between RAS mutations and poor outcome after resection of CLM, strengthening the evidence of RAS mutations as molecular biomarkers in all patients with resectable CLM (Brunsell TH et al. 2019, unpublished data and Berg KCG et al. 2019, unpublished data). RAS mutations may not hold the same prognostic value in unresectable metastatic colorectal cancer as in resectable CLM (24), but this remains somewhat controversial (25). One possible explanation for the observed difference between unresectable metastatic colorectal cancer and resectable CLM may be the impact of intercurrent prognostic factors in multivariable independency analysis. In unresectable patients, the presence of disease may be a strong driver of prognosis, rendering less powerful factors insignificant in analysis. This is likely similar for the classical clinicopathological factors as for RAS mutations.
It has been hypothesized that RAS-mutated metastases have a more aggressive growth pattern than RAS wild-type metastases (26). Investigators have explored this in the context of an unfree resection margin after resection, higher rates of local recurrence after ablation, and survival after reresection (27-29). All outcomes were found negatively associated with RAS-mutated metastases. Whether these findings can be explained by a more invasive and migratory tumor biology of the mutated metastases, or this finding could be a result of poorer response to preoperative chemotherapy or any other factor, remains uncertain. One study described a shorter median resection margin in RAS-mutated CLM irrespective of the size of the metastases and discussed theories of different types of tumor growth and microscopic tumor deposit (27). Another study found anatomic resection may be better than non-anatomic resection for RAS-mutated CLM to clear a larger margin (30). However, the molecular impact on growth pattern, micrometastases, and tumor microenvironment should be approached with caution in studies powered and designed for a clinical outcome. Furthermore, while RAS mutations may be associated with recurrence, the intrahepatic site of the recurrence is poorly described. Parenchymal sparing liver resection, when possible, is still recommended to allow for reresections (31,32).
There are subfamilies of the RAS gene. KRAS is the most common, typically detected in 20% to 50% of colorectal metastases upon resection. NRAS is less common and usually present in less than 5% (19). The subfamilies are regulated similarly, but their proteins likely to act at different locations within the cell. There are publications suggesting all RAS mutations should be assessed similarly (33) and have the same impact on outcome. However, there has been no solid conclusion because of the low frequency of these mutations. Furthermore, in colorectal cancer, mutations in the RAS genes most commonly occur in codons 12, 13, 61 and 146. Investigators have suggested different impacts on survival for the different codon mutations (34), but this finding could not be validated in another patient cohort and therefore remains uncertain (35).
Clinical significance of BRAF mutations in patients with resectable CLM and its limitations
BRAF mutations occur in 7% to 10% of metastatic colorectal cancer. In cohorts of patients with resectable metastatic disease, often less than 5% have BRAF mutations, which is likely a result of selection (36). Due to the low frequency of these mutations, it has been difficult to perform retrospective or prospective analysis to determine the impact on outcome, but BRAF mutations have been associated with poor survival after resection of CLM. BRAF-mutated tumors more often present with peritoneal metastases, and it could be that the biology of the disease renders the patients less likely candidates for liver resection (37). Other studies, however, found less extrahepatic disease in BRAF-mutated tumors (36).
While most previous studies have highlighted significantly worse survival of patients with BRAF mutations, emerging evidence has suggested that there seem to be several groups of patients presenting acceptable clinical outcomes even with BRAF-mutated status. Recently, a case-matched comparison was performed between BRAF wild and BRAF-mutated CLM using a large cohort from 24 centers. The researchers found that surgical treatment for resectable BRAF-mutated CLM was not associated with increased risk of recurrence compared with those with wild type BRAF, but BRAF mutation was associated with worse survival after development of recurrence (38). Another study has reported that while BRAF V600E mutations were associated with worse survival outcomes in general, patients with deficient mismatch repair tumor and/or resectable disease experienced a longer survival than expected (39). A large cohort study from China also confirmed that BRAF mutation was not associated with worse survival in stage I–III colorectal cancer, while it was an independent prognostic factor in stage IV colorectal cancer (40). As such, although BRAF mutation could be an independent prognostic factor, there has been no sufficient evidence for precluding surgery for BRAF-mutated CLMs, and aggressive multidisciplinary treatment approach remains a mainstay regardless of the mutational status in BRAF.
From single mutation to molecular footprints in resectable CLM
Despite extensive efforts to investigate molecular drivers that determine the fate of patients with CLM in actual clinical settings, no single molecular biomarker has been found to be suitable for excluding patients with CLM from liver resection. Because the overall picture of oncological molecular status seems to be complex and not well understood so far, it remains difficult to base clinical decisions entirely on this data in patients with CLM. Recently, several groups have reported that combination of mutational status in several molecular markers may better predict clinical outcomes of patients with CLM (41-45). Kawaguchi et al. reported the clinical impact of coexisting mutations in RAS, TP53, and SMAD4 and showed that RAS mutation status alone is not sufficient for precisely predicting prognosis after CLM resection (46).
In addition to these conventional viewpoints regarding the molecular status in tumor tissue, Nishioka et al. has recently reported that genetic background of the host could also be a new prognostic marker for CLM (47). In a comprehensive screening of 578 cancer-related genes, they found that MICA variant, an MHC class I chain-related gene family which is associated with innate immune response, shows significant correlation with response to chemotherapy and recurrence-free survival after CLM resection. Because somatic mutation is quite rare in the MICA gene and very high concordance rate was confirmed between the normal liver tissue and tumors, these results suggest that immune profiles in each host could be a new target of research in the field of biomarkers of CLM. However, we are still at the entrance of the vast genetic and molecular backgrounds of CLMs. Further studies are needed to define an optimal combination of genes to be screened for clinical management.
Spatiotemporal heterogeneity in patients with resectable CLM
It has been reported that clonal heterogeneity and genetic diversity exist within the same colorectal cancer sample (i.e., intratumoral heterogeneity) (48). Del Carmen et al. investigated mutational profiles in primary tumors, lymph nodes and liver metastases from 26 untreated metastatic colorectal cancer patients and confirmed the presence of different mutational profiles among primary tumors, lymph node metastases and liver metastases (49). These findings are clinically important because both intratumoral and intertumoral heterogeneity could affect the response to targeted therapies.
Routine clinical RAS testing is performed by Sanger sequencing or polymerase chain reaction. With such conventional methods, however, it is difficult to identify spatial heterogeneity of tumor (i.e., intratumoral or intertumoral heterogeneity) or genetic profile alterations that are usually caused by anticancer agents (temporal heterogeneity) during the clinical course. Therefore, more sophisticated, sensitive tests including liquid biopsy and a strategy to screening the refractory nature of tumors would be needed to better characterize tumor biology and alter clinical management as appropriate.
Impact of treatment on molecular profiles of cancer
With introduction of modern chemotherapy and multidisciplinary treatment, traditional clinicopathological factors are losing their weight in prediction of prognostic outcomes (16). For example, response to chemotherapy may be more important than size and number of CLM. Synchronous CLM responding to preoperative chemotherapy may prognostically be in a better place than metachronous CLM emerging after adjuvant chemotherapy for primary lesion. Also, RAS mutation is a potent prognostic factor as we discussed earlier.
Importantly, use of chemotherapy might provide a selection pressure. One study reported that the mutational status of KRAS may not be altered by treatment with oxaliplatin-based modern chemotherapy (50), while another study reported higher rates of somatic gene mutations in metachronous CLM after oxaliplatin based adjuvant treatment for the primary colorectal cancer (51). These results suggest that modern chemotherapy may prevent recurrence in KRAS wild-type patients, favoring chemotherapy-resistant KRAS-mutated subsets to form recurrence.
Similar concepts could be applied in the setting of preoperative chemotherapy. Given that complete removal of cancerous tissue may provide a chance of cure even for a patient with stage IV colorectal cancer, the goal of preoperative chemotherapy may not be cytoreduction of tumor. An important purpose of preoperative chemotherapy is, instead, improved selection of patients who will truly benefit from surgery. Single somatic mutations do not dictate the full phenotype of a tumor, thus preoperative observation under chemotherapy may offer important information on tumor biology and curative potential of patients, regardless of known biologic markers including RAS mutational status.
Increased knowledge about molecular biomarkers and understanding of tumor biology may explain differences between the expected and the observed outcome after resection of CLM. This information is important for surgeons as this may aid better patient selection and surgery. However, we are only in the early days of understanding how to use this information in clinical decision making.
Each newly proposed molecular biomarker should be validated in prospective patient series to reduce the risk of selection bias. Furthermore, when expanding the panel of molecular biomarkers available in resectable CLM, parallel research should be aimed to explain molecular mechanisms and how the data can be translated to clinically meaningful information (52).
Personalized medicine will continue to evolve, especially in the field of medical oncology, but also in surgical oncology. Molecular biomarkers are included in the overall assessment of patients before resection of CLM. So far, no biomarker has been found suited to exclude patients from surgery, and selection to surgery is still largely based on personal judgement by the surgeons. While it is important to approach new information about molecular biomarkers with caution, it is likely that in the future, genomic analysis will determine which patient is amenable to surgery or not, the timing of surgery versus other modalities, as well as how to approach the metastases technically.
Conflicts of Interest: The authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
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