Gene mutation and surgical technique: suggestion or more?

Abstract

Advancements in chemotherapy and molecular targeted therapy have improved long-term outcomes for patients with resectable colorectal liver metastases (CLM). RAS mutation status was an original focus as a molecular biomarker as it predicted treatments response to anti-epidermal growth factor receptor agents. More recently, studies have incorporated somatic mutation data in analyses pertaining to surgical outcomes and prognosis. This evidenced-based review covers the implications of somatic mutations in patients undergoing resection of CLM.

1. Introduction

Colorectal liver metastases (CLM) are found in approximately 15–30% of patients with colorectal cancer [1]. Liver resection has a survival benefit over chemotherapy alone and provides 5-year overall survival (OS) rates that range from 40% to 58% [2–4]. Modern chemotherapy and molecular targeted therapy can downsize CLM and have increased the number of patients eligible for curative resection [5]. Indeed, chemotherapy regimens that include anti-epidermal growth factor receptor (EGFR) agents have improved long-term outcomes in patients with unresectable metastases from colorectal cancer [6]. However, it was quickly noted that patients with mutations in the RAS gene family (KRAS, NRAS, and HRAS) exhibited lack of response to anti-EGRF therapy [7–9]. Subsequent studies found association between mutations in RAS and BRAF and worse prognosis after CLM resection. With the recent development of next generation sequencing, testing of multiple somatic mutations can occur in the context of clinical practice. This article reviews the association of somatic gene mutations with prognosis and surgical outcomes after CLM resection to facilitate better clinical decision-making.

2. Common somatic gene mutation and prognosis

2.1. RAS mutation

The RAS oncogene (KRAS, NRAS, and HRAS) is a key member of the mitogen-activated protein kinase (MAPK) pathway and contributes to deregulation of tumor-cell growth, programmed cell death and invasiveness, and induction of new blood-vessel formation [10]. An important clinical implication of the RAS mutation is resistance to anti-EGFR therapy [11]. EGFR belongs to a family of receptor tyrosine kinases that includes three other members (erbB2/HER-2, erbB3/HER-3, and erB4/HER-4) [12, 13]. The binding of epidermal growth factor or other ligands to EGFR initiates a mitogenic signaling cascade through the MAPK signaling pathway and the phosphatidylinositol 3-kinase (PI3K) signaling pathway [11]. Mutations in RAS result in continuous activation of the downstream MAPK signaling pathway, even if the EGFR is pharmacologically blocked [7, 14].

Recently, studies have reported an association of RAS mutation with prognosis in patients undergoing CLM resection (Table 1). Based on these series, anywhere from 15 to 50% of patients have a RAS mutation. Many studies report that RAS mutant patients have shorter OS and recurrence-free survival (RFS) than RAS wild-type patients. However, the prognostic impact of RAS mutation is inconsistent across the literature. Recently, our group demonstrated that a “triple mutation” in RAS, TP53, and SMAD4 was independently associated with worse OS and RFS in 507 patients undergoing CLM resection [15]. The study showed that a subset of patients with only a RAS mutation has similar long-term outcomes as RAS wild-type patients. OS and RFS in patients with RAS mutation and wild-type TP53 and SMAD4 were not significantly different from OS and RFS in patients with RAS wild-type. For example, the median OS for patients with RAS mutation and wild-type TP53 and SMAD4 was 7.3 years compared to 7.0 years for RAS wild-type patients (P = 0.858). This finding may explain the inconsistency in terms of long-term outcomes in patients with RAS mutation, and suggests that information regarding RAS mutation alone is perhaps insufficient.

Table 1.

Studies of RAS mutation in patients who underwent resection of colorectal liver metastases^*

Reference	Year	Gene analyzed	No. of patients	Frequency	Association of RAS (KRAS) mutation with prognosis
					OS	RFS
Nash et al. [65]	2010	KRAS	188	51 (27%)	Worse	-
Teng et al. [24]	2012	KRAS	292	111 (38%)	No association	-
Vauthey et al. [26]	2013	RAS	193	34 (17.6%)	Worse	Worse
Karagkounis et al.[66]	2013	KRAS	202	58 (29%)	Worse	Worse
	2014	KRAS	154	43 (28%)	No association	-
Lin et al. [67]	2015	RAS	309	160 (52%)	No association	No association
Scirripa et al.[22]	2016	KRAS	512	190 (37%)	-	No association
Margonis et al. [68]	2016	RAS	633	229 (36%)	Worse	-
Brudvik et al. [50]	2017	RAS	342	19 (44%)	Worse	Worse
Amikura et al. [69]	2017	KRAS	300	110 (37%)	Worse	-
Wang et al. [70]	2019	RAS	507	257 (51%)	Worse	Worse
Kawaguchi et al. [15]

Abbreviations: OS, overall survival; RFS, recurrence-free survival.

^*More than 150 patients and a Cox proportional hazards model analysis

2.2. BRAF mutation

Similar to RAS, the BRAF oncogene is an important member of the MAPK pathway [16] and a mutation in BRAF results in continuous activation of the downstream MAPK signaling pathway [17]. BRAF is mutated in approximately 10% of all patients with colorectal cancer [18]. The prognostic role of a BRAF mutation in patients with colorectal cancer is well established and associated with poor survival outcomes [19, 20]. Based on surgical series, BRAF is mutated in only 1.0-6.1% of patients undergoing CLM resection, likely given its associated poor prognosis [21–26]. Similar to patients with colorectal cancer, BRAF mutant patients undergoing CLM resection have been shown to have shorter survival than BRAF wild-type patients [21–25, 27]. It should be noted that the single institution studies have been able to analyze anywhere from three to twelve patients with BRAF mutations because of the low mutation frequency in this patient cohort [22–25]. Recently, two multi-institutional studies analyzed 35 BRAF mutant patients out of 1497 total patients [27] and 45 BRAF mutant patients of 853 patients [21] (Table 2). Both studies showed that BRAF mutant patients had significantly worse OS and RFS than BRAF wild-type patients [21, 27].

Table 2.

Studies of BRAF mutation in patients who underwent resection of colorectal liver metastases^*

Reference	Year	No. of patients	Frequency	Multivariable analysis	Association of BRAF mutation with prognosis
					OS	RFS
Gagniere et al.[27]	2018	1497	35 (2%)	No	Worse	Worse
Margonis et al.[21]	2018	853	43 (5%)	Yes	Worse†	Worse†

Abbreviations: OS, overall survival; RFS, recurrence-free survival.

^*More than 20 patients.

^†V600E BRAF vs. wild-type BRAF

Of all the BRAF mutations, 80% were V600E (1799T>A) [28]. For patients with unresectable colorectal cancer, two multi-institution studies showed that the non-V600E BRAF mutation is a distinct molecular subset compared to the V600E BRAF mutation [29,30]. Patients with a V600E BRAF mutation had a worse prognosis; however, patients with non-V600E BRAF mutations had a similar survival to patients with BRAF wild-type [29, 30]. The rarity of BRAF mutation (all, 10%; V600E, 8%; non-V600E, 2%) is a barrier to ensuring statistical power and avoiding the type II error in clinical studies. To detect a difference of 5% between BRAF wild-type and non-V600E BRAF mutation in patients with colorectal metastases, more than 18,000 events may be needed based on the sample size analysis reported by Lakatos [31] using the following parameters (alpha, 0.05; beta, 0.20; non-V600E BRAF mutation, 2%; 5-year OS in BRAF wild-type patients, 30%; non-V600E BRAF mutant patients, 25%) [30].

2.3. TP53 mutation

TP53 is a tumor suppressor gene in the p53 pathway and encodes p53 protein. Malignancy-associated stress signals activate p53, which inhibits tumor-cell growth either through cell-cycle arrest or induction of apoptosis [32, 33]. TP53 is mutated in approximately 40–70% of patients undergoing CLM resection (Table 3). Currently, the literature remains divided as to the prognostic role of TP53 in patients undergoing CLM resection. In 1999, Tullo et al. reported that patients with a TP53 mutation had shorter RFS than TP53 wild-type patients [34]. In contrast, in 2000, Yang et al. reported that OS and RFS were better in TP53 mutant patients than in TP53 wild-type patients [35]. Subsequently, four studies have shown worse OS and/or RFS in TP53 mutant patients compared to TP53 wild-type patients [15, 36–38], whereas, in five studies, TP53 mutation was not significantly associated with prognosis [25, 39–42].

Table 3.

Studies of other somatic gene mutations in patients who underwent resection of colorectal liver metastases

Gene analyzed	Reference	Year	No. of patients	Frequency	Multivariable analysis	Association of gene mutation with prognosis
						OS	RFS
TP53	Tullo et al. [34]	1999	40	19 (48%)	No	-	Worse
	Yang et al. [35]	2001	39	16 (41%)	No	Better	Better
	Saw et al. [39]	2002	60	35 (58%)	No	No association	-
	De Jong et al[40]	2005	44	16 (36%)	No	No association	No association
	Mollevi et al. [36]	2007	91	46 (51%)	Yes	Worse	-
	Pilat et al. [38]	2015	76	42 (55%)	No	Worse*	-
	Loes et al. [25]	2016	164	99 (60%)	Yes	No association	No association
	Fankel et al. [41]	2017	165	95 (58%)	No	No association	-
	Chun et al. [42]	2019	401	263 (66%)	No	No association	-
	Kawaguchi et al. [15]	2019	507	359 (71%)	Yes	Worse	Worse
PIK3CA	Loes et al. [25]	2016	164	22 (13%)	Yes	No association	No association
	Fankel et al. [41]	2017	165	20 (12%)	No	No association	No association
	Kawaguchi et al. [15]	2019	507	80 (16%)	Yes	No association	No association
SMAD4	Mizuno et al. [47]	2018	278	37 (13%)	Yes	Worse	-
	Kawaguchi et al. [15]	2019	507	56 (11%)	Yes	Worse	Worse

Abbreviations: OS, overall survival; RFS, recurrence-free survival.

^*51 patients who received preoperative chemotherapy

2.4. PIK3CA mutation and prognosis

The PIK3CA gene encodes a catalytic subunit of class IA PI3Ks [43]. PI3Ks activate serine/threonine-protein kinases and other downstream effector pathways. Serine/threonine-protein kinases activate the mammalian target of rapamycin. Through these activation processes, the PI3K signaling pathway play a key regulatory role in cell survival, proliferation, angiogenesis and differentiation [43, 44]. PIK3CA is mutated in approximately 12–13% of CLM patients (Table 3). However, the prognostic role of PIK3CA mutation is not well described. Two studies have reported that OS and RFS did not significantly differ between PIK3CA mutant and PIK3CA wild-type patients. The previously mentioned study of 507 patients from our institution also showed that PIK3CA mutation status was not associated with OS or RFS [15].

2.5. SMAD4 mutation and prognosis

SMAD4 is a tumor suppressor gene in the transforming growth factor-β pathway, involved in the regulation of cell proliferation, differentiation, migration, and apoptosis [45, 46]. SMAD4 is mutated in approximately 10% of patients undergoing CLM resection [42, 47]. Two studies have shown a negative prognostic role of SMAD4 in patients undergoing CLM resection with worse OS and/or RFS than SMAD4 wild-type patients (Table 3) [15, 47].

3. Association of RAS mutation with surgical outcomes

RAS mutation status has been widely tested because of its clinical relevance in regards to the use of anti-EGFR therapy. As such, studies have also reported the associations of RAS mutation with various surgical outcomes. The following section summarizes the implication of RAS mutation status in regards to surgical margin, ablation margin, repeat hepatectomy, and two-stage hepatectomy.

3.1. Resection margin

A positive resection margin is associated with worse prognosis in the era of modern preoperative chemotherapy [48]. Two studies have investigated the impact of RAS mutation status on surgical margin [49, 50]. Brudvik et al. reported that independent predictors for positive resection margin were RAS mutation (hazard ratio [HR] 2.44, 95 % confidence interval [CI] 1.30–4.58, P = 0.005) and carcinoembryonic antigen level ≥ 4.5 ng/mL (HR 95 % CI 1.09–3.89, P = 0.026) [50]. In patients who developed liver-first recurrence, the median width of the resection margin was significantly smaller in RAS mutant patients than in RAS wild-type patients: 4 mm (0–70 mm) vs. 7 mm (0–67 mm), P = 0.031. Margonis et al. also demonstrated a difference in the effect of surgical margin on surgical outcomes between KRAS mutant and KRAS wild-type patients [49]. For KRAS wild-type patients, a resection margin ≥ 1 mm was associated with better OS than a resection margin < 1 mm. In contrast, for KRAS mutant patients, OS did not differ significantly between a resection margin < 1 mm and ≥ 1 mm. These studies show that the prognostic effect of surgical margin may differ between patients with RAS mutation and those who are RAS wild-type.

3.2. Ablation margin

Three studies have described an association between RAS mutation and ablation margin. Odisio et al. showed that local tumor progression-free survival after percutaneous ablation for CLM was worse in patients with RAS mutation (35% at 3 years) than in those who were RAS wild-type (71% at 3 years) (P < 0.001). Of 25 ablated CLMs with local tumor progression, patients with RAS mutation had earlier progression than patients with RAS wild-type. In a series of 218 ablated CLMs, Calandri et al. showed that RAS mutation status and ablation margin ≤ 10 mm were associated with local tumor progression-free survival: RAS mutation, HR 2.85, 95% CI 1.74–4.69, P < 0.001; ablation margin ≤ 10mm, HR 1.80, 95% CI 1.11–2.89, P = 0.017. Finally, Jian et al. analyzed 154 ablated CLM and also showed that KRAS mutation status and ablation margin were associated with local tumor progression [51].

3.3. Repeat hepatectomy

Liver resection has been regarded as a gold standard for patients with colorectal liver metastases. However, most patients experience recurrence [2–4, 52]. Studies have shown that repeat hepatectomy for recurrence of liver metastases can improve survival in selected patients [53, 54]. Denbo et al. reported that the median RFS after repeat hepatectomy for recurrent CLM is lower in RAS mutant patients than in RAS wild-type patients: 6.1 months vs. 12.2 months, P = 0.03. RAS mutation was an independent risk factor for both OS and RFS in patients who underwent repeat hepatectomy for recurrent CLM (OS: HR, 1.69, 95% CI, 1.03–2.72, P = 0.04; RFS: HR, 2.11, 95% CI, 1.11–3.98, P = 0.02). This study suggests that RAS mutation status can be used for decision-making regarding the use of repeat resection or medical therapy in patients who experience recurrence after initial CLM resection.

3.4. Two-stage hepatectomy

Two-stage hepatectomy for bilateral CLMs was described in the early 2000s as a technique for improving resectability [55, 56] because patients with bilateral CLM were often excluded from curative intent resection due to an insufficient future liver remnant [57]. Passot et al. showed the importance of RAS mutation status in regards to patient selection for two-stage hepatectomy [58]. In this series, the 5-year OS rate was 67% in patients with RAS wild-type, compared to only 12% in patients with a RAS mutation.

3.5. Repeat surgery for recurrence after two-stage hepatectomy

Recurrence after two-stage hepatectomy is frequent because this strategy is generally used for patients with multiple bilateral CLMs [59, 60]. A recent study by Lillemoe et al. assessed the feasibility and safety of repeat surgical resection for recurrence after two-stage hepatectomy for CLM [61]. In 83 patients who developed recurrence after two-stage hepatectomy, 31 patients (37%) underwent resection for recurrence. RAS mutation and first recurrence in multiple sites were associated with worse survival. Specifically, RAS mutant patients undergoing repeat surgery for recurrence had shorter OS than RAS wild-type patients undergoing repeat surgery (5-year OS: 38% vs. 86%, P = 0.019). In contrast, for patients who did not undergo resection for recurrence after two-stage hepatectomy, OS did not differ significantly between RAS mutant patients and RAS wild-type patients (P = 0.517) [61]. Thus, RAS mutation status remains an important prognostic factor in advanced disease and should be considered when determining treatment.

3.6. Synchronous liver and lung metastases

Lung metastases are the most frequent type of extrahepatic metastasis of colorectal cancer [62]. As such, for patients with both lung and liver metastases, clarifying the impact of the lung metastases is key to maximize the benefit of CLM resection. Patients with a RAS mutation have a higher propensity for developing lung metastases than patients with RAS wild-type [26, 63]. Mise et al. demonstrated that in patients undergoing resection of CLM without resection of lung metastases, KRAS mutation (HR, 2.10, 95% CI, 1.21–3.64, P < 0.001) and rectal primary tumor (HR, 1.72, 95% CI, 1.02–3.64, P = 0.039) were associated with worse OS [64]. The authors showed that the 3-year OS rate for patients with no risk factors (KRAS wild-type and colon primary tumor) was 76.9%, compared to 36.7% for patients with one risk factor and 13.5% for patients with two risk factors.

4. Conclusions

Mutations in the RAS oncogene family were the original focus of genetic sequencing in patients with CLM due to the clinical relevance in regards to anti-EGFR therapy resistance. Recently, the association of RAS mutation with prognosis after CLM resection has been increasingly reported, with most studies reporting substantially shorter OS in RAS mutant patients compared to RAS wild-type patients. RAS mutation status has also been evaluated in the context of other parameters related to CLM resection. Studies have found associations with RAS mutations and surgical margin, ablation margin, and long-term outcomes after repeat hepatectomy and two-stage hepatectomy. Similar to patients with primary colorectal cancer, mutations in BRAF are also associated with a poor prognosis. However, it should be noted that BRAF mutations are rare in this patient population (at most 5% of patients undergoing CLM resection), making it difficult to evaluate its prognostic importance. Finally, TP53, APC, PIK3CA, and SMAD4 are commonly mutated in patients undergoing CLM resection. As genetic sequencing becomes more accessible, more data will arise regarding the prognostic implication of these mutations. Continued advancements in the realm of tumor biology based on next generation sequencing will further improve outcomes and clinical decision making for patients with CLM.

Table 4.

RAS mutation and surgical outcomes in patients undergoing resection of colorectal liver metastases

Reference	Year	Gene analyzed	No. of patients	Frequency	Findings
Surgical margin
Brudvik et al. [50]	2016	RAS	633	229 (36%)	RAS mutation is associated with positive and closer surgical margin.
Margonis et al. [49]	2016	KRAS	411	153 (37%)	OS in RAS mutant patients was similar between R0 and R1 resections.
Ablation margin
Odisio et al. [71]	2017	RAS	92	36 (39%)	LTPFS after ablation was worse in RAS mutant patients.
Calandri et al. [72]	2018	RAS	136	54 (40%)	RAS and margin > 10 mm are predictors for LTPFS.
Jian et al.[51]	2019	KRAS	76	38 (50%)	KRAS and margin are predictors for LTPFS
Repeat hepatectomy
Denbo et al.[73]	2017	RAS	98	34 (35%)	RAS mutation was associated with worse OS and RFS after repeat hepatectomy
Two-stage hepatectomy
Passot et al. [58]	2016	RAS	93	40 (43%)	RAS mutation was associated with worse OS and RFS in patients undergoing two-stage hepatectomy.
Repeat surgery for recurrence after two-stage hepatectomy
Lillemoe et al. [61]	2018	RAS	83	36 (46%)	RAS mutation was associated with worse OS in patients undergoing resection after two-stage hepatectomy.
Synchronous liver and lung metastases
Mise et al. [64]	2015	KRAS	98	44 (45%)	KRAS mutation was associated with worse OS in patients undergoing CLM resection without resection of synchronous lung metastases.

Abbreviations: OS, overall survival; LTPFS, local tumor progression-free survival; RFS, recurrence-free survival; CLM, colorectal liver metastases.

ABBREVIATIONS

CLM

colorectal liver metastases

OS

overall survival

EGFR

epidermal growth factor receptor

MAPK

mitogen-activated protein kinase

PI3K

phosphatidylinositol 3-kinase

RFS

recurrence-free survival

HR

hazard ratio

CI

confidence interval