Mutations in specific codons of the KRAS oncogene are associated with variable resistance to neoadjuvant chemoradiation therapy in patients with rectal adenocarcinoma

Abstract

Background

Mutations in KRAS and TP53 are common in colorectal carcinogenesis and are associated with resistance to therapy. Rectal cancers carrying both mutations are less likely to respond to neoadjuvant chemoradiation therapy (CRT) compared to wildtype tumors. Codon-specific KRAS mutations are associated with variable resistance to targeted therapies, but their association with rectal cancer response to CRT remains unclear. Our objective was to establish a correlation between specific KRAS mutations and rectal cancer response to CRT, and investigate if the correlation was related to a different association between KRAS and TP53 mutations.

Methods

One hundred forty-eight stage II–III rectal cancer patients underwent pre-operative CRT followed by surgery. DNA was extracted from pretreatment tumor biopsies and paired normal surgical tissues and KRAS and TP53 genotyping was performed. Specific KRAS mutations were then correlated with tumor response, and with concurrent TP53 mutation.

Results

Sixty patients had KRAS mutation; 12 in codon 13, and 48 in other locations. Eighty patients had TP53 mutation; 27 had concurrent KRAS/TP53 mutations. Tumors with any KRAS mutation were less likely to have a pCR compared to wildtype KRAS (p=0.006). Specifically, no tumors with KRAS codon 13 mutations had a pCR (p=0.03). Tumors with KRAS codon 13 mutations also had a higher incidence of concurrent TP53 mutation compared to tumors with other KRAS mutations (p=0.02).

Conclusions

Mutations in different KRAS codons may have different effects on rectal cancer resistance to CRT. This variable resistance may be related to a different frequency of TP53 mutations in KRAS mutant tumors.

Introduction

Mutation in the KRAS oncogene is a common event in colorectal carcinogenesis. [1] KRAS mutation causes constitutive activation of the KRAS protein and results in dysregulation of downstream processes and uncontrolled cell growth. [2] The presence of KRAS mutation has been associated with aggressive tumor behavior such as accelerated tumor growth, early formation of distant metastasis, and resistance to anti-epidermal growth factor receptor (EGFR)-based regimens in metastatic colorectal cancer patients. [3–5]

Most KRAS mutations (85%) in colorectal cancers occur in codon 12 or codon 13. [2, 3, 5] Other KRAS mutations occur more rarely and are found in only 1% to 3% of tumors. [4] Recent evidence suggests that not all KRAS mutations are alike, and that codon-specific KRAS mutations may affect different signaling pathways, resulting in variant tumor phenotypes. [6–9] Colorectal cancer patients with KRAS codon 13 mutations have different survival after cetuximab compared to other KRAS mutations, [10] and studies in colorectal and lung cancers have shown that patients with different codon 12 mutations have disparate prognosis. [11] Specifically, patients with KRAS G12V mutations have a worse prognosis than patients with KRAS G12D mutations, [2, 3, 11] suggesting that differences in single amino acids may dictate specific oncogenic characteristics of KRAS mutations.

We recently reported that rectal cancers with KRAS mutation are less likely to develop a pathologic complete response (pCR) to neoadjuvant chemoradiation (CRT) compared to tumors with wildtype KRAS. [12] Interestingly, we also found that none of the tumors with concurrent mutations in the TP53 tumor suppressor gene and KRAS develop a pCR after neoadjuvant CRT. In this study we aimed to establish a potential correlation between specific KRAS mutations and rectal cancer response to CRT, and to investigate if that correlation could be related to a different association between the KRAS and TP53 mutations.

Methods

Patients and treatment

This study included 148 patients with Stage II/III rectal cancer enrolled in a prospective multicenter clinical trial investigating the effect of increasing the CRT-to-surgery time interval, and adding chemotherapy during the waiting period (ClinicalTrials.org Identifier: NCT00335816). This trial was designed as a series of sequential Phase II trials or study groups (SGs) each with a progressively longer CRT-to-surgery time interval and increasing cycles of pre-operative chemotherapy. This study was approved by an Institutional Review Board (IRB) at each participating institution as well as a central IRB, and informed written consent was obtained from each patient prior to enrollment in the trial. Patients in the present study were pooled from SG1 (n=60), SG2 (n=67) and SG3 (n=21).

Patients in all SGs were treated with CRT; 5-Fluorouracil (FU) 225 mg/m²/day for 7 days in continuous infusion, and a total of 50.4Gy radiation. Patients in SG1 underwent TME an average of 6 weeks after completing CRT (standard of care). Following CRT, patients in SG2 and SG3 with signs of stable disease or disease progression compared with baseline staging had surgery without further delay. All other patients received 2 and 4 cycles of additional chemotherapy (modified FOLFOX-6), respectively; leucovorin 200 mg/m² or 400 mg/m² plus oxaliplatin 85 mg/m² by 2h infusion, followed by bolus of 5-FU 400 mg/m² and a 46h infusion of 5-FU 2,400 mg/m². Patients in SG2 and SG3 underwent TME an average of 11 and 16 weeks after completing CRT. Further details of this trial are presented elsewhere. [13]

Pathologic staging was performed according to American Joint Committee on Cancer (AJCC) guidelines. [14] Histological sections were stained and examined by two independent blinded pathologists. A pCR was defined as the absence of viable tumor in the entire resected surgical specimen and in the regional lymph nodes.

Sample preparation, DNA extraction, and mutation analysis

Tumor DNA from each patient was obtained from pretreatment biopsy tissue. Tumor cells were isolated using microdissection and genomic DNA was extracted as described previously. [12] Standard polymerase chain reaction (PCR) analysis was performed to detect specific mutations in KRAS (exons 2 and 3, which contain the most frequently reported KRAS mutations) and TP53 (exons 4–8) using established primers and PCR conditions. [12] Primer sequences are shown in Supplementary Table 1. KRAS and TP53 mutation status was confirmed by two independently-derived PCR products.

Statistical analysis

Patients were stratified by KRAS mutation status, and clinicopathologic characteristics and tumor response (pCR versus non-pCR) were compared between the two groups. Patients were then stratified by tumor response and univariate analysis was performed using the Fisher Exact Test to determine if specific KRAS mutations correlated with tumor response. No adjustments were made for multiple comparisons. Correlations were considered significant for two-sided p-values of <0.05. Finally, univariate analysis was performed using the Fisher Exact Test to determine if specific KRAS mutations correlated with TP53 mutations. A p-value of <0.05 was considered statistically significant.

Results

Patient demographics and tumor response

The clinicopathologic characteristics of the 148 patients are shown in Table 1. Eighty-five out of 148 (57%) patients were male and the average age was 57. Almost half of the patients (69 out of 148; 47%) had tumors located in the middle rectum. Initial clinical staging (cT and cN) showed that most patients were cT3 (135 of 148; 91%) and cN1 (106 out of 148; 72%). There was no statistically significant difference in pre-treatment clinical stage between SGs. Evaluation of surgical specimens after CRT revealed that 37 out of 148 (25%) patients had a pCR. There was no statistically significant difference in pCR rate between SGs.

Table 1.

Patient demographics

Factors	Total n=148	KRASwt1 n=88 (59%)	KRASmut2 n=60 (41%)	p-value
Age, years, median (range)	57 (25–87)	57 (32–85)	57 (25–87)	1.00
Gender				0.13
Male	85 (57%)	46 (52%)	39 (65%)
Female	63 (43%)	42 (48%)	21 (35%)
Pre-treatment T stage
cT2	11 (7%)	7 (8%)	4 (7%)	0.75
cT3	135 (91%)	79 (90%)	56 (93%)	0.47
cT4	2 (2)	2 (2%)	0 (0%)	0.32
Pre-treatment N stage				0.37
cN0	34 (23%)	18 (20%)	16 (27%)
cN+	114 (77%)	70 (80%)	44 (73%)
Tumor location3
Lower	46 (31%)	25 (28%)	21 (35%)	0.38
Middle	69 (47%)	42 (48%)	27 (45%)	0.70
Upper	13 (9%)	9 (10%)	4 (7%)	0.47
Tumor response				0.006
pCR4	37 (25%)	29 (33%)	8 (13%)
Non-pCR	111 (75%)	59 (67%)	52 (87%)

¹wt: wildtype

²mut: mutant.

³The tumor location for 20 patients was not recorded and was therefore not included in the analysis for tumor location.

⁴pCR: pathologic complete response.

Prevalence of KRAS mutation

The results of the KRAS mutation analysis for the 148 patients are shown in Table 2. Overall, 60 out of 148 (41%) patients had KRAS mutation; 40 of these 60 patients (67%) had mutations in codon 12, 12 (20%) in codon 13, 5 (8%) in codon 61, and 1 (2%) patient each in codons 6, 22 and 64. The most common mutation in KRAS codon 13 and the second most common KRAS mutation overall was G13D, detected in 11 out of 60 (18%) patients. One other patient carried a KRAS codon 13 mutation in G13C. The most common mutation identified was G12D in codon 12; it accounted for over half the KRAS mutations identified (31 out of 60; 52%). Other codon 12 mutations detected were: G12V in 7 (12%) patients, G12A in 1 (2%) patient and G12S in 1 (2%) patient. Mutations in the other codons were detected with a much lower prevalence in our patient population. Five out of 60 patients had mutations in codon 61; Q61L and Q61H, each detected in 2 (3%) patients and one patient had a 4 base-pair deletion, which spanned across codons 60 and 61. The remaining KRAS mutations were in codon 6 (L6F, one patient), codon 22 (6 base-pair insertion, one patient) and lastly, codon 64 (Y64H, one patient). The distribution of KRAS mutations among the three SGs was random, and the differences in the frequency of KRAS mutations and codon-specific mutations between SGs were not significant.

Table 2.

Prevalence of specific KRAS mutations

KRAS mutation	n=60
Codon 12	40 (67%)
G12D	31 (52%)
G12V	7 (12%)
G12A	1(2%)
G12S	1 (2%)
Codon 13	12 (20%)
G13D	11 (18%)
G13C	1 (2%)
Codon 61	5 (8%)
Q61L	2 (3%)
Q61H	2 (3%)
4 base-pair deletion	1 (2%)
Codon 6	1 (2%)
L6F
Codon 22	1 (2%)
6 base-pair insertion
Codon 64	1(2%)
Y64H

KRAS mutation and tumor response

The association of KRAS mutation with pCR is shown in Tables 1 and 3. Overall, tumors with any KRAS mutation were less likely to have a pCR compared to patients with wildtype KRAS; 8 (13%) versus 29 (33%) patients, respectively, p=0.006 (Table 1). Specifically, none of the tumors with KRAS mutation in codon 13 had a pCR (p=0.03) (Table 3).

Table 3.

KRAS mutations and tumor response

KRAS Status	n=148	pCR1	Non-pCR	p-value
Wildtype	88	29 (33%)	59 (67%)
Codon 13 mutation	12	0 (0%)	12 (100%)	0.03*
Other KRAS mutations2	48	8 (17%)	40 (83%)

^*KRAS codon 13 mutation correlates significantly with resistance to CRT compared to all other KRAS mutations.

¹pCR: pathologic complete response.

²Other KRAS mutations: Mutations in codons 12, 61, 6, 22, and 64 as listed in Table 2.

Correlation of KRAS with concurrent TP53 mutation

Eighty out of 148 (54%) patients had a mutation in TP53 and 27 patients had concurrent KRAS/TP53 mutations (Table 4). Patients with any mutation in KRAS had a lower prevalence of TP53 mutation compared to patients with wildtype KRAS, although this difference was not statistically significant (p=0.07). In patients with KRAS mutation, the presence of KRAS codon 13 mutation was strongly associated with concurrent TP53 mutation compared to all other KRAS mutations; 9 out of 12 (75%) patients with KRAS codon 13 mutations had concurrent TP53 mutations compared to 18 out of 48 (37%) patients with any other KRAS mutation (p=0.02).

Table 4.

Correlation of KRAS mutations with concurrent TP53 gene mutation

KRAS Status	Total n=148	TP53wt1 n=68	TP53mut2 n=80	p-value
Wildtype	88	35 (40%)	53 (60%)
Codon 13 mutation	12	3 (25%)	9 (75%)	0.02*
Other KRAS mutations3	48	30 (63%)	18 (37%)

^*KRAS codon 13 mutation correlates significantly TP53 mutation compared to other KRAS mutations.

¹wt: wildtype

²mut: mutant

³Other KRAS mutations: Mutations in codons 12, 61, 6, 22, and 64 as listed in Table 2.

Discussion

The current study confirms in a larger series our previous observation that rectal cancer patients with tumors carrying any KRAS mutation are less likely to have a pCR in response to CRT compared to patients with wildtype KRAS. In addition, we show that tumors with KRAS codon 13 mutation are particularly resistant to CRT as none of them achieve a pCR. We also found that while tumors with KRAS mutation tend to have lower frequency of TP53 mutation compared to tumors with wildtype KRAS, patients with specific KRAS codon 13 mutation have a higher frequency of concurrent TP53 mutation.

Several studies have demonstrated an association between KRAS mutation and lower tumor response and poor survival in patients with metastatic colorectal cancer treated with cetuximab-based regimens. [15–18] However, only one study has investigated the association of specific KRAS mutation with tumor response in patients with metastatic colorectal cancer treated with best supportive care with or without cetuximab. [10] This study found that patients with the KRAS G13D mutation treated with best supportive care (fluoropyrimidine, oxaliplatin or irinotecan) had worse overall survival compared to patients with other KRAS mutations or wildtype KRAS in univariate analysis but not in multivariate analysis. These data support our findings of less responsiveness to fluoropyrimidine-based CRT plus oxaliplatin chemotherapy in tumors carrying KRAS codon 13 mutation. However, the same KRAS codon 13 mutation was associated with improved response and survival compared to other KRAS mutations in chemotherapy-refractory metastatic colorectal cancer patients treated with cetuximab. [10] A more recent retrospective analysis also indicated that patients with metastatic colorectal cancer carrying the KRAS G13D mutation were less likely to respond to first-line chemotherapy and had a shorter progression-free and overall survival compared to patients with wild-type and other KRAS mutations. [19]

A number of studies have investigated the specific association of KRAS mutations and rectal cancer response to radiation with different results, but only one study has examined the association of specific KRAS mutations and rectal cancer response to CRT. [20] Gaedcke et al. screened 95 patients with rectal cancer treated with 5-fluorouracil and oxaliplatin-based CRT for mutations in codons 12, 13, and 61 of the KRAS gene and found no difference in response rate between tumors with KRAS mutations, combined or individually, and wildtype tumors. [20] The discrepancy with our results could be explained by the smaller sample size of their series and the use of a five-point tumor regression grade as a measure of response rather than pCR.

Persistent activation of KRAS affects numerous downstream pathways that play a role in cancer progression. [21] Several studies suggest that the specific location of KRAS mutation within the gene can affect different downstream pathways, which ultimately could affect tumor response to therapy. [8–10] Guerrero et al. examined differences between KRAS codon 12 and codon 13 mutations on downstream signaling in transfected NIH3T3 fibroblasts and noted similar KRAS activity and upregulation of the MAPK pathway, but differential activation of the AKT, c-Jun N-terminal kinase (JNK), and focal adhesion kinase (FAK) pathways. The functional consequences of differential downstream signaling manifested as altered apoptotic and mitotic rates. [8, 9] Divergent signal transduction between KRAS codon 12 and 13 mutations was also demonstrated in in vitro studies by De Roock et al. In the presence of cetuximab, SW48 colorectal cancer cells transfected with KRAS G12V were still able to signal through to extracellular signal-regulated kinase (ERK), whereas KRAS G13D transfected cells did not activate ERK signaling. [10] Other studies have reported an association between the activation or attenuation of several signaling pathways including AKT, JNK, and FAK, and resistance to several chemotherapeutic agents and radiation in multiple cancers. [22–24] Thus, utilization of alternative downstream pathways may provide an avenue of resistance to CRT in rectal cancer patients with different KRAS codon mutations.

Our current understanding of colorectal carcinogenesis suggests that colorectal cancer develops as a result of the accumulation of genetic alterations in oncogenes and tumor suppressor genes, and that it is the number rather than the specific mutations that is important for the development of the cancer. The most commonly mutated genes in colorectal cancer are APC, TP53, and KRAS, and while APC mutations are common in tumors with TP53 or KRAS mutations, the combination of all three mutations in the same tumor is exceedingly rare, suggesting that the combination of mutations in a specific tumor may not be random. [25] The mutual exclusivity of KRAS and BRAF mutations in the same tumor seems to be another example of the non-random distribution of mutations in colorectal cancer. [26] Our findings are consistent with these observations and suggest that a non-random combination of mutations may define subtypes of cancer with different biological behavior and even response to therapy.

There are limitations to our study that deserve mention. We had a relatively small sample size, which could account for the low frequency of codon 13 mutations. Additionally, the patient group was not completely homogeneous; there were three treatment arms in our study, and while all patients received pre-operative CRT, some patients also received additional chemotherapy. External validation of our findings will therefore be important to further substantiate our findings.

In conclusion, we demonstrate that rectal cancer patients with KRAS codon 13 mutations are resistant to neoadjuvant CRT and do not achieve a pCR. Furthermore, the presence of KRAS codon 13 mutation correlates with TP53 mutation. While it will be important to validate our findings in an independent large patient cohort, screening for these mutations may help to select effective treatments for patients carrying these mutations.

Synopsis.

Rectal cancer response to neoadjuvant chemoradiation therapy may be affected by specific KRAS mutations. We show that rectal cancers harboring KRAS codon 13 mutations are resistant to neoadjuvant chemoradiation therapy and do not develop a pathologic complete response.