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Asia-Pacific Journal of Clinical Oncology 2015; 11: 160–169
doi: 10.1111/ajco.12342
ORIGINAL ARTICLE
Elucidating the prognostic significance of KRAS, NRAS, BRAF and PIK3CA mutations in Chinese patients with metastatic colorectal cancer Brigette B MA,1 Frankie MO,1 Joanna H TONG,2 Ashley WONG,1 SC Cesar WONG,1,3 Wing M HO,1 Cherry WU,4 Polly WY LAM,5 KF CHAN,6 Timothy SK CHAN,7 Wilson MS TSUI,8 Alex KH TSANG,9 Mandy NS FUNG,10 Anthony TC CHAN1 and Ka Fai TO2 1
State Key Laboratory in Oncology in South China, Sir YK Pao Centre for Cancer, Department of Clinical Oncology, Hong Kong Cancer Institute and the Chinese University of Hong Kong, 2Department of Anatomical and Cellular Pathology, Chinese University of Hong Kong, 3Department of Health Technology and Informatics, Hong Kong Polytechnic University, 4 Department of Pathology, Alice Ho Miu Ling Nethersole Hospital, 5Department of Pathology, Queen Elizabeth Hospital, 6 Department of Pathology, Tuen Mun Hospital, 7Department of Pathology, Kwong Wah Hospital, 8Department of Pathology, Caritas Medical Centre, 9Department of Pathology, Yan Chai Hospital, 10Department of Pathology, United Christian Hospital, Hong Kong
Abstract Aim: The prognostic significance of KRAS, NRAS, PIK3CA and BRAF mutations was evaluated in Chinese patients with metastatic colorectal cancer (CRC). Method: Tumor samples from 183 patients were retrospectively tested for KRAS, NRAS, PIK3CA and BRAF mutations. Multivariate analysis was performed to determine the relationship between mutational status, drug response and survival. Result: Over 70% of patients received two or more lines of chemotherapy, 50% had cetuximab and 18% had bevacizumab. The prevalence of KRAS, NRAS, BRAF and PIK3CA mutations was 45, 3.2, 5 and 20%, respectively. For the entire cohort, the median overall survival was 24 months (95% confidence interval [CI] = 20.4–26.4 months). Of the genes tested, only KRAS mutation was an independent prognostic factor with a multivariate hazard ratio of 1.5 (95% CI = 1.05–2.16, P = 0.03). In the subgroup of patients who received cetuximab-based therapy in the first-line setting, KRAS mutation was associated with a lack of response to chemotherapy (28% vs 66%, chi-square, P = 0.01). Patients with KRAS mutant tumors (or KRAS wild-type tumors that harbored BRAF and/or PIK3CA mutations) tended to have lower response rates to chemotherapy and/or cetuximab (P = not significant). The number of NRAS mutant cases was too small to allow any statistical analysis. Conclusion: The prevalence of KRAS, NRAS, BRAF and PIK3CA mutations in this cohort is consistent with reports from non-Asian populations, and KRAS mutation has both prognostic and predictive significance in Chinese patients with metastatic CRC. Key words: BRAF, Chinese, KRAS, NRAS, PIK3CA mutations.
Correspondence: Professor Brigette B Ma MD, Department of Clinical Oncology, Basement, LKS Specialist Clinic, Prince of Wales Hospital, Shatin, New Territories, Hong Kong. Email: [email protected] Conflict of interest: none Accepted for publication 11 January 2015.
© 2015 Wiley Publishing Asia Pty Ltd
INTRODUCTION The incidence of colorectal cancer (CRC) is rising in urbanized regions of Asia such as Hong Kong, where CRC is the second most prevalent cancer.1 In Hong Kong, over 50% of patients with CRC present with
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stage III–IV disease where systemic therapy plays an important role in management.1 The use of antibodies against the epidermal growth factor receptor (EGFR) has improved the survival of patients with metastatic CRC (mCRC),2–7 and KRAS mutation status is now routinely used in practice to select patients for this therapy. It is well recognized that the presence of KRAS mutations found in exon 2 (codon 12/13) is associated with resistance to EGFR antibodies, but these mutations probably account for less than 40% of nonresponders to EGFR antibodies.8 Rarer mutations such as NRAS mutations in exon 2 (codon 12/13) have also been shown in recent prospective phase III trials to predict poor response to EGFR antibodies,9–11 while the clinical significance of other uncommon KRAS mutations at exons 3 and 4 has recently come to light. In the PRIME study, patients with mutation of KRAS (exon 2, 3 or 4) or NRAS (exon 2, 3 or 4) did not benefit from the addition of panitumumab to chemotherapy.5 Furthermore, activating mutations of other oncogenic pathways that are located downstream to the EGFR, such as the BRAF and PIK3CA mutations, have been implicated in mediating resistance to EGFR antibodies in CRC.12–15 Population-based differences in response to anticancer therapy and in the expression of predictive biomarkers have been well described for CRC and other cancers.16 One of the best known examples is the higher prevalence of activating mutation of the EGFR tyrosine kinase domain in Northeast Asians with lung adenocarcinoma than other ethnic groups.16 In CRC, it is unclear whether ethnic variation exists in the prevalence and clinical significance of KRAS, NRAS, BRAF and PIK3CA expression. In this study, the prognostic and predictive significance of these mutations in Chinese patients who received chemotherapy with or without cetuximab for mCRC was evaluated retrospectively. The primary objective of this study was to investigate the relationship between mutational status and survival and drug response, and the secondary objective was to investigate the relationship between mutational status and treatment outcome in a subgroup of patients who received cetuximab-based therapy.
METHOD
tumors available for mutational analysis. Some of the patients selected for this study were treated before KRAS mutation testing was made mandatory at our institution before starting cetuximab-based treatment in the late 2009. Patients were treated according to departmental protocol or in consenting subjects, as part of a clinical trial protocol. For patients who were treated outside a clinical trial, the types of chemotherapeutic regimens used are outlined in Supporting Information Table S1. Response to treatment was assessed at regular intervals with computed tomography scans (sometimes chest X-rays, ultrasound or positron emission tomography (PFT) scans) of target lesions, as well as monitoring of serum carcinoembryonic antigen level. Antibodies such as cetuximab and bevacizumab are available as self-financed items (without government reimbursement) in patients who receive treatment in public hospitals in Hong Kong; therefore these items were not available to all patients in this cohort. This study was approved by the New Territories East Cluster-Chinese University of Hong Kong Ethics committee.
Study endpoints and statistical analysis The relationship between expression of KRAS, NRAS, BRAF and PIK3CA mutations and clinical outcome (i.e. overall survival [OS] and response to chemotherapy) was studied. The chi-square test was used to evaluate any correlation between mutational status and response to treatment. Survival curves were computed using the Kaplan–Meier method and compared using the logrank test. The influence of cofactors (such as age, sex, number of lines of chemotherapy received, use of bevacizumab, the number of sites of metastases) on response and survival was analyzed using the Cox proportional hazard model. OS was defined as the period from the date of initial diagnosis of metastatic disease to the date of death from any cause or the date of last follow-up in the outpatient clinics. Response to chemotherapy was defined based on the RECIST (Response Evaluation Criteria in Solid Tumors) criteria, objective response is defined as the sum of partial response (PR) and complete response (CR). Survival and response were determined for the entire cohort and for a subgroup of patients who received cetuximab.
Patient selection and management The medical records of 244 patients with mCRC who attended the outpatient clinics with the intent of undergoing chemotherapy at the Prince of Wales Hospital from 2008 to 2012 were identified retrospectively. Of these patients, 183 had archived, paraffin-embedded
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Mutational analysis The presence of mutations in KRAS (codon 12/13 at exon 2; codon 59/61 at exon 3, codon 117/146 at exon 4), NRAS (codon 12/13 at exon 2, codon 59/61 at exon 3, codon 117/146 at exon 4), PIK3CA (exons 9 and 20)
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and BRAF (V600E) in these samples was evaluated with PCR-direct sequencing in this order of priority given the limited amount of archived tissues available. Tumor cells were isolated from paraffin sections by manual microdissection. Genomic DNA was extracted for the PCR amplification. The PCR products were purified and sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA) and analyzed by the Life Technologies New York, Hong Kong 3500 Genetic Analyzer. Positive and negative controls were included. The data were collected and analyzed using Applied Biosystems sequencing analysis software. The DNA sequence was compared with consensus coding sequence of KRAS, NRAS, PIK3CA and BRAF from the Consensus Coding Sequence (CCDS).
RESULT Patient characteristics and treatment outcome There were 183 Chinese patients who had archived tumor samples available for mutational testing. The median age was 58 years (range 24–83 years) and there were 79 females and 104 males. The location of the primary tumor was colon in 78% and “rectum or others” in 22% of patients, and 26% of patients had metastases confined to the liver. A total of 174 (95%) out of 183 patients received treatment with any drug therapy (chemotherapy and/or targeted therapy) for mCRC, and the percentages of patients who had 1, 2 and ≥3 line(s) of chemotherapy for mCRC were 21, 42 and 32%, respectively. Of these patients, 50% (91) received cetuximab in any line of therapy either as monotherapy or in combination with chemotherapy for mCRC, and 18% had bevacizumab. For the cetuximabtreated subgroup, the commonest chemotherapeutic agent(s) which were coadministered with cetuximab were oxaliplatin-based regimen in 68% of patients treated in the first-line setting, and irinotecan-based regimen in over 80% of patients treated in subsequent lines (Supporting Information Table S1). None of those cetuximab-treated patients ever received bevacizumab for mCRC. At the time of data analysis, the median OS for the entire cohort is 24 months (95% confidence interval [CI] = 20.4–26.4 months), 68% of patients have died and 32% were alive with cancer. For the subgroup of cetuximab-treated patients, the overall response rates (PR and CR) to cetuximab-based therapy in the first-, second- and third-line settings were 56, 37 and 15%,
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respectively (Table 1). For the subgroup of patients who never received any cetuximab and had archived tumors available for mutational testing, the overall response rates to chemotherapy in the first-, second- and thirdline settings were 25% (21 of 84 patients in total), 11% (6 of 52 patients) and 0% (10 patients), respectively.
Result of mutational analyses For KRAS mutations, 55% of the 183 tumors were wild type and 45% were mutant (Fig. 1a). For the cetuximabtreated subgroup of 91 patients, 72% were wild type and 28% were mutant (Fig. 1b). The commonest subtype of KRAS mutations was located in codon 12 such as G12D (36%), G12V (22%), G12S (7%), G12C (5%) and G12A (4%), followed by codon 13 mutation such as G13D (16%), and then codon 61 such as Q61H (5%). Other rarer subtypes of KRAS mutations included those located at exon 4 such as A146T or A146V (5%), and a case of exon 4 mutation at codon 117.17–19 NRAS mutations were identified in six patients (3%), which were located in codon 12 (G12D, G12A, G12V), codon 13 (G13R) and codon 61 (Q61L). For the entire cohort, BRAF mutations detected in nine patients (5%) were found to have the commonest V600E mutations. The tissues of 64 patients were available for analysis of PIK3CA mutation, and 13 (20%) samples were found to be mutant. For the cetuximabtreated subgroup, BRAF mutation and PIK3CA mutations were found in 3 and 10% of patients, respectively. Only two patients with NRAS mutation-expressing tumors received cetuximab. In the KRAS wild-type subgroup of patients, six had BRAF mutations alone, six had PIK3CA mutation alone, and one had both BRAF and PIK3CA mutations.
Prognostic significance of KRAS, BRAF and PIK3CA mutations in the entire cohort The relationship between specific mutations and clinical outcome in terms of survival and response to chemotherapy was investigated in 183 patients with archived tumors. The presence of KRAS mutation was associated with inferior OS with a hazard ratio (HR) of 1.55 (95% CI = 1.08–2.23, P < 0.02) (Fig. 2). Multivariate analysis showed that both KRAS mutation and previous use of bevacizumab were independent prognostic factors with the respective HRs of 1.5 (95% CI = 1.05–2.16, P = 0.03) and 0.51 (95% CI = 0.3–0.87, P = 0.01). No correlation with response to chemotherapy was observed. The nine patients with the rarer KRAS mutations (exons 3, 4) had multiple sites of metastases and did not respond (PR or CR) to drug therapy. Thirteen
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Table 1
Relationship between KRAS mutational status and best response to cetuximab-treated 91 patients
Best radiological response (a) First-line setting (n = 25) Partial response Stable disease Complete response Progressive disease Not evaluable Overall responses Total number of patients Overall response rate Best radiological response (b) Second-line setting (n = 28) Partial response Stable disease Complete response Progressive disease Not evaluable Overall responses Total number of patients Overall response rate Best radiological response (c) Third-line setting (n = 40) Partial response Stable disease Complete response Progressive disease Not evaluable Overall number of responses Total number of patients Overall response rate †
All
KRAS wild type†
KRAS mutant†
11 5 3 3 3 14 25 56%
9 2 3 1 2 12 18 66%
2 3 0 2 0 2 7 28%
All
KRAS wild type†
KRAS mutant†
8 10 2 5 2 10 27 37%
7 7 2 4 1 9 21 43%
1 3 0 1 1 1 6 16%
All
KRAS wild type
KRAS mutant
4 9 1 17 9 5 40 15%
4 6 1 7 8 5 26 19%
0 3 0 10 1 0 14 0
Number of patients.
patients had the G13D mutations which have been associated with response to cetuximab-based therapy.20 Only three of these patients received cetuximab and none of them responded. Of the six patients with NRAS mutations, three received cetuximab and chemotherapy in combination at different stages of their treatment, of whom two did not respond and one had a PR. The other patients did not receive cetuximab. Therefore, the number of NRAS mutant cases was too low to allow any statistical analyses. The prevalence of BRAF mutation was low (5%) in this cohort and thus no statistically significant correlation was identified. Of the nine patients whose tumors were BRAF mutant, six had multiple sites of metastases, eight had died of mCRC, and one was alive at the time of data analysis. One of nine patients who were treated in the first-line setting responded to chemotherapy, while
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none of the patients who were treated in the second- and third-line settings responded. Of the 13 patients whose tumors were PIK3CA mutant, 7 also had coexisting KRAS mutations and 1 had coexisting BRAF mutation. At the time of data analysis, nine patients had died, two were alive, and two were lost to follow-up. Three of 13 patients who were treated in the first-line setting responded to chemotherapy, while 1 of 11 patients who were treated in the first-line setting responded to chemotherapy. There was no correlation between the presence of BRAF and/or PIK3CA mutations and OS or objective response in all 183 patients. Seven patients had co-expression of KRAS and PIK3CA mutations, and one patient co-expressed BRAF and PIK3CA mutations. These patients all had multiple sites of metastases and all of them had died at the time of data analysis.
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Figure 1 (a) Result of mutational analysis for the entire cohort of 183 patients. (b) Result of mutational analysis for the cetuximab-treated subgroup.
For the subgroup of patients (n = 7) with KRAS wildtype tumor that harbors either a BRAF and/or PIK3CA mutation, 6 with BRAF mutations have died and 5 with PIK3CA mutations have also died. The patient with both BRAF and PIK3CA mutations had multiple sites of metastases and never responded to chemotherapy with or without cetuximab. Only one patient who had BRAF mutation alone had a PR to first-line chemotherapy, the rest of the BRAF and PIK3CA mutant groups never responded to drug therapy.
Prognostic significance of KRAS, BRAF and PIK3CA mutations in the cetuximab-treated cohort
Figure 2 Correlation between KRAS mutational status and ) mutant; ( ) wild overall survival for all 183 patients. ( type.
As outlined in Table 1, the overall response rates of cetuximab-treated patients appeared to be higher in patients with KRAS wild-type tumors than those with KRAS mutant tumors in the first-, second- and third-line settings; the differences did not reach statistical significance. With regard to survival, KRAS mutation was associated with inferior OS with a HR of 2.2 (95% CI = 1.27–3.78, P < 0.005), see Figure 3a. However, this did not reach statistical significance in multivariate
analysis where the number of lines of prior therapy was found to be the only independent prognostic factor. KRAS mutation status did not correlate with drug response in all 91 cetuximab-treated patients. In a subgroup analysis of the 25 patients who received cetuximab-based therapy in the first-line setting, KRAS mutation was associated with a lack of response
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In the cetuximab-treated subgroup, the number of patients with PIK3CA mutant, NRAS mutant or BRAF mutant tumors was too small to allow any meaningful statistical correlation with survival and treatment response.
DISCUSSION
Figure 3 (a) Correlation between KRAS mutational status and overall survival for 91 cetuximab-treated patients. (b) Correlation between KRAS mutational status and overall ) survival for 92 patients who never received cetuximab. ( ) wild type. mutant; (
to therapy in both univariate and multivariate analyses (chi-square, P = 0.02, Table 1a). A statistically significant association with OS was also found in this small subgroup (n = 25), with a HR of 6.9 (95% CI 1.8–26.8, P = 0.005). No association was found in patients who were treated in the second- or third-line setting. Conversely, in the subgroup of patients who received any line of therapy without cetuximab and had KRAS mutation status determined (n = 92), there was no statistical association between KRAS mutation and response to chemotherapy or OS (Fig. 3b). Among the 52 patients who underwent second-line chemotherapy and never received cetuximab, there was a significant association between KRAS mutation and inferior OS, with a HR of 2.88 (95% CI = 1.16–7.14, P = 0.02) in a multivariate analysis. The KRAS G13D mutation has been linked to better clinical response than other types of KRAS mutations.20 Of the 13 patients whose tumors harbored the G13D mutation, 3 had cetuximab and chemotherapy in combination and all of them did not respond to treatment.
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In this cohort of Chinese patients with mCRC who received systemic treatment at a tertiary hospital, the prevalence of KRAS and BRAF mutations is consistent with most reports from other populations (Table 2). Similarly, as reported in the literature, G12D and G12V mutations account for over 50% of all KRAS mutation subtypes found in the current cohort. No apparent ethnic differences can be found in the prevalence and subtyping of KRAS mutations.28–30 The 20% prevalence rate of PIK3CA mutation reported in this study lies on the higher end of the relatively wide range of rates reported in the literature (4.7–17%).12,21,26,31 The prevalence of NRAS mutation in our study (3%) lies toward the lower end of the range of rates reported in the literature to date between 2.6% and 7%.10,12,32 KRAS mutation was found to be a negative prognostic factor in patients with mCRC irrespective of whether anti-EGFR antibodies were used. This seems to be supported by a subgroup analysis of patients who underwent second-line chemotherapy, but never received cetuximab. KRAS mutation was associated with poorer OS in a multivariate analysis. As some of the patients in this study were treated before KRAS and NRAS mutation testing became mandatory, this study’s result is consistent with the existing literature that patients with KRAS mutant tumors had poorer OS than those with wild-type tumors following cetuximab-based therapy. In a recently published meta-analysis of over 2300 patients treated with EGFR antibodies for mCRC, the progression free survival (PFS) (HR of 2.19) and overall survival (OS) (HR of 1.78) were shorter among patients with KRAS mutations compared with those without.33 The HR for OS of 2.2 (95% CI = 1.27–3.78, P < 0.005, Fig. 3a) reported in our cetuximab-treated cohort is consistent with the conclusion in this meta-analysis.33 This may also apply to objective response rate; however, the difference observed in this study was not statistically significant for the cohort of 91 patients. Only a difference in poorer response rate was observed in the small subgroup of 25 patients who were treated in the firstline setting. This is probably due to the relatively small sample size and also to the fact that 26 of 91 (28%) cetuximab-treated patients were treated before the time
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Table 2
BB Ma et al.
Prevalence of KRAS, BRAF and PIK3CA mutations in different populations
First author Current study Mao et al.21 Gao et al.22
Sample/ethnicity 183/Chinese (91 cetuximab treated) 69/Chinese 273/Chinese (59 cetuximab treated) 90/Chinese (cetuximab treated) 314/Chinese
NRAS mutation
KRAS mutation 45% (G12D, 36%)
3.2%
PIK3CA mutation 20%
BRAF mutation
Correlation with outcome
5%
KRAS correlates with survival and RR Not done KRAS correlates with RR and survival KRAS correlates with RR and survival KRAS correlates with survival KRAS correlates with RR and survival All three mutations correlate with RR and survival Both mutations correlate with survival All mutations correlate with RR —
43.9% (V14G, 26.7%) 38.5% (G12D, 41%)
— —
8.2% —
25.4% 5.1%
33.3% (G12A, 58.6%)
—
—
—
20.7%
—
—
3.8%
66/Korean (cetuximab treated) 43/Japanese (cetuximab treated)
40.9% (G12D, 78%)
—
—
0%
28% (G12D, 14%)
—
4.7%
4.7%
Nakanishi et al.27
254/Japanese
33.5%
—
—
6.7%
De Roock et al.12
1022/European
40%
2.6%
14.5%
4.7%
Imamura et al.28
1261/ United States
36% G12D (36%) G12V (21%)
—
—
—
Li et al.23 Liou et al.24 Sohn et al.25 Soeda et al.26
RR, response rate.
when KRAS mutation testing was considered mandatory, thus KRAS mutant patients were inadvertently treated with cetuximab. Although the number of patients who had NRAS mutation and rarer KRAS mutations was small in the current study, it seemed that most patients with these mutations did not respond to treatment. This is consistent with findings in Western populations that NRAS mutation and rarer KRAS mutations are associated with worse prognosis and poor/no response to EGFR antibodies.9–11,20 The prognostic role of KRAS mutation in CRC remains controversial among patients who are not treated with anti-EGFR antibodies in the adjuvant and palliative setting. The QUASAR study is the largest study to date that showed KRAS mutation to be a marker of increased risk of disease recurrence following adjuvant chemotherapy in stage II–III CRC.34 This relationship may depend on the mismatch repair (MMR) status of the patient’s tumor, as suggested by the N0147 study which found that patients with KRAS mutant tumors had lower disease-free survival following adjuvant FOLFOX if their tumors were also proficient MMR, but not if their tumors were deficient MMR (dMMR).35 In the current study, KRAS mutation was found to be prognostic in patients who underwent oxa-
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liplatin and irinotecan-based chemotherapy, irrespective of whether cetuximab was used. To date, the results from previous studies on the prognostic (as opposed to predictive) significance of KRAS mutation in mCRC remain conflicting. The MRC (Medical Research Council) FOCUS study found that KRAS mutation is a poor prognostic factor for OS, but not a predictive factor of response to chemotherapy in mCRC.36 Similarly, a Spanish study found that KRAS mutation was associated with poorer OS in patients treated with bevacizumab-XELOX. In contrast, an Australian study failed to find KRAS mutation as a prognostic factor in bevacizumab-treated patients.13 It is interesting to note that none of these studies in metastatic cohorts examined MMR status as a cofactor in this association, and it is possible that the prognostic value of KRAS mutation could be dependent on the MMR status as well in mCRC. The relevance of MMR status warrants further investigation. The role of KRAS mutation as a predictive factor to EGFR antibody therapy is well established, but its role in predicting clinical benefit from palliative or adjuvant chemotherapy remains unproven. A recent study in vitro suggests that KRAS mutation may predict oxaliplatin sensitivity in CRC cells by down-regulating the level
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of the excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1) enzyme.37 However, the FOCUS study as mentioned above did not find a correlation between KRAS mutation and response to chemotherapy.36 The current study’s result is consistent with this finding.36 Activating mutations of BRAF and PIK3CA mutations have been implicated in resistance toward EGFR antibodies. De Roock et al. found that in KRAS wildtype patients with mCRC, the presence of BRAF and/or PIK3CA is associated with very low response to EGFR antibodies.12 In particular, PIK3CA exon 9 mutations had no predictive effect, whereas exon 20 mutations were associated with worse outcome compared with wild types.12 It is interesting to note that in the current study, only one of the seven KRAS wild-type patients whose tumors harbor BRAF and/or PIK3CA mutations had an objective response to chemotherapy alone. A pooled analysis of the phase III CRYSTAL and OPUS study has suggested that BRAF mutation is more of a negative prognostic factor of OS than a predictive factor of response to cetuximab-based therapy in the metastatic setting.38 This is supported by other studies which found that the prognostic nature of BRAF mutation is independent of the usage of EGFR antibodies.39,40 Adjuvant studies in stage II–III CRC also support BRAF mutation to be a negative prognostic factor for OS in dMMR tumors.41 The evidence with PIK3CA mutation is less clear, as one study suggests that only exon 20 mutations are prognostic,12 while other studies found that it may not be a major determinant of response to anti-EGFR therapy.40,42 A recently published study suggests that patients with PIK3CA mutant tumors may have better survival if they reported a regular use of aspirin.31 Routine testing of PIK3CA and BRAF mutations prior to EGFR antibody therapy for mCRC has not been recommended by the European Society of Medical Oncology and the Evaluation of Genomic Applications in Practice and Prevention Working Group.43,44 A large US study found that routine testing of KRAS and BRAF mutations may improve the cost-effectiveness of anti-EGFR therapy; however, the incremental costeffectiveness ratio remains above an acceptable level.45 Given the current data,9,10 the use of panitumumab and cetuximab has now been restricted to patients with RAS wild-type (i.e. KRAS and NRAS wild type) mCRC since September 2013 and January 2014, respectively, by the European Medicines Agency. In Hong Kong, the cost of KRAS mutation testing is largely reimbursed by the government in public hospitals, and both KRAS and
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NRAS testing is now routinely performed prior to starting EGFR antibody therapy in patients with mCRC. In conclusion, this study found that the prevalence of KRAS, NRAS, BRAF and PIK3CA mutations and KRAS mutation subtypes in local Chinese population in Hong Kong does not differ significantly from other populations. Patients with tumors that harbor KRAS mutation have poorer OS and lower response to chemotherapy with or without anti-EGFR therapy, although the latter did not reach statistical significance. Further studies are needed before a broader application of BRAF and PIK3CA mutation testing can be advocated.
ACKNOWLEDGMENT This study was supported by the Chinese University Direct Grant for Research (reference 2008.1.026).
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23 Li FH, Shen L, Li ZH et al. Impact of KRAS mutation and PTEN expression on cetuximab-treated colorectal cancer. World J Gastroenterol 2010; 16: 5881–8. 24 Liou JM, Wu MS, Shun CT et al. Mutations in BRAF correlate with poor survival of colorectal cancers in Chinese population. Int J Colorectal Dis 2011; 26(11): 1387–95. 25 Sohn BS, Kim TW, Lee JL et al. The role of KRAS mutations in predicting the efficacy of cetuximab-plusirinotecan therapy in irinotecan-refractory Korean metastatic colorectal cancer patients. Oncology 2009; 77: 224–30. 26 Soeda H, Shimodaira H, Watanabe M et al. Clinical usefulness of KRAS, BRAF, and PIK3CA mutations as predictive markers of cetuximab efficacy in irinotecanand oxaliplatin-refractory Japanese patients with metastatic colorectal cancer. Int J Clin Oncol 2013; 18(4): 670–7. 27 Nakanishi R, Harada J, Tuul M et al. Prognostic relevance of KRAS and BRAF mutations in Japanese patients with colorectal cancer. Int J Clin Oncol 2013; 18(6): 1042–8. 28 Imamura Y, Morikawa T, Liao X et al. Specific mutations in KRAS codons 12 and 13, and patient prognosis in 1075 BRAF wild-type colorectal cancers. Clin Cancer Res 2012; 18: 4753–63. 29 Sylvester BE, Huo D, Khramtsov A et al. Molecular analysis of colorectal tumors within a diverse patient cohort at a single institution. Clin Cancer Res 2011; 18: 350–9. 30 Peeters M, Douillard JY, Van Cutsem E et al. Mutant KRAS codon 12 and 13 alleles in patients with metastatic colorectal cancer: assessment as prognostic and predictive biomarkers of response to panitumumab. J Clin Oncol 2013; 31: 759–65. 31 Liao X, Lochhead P, Nishihara R et al. Aspirin use, tumor PIK3CA mutation, and colorectal-cancer survival. N Engl J Med 2013; 367: 1596–606. 32 Irahara N, Baba Y, Nosho K et al. NRAS mutations are rare in colorectal cancer. Diagn Mol Pathol 2010; 19: 157–63. 33 Therkildsen C, Bergmann TK, Henrichsen-Schnack T, Ladelund S, Nilbert M. The predictive value of KRAS, NRAS, BRAF, PIK3CA and PTEN for anti-EGFR treatment in metastatic colorectal cancer: a systematic review and meta-analysis. Acta Oncol 2014; 53(7): 852–64. 34 Hutchins G, Southward K, Handley K et al. Value of mismatch repair, KRAS, and BRAF mutations in predicting recurrence and benefits from chemotherapy in colorectal cancer. J Clin Oncol 2012; 29: 1261–70. 35 Sinicrope FMM, Smyrk T, Thibodeau S et al. Prognostic impact of BRAF and KRAS mutations and their relationship to DNA mismatch repair (MMR) status in 2,686 stage III colon cancer patients (pts) treated in a phase III study of adjuvant FOLFOX with or without cetuximab: NCCTG N0147. J Clin Oncol 2012; 30 (suppl; abstr 3514).
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36 Richman SD, Seymour MT, Chambers P et al. KRAS and BRAF mutations in advanced colorectal cancer are associated with poor prognosis but do not preclude benefit from oxaliplatin or irinotecan: results from the MRC FOCUS trial. J Clin Oncol 2009; 27: 5931–7. 37 Lin YL, Liau JY, Yu SC et al. KRAS mutation is a predictor of oxaliplatin sensitivity in colon cancer cells. PLoS ONE 2013; 7: e50701. 38 Bokemeyer C, Van Cutsem E, Rougier P et al. Addition of cetuximab to chemotherapy as first-line treatment for KRAS wild-type metastatic colorectal cancer: pooled analysis of the CRYSTAL and OPUS randomised clinical trials. Eur J Cancer 2012; 48: 1466–75. 39 Souglakos J, Philips J, Wang R et al. Prognostic and predictive value of common mutations for treatment response and survival in patients with metastatic colorectal cancer. Br J Cancer 2009; 101: 465–72. 40 Tol J, Dijkstra JR, Klomp M et al. Markers for EGFR pathway activation as predictor of outcome in metastatic colorectal cancer patients treated with or without cetuximab. Eur J Cancer 2012; 46: 1997–2009. 41 Roth AD, Tejpar S, Delorenzi M et al. Prognostic role of KRAS and BRAF in stage II and III resected colon cancer: results of the translational study on the PETACC-3, EORTC 40993, SAKK 60-00 trial. J Clin Oncol 2010; 28: 466–74. 42 Prenen H, De Schutter J, Jacobs B et al. PIK3CA mutations are not a major determinant of resistance to the epidermal
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growth factor receptor inhibitor cetuximab in metastatic colorectal cancer. Clin Cancer Res 2009; 15: 3184–8. 43 Van Cutsem E, Dicato M, Arber N et al. Molecular markers and biological targeted therapies in metastatic colorectal cancer: expert opinion and recommendations derived from the 11th ESMO/World Congress on Gastrointestinal Cancer, Barcelona, 2009. Ann Oncol 2010; 21 (Suppl 6): vi1–10. 44 Calonge N, Fisher NL, Berg AO et al. Recommendations from the EGAPP Working Group: can testing of tumor tissue for mutations in EGFR pathway downstream effector genes in patients with metastatic colorectal cancer improve health outcomes by guiding decisions regarding anti-EGFR therapy? Genet Med 2013; 15(7): 517–27. 45 Behl AS, Goddard KA, Flottemesch TJ et al. Costeffectiveness analysis of screening for KRAS and BRAF mutations in metastatic colorectal cancer. J Natl Cancer Inst 2012; 104: 1785–95.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1 (a) Details on drug treatment – for the subgroup of cetuximab-treated patients. (b) Details on drug treatment – for patients who never had cetuximab.
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doi: 10.1111/ajco.12342
ORIGINAL ARTICLE
Elucidating the prognostic significance of KRAS, NRAS, BRAF and PIK3CA mutations in Chinese patients with metastatic colorectal cancer Brigette B MA,1 Frankie MO,1 Joanna H TONG,2 Ashley WONG,1 SC Cesar WONG,1,3 Wing M HO,1 Cherry WU,4 Polly WY LAM,5 KF CHAN,6 Timothy SK CHAN,7 Wilson MS TSUI,8 Alex KH TSANG,9 Mandy NS FUNG,10 Anthony TC CHAN1 and Ka Fai TO2 1
State Key Laboratory in Oncology in South China, Sir YK Pao Centre for Cancer, Department of Clinical Oncology, Hong Kong Cancer Institute and the Chinese University of Hong Kong, 2Department of Anatomical and Cellular Pathology, Chinese University of Hong Kong, 3Department of Health Technology and Informatics, Hong Kong Polytechnic University, 4 Department of Pathology, Alice Ho Miu Ling Nethersole Hospital, 5Department of Pathology, Queen Elizabeth Hospital, 6 Department of Pathology, Tuen Mun Hospital, 7Department of Pathology, Kwong Wah Hospital, 8Department of Pathology, Caritas Medical Centre, 9Department of Pathology, Yan Chai Hospital, 10Department of Pathology, United Christian Hospital, Hong Kong
Abstract Aim: The prognostic significance of KRAS, NRAS, PIK3CA and BRAF mutations was evaluated in Chinese patients with metastatic colorectal cancer (CRC). Method: Tumor samples from 183 patients were retrospectively tested for KRAS, NRAS, PIK3CA and BRAF mutations. Multivariate analysis was performed to determine the relationship between mutational status, drug response and survival. Result: Over 70% of patients received two or more lines of chemotherapy, 50% had cetuximab and 18% had bevacizumab. The prevalence of KRAS, NRAS, BRAF and PIK3CA mutations was 45, 3.2, 5 and 20%, respectively. For the entire cohort, the median overall survival was 24 months (95% confidence interval [CI] = 20.4–26.4 months). Of the genes tested, only KRAS mutation was an independent prognostic factor with a multivariate hazard ratio of 1.5 (95% CI = 1.05–2.16, P = 0.03). In the subgroup of patients who received cetuximab-based therapy in the first-line setting, KRAS mutation was associated with a lack of response to chemotherapy (28% vs 66%, chi-square, P = 0.01). Patients with KRAS mutant tumors (or KRAS wild-type tumors that harbored BRAF and/or PIK3CA mutations) tended to have lower response rates to chemotherapy and/or cetuximab (P = not significant). The number of NRAS mutant cases was too small to allow any statistical analysis. Conclusion: The prevalence of KRAS, NRAS, BRAF and PIK3CA mutations in this cohort is consistent with reports from non-Asian populations, and KRAS mutation has both prognostic and predictive significance in Chinese patients with metastatic CRC. Key words: BRAF, Chinese, KRAS, NRAS, PIK3CA mutations.
Correspondence: Professor Brigette B Ma MD, Department of Clinical Oncology, Basement, LKS Specialist Clinic, Prince of Wales Hospital, Shatin, New Territories, Hong Kong. Email: [email protected] Conflict of interest: none Accepted for publication 11 January 2015.
© 2015 Wiley Publishing Asia Pty Ltd
INTRODUCTION The incidence of colorectal cancer (CRC) is rising in urbanized regions of Asia such as Hong Kong, where CRC is the second most prevalent cancer.1 In Hong Kong, over 50% of patients with CRC present with
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stage III–IV disease where systemic therapy plays an important role in management.1 The use of antibodies against the epidermal growth factor receptor (EGFR) has improved the survival of patients with metastatic CRC (mCRC),2–7 and KRAS mutation status is now routinely used in practice to select patients for this therapy. It is well recognized that the presence of KRAS mutations found in exon 2 (codon 12/13) is associated with resistance to EGFR antibodies, but these mutations probably account for less than 40% of nonresponders to EGFR antibodies.8 Rarer mutations such as NRAS mutations in exon 2 (codon 12/13) have also been shown in recent prospective phase III trials to predict poor response to EGFR antibodies,9–11 while the clinical significance of other uncommon KRAS mutations at exons 3 and 4 has recently come to light. In the PRIME study, patients with mutation of KRAS (exon 2, 3 or 4) or NRAS (exon 2, 3 or 4) did not benefit from the addition of panitumumab to chemotherapy.5 Furthermore, activating mutations of other oncogenic pathways that are located downstream to the EGFR, such as the BRAF and PIK3CA mutations, have been implicated in mediating resistance to EGFR antibodies in CRC.12–15 Population-based differences in response to anticancer therapy and in the expression of predictive biomarkers have been well described for CRC and other cancers.16 One of the best known examples is the higher prevalence of activating mutation of the EGFR tyrosine kinase domain in Northeast Asians with lung adenocarcinoma than other ethnic groups.16 In CRC, it is unclear whether ethnic variation exists in the prevalence and clinical significance of KRAS, NRAS, BRAF and PIK3CA expression. In this study, the prognostic and predictive significance of these mutations in Chinese patients who received chemotherapy with or without cetuximab for mCRC was evaluated retrospectively. The primary objective of this study was to investigate the relationship between mutational status and survival and drug response, and the secondary objective was to investigate the relationship between mutational status and treatment outcome in a subgroup of patients who received cetuximab-based therapy.
METHOD
tumors available for mutational analysis. Some of the patients selected for this study were treated before KRAS mutation testing was made mandatory at our institution before starting cetuximab-based treatment in the late 2009. Patients were treated according to departmental protocol or in consenting subjects, as part of a clinical trial protocol. For patients who were treated outside a clinical trial, the types of chemotherapeutic regimens used are outlined in Supporting Information Table S1. Response to treatment was assessed at regular intervals with computed tomography scans (sometimes chest X-rays, ultrasound or positron emission tomography (PFT) scans) of target lesions, as well as monitoring of serum carcinoembryonic antigen level. Antibodies such as cetuximab and bevacizumab are available as self-financed items (without government reimbursement) in patients who receive treatment in public hospitals in Hong Kong; therefore these items were not available to all patients in this cohort. This study was approved by the New Territories East Cluster-Chinese University of Hong Kong Ethics committee.
Study endpoints and statistical analysis The relationship between expression of KRAS, NRAS, BRAF and PIK3CA mutations and clinical outcome (i.e. overall survival [OS] and response to chemotherapy) was studied. The chi-square test was used to evaluate any correlation between mutational status and response to treatment. Survival curves were computed using the Kaplan–Meier method and compared using the logrank test. The influence of cofactors (such as age, sex, number of lines of chemotherapy received, use of bevacizumab, the number of sites of metastases) on response and survival was analyzed using the Cox proportional hazard model. OS was defined as the period from the date of initial diagnosis of metastatic disease to the date of death from any cause or the date of last follow-up in the outpatient clinics. Response to chemotherapy was defined based on the RECIST (Response Evaluation Criteria in Solid Tumors) criteria, objective response is defined as the sum of partial response (PR) and complete response (CR). Survival and response were determined for the entire cohort and for a subgroup of patients who received cetuximab.
Patient selection and management The medical records of 244 patients with mCRC who attended the outpatient clinics with the intent of undergoing chemotherapy at the Prince of Wales Hospital from 2008 to 2012 were identified retrospectively. Of these patients, 183 had archived, paraffin-embedded
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Mutational analysis The presence of mutations in KRAS (codon 12/13 at exon 2; codon 59/61 at exon 3, codon 117/146 at exon 4), NRAS (codon 12/13 at exon 2, codon 59/61 at exon 3, codon 117/146 at exon 4), PIK3CA (exons 9 and 20)
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and BRAF (V600E) in these samples was evaluated with PCR-direct sequencing in this order of priority given the limited amount of archived tissues available. Tumor cells were isolated from paraffin sections by manual microdissection. Genomic DNA was extracted for the PCR amplification. The PCR products were purified and sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA) and analyzed by the Life Technologies New York, Hong Kong 3500 Genetic Analyzer. Positive and negative controls were included. The data were collected and analyzed using Applied Biosystems sequencing analysis software. The DNA sequence was compared with consensus coding sequence of KRAS, NRAS, PIK3CA and BRAF from the Consensus Coding Sequence (CCDS).
RESULT Patient characteristics and treatment outcome There were 183 Chinese patients who had archived tumor samples available for mutational testing. The median age was 58 years (range 24–83 years) and there were 79 females and 104 males. The location of the primary tumor was colon in 78% and “rectum or others” in 22% of patients, and 26% of patients had metastases confined to the liver. A total of 174 (95%) out of 183 patients received treatment with any drug therapy (chemotherapy and/or targeted therapy) for mCRC, and the percentages of patients who had 1, 2 and ≥3 line(s) of chemotherapy for mCRC were 21, 42 and 32%, respectively. Of these patients, 50% (91) received cetuximab in any line of therapy either as monotherapy or in combination with chemotherapy for mCRC, and 18% had bevacizumab. For the cetuximabtreated subgroup, the commonest chemotherapeutic agent(s) which were coadministered with cetuximab were oxaliplatin-based regimen in 68% of patients treated in the first-line setting, and irinotecan-based regimen in over 80% of patients treated in subsequent lines (Supporting Information Table S1). None of those cetuximab-treated patients ever received bevacizumab for mCRC. At the time of data analysis, the median OS for the entire cohort is 24 months (95% confidence interval [CI] = 20.4–26.4 months), 68% of patients have died and 32% were alive with cancer. For the subgroup of cetuximab-treated patients, the overall response rates (PR and CR) to cetuximab-based therapy in the first-, second- and third-line settings were 56, 37 and 15%,
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respectively (Table 1). For the subgroup of patients who never received any cetuximab and had archived tumors available for mutational testing, the overall response rates to chemotherapy in the first-, second- and thirdline settings were 25% (21 of 84 patients in total), 11% (6 of 52 patients) and 0% (10 patients), respectively.
Result of mutational analyses For KRAS mutations, 55% of the 183 tumors were wild type and 45% were mutant (Fig. 1a). For the cetuximabtreated subgroup of 91 patients, 72% were wild type and 28% were mutant (Fig. 1b). The commonest subtype of KRAS mutations was located in codon 12 such as G12D (36%), G12V (22%), G12S (7%), G12C (5%) and G12A (4%), followed by codon 13 mutation such as G13D (16%), and then codon 61 such as Q61H (5%). Other rarer subtypes of KRAS mutations included those located at exon 4 such as A146T or A146V (5%), and a case of exon 4 mutation at codon 117.17–19 NRAS mutations were identified in six patients (3%), which were located in codon 12 (G12D, G12A, G12V), codon 13 (G13R) and codon 61 (Q61L). For the entire cohort, BRAF mutations detected in nine patients (5%) were found to have the commonest V600E mutations. The tissues of 64 patients were available for analysis of PIK3CA mutation, and 13 (20%) samples were found to be mutant. For the cetuximabtreated subgroup, BRAF mutation and PIK3CA mutations were found in 3 and 10% of patients, respectively. Only two patients with NRAS mutation-expressing tumors received cetuximab. In the KRAS wild-type subgroup of patients, six had BRAF mutations alone, six had PIK3CA mutation alone, and one had both BRAF and PIK3CA mutations.
Prognostic significance of KRAS, BRAF and PIK3CA mutations in the entire cohort The relationship between specific mutations and clinical outcome in terms of survival and response to chemotherapy was investigated in 183 patients with archived tumors. The presence of KRAS mutation was associated with inferior OS with a hazard ratio (HR) of 1.55 (95% CI = 1.08–2.23, P < 0.02) (Fig. 2). Multivariate analysis showed that both KRAS mutation and previous use of bevacizumab were independent prognostic factors with the respective HRs of 1.5 (95% CI = 1.05–2.16, P = 0.03) and 0.51 (95% CI = 0.3–0.87, P = 0.01). No correlation with response to chemotherapy was observed. The nine patients with the rarer KRAS mutations (exons 3, 4) had multiple sites of metastases and did not respond (PR or CR) to drug therapy. Thirteen
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Table 1
Relationship between KRAS mutational status and best response to cetuximab-treated 91 patients
Best radiological response (a) First-line setting (n = 25) Partial response Stable disease Complete response Progressive disease Not evaluable Overall responses Total number of patients Overall response rate Best radiological response (b) Second-line setting (n = 28) Partial response Stable disease Complete response Progressive disease Not evaluable Overall responses Total number of patients Overall response rate Best radiological response (c) Third-line setting (n = 40) Partial response Stable disease Complete response Progressive disease Not evaluable Overall number of responses Total number of patients Overall response rate †
All
KRAS wild type†
KRAS mutant†
11 5 3 3 3 14 25 56%
9 2 3 1 2 12 18 66%
2 3 0 2 0 2 7 28%
All
KRAS wild type†
KRAS mutant†
8 10 2 5 2 10 27 37%
7 7 2 4 1 9 21 43%
1 3 0 1 1 1 6 16%
All
KRAS wild type
KRAS mutant
4 9 1 17 9 5 40 15%
4 6 1 7 8 5 26 19%
0 3 0 10 1 0 14 0
Number of patients.
patients had the G13D mutations which have been associated with response to cetuximab-based therapy.20 Only three of these patients received cetuximab and none of them responded. Of the six patients with NRAS mutations, three received cetuximab and chemotherapy in combination at different stages of their treatment, of whom two did not respond and one had a PR. The other patients did not receive cetuximab. Therefore, the number of NRAS mutant cases was too low to allow any statistical analyses. The prevalence of BRAF mutation was low (5%) in this cohort and thus no statistically significant correlation was identified. Of the nine patients whose tumors were BRAF mutant, six had multiple sites of metastases, eight had died of mCRC, and one was alive at the time of data analysis. One of nine patients who were treated in the first-line setting responded to chemotherapy, while
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none of the patients who were treated in the second- and third-line settings responded. Of the 13 patients whose tumors were PIK3CA mutant, 7 also had coexisting KRAS mutations and 1 had coexisting BRAF mutation. At the time of data analysis, nine patients had died, two were alive, and two were lost to follow-up. Three of 13 patients who were treated in the first-line setting responded to chemotherapy, while 1 of 11 patients who were treated in the first-line setting responded to chemotherapy. There was no correlation between the presence of BRAF and/or PIK3CA mutations and OS or objective response in all 183 patients. Seven patients had co-expression of KRAS and PIK3CA mutations, and one patient co-expressed BRAF and PIK3CA mutations. These patients all had multiple sites of metastases and all of them had died at the time of data analysis.
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Figure 1 (a) Result of mutational analysis for the entire cohort of 183 patients. (b) Result of mutational analysis for the cetuximab-treated subgroup.
For the subgroup of patients (n = 7) with KRAS wildtype tumor that harbors either a BRAF and/or PIK3CA mutation, 6 with BRAF mutations have died and 5 with PIK3CA mutations have also died. The patient with both BRAF and PIK3CA mutations had multiple sites of metastases and never responded to chemotherapy with or without cetuximab. Only one patient who had BRAF mutation alone had a PR to first-line chemotherapy, the rest of the BRAF and PIK3CA mutant groups never responded to drug therapy.
Prognostic significance of KRAS, BRAF and PIK3CA mutations in the cetuximab-treated cohort
Figure 2 Correlation between KRAS mutational status and ) mutant; ( ) wild overall survival for all 183 patients. ( type.
As outlined in Table 1, the overall response rates of cetuximab-treated patients appeared to be higher in patients with KRAS wild-type tumors than those with KRAS mutant tumors in the first-, second- and third-line settings; the differences did not reach statistical significance. With regard to survival, KRAS mutation was associated with inferior OS with a HR of 2.2 (95% CI = 1.27–3.78, P < 0.005), see Figure 3a. However, this did not reach statistical significance in multivariate
analysis where the number of lines of prior therapy was found to be the only independent prognostic factor. KRAS mutation status did not correlate with drug response in all 91 cetuximab-treated patients. In a subgroup analysis of the 25 patients who received cetuximab-based therapy in the first-line setting, KRAS mutation was associated with a lack of response
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In the cetuximab-treated subgroup, the number of patients with PIK3CA mutant, NRAS mutant or BRAF mutant tumors was too small to allow any meaningful statistical correlation with survival and treatment response.
DISCUSSION
Figure 3 (a) Correlation between KRAS mutational status and overall survival for 91 cetuximab-treated patients. (b) Correlation between KRAS mutational status and overall ) survival for 92 patients who never received cetuximab. ( ) wild type. mutant; (
to therapy in both univariate and multivariate analyses (chi-square, P = 0.02, Table 1a). A statistically significant association with OS was also found in this small subgroup (n = 25), with a HR of 6.9 (95% CI 1.8–26.8, P = 0.005). No association was found in patients who were treated in the second- or third-line setting. Conversely, in the subgroup of patients who received any line of therapy without cetuximab and had KRAS mutation status determined (n = 92), there was no statistical association between KRAS mutation and response to chemotherapy or OS (Fig. 3b). Among the 52 patients who underwent second-line chemotherapy and never received cetuximab, there was a significant association between KRAS mutation and inferior OS, with a HR of 2.88 (95% CI = 1.16–7.14, P = 0.02) in a multivariate analysis. The KRAS G13D mutation has been linked to better clinical response than other types of KRAS mutations.20 Of the 13 patients whose tumors harbored the G13D mutation, 3 had cetuximab and chemotherapy in combination and all of them did not respond to treatment.
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In this cohort of Chinese patients with mCRC who received systemic treatment at a tertiary hospital, the prevalence of KRAS and BRAF mutations is consistent with most reports from other populations (Table 2). Similarly, as reported in the literature, G12D and G12V mutations account for over 50% of all KRAS mutation subtypes found in the current cohort. No apparent ethnic differences can be found in the prevalence and subtyping of KRAS mutations.28–30 The 20% prevalence rate of PIK3CA mutation reported in this study lies on the higher end of the relatively wide range of rates reported in the literature (4.7–17%).12,21,26,31 The prevalence of NRAS mutation in our study (3%) lies toward the lower end of the range of rates reported in the literature to date between 2.6% and 7%.10,12,32 KRAS mutation was found to be a negative prognostic factor in patients with mCRC irrespective of whether anti-EGFR antibodies were used. This seems to be supported by a subgroup analysis of patients who underwent second-line chemotherapy, but never received cetuximab. KRAS mutation was associated with poorer OS in a multivariate analysis. As some of the patients in this study were treated before KRAS and NRAS mutation testing became mandatory, this study’s result is consistent with the existing literature that patients with KRAS mutant tumors had poorer OS than those with wild-type tumors following cetuximab-based therapy. In a recently published meta-analysis of over 2300 patients treated with EGFR antibodies for mCRC, the progression free survival (PFS) (HR of 2.19) and overall survival (OS) (HR of 1.78) were shorter among patients with KRAS mutations compared with those without.33 The HR for OS of 2.2 (95% CI = 1.27–3.78, P < 0.005, Fig. 3a) reported in our cetuximab-treated cohort is consistent with the conclusion in this meta-analysis.33 This may also apply to objective response rate; however, the difference observed in this study was not statistically significant for the cohort of 91 patients. Only a difference in poorer response rate was observed in the small subgroup of 25 patients who were treated in the firstline setting. This is probably due to the relatively small sample size and also to the fact that 26 of 91 (28%) cetuximab-treated patients were treated before the time
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Table 2
BB Ma et al.
Prevalence of KRAS, BRAF and PIK3CA mutations in different populations
First author Current study Mao et al.21 Gao et al.22
Sample/ethnicity 183/Chinese (91 cetuximab treated) 69/Chinese 273/Chinese (59 cetuximab treated) 90/Chinese (cetuximab treated) 314/Chinese
NRAS mutation
KRAS mutation 45% (G12D, 36%)
3.2%
PIK3CA mutation 20%
BRAF mutation
Correlation with outcome
5%
KRAS correlates with survival and RR Not done KRAS correlates with RR and survival KRAS correlates with RR and survival KRAS correlates with survival KRAS correlates with RR and survival All three mutations correlate with RR and survival Both mutations correlate with survival All mutations correlate with RR —
43.9% (V14G, 26.7%) 38.5% (G12D, 41%)
— —
8.2% —
25.4% 5.1%
33.3% (G12A, 58.6%)
—
—
—
20.7%
—
—
3.8%
66/Korean (cetuximab treated) 43/Japanese (cetuximab treated)
40.9% (G12D, 78%)
—
—
0%
28% (G12D, 14%)
—
4.7%
4.7%
Nakanishi et al.27
254/Japanese
33.5%
—
—
6.7%
De Roock et al.12
1022/European
40%
2.6%
14.5%
4.7%
Imamura et al.28
1261/ United States
36% G12D (36%) G12V (21%)
—
—
—
Li et al.23 Liou et al.24 Sohn et al.25 Soeda et al.26
RR, response rate.
when KRAS mutation testing was considered mandatory, thus KRAS mutant patients were inadvertently treated with cetuximab. Although the number of patients who had NRAS mutation and rarer KRAS mutations was small in the current study, it seemed that most patients with these mutations did not respond to treatment. This is consistent with findings in Western populations that NRAS mutation and rarer KRAS mutations are associated with worse prognosis and poor/no response to EGFR antibodies.9–11,20 The prognostic role of KRAS mutation in CRC remains controversial among patients who are not treated with anti-EGFR antibodies in the adjuvant and palliative setting. The QUASAR study is the largest study to date that showed KRAS mutation to be a marker of increased risk of disease recurrence following adjuvant chemotherapy in stage II–III CRC.34 This relationship may depend on the mismatch repair (MMR) status of the patient’s tumor, as suggested by the N0147 study which found that patients with KRAS mutant tumors had lower disease-free survival following adjuvant FOLFOX if their tumors were also proficient MMR, but not if their tumors were deficient MMR (dMMR).35 In the current study, KRAS mutation was found to be prognostic in patients who underwent oxa-
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liplatin and irinotecan-based chemotherapy, irrespective of whether cetuximab was used. To date, the results from previous studies on the prognostic (as opposed to predictive) significance of KRAS mutation in mCRC remain conflicting. The MRC (Medical Research Council) FOCUS study found that KRAS mutation is a poor prognostic factor for OS, but not a predictive factor of response to chemotherapy in mCRC.36 Similarly, a Spanish study found that KRAS mutation was associated with poorer OS in patients treated with bevacizumab-XELOX. In contrast, an Australian study failed to find KRAS mutation as a prognostic factor in bevacizumab-treated patients.13 It is interesting to note that none of these studies in metastatic cohorts examined MMR status as a cofactor in this association, and it is possible that the prognostic value of KRAS mutation could be dependent on the MMR status as well in mCRC. The relevance of MMR status warrants further investigation. The role of KRAS mutation as a predictive factor to EGFR antibody therapy is well established, but its role in predicting clinical benefit from palliative or adjuvant chemotherapy remains unproven. A recent study in vitro suggests that KRAS mutation may predict oxaliplatin sensitivity in CRC cells by down-regulating the level
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of the excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1) enzyme.37 However, the FOCUS study as mentioned above did not find a correlation between KRAS mutation and response to chemotherapy.36 The current study’s result is consistent with this finding.36 Activating mutations of BRAF and PIK3CA mutations have been implicated in resistance toward EGFR antibodies. De Roock et al. found that in KRAS wildtype patients with mCRC, the presence of BRAF and/or PIK3CA is associated with very low response to EGFR antibodies.12 In particular, PIK3CA exon 9 mutations had no predictive effect, whereas exon 20 mutations were associated with worse outcome compared with wild types.12 It is interesting to note that in the current study, only one of the seven KRAS wild-type patients whose tumors harbor BRAF and/or PIK3CA mutations had an objective response to chemotherapy alone. A pooled analysis of the phase III CRYSTAL and OPUS study has suggested that BRAF mutation is more of a negative prognostic factor of OS than a predictive factor of response to cetuximab-based therapy in the metastatic setting.38 This is supported by other studies which found that the prognostic nature of BRAF mutation is independent of the usage of EGFR antibodies.39,40 Adjuvant studies in stage II–III CRC also support BRAF mutation to be a negative prognostic factor for OS in dMMR tumors.41 The evidence with PIK3CA mutation is less clear, as one study suggests that only exon 20 mutations are prognostic,12 while other studies found that it may not be a major determinant of response to anti-EGFR therapy.40,42 A recently published study suggests that patients with PIK3CA mutant tumors may have better survival if they reported a regular use of aspirin.31 Routine testing of PIK3CA and BRAF mutations prior to EGFR antibody therapy for mCRC has not been recommended by the European Society of Medical Oncology and the Evaluation of Genomic Applications in Practice and Prevention Working Group.43,44 A large US study found that routine testing of KRAS and BRAF mutations may improve the cost-effectiveness of anti-EGFR therapy; however, the incremental costeffectiveness ratio remains above an acceptable level.45 Given the current data,9,10 the use of panitumumab and cetuximab has now been restricted to patients with RAS wild-type (i.e. KRAS and NRAS wild type) mCRC since September 2013 and January 2014, respectively, by the European Medicines Agency. In Hong Kong, the cost of KRAS mutation testing is largely reimbursed by the government in public hospitals, and both KRAS and
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NRAS testing is now routinely performed prior to starting EGFR antibody therapy in patients with mCRC. In conclusion, this study found that the prevalence of KRAS, NRAS, BRAF and PIK3CA mutations and KRAS mutation subtypes in local Chinese population in Hong Kong does not differ significantly from other populations. Patients with tumors that harbor KRAS mutation have poorer OS and lower response to chemotherapy with or without anti-EGFR therapy, although the latter did not reach statistical significance. Further studies are needed before a broader application of BRAF and PIK3CA mutation testing can be advocated.
ACKNOWLEDGMENT This study was supported by the Chinese University Direct Grant for Research (reference 2008.1.026).
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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1 (a) Details on drug treatment – for the subgroup of cetuximab-treated patients. (b) Details on drug treatment – for patients who never had cetuximab.
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