RESEARCH ARTICLE For reprint orders, please contact: [email protected]
Prognostic role of KRAS, NRAS, BRAF and PIK3CA mutations in advanced colorectal cancer Luisa Foltran*,1,2, Giovanna De Maglio3, Nicoletta Pella1, Paola Ermacora1, Giuseppe Aprile1, Elena Masiero3, Mariella Giovannoni1, Emiliana Iaiza1, Giovanni Gerardo Cardellino1, Stefania Eufemia Lutrino1, Micol Mazzer1,4, Manuela Giangreco5, Federica Edith Pisa5, Stefano Pizzolitto3 & Gianpiero Fasola1 ABSTRACT Aim: To explore the prognostic value of extended mutational profiling for metastatic colorectal cancer (mCRC). Materials & methods: We retrospectively reviewed survival results of 194 mCRC patients that were assigned to four molecular subgroups: BRAF mutated; KRAS mutated codons 12-13 only; any of KRAS codons 61-146, PIK3CA or NRAS mutations and all wild-type. Point mutations were investigated by pyrosequencing. Results: BRAF (5.2%) and KRAS 12-13 (31.9%) mutations were associated with poorer survival (HR 2.8 and 1.76, respectively). Presenting with right-sided colon cancer, not resected primary tumor, WBC >10 × 109/l, receiving less chemotherapy or no bevacizumab were all associated with inferior outcome. The all-wild-type subgroup (39.2%) reported the longest survival. Conclusion: Extended mutational profile combined with clinical factors may impact on survival in mCRC. In the last years, the research on predictive and prognostic biomarkers has dramatically increased providing useful information for the management of colorectal cancer (CRC) patients. Signaling pathway activated by EGF receptor (EGFR) has been widely investigated and KRAS-mutation status is currently a validated predictive biomarker for anti-EGFR therapy with cetuximab or panitumumab. Since 2008, KRAS mutations on codons 12 and 13 in exon 2 have been established as predictive biomarkers of resistance to anti-EGFR antibodies [1–4] . Soon after, KRAS mutations on codons 61 and 146 have been reported to limit the activity of EGFR inhibitors [5,6] . More recently, the influence of other KRAS mutations, such as exon 3 and 4, on treatment efficacy of cetuximab [7] and panitumumab [8] has been demonstrated. The prognostic role of KRAS mutation, however, remains controversial. A retrospective analysis of CO.17 trial [1] showed that KRAS had limited prognostic value in chemotherapy-resistant mCRC patients. Phipps et al. [9] reported a correlation of KRAS mutation and poorer disease-specific survival in a large CRC series, but the association was not confirmed in those patients who presented with advanced disease. Conversely, KRAS mutation was a poor prognostic factor in Medical
KEYWORDS
• colorectal cancer • EGFR pathway • mutational profiling • prognostic factors
Department of Oncology, University Hospital ‘S Maria della Misericordia’, Piazzale S Maria della Misericordia 15, Udine, Italy Department of Oncology, General Hospital ‘S Maria degli Angeli’, Via Montereale 24, Pordenone, Italy 3 Department of Pathology, University Hospital ‘S Maria della Misericordia’, Piazzale S Maria della Misericordia 15, Udine, Italy 4 Hospice ‘Casa dei Gelsi’, ULSS 9, Via Fossagera 4/c, Treviso, Italy 5 Institute of Hygiene & Clinical Epidemiology, University Hospital ‘S Maria della Misericordia’, Piazzale S Maria della Misericordia 15, Udine, Italy *Author for correspondence: Tel.: + 39 0434 399652; Fax: +39 0434 399187; [email protected] 1 2
10.2217/FON.14.279 © 2015 Future Medicine Ltd
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Research Article Foltran, De Maglio, Pella et al. Research Council (MRC) FOCUS trial [10] which compared treatment sequences to a combination of chemotherapies in advanced CRC patients. Moreover, Yokota et al. [11] showed a trend toward poor OS for patients with KRAS 13 mutations. Also BRAF, NRAS and PIK3CA mutations may negatively impact on the response to EGFR inhibitors [6] . Patients bearing BRAF (4.7%), PIK3CA exon 20 (3%) or NRAS mutations (2%) had lower response rates (RR) to cetuximab plus chemotherapy. In the same series, BRAF and PIK3CA exon 20 mutations were also significantly associated with shorter survival. Interestingly, the all-wild-type population reported the highest objective response rates. BRAF mutation, more frequently reported in right-sided, mucinous histotype or poorly differentiated CRC, is widely accepted as a strong negative prognostic factor in the advanced disease stage [10–14] . NRAS, a member of RAS oncogene family, is rarely mutated in CRC [15] . NRAS exon 2, 3, 4 mutations have recently demonstrated to be predictors of resistance to anti-EGFR therapies [7,8] . Moreover, NRAS plus KRAS mutations correlate with poor survival in patients undergoing curative resection of liver metastases [16] . PIK3CA mutations on exon 9 and 20 may have a prognostic value, as suggested in resectable stage I–III colorectal cancers [17] . PIK3CA mutations often coexist with KR AS mutations [18] and predict resistance to anti-EGFR therapy [19] . Evidence is emerging about the usefulness of a full molecular profile for treatment strategy in CRC. The all-wild-type patients (KRAS, BRAF, NRAS and PI3KCA-exon 20 wild-type) reached the highest overall response rate from anti-EGFR therapy in chemorefractory setting [6] . Our retrospective cohort analysis aimed to explore the prognostic effect of gene profiling of EGFR pathway in patients treated for mCRC. We sought to verify if combining an extended mutational analysis (KRAS, BRAF, NRAS and PI3KCA) with baseline clinical features may give more accurate prognostic information and allow us to identify different subgroups of patients. Materials & methods ●●Study population
A total of 194 patients treated with systemic chemotherapy for mCRC at the University Hospital, Udine, Italy between 1 January
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2004 and 31 January 2010 were retrospectively reviewed. Each patient included in the analysis should have available and adequate tumor specimens for molecular analysis. During the study, nearly 27 patients (15%) were excluded because of unavailability of tumor specimens. Also, eight patients (4%) were not included in the analysis because they never received chemotherapy in metastatic setting due to poor prognosis. Clinical data consisted of age, sex, previous resection and site of primary tumor, number and type of received treatments, exposure to EGFR inhibitors or antiangiogenics, type and date of metastasectomy, start date of first-line chemotherapy and death date. Since all patients had stage IV colon cancer, TNM and Union for International Cancer Control staging system were not included because of marginal importance. Information was all retrieved from patient electronic medical records and registered in an Excel database created ad hoc. Validated baseline clinical parameters such as WBC count, alkaline phosphatase, Eastern Cooperative Oncology Group (ECOG) performance status and number of metastatic sites [20] were collected. This retrospective study was approved by the Investigational Review Board of the University Hospital of Udine, Italy and it was notified to the local Ethical Committee. Because of the retrospective nature of this study, informed consent was waived. However, all included patients have given their authorization to access sensitive data for clinical and research purposes. ●●Tissues & pyrosequencing technique
Genomic DNA was extracted from formalinfixed-paraffin-embedded tissues by mean of commercially available kit (QIAamp DNA Mini kit, Qiagen, Germany) either of surgery primary CRC samples, resected metastasis or small endoscopic biopsies. After hematoxylineosin slides revision of all cases, macrodissection was performed in order to obtain a neoplastic cell enrichment of at least 50%, limiting contamination of not neoplastic tissue, necrotic or hemorrhagic areas. Genotyping was carried out on the pyrosequencing platform PyroMark™Q96 ID instrument (Qiagen, Germany) with commercially available kits Anti-EGFR MoAb response ® (Diatech Pharmacogenetics, Italy), according to manufacturer’s instruction. Point mutations (Figure 1) were detected for KRAS codons 12-13 (exon 2), 61 (exon 3) and
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Molecular profile & survival in colorectal cancer
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Figure 1. Pyrograms of wild-type and mutant KRAS. Pyrograms showing (A) KRAS wild-type, (B) p.Gly12Asp mutated and (C) p.Gly13Asp mutated samples.
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Research Article Foltran, De Maglio, Pella et al. 146 (exon 4), NRAS codons 12- 13 (exon 2), 61 (exon 3), BRAF exon 15 and PIK3CA exons 9 and 20. ●●Statistical analysis
Because this analysis was exploratory in nature, no sample size was calculated. However, our sample reflected the feasibility of the study based on availability of clinical and molecular data of 194 mCRC patients treated at our Institution over the study period. Descriptive analysis for clinical and molecular data was performed. Frequency distributions for categorical variables and mean with standard deviation, 25th and 75th percentiles for continuous variables were calculated. We estimated the overall survival (OS) from start date of chemotherapy for each mutation using uni- and multivariate Cox’s proportional hazard regression model. The selection of covariates in the final model was based both on statistical significance and clinical relevance. To explore differences of survival, we created four molecular subgroups: BRAF mutated; KRAS mutated codons 12 or 13 only; any of KRAS codons 61-146, PIK3CA exon 9–20 or NRAS cod 12-13-61 mutations and all-wildtype. According to literature [6,10] we took the all-wild-type population as reference category and we studied outcomes in BRAF-mutated and KRAS 12–13 mutated patients. Moreover, we investigated the prognostic role of the other rarest mutations taking these together. p-values < 0.05 were considered statistically significant. Results ●●Characteristics of patients
Our study population was predominantly male (68%), greater than 65 years (58%), ECOG performance status 0 at baseline (60%), left colon or rectal cancer (76%). Most of the patients underwent primary tumor resection, while a third had metastasectomy. Overall, 83 patients (43%) were treated with anti-EGFR cetuximab or panitumumab, while 72 patients (37%) received antiangiogenic therapy with bevacizumab for mCRC (Table 1) . ●●Frequency of mutations
Out of 194 tumors, 76 (39.2%) were all wildtype, defined as no mutation in KRAS codons 12–13 (exon 2), 61 (exon 3) and 146 (exon 4), NRAS codons 12–13 (exon 2), 61 (exon 3),
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BRAF exon 15 and PIK3CA exons 9 and 20. KRAS mutations in codons 12 or 13 were exclusively detected in 62 (31.9%) and BRAF V600E mutation in 10 (5.2%). KRAS codons 61-146, NRAS and PIK3CA mutations were found in 46 samples (23.7%) and analyzed as a pooled category (Table 1) . Distribution of mutations detected in KRAS, BRAF, NRAS and PIK3CA genes is summarized in Table 2. To be noted that KRAS, BRAF and NRAS mutations were all mutually exclusive in our series, while 23 patients (11.9%) carried both KRAS and PIK3CA mutations, as shown in Figure 2. ●●Results of statistical analysis
Some clinical features were associated with poorer survival in univariate analysis (Table 1) . Similarly, the three mutational subgroups (KRAS 12 or 13 mutated; BRAF mutated; and any of KRAS 61-146, NRAS or PIK3CA mutated) reported inferior outcome compared with the all-wild-type subgroup. Patients with any mutation of the oncogenes had poorer survival compared with those all-wild-type (p < 0.001; data not shown). A multivariate analysis was performed (Table 3) . BRAF mutation as well as KRAS codons 12 or 13 mutations emerged as prognostic factors. Particularly BRAF-mutated patients had a risk of death three-times and those KRAS 12-13 mutated almost two-times greater than all-wild-type reference group. Moreover, some clinical variables have been demonstrated to correlate with poorer OS, such as lack of exposure to bevacizumab, receiving less lines of chemotherapy and having primary tumor not resected. Right-sided tumor and elevated WBC count have been confirmed to be associated with poor prognosis. Compared with the all-wild-type, the other three molecular subgroups had worse outcome. The all-wild-type population had the longest median OS (27.7 months), while BRAFmutated patients had the shortest (7.6 months). Kaplan–Meier survival curves are reported for the four subgroups in Figure 3. Discussion This retrospective cohort study confirmed that mutational profiling may help in understanding biology and defining prognosis of advanced CRC. In our study, the frequencies of oncogene mutations were similar to those previously
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Table 1. Socio-demographic, clinical and molecular characteristics of patients. Feature
n
%
Median age (years), 67 (range: 60–73): – ≥65 years – >65 years† Gender: – Female – Male Tumor location: – Left colon – Right colon† – Rectum Anti-EGFR treatment: – No – Yes Bevacizumab treatment: – Yes – No† Number of lines of treatment: – ≥3 – 1† – 2† Primary tumor resection: – Yes – No† Metastasectomy: – No – Yes ECOG PS: –0 – 1–2† WBC count: – ≤10 × 109/l – >10 × 109/l† – NR Alkalyne phosphatase: – ≤300 U/l – >300 U/l† – NR Number of metastatic sites: –1 –2 – ≥3 Mutation: – All wild-type – BRAF mutated† – KRAS 12–13 only mutated† – Any of KRAS 61–146, NRAS, PIK3CA mutations†
81 113 62 132 92 46 55 111 83 72 122 85 46 63 143 51 144 50 116 78 164 26 4 180 8 6 84 88 22 76 10 62 46
42 58 32 68 48 24 28 57 43 37 63 44 24 32 74 26 74 26 60 40 86 14 96 4 43 46 11 39.2 5.2 31.9 23.7
Features associated with poorer outcome in univariate analysis (p-value < 0.05)
†
reported [2–4,6,21] . Interestingly, our all wild-type population (KRAS-, BRAF-, NRAS-, PI3KCAnot mutated) reported the longest survival.
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Strong prognostic effect of KRAS, BRAF and NRAS mutations was previously reported by Maughan et al. [22] in COIN trial. In that study,
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Research Article Foltran, De Maglio, Pella et al. Table 2. Absolute frequencies of molecular mutations. Gene mutations
Frequency (n)
KRAS Codon 12 p.G12D p.G12V p.G12A p.G12S p.G12C p.G12R Codon 13 p.G13D Codon 61 p.Q61H p.Q61R Codon 146 p.A146T PIK3CA Exon 9 p.E545K p.E542K p.E545G p.Q546K p.Q546P Exon 20 p.H1047R p.G1049R NRAS Codon 12 p.G12D p.G12A Codon 13 p.G13R Codon 61 p.Q61L p.Q61R BRAF p.V600E All wild-type
92/194 65 (70.7%) 26 19 10 5 4 1 18 (19.6%) 18 5 (5.4%) 4 1 4 (4.3%) 4 32/194 25 (78.1%) 18 4 1 1 1 7 (21.9%) 6 1 7/194 3 (42.8%) 2 1 1 (14.3%) 1 3 (42.9%) 2 1 10/194 10 76/194 (39.2%)
the OS was shorter for patients with any mutation of the three oncogenes compared with all wild-type, irrespective of treatment received. Although mutation on KRAS codon 12 was associated with poor prognosis in the RASCAL II study [23] and KRAS codon 12-mutated alleles were associated with stronger transforming activity in vitro than KRAS codon 13 mutant alleles [24] , we studied the prognostic role of KRAS codons 12 and 13 mutations taken together. The negative effect on survival of KRAS mutation has been shown in our analysis. In the multivariate analysis, KRAS mutations in
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codons 12 and 13 were associated with shorter survival if compared with the all-wild-type (KRAS, NRAS, BRAF and PIK3CA) molecular subgroup used as reference. These results are in line with MRC FOCUS trial [10] where negative effect on outcome for KRAS codons 12, 13 and 61 mutations has been shown. It was also demonstrated that KRAS codons 12–13 mutated patients undergoing liver surgery for CRC metastases had worse survival [25] . By contrast, Douillard et al. [8] did not show a negative prognostic role of KRAS mutation in first-line treatment for mCRC. Similar results were reported
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Molecular profile & survival in colorectal cancer in a retrospective analysis of three randomized Phase III trials [26] . Our study confirmed the negative prognostic role of BRAF mutation (HR: 2.8; 95% CI: 1.12–6.95; p = 0.027). Patients with BRAF V600E mutation had a median survival of 7.6 months (95% CI: 0.5–11.7), significantly shorter compared with that reported by the allwild-type population (27.7 months; 95% CI: 21.4–36.6). These results are consistent with those of previous studies [5,8,11,13] . Mutations in any other oncogenes (KRAS 61–146, NRAS, PIK3CA) were not significantly associated with survival, albeit the limited incidence of these mutations may preclude conclusions on their prognostic value. Our analysis did not detect other mutations of RAS pathway which may give more information on outcome, as recently reported [16] . Also, the prognostic impact of microsatellite instability (MSI) was not
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explored in our population. MSI has been recognized as an independent good prognostic factor in early-stage colon cancer [27,28] , but it remains uncertain in mCRC [29,30] . MSI is rare (4%) in mCRC [29] . BRAF mutation has been associated with MSI high. Goldstein et al. [31] showed that BRAF mutation was a poor prognostic factor also in MSI-high mCRC. Therefore, the metastatic setting of our study cohort, the limited sample size and the exiguous number of BRAF-mutant patients could limit the power of the MSI analysis. In addition, our study suggested right colon cancer as a prognostic factor. Notably, most of BRAF-mutated samples (7/10) in this subgroup were right-sided colon cancers. BRAF mutation, which is a negative prognostic factor, was associated with right colon cancer also in previous studies [11,32] . Although the prognostic value of tumor location remains controversial [33,34] , some studies indicated right colon cancer as a different
KRAS codons 12–13; 62 (31.9%) KRAS codons 12–13/ PIK3CA; 21 (10.9%) PIK3CA; 9 (4.6%)
KRAS codons 61,146; 7 (3.6%) KRAS codons 61,146/PIK3CA; 2 (1%)
BRAF; 10 (5.2%)
NRAS; 7 (3.6%)
Figure 2. Set diagram illustrates the distribution of molecular mutations in the study population.
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Research Article Foltran, De Maglio, Pella et al. Table 3. Results of multivariate analysis for overall survival. Variable
Multivariate analysis
BRAF mutated vs all-wild-type KRAS 12-13 mutated vs all-wild-type† Any of KRAS 61 -146, NRAS, PIK3CA mutated vs all-wild-type Sex (male vs female) Age (>65 vs ≤65 years) Tumor location (right vs left colon)† Anti-EGFR treatment (no vs yes) Bevacizumab treatment (no vs yes)† Number of lines of treatment (1 vs ≥3)† Number of lines of treatment (2 vs ≥3)† Resection of primary tumour (no vs yes)† Metastasectomy (no vs yes) ECOG performance status (≥1 vs 0) Alkaline phosphatase (>300 vs ≤300) WBC count (>10,000 vs ≤10,000)† Number of metastatic sites (2 vs 1) Number of metastatic sites (≥3 vs 1) †
Hazard ratio (95% CI)
p-value
2.80 (1.12–6.95) 1.76 (1.14–2.72) 1.2 (0.78–1.85)
0.027† 0.01† 0.41
1.07 (0.72–1.6) 1.37 (0.95–1.98) 1.80 (1.15–2.82) 0.78 (0.50–1.21) 1.44 (1.01–2.07) 2.25 (1.38–3.65) 1.67 (1.11–2.54) 2.05 (1.38–3.04) 1.18 (0.79–1.78) 1.27 (0.87–1.85) 1.88 (0.86–4.12) 2.29 (1.37–3.83) 0.94 (0.64–1.37) 1.11 (0.63–1.96)
0.73 0.09 0.01† 0.27 0.047† 0.001† 0.015† 2) in our cohort reported improved survival compared with those who received less chemotherapy, as Grothey et al. [39] elegantly demonstrated.
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Furthermore, we suggested that treatment with bevacizumab may correlate with OS. This is consistent with results of several randomized controlled trials and meta-analyses. Recently, a pooled analysis showed that bevacizumab with chemotherapy compared with chemotherapy alone improved outcomes in patients with mCRC [40] . Since RAS mutations are predictive biomarkers of resistance to anti-EGFR antibodies, we explored the impact of anti-EGFR treatment on prognosis of our population. In the multivariate analysis, no association of anti-EGFR treatment (no vs yes) with OS (HR: 0.78; 95% CI: 0.50–1.21) was found. This study has some limitations. It is retrospective and exploratory in nature. The analysis was conducted at a single center and the limited sample size prevents us from drawing definite conclusions. Also, our study population may be considered a little outdated: patients were treated for mCRC from 2004 to 2010. Indeed, a small proportion of patients received bevacizumab or anti-EGFR therapy. However, this could be considered a strength since the prognostic effect of the molecular characterization is not confounded with its potential negative predictive value. A Cox regression model was also applied: it showed that anti-EGFR treatment did not affect the survival in the four molecular subgroups, particularly in the all-wild-type population (p = 0.14).
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Molecular profile & survival in colorectal cancer Moreover, the four molecular subgroups were arbitrarily created: the reasons for this choice are subsequently explained. First, this retrospective analysis was conceived in early 2012 when most available studies suggested the predictive and prognostic role of KRAS codons 12 and 13 and BRAF V600E mutations. At that point, we had few data on other rare mutations such as NRAS, PIK3CA or KRAS 61-146. Therefore we decided to analyze KRAS codons 12 and 13 mutations and BRAF mutations separately from the other mutations. Second, the predictive and possibly prognostic value of RAS in colorectal cancer is extremely recent [7–8,16] . For this reason, at the time of study design, KRAS was not combined with NRAS mutations. Most importantly, we aimed to explore the prognostic role of the all-wild-type subgroup since De Roock et al. [6] showed that mCRC patients bearing tumors with no mutations reported the best outcomes. Finally, the other mutations (KRAS 61–146, NRAS and PIK3CA) Mutational status BRAF mutation KRAS mutation 12–13 only Any of KRAS 61–146, NRAS, PIK3CA mutation All wild-type (reference category)
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were very few in number, and we decided to put these together in an arbitrary group. Nevertheless, the differences in median survival in the four molecular subgroups are both biologically and clinically meaningful. Basically, the all-wild-type patients had the best prognosis while the BRAF-mutated patients had the worst outcome. Undoubtedly, the role of RAS mutations and PIK3CA mutations are worthy of further investigation. In short, mCRC patients may have different tumors and require different treatment approaches in practice. Clinical information integrated with molecular data allows us to better select patients for treatment strategy and it may help stratification in clinical trials. Conclusion Our retrospective analysis identified distinct molecular prognostic subgroups of patients. Our analysis suggests that extended mutational Median survival (months) 7.6 16.7 22 27.7
1.0
95% CI 0.5–11.7 13.6–21.7 15.9–24.4 21.4–36.6
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Figure 3. Median survival in the four molecular subgroups. Kaplan–Meier analysis. Log-rank test: p < 0.0001.
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Research Article Foltran, De Maglio, Pella et al. profiling of advanced CRC allows better understanding of tumor biology and may drive patient stratification both in clinical practice and in the design of clinical trials. Future perspective In the last 20 years, intense translational and clinical research led to the development of new targeted therapies in mCRC patients. The study of predictive and prognostic biomarkers provided useful information on tumor biology and guided treatment choice in CRC. We aimed to explore the prognostic value of an extensive molecular profile in colorectal cancer (KRAS, NRAS, BRAF, PIK3CA). Our analysis
confirmed the negative prognostic role of BRAF mutation and showed KRAS 12-13 codons mutation as a prognostic factor. Above all, the study demonstrated clinically meaningful differences of survival across the four molecular subgroups. The all-wild-type population had the longest survival while the other mutational subgroups reported inferior outcomes. Some clinical factors were found to be associated with poorer survival. Clinical variables such as right-sided colon cancer, not resected primary tumor, receiving less chemotherapy and no bevacizumab need to be further investigated. In the era of personalized medicine, it may be assumed that an accurate molecular selection of
EXECUTIVE SUMMARY Aim of the study ●●
The usefulness of a full molecular profile for treatment strategy in colorectal cancer has been shown.
●●
This study aimed to investigate the prognostic effect of extensive molecular profiling integrated with clinical factors in metastatic colorectal cancer (mCRC).
Materials & methods ●●
Socio-demographic, molecular and clinical data of 194 patients treated for mCRC were retrospectively reviewed.
●●
Point mutations were detected by pyrosequencing for KRAS codons 12, 13, 61 and 146, BRAF exon 15, NRAS codons 12, 13, 61 and PIK3CA exons 9 and 20.
●●
To explore differences of survival, four molecular subgroups were created: --
BRAF mutated;
--
KRAS-mutated codons 12 and 13;
--
Any of KRAS codons 61-146, PIK3CA or NRAS;
--
All-wild-type (reference category).
Results
BRAF mutation as well as KRAS codons 12 or 13 mutations were associated with increased risk of death compared with
●●
the all-wild-type reference group.
Some clinical variables correlated with poorer overall survival:
●● --
Lack of exposure to bevacizumab;
--
Receiving less lines of chemotherapy;
--
Having primary tumor not resected;
--
Right-sided tumor location;
--
Elevated white blood cell count (>10 × 109/l).
●●
The all-wild-type population had the longest survival (27.7 months), while BRAF mutated the shortest (7.6 months). Patients with KRAS 12-13 mutation and those with the other rarest mutations reported poorer outcome compared with the all wild-type (16.7 and 22 months, respectively).
Conclusion ●●
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Extensive molecular profiling added to clinical factors have a prognostic impact in mCRC and require further study.
Future Oncol. (2015) 11(4)
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Molecular profile & survival in colorectal cancer CRC patients combined with clinical features allows us to better identify subgroup of patients with different prognoses and to drive selection and stratification of patients in large prospective clinical trials.
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options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or
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prognostic role of KRAS, BRAF, PIK3CA and PTEN in colorectal cancer. Br. J. Cancer 108(10), 2153–2163 (2013). •• The study analyzed KRAS, BRAF, PIK3CA and PTEN mutations combined as a Quadruple Index. Authors concluded that molecular subgroups based on KRAS and BRAF mutations have a prognostic impact in CRC. 15 Irahara N, Baba Y, Nosho K et al. NRAS
mutations are rare in colorectal cancer. 19(3), 157–163 (2010). 16 Vauthey JN, Zimmitti G, Kopetz SE et al.
RAS mutation status predicts survival and patterns of recurrence in patients undergoing hepatectomy for colorectal liver metastases. Ann. Surg. 258(4), 619–626 (2013). 17 Ogino S, Nosho K, Kirkner GJ et al. PIK3CA
mutation is associated with poor prognosis among patients with curatively resected colon cancer. J. Clin. Oncol. 27(9), 1477–1484 (2009). 18 Janku F, Lee JJ, Tsimberidou AM et al.
11 Yokota T, Ura T, Shibata N et al. BRAF
PIK3CA mutations frequently coexist with RAS and BRAF mutations in patients with advanced cancers. PLoS ONE 6(7), e22769 (2011).
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12 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 101(3), 465–472 (2009).
19 Perrone F, Lampis A, Orsenigo M et al.
PI3KCA/PTEN deregulation contributes to impaired responses to cetuximab in metastatic colorectal cancer patients. Ann. Oncol. 20(1), 84–90 (2009). 20 Köhne CH, Cunningham D, Di Costanzo F
et al. Clinical determinants of survival in patients with 5-fluorouracil-based treatment for metastatic colorectal cancer: results of a multivariate analysis of 3825 patients. Ann. Oncol. 13(2), 308–317 (2002).
13 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 46(11), 1997–2009 (2010).
•
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et al. Kirsten ras mutations in patients with colorectal cancer: the ‘RASCAL II’ study. Br. J. Cancer 85(5), 692–696 (2001). 24 Smith G, Bounds R, Wolf H, Steele RJ,
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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. 31(6), 759–765 (2013). 27 Merok MA, Ahlquist T, Røyrvik EC et al.
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retrospective analysis of metastatic colorectal cancer (CRC) with high-level microsatellite instability (MSI-H). Ann. Oncol. 25(5), 1032–1038 (2014). 32 Gonsalves WI, Mahoney MR, Sargent DJ
et al. Patient and tumor characteristics and BRAF and KRAS mutations colon cancer, NCCTG/Alliance N0147. J. Natl Cancer Inst. 106(7), doi:10.1093/jnci/dju106 (2014) (Epub ahead of print). 33 Hansen IO, Jess P. Possible better long-term
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Comparison of 17,641 patients with right- and left-sided colon cancer: differences in epidemiology, perioperative course, histology, and survival. Dis. Colon Rectum 53(1), 57–64 (2010). 36 Missiaglia E, Jacobs B, D’Ario G et al. Distal
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Sørensen HT, Nørgaard M. The impact of comorbidity on cancer survival: a review. Clin. Epidemiol. 5(1), 3–29 (2013). 38 Kumar R, Price TJ, Beeke C et al. Colorectal
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relationship between tumour site, clinicopathological characteristics and
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Prognostic role of KRAS, NRAS, BRAF and PIK3CA mutations in advanced colorectal cancer Luisa Foltran*,1,2, Giovanna De Maglio3, Nicoletta Pella1, Paola Ermacora1, Giuseppe Aprile1, Elena Masiero3, Mariella Giovannoni1, Emiliana Iaiza1, Giovanni Gerardo Cardellino1, Stefania Eufemia Lutrino1, Micol Mazzer1,4, Manuela Giangreco5, Federica Edith Pisa5, Stefano Pizzolitto3 & Gianpiero Fasola1 ABSTRACT Aim: To explore the prognostic value of extended mutational profiling for metastatic colorectal cancer (mCRC). Materials & methods: We retrospectively reviewed survival results of 194 mCRC patients that were assigned to four molecular subgroups: BRAF mutated; KRAS mutated codons 12-13 only; any of KRAS codons 61-146, PIK3CA or NRAS mutations and all wild-type. Point mutations were investigated by pyrosequencing. Results: BRAF (5.2%) and KRAS 12-13 (31.9%) mutations were associated with poorer survival (HR 2.8 and 1.76, respectively). Presenting with right-sided colon cancer, not resected primary tumor, WBC >10 × 109/l, receiving less chemotherapy or no bevacizumab were all associated with inferior outcome. The all-wild-type subgroup (39.2%) reported the longest survival. Conclusion: Extended mutational profile combined with clinical factors may impact on survival in mCRC. In the last years, the research on predictive and prognostic biomarkers has dramatically increased providing useful information for the management of colorectal cancer (CRC) patients. Signaling pathway activated by EGF receptor (EGFR) has been widely investigated and KRAS-mutation status is currently a validated predictive biomarker for anti-EGFR therapy with cetuximab or panitumumab. Since 2008, KRAS mutations on codons 12 and 13 in exon 2 have been established as predictive biomarkers of resistance to anti-EGFR antibodies [1–4] . Soon after, KRAS mutations on codons 61 and 146 have been reported to limit the activity of EGFR inhibitors [5,6] . More recently, the influence of other KRAS mutations, such as exon 3 and 4, on treatment efficacy of cetuximab [7] and panitumumab [8] has been demonstrated. The prognostic role of KRAS mutation, however, remains controversial. A retrospective analysis of CO.17 trial [1] showed that KRAS had limited prognostic value in chemotherapy-resistant mCRC patients. Phipps et al. [9] reported a correlation of KRAS mutation and poorer disease-specific survival in a large CRC series, but the association was not confirmed in those patients who presented with advanced disease. Conversely, KRAS mutation was a poor prognostic factor in Medical
KEYWORDS
• colorectal cancer • EGFR pathway • mutational profiling • prognostic factors
Department of Oncology, University Hospital ‘S Maria della Misericordia’, Piazzale S Maria della Misericordia 15, Udine, Italy Department of Oncology, General Hospital ‘S Maria degli Angeli’, Via Montereale 24, Pordenone, Italy 3 Department of Pathology, University Hospital ‘S Maria della Misericordia’, Piazzale S Maria della Misericordia 15, Udine, Italy 4 Hospice ‘Casa dei Gelsi’, ULSS 9, Via Fossagera 4/c, Treviso, Italy 5 Institute of Hygiene & Clinical Epidemiology, University Hospital ‘S Maria della Misericordia’, Piazzale S Maria della Misericordia 15, Udine, Italy *Author for correspondence: Tel.: + 39 0434 399652; Fax: +39 0434 399187; [email protected] 1 2
10.2217/FON.14.279 © 2015 Future Medicine Ltd
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Research Article Foltran, De Maglio, Pella et al. Research Council (MRC) FOCUS trial [10] which compared treatment sequences to a combination of chemotherapies in advanced CRC patients. Moreover, Yokota et al. [11] showed a trend toward poor OS for patients with KRAS 13 mutations. Also BRAF, NRAS and PIK3CA mutations may negatively impact on the response to EGFR inhibitors [6] . Patients bearing BRAF (4.7%), PIK3CA exon 20 (3%) or NRAS mutations (2%) had lower response rates (RR) to cetuximab plus chemotherapy. In the same series, BRAF and PIK3CA exon 20 mutations were also significantly associated with shorter survival. Interestingly, the all-wild-type population reported the highest objective response rates. BRAF mutation, more frequently reported in right-sided, mucinous histotype or poorly differentiated CRC, is widely accepted as a strong negative prognostic factor in the advanced disease stage [10–14] . NRAS, a member of RAS oncogene family, is rarely mutated in CRC [15] . NRAS exon 2, 3, 4 mutations have recently demonstrated to be predictors of resistance to anti-EGFR therapies [7,8] . Moreover, NRAS plus KRAS mutations correlate with poor survival in patients undergoing curative resection of liver metastases [16] . PIK3CA mutations on exon 9 and 20 may have a prognostic value, as suggested in resectable stage I–III colorectal cancers [17] . PIK3CA mutations often coexist with KR AS mutations [18] and predict resistance to anti-EGFR therapy [19] . Evidence is emerging about the usefulness of a full molecular profile for treatment strategy in CRC. The all-wild-type patients (KRAS, BRAF, NRAS and PI3KCA-exon 20 wild-type) reached the highest overall response rate from anti-EGFR therapy in chemorefractory setting [6] . Our retrospective cohort analysis aimed to explore the prognostic effect of gene profiling of EGFR pathway in patients treated for mCRC. We sought to verify if combining an extended mutational analysis (KRAS, BRAF, NRAS and PI3KCA) with baseline clinical features may give more accurate prognostic information and allow us to identify different subgroups of patients. Materials & methods ●●Study population
A total of 194 patients treated with systemic chemotherapy for mCRC at the University Hospital, Udine, Italy between 1 January
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2004 and 31 January 2010 were retrospectively reviewed. Each patient included in the analysis should have available and adequate tumor specimens for molecular analysis. During the study, nearly 27 patients (15%) were excluded because of unavailability of tumor specimens. Also, eight patients (4%) were not included in the analysis because they never received chemotherapy in metastatic setting due to poor prognosis. Clinical data consisted of age, sex, previous resection and site of primary tumor, number and type of received treatments, exposure to EGFR inhibitors or antiangiogenics, type and date of metastasectomy, start date of first-line chemotherapy and death date. Since all patients had stage IV colon cancer, TNM and Union for International Cancer Control staging system were not included because of marginal importance. Information was all retrieved from patient electronic medical records and registered in an Excel database created ad hoc. Validated baseline clinical parameters such as WBC count, alkaline phosphatase, Eastern Cooperative Oncology Group (ECOG) performance status and number of metastatic sites [20] were collected. This retrospective study was approved by the Investigational Review Board of the University Hospital of Udine, Italy and it was notified to the local Ethical Committee. Because of the retrospective nature of this study, informed consent was waived. However, all included patients have given their authorization to access sensitive data for clinical and research purposes. ●●Tissues & pyrosequencing technique
Genomic DNA was extracted from formalinfixed-paraffin-embedded tissues by mean of commercially available kit (QIAamp DNA Mini kit, Qiagen, Germany) either of surgery primary CRC samples, resected metastasis or small endoscopic biopsies. After hematoxylineosin slides revision of all cases, macrodissection was performed in order to obtain a neoplastic cell enrichment of at least 50%, limiting contamination of not neoplastic tissue, necrotic or hemorrhagic areas. Genotyping was carried out on the pyrosequencing platform PyroMark™Q96 ID instrument (Qiagen, Germany) with commercially available kits Anti-EGFR MoAb response ® (Diatech Pharmacogenetics, Italy), according to manufacturer’s instruction. Point mutations (Figure 1) were detected for KRAS codons 12-13 (exon 2), 61 (exon 3) and
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Molecular profile & survival in colorectal cancer
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Figure 1. Pyrograms of wild-type and mutant KRAS. Pyrograms showing (A) KRAS wild-type, (B) p.Gly12Asp mutated and (C) p.Gly13Asp mutated samples.
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Research Article Foltran, De Maglio, Pella et al. 146 (exon 4), NRAS codons 12- 13 (exon 2), 61 (exon 3), BRAF exon 15 and PIK3CA exons 9 and 20. ●●Statistical analysis
Because this analysis was exploratory in nature, no sample size was calculated. However, our sample reflected the feasibility of the study based on availability of clinical and molecular data of 194 mCRC patients treated at our Institution over the study period. Descriptive analysis for clinical and molecular data was performed. Frequency distributions for categorical variables and mean with standard deviation, 25th and 75th percentiles for continuous variables were calculated. We estimated the overall survival (OS) from start date of chemotherapy for each mutation using uni- and multivariate Cox’s proportional hazard regression model. The selection of covariates in the final model was based both on statistical significance and clinical relevance. To explore differences of survival, we created four molecular subgroups: BRAF mutated; KRAS mutated codons 12 or 13 only; any of KRAS codons 61-146, PIK3CA exon 9–20 or NRAS cod 12-13-61 mutations and all-wildtype. According to literature [6,10] we took the all-wild-type population as reference category and we studied outcomes in BRAF-mutated and KRAS 12–13 mutated patients. Moreover, we investigated the prognostic role of the other rarest mutations taking these together. p-values < 0.05 were considered statistically significant. Results ●●Characteristics of patients
Our study population was predominantly male (68%), greater than 65 years (58%), ECOG performance status 0 at baseline (60%), left colon or rectal cancer (76%). Most of the patients underwent primary tumor resection, while a third had metastasectomy. Overall, 83 patients (43%) were treated with anti-EGFR cetuximab or panitumumab, while 72 patients (37%) received antiangiogenic therapy with bevacizumab for mCRC (Table 1) . ●●Frequency of mutations
Out of 194 tumors, 76 (39.2%) were all wildtype, defined as no mutation in KRAS codons 12–13 (exon 2), 61 (exon 3) and 146 (exon 4), NRAS codons 12–13 (exon 2), 61 (exon 3),
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BRAF exon 15 and PIK3CA exons 9 and 20. KRAS mutations in codons 12 or 13 were exclusively detected in 62 (31.9%) and BRAF V600E mutation in 10 (5.2%). KRAS codons 61-146, NRAS and PIK3CA mutations were found in 46 samples (23.7%) and analyzed as a pooled category (Table 1) . Distribution of mutations detected in KRAS, BRAF, NRAS and PIK3CA genes is summarized in Table 2. To be noted that KRAS, BRAF and NRAS mutations were all mutually exclusive in our series, while 23 patients (11.9%) carried both KRAS and PIK3CA mutations, as shown in Figure 2. ●●Results of statistical analysis
Some clinical features were associated with poorer survival in univariate analysis (Table 1) . Similarly, the three mutational subgroups (KRAS 12 or 13 mutated; BRAF mutated; and any of KRAS 61-146, NRAS or PIK3CA mutated) reported inferior outcome compared with the all-wild-type subgroup. Patients with any mutation of the oncogenes had poorer survival compared with those all-wild-type (p < 0.001; data not shown). A multivariate analysis was performed (Table 3) . BRAF mutation as well as KRAS codons 12 or 13 mutations emerged as prognostic factors. Particularly BRAF-mutated patients had a risk of death three-times and those KRAS 12-13 mutated almost two-times greater than all-wild-type reference group. Moreover, some clinical variables have been demonstrated to correlate with poorer OS, such as lack of exposure to bevacizumab, receiving less lines of chemotherapy and having primary tumor not resected. Right-sided tumor and elevated WBC count have been confirmed to be associated with poor prognosis. Compared with the all-wild-type, the other three molecular subgroups had worse outcome. The all-wild-type population had the longest median OS (27.7 months), while BRAFmutated patients had the shortest (7.6 months). Kaplan–Meier survival curves are reported for the four subgroups in Figure 3. Discussion This retrospective cohort study confirmed that mutational profiling may help in understanding biology and defining prognosis of advanced CRC. In our study, the frequencies of oncogene mutations were similar to those previously
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Molecular profile & survival in colorectal cancer
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Table 1. Socio-demographic, clinical and molecular characteristics of patients. Feature
n
%
Median age (years), 67 (range: 60–73): – ≥65 years – >65 years† Gender: – Female – Male Tumor location: – Left colon – Right colon† – Rectum Anti-EGFR treatment: – No – Yes Bevacizumab treatment: – Yes – No† Number of lines of treatment: – ≥3 – 1† – 2† Primary tumor resection: – Yes – No† Metastasectomy: – No – Yes ECOG PS: –0 – 1–2† WBC count: – ≤10 × 109/l – >10 × 109/l† – NR Alkalyne phosphatase: – ≤300 U/l – >300 U/l† – NR Number of metastatic sites: –1 –2 – ≥3 Mutation: – All wild-type – BRAF mutated† – KRAS 12–13 only mutated† – Any of KRAS 61–146, NRAS, PIK3CA mutations†
81 113 62 132 92 46 55 111 83 72 122 85 46 63 143 51 144 50 116 78 164 26 4 180 8 6 84 88 22 76 10 62 46
42 58 32 68 48 24 28 57 43 37 63 44 24 32 74 26 74 26 60 40 86 14 96 4 43 46 11 39.2 5.2 31.9 23.7
Features associated with poorer outcome in univariate analysis (p-value < 0.05)
†
reported [2–4,6,21] . Interestingly, our all wild-type population (KRAS-, BRAF-, NRAS-, PI3KCAnot mutated) reported the longest survival.
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Strong prognostic effect of KRAS, BRAF and NRAS mutations was previously reported by Maughan et al. [22] in COIN trial. In that study,
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Research Article Foltran, De Maglio, Pella et al. Table 2. Absolute frequencies of molecular mutations. Gene mutations
Frequency (n)
KRAS Codon 12 p.G12D p.G12V p.G12A p.G12S p.G12C p.G12R Codon 13 p.G13D Codon 61 p.Q61H p.Q61R Codon 146 p.A146T PIK3CA Exon 9 p.E545K p.E542K p.E545G p.Q546K p.Q546P Exon 20 p.H1047R p.G1049R NRAS Codon 12 p.G12D p.G12A Codon 13 p.G13R Codon 61 p.Q61L p.Q61R BRAF p.V600E All wild-type
92/194 65 (70.7%) 26 19 10 5 4 1 18 (19.6%) 18 5 (5.4%) 4 1 4 (4.3%) 4 32/194 25 (78.1%) 18 4 1 1 1 7 (21.9%) 6 1 7/194 3 (42.8%) 2 1 1 (14.3%) 1 3 (42.9%) 2 1 10/194 10 76/194 (39.2%)
the OS was shorter for patients with any mutation of the three oncogenes compared with all wild-type, irrespective of treatment received. Although mutation on KRAS codon 12 was associated with poor prognosis in the RASCAL II study [23] and KRAS codon 12-mutated alleles were associated with stronger transforming activity in vitro than KRAS codon 13 mutant alleles [24] , we studied the prognostic role of KRAS codons 12 and 13 mutations taken together. The negative effect on survival of KRAS mutation has been shown in our analysis. In the multivariate analysis, KRAS mutations in
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codons 12 and 13 were associated with shorter survival if compared with the all-wild-type (KRAS, NRAS, BRAF and PIK3CA) molecular subgroup used as reference. These results are in line with MRC FOCUS trial [10] where negative effect on outcome for KRAS codons 12, 13 and 61 mutations has been shown. It was also demonstrated that KRAS codons 12–13 mutated patients undergoing liver surgery for CRC metastases had worse survival [25] . By contrast, Douillard et al. [8] did not show a negative prognostic role of KRAS mutation in first-line treatment for mCRC. Similar results were reported
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Molecular profile & survival in colorectal cancer in a retrospective analysis of three randomized Phase III trials [26] . Our study confirmed the negative prognostic role of BRAF mutation (HR: 2.8; 95% CI: 1.12–6.95; p = 0.027). Patients with BRAF V600E mutation had a median survival of 7.6 months (95% CI: 0.5–11.7), significantly shorter compared with that reported by the allwild-type population (27.7 months; 95% CI: 21.4–36.6). These results are consistent with those of previous studies [5,8,11,13] . Mutations in any other oncogenes (KRAS 61–146, NRAS, PIK3CA) were not significantly associated with survival, albeit the limited incidence of these mutations may preclude conclusions on their prognostic value. Our analysis did not detect other mutations of RAS pathway which may give more information on outcome, as recently reported [16] . Also, the prognostic impact of microsatellite instability (MSI) was not
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explored in our population. MSI has been recognized as an independent good prognostic factor in early-stage colon cancer [27,28] , but it remains uncertain in mCRC [29,30] . MSI is rare (4%) in mCRC [29] . BRAF mutation has been associated with MSI high. Goldstein et al. [31] showed that BRAF mutation was a poor prognostic factor also in MSI-high mCRC. Therefore, the metastatic setting of our study cohort, the limited sample size and the exiguous number of BRAF-mutant patients could limit the power of the MSI analysis. In addition, our study suggested right colon cancer as a prognostic factor. Notably, most of BRAF-mutated samples (7/10) in this subgroup were right-sided colon cancers. BRAF mutation, which is a negative prognostic factor, was associated with right colon cancer also in previous studies [11,32] . Although the prognostic value of tumor location remains controversial [33,34] , some studies indicated right colon cancer as a different
KRAS codons 12–13; 62 (31.9%) KRAS codons 12–13/ PIK3CA; 21 (10.9%) PIK3CA; 9 (4.6%)
KRAS codons 61,146; 7 (3.6%) KRAS codons 61,146/PIK3CA; 2 (1%)
BRAF; 10 (5.2%)
NRAS; 7 (3.6%)
Figure 2. Set diagram illustrates the distribution of molecular mutations in the study population.
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Research Article Foltran, De Maglio, Pella et al. Table 3. Results of multivariate analysis for overall survival. Variable
Multivariate analysis
BRAF mutated vs all-wild-type KRAS 12-13 mutated vs all-wild-type† Any of KRAS 61 -146, NRAS, PIK3CA mutated vs all-wild-type Sex (male vs female) Age (>65 vs ≤65 years) Tumor location (right vs left colon)† Anti-EGFR treatment (no vs yes) Bevacizumab treatment (no vs yes)† Number of lines of treatment (1 vs ≥3)† Number of lines of treatment (2 vs ≥3)† Resection of primary tumour (no vs yes)† Metastasectomy (no vs yes) ECOG performance status (≥1 vs 0) Alkaline phosphatase (>300 vs ≤300) WBC count (>10,000 vs ≤10,000)† Number of metastatic sites (2 vs 1) Number of metastatic sites (≥3 vs 1) †
Hazard ratio (95% CI)
p-value
2.80 (1.12–6.95) 1.76 (1.14–2.72) 1.2 (0.78–1.85)
0.027† 0.01† 0.41
1.07 (0.72–1.6) 1.37 (0.95–1.98) 1.80 (1.15–2.82) 0.78 (0.50–1.21) 1.44 (1.01–2.07) 2.25 (1.38–3.65) 1.67 (1.11–2.54) 2.05 (1.38–3.04) 1.18 (0.79–1.78) 1.27 (0.87–1.85) 1.88 (0.86–4.12) 2.29 (1.37–3.83) 0.94 (0.64–1.37) 1.11 (0.63–1.96)
0.73 0.09 0.01† 0.27 0.047† 0.001† 0.015† 2) in our cohort reported improved survival compared with those who received less chemotherapy, as Grothey et al. [39] elegantly demonstrated.
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Furthermore, we suggested that treatment with bevacizumab may correlate with OS. This is consistent with results of several randomized controlled trials and meta-analyses. Recently, a pooled analysis showed that bevacizumab with chemotherapy compared with chemotherapy alone improved outcomes in patients with mCRC [40] . Since RAS mutations are predictive biomarkers of resistance to anti-EGFR antibodies, we explored the impact of anti-EGFR treatment on prognosis of our population. In the multivariate analysis, no association of anti-EGFR treatment (no vs yes) with OS (HR: 0.78; 95% CI: 0.50–1.21) was found. This study has some limitations. It is retrospective and exploratory in nature. The analysis was conducted at a single center and the limited sample size prevents us from drawing definite conclusions. Also, our study population may be considered a little outdated: patients were treated for mCRC from 2004 to 2010. Indeed, a small proportion of patients received bevacizumab or anti-EGFR therapy. However, this could be considered a strength since the prognostic effect of the molecular characterization is not confounded with its potential negative predictive value. A Cox regression model was also applied: it showed that anti-EGFR treatment did not affect the survival in the four molecular subgroups, particularly in the all-wild-type population (p = 0.14).
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Molecular profile & survival in colorectal cancer Moreover, the four molecular subgroups were arbitrarily created: the reasons for this choice are subsequently explained. First, this retrospective analysis was conceived in early 2012 when most available studies suggested the predictive and prognostic role of KRAS codons 12 and 13 and BRAF V600E mutations. At that point, we had few data on other rare mutations such as NRAS, PIK3CA or KRAS 61-146. Therefore we decided to analyze KRAS codons 12 and 13 mutations and BRAF mutations separately from the other mutations. Second, the predictive and possibly prognostic value of RAS in colorectal cancer is extremely recent [7–8,16] . For this reason, at the time of study design, KRAS was not combined with NRAS mutations. Most importantly, we aimed to explore the prognostic role of the all-wild-type subgroup since De Roock et al. [6] showed that mCRC patients bearing tumors with no mutations reported the best outcomes. Finally, the other mutations (KRAS 61–146, NRAS and PIK3CA) Mutational status BRAF mutation KRAS mutation 12–13 only Any of KRAS 61–146, NRAS, PIK3CA mutation All wild-type (reference category)
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were very few in number, and we decided to put these together in an arbitrary group. Nevertheless, the differences in median survival in the four molecular subgroups are both biologically and clinically meaningful. Basically, the all-wild-type patients had the best prognosis while the BRAF-mutated patients had the worst outcome. Undoubtedly, the role of RAS mutations and PIK3CA mutations are worthy of further investigation. In short, mCRC patients may have different tumors and require different treatment approaches in practice. Clinical information integrated with molecular data allows us to better select patients for treatment strategy and it may help stratification in clinical trials. Conclusion Our retrospective analysis identified distinct molecular prognostic subgroups of patients. Our analysis suggests that extended mutational Median survival (months) 7.6 16.7 22 27.7
1.0
95% CI 0.5–11.7 13.6–21.7 15.9–24.4 21.4–36.6
+ with censure
Probability of survival
0.8
0.6 0.5 0.4
7.6 16.7 months months
22 months
27.7 months
0.2
0.0 0
20
40
60
80
100
Survival time (months) BRAF_mut any_KRAS_61_146_NRAS_PIK3CA
KRAS_12_13 wild_type
Figure 3. Median survival in the four molecular subgroups. Kaplan–Meier analysis. Log-rank test: p < 0.0001.
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Research Article Foltran, De Maglio, Pella et al. profiling of advanced CRC allows better understanding of tumor biology and may drive patient stratification both in clinical practice and in the design of clinical trials. Future perspective In the last 20 years, intense translational and clinical research led to the development of new targeted therapies in mCRC patients. The study of predictive and prognostic biomarkers provided useful information on tumor biology and guided treatment choice in CRC. We aimed to explore the prognostic value of an extensive molecular profile in colorectal cancer (KRAS, NRAS, BRAF, PIK3CA). Our analysis
confirmed the negative prognostic role of BRAF mutation and showed KRAS 12-13 codons mutation as a prognostic factor. Above all, the study demonstrated clinically meaningful differences of survival across the four molecular subgroups. The all-wild-type population had the longest survival while the other mutational subgroups reported inferior outcomes. Some clinical factors were found to be associated with poorer survival. Clinical variables such as right-sided colon cancer, not resected primary tumor, receiving less chemotherapy and no bevacizumab need to be further investigated. In the era of personalized medicine, it may be assumed that an accurate molecular selection of
EXECUTIVE SUMMARY Aim of the study ●●
The usefulness of a full molecular profile for treatment strategy in colorectal cancer has been shown.
●●
This study aimed to investigate the prognostic effect of extensive molecular profiling integrated with clinical factors in metastatic colorectal cancer (mCRC).
Materials & methods ●●
Socio-demographic, molecular and clinical data of 194 patients treated for mCRC were retrospectively reviewed.
●●
Point mutations were detected by pyrosequencing for KRAS codons 12, 13, 61 and 146, BRAF exon 15, NRAS codons 12, 13, 61 and PIK3CA exons 9 and 20.
●●
To explore differences of survival, four molecular subgroups were created: --
BRAF mutated;
--
KRAS-mutated codons 12 and 13;
--
Any of KRAS codons 61-146, PIK3CA or NRAS;
--
All-wild-type (reference category).
Results
BRAF mutation as well as KRAS codons 12 or 13 mutations were associated with increased risk of death compared with
●●
the all-wild-type reference group.
Some clinical variables correlated with poorer overall survival:
●● --
Lack of exposure to bevacizumab;
--
Receiving less lines of chemotherapy;
--
Having primary tumor not resected;
--
Right-sided tumor location;
--
Elevated white blood cell count (>10 × 109/l).
●●
The all-wild-type population had the longest survival (27.7 months), while BRAF mutated the shortest (7.6 months). Patients with KRAS 12-13 mutation and those with the other rarest mutations reported poorer outcome compared with the all wild-type (16.7 and 22 months, respectively).
Conclusion ●●
638
Extensive molecular profiling added to clinical factors have a prognostic impact in mCRC and require further study.
Future Oncol. (2015) 11(4)
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Molecular profile & survival in colorectal cancer CRC patients combined with clinical features allows us to better identify subgroup of patients with different prognoses and to drive selection and stratification of patients in large prospective clinical trials.
Research Article
options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or
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