IJC International Journal of Cancer

Role of NRAS mutations as prognostic and predictive markers in metastatic colorectal cancer Marta Schirripa1, Chiara Cremolini1, Fotios Loupakis1, Manfredi Morvillo1, Francesca Bergamo2, Federica Zoratto1, Lisa Salvatore1, Carlotta Antoniotti1, Federica Marmorino1, Elisa Sensi3, Cristiana Lupi3, Gabriella Fontanini3, Veronica De Gregorio1, Riccardo Giannini3, Fulvio Basolo3, Gianluca Masi1 and Alfredo Falcone1 1

Unit of Medical Oncology 2, University Hospital of Pisa, Pisa, Italy Unit of Medical Oncology 1, Oncology Institute of Veneto, Padua, Italy 3 Unit of Pathology, Department of Surgery, University Hospital of Pisa, Pisa, Italy

NRAS mutations occur in 3–5% of colorectal cancer. Differently from KRAS and BRAF mutations, the role of NRAS mutations as prognostic and predictive markers in metastatic colorectal cancer (mCRC) has been investigated to a lesser extent. A retrospective series suggested the role of NRAS mutations as predictors of resistance to anti-EGFR monoclonal antibodies (MoAbs) in chemo-refractory patients with mCRC. In our study, KRAS codons 12, 13, 61 and BRAF codon 600 mutational status were evaluated in mCRCs referred to our Institution from 2009 to 2012. NRAS codons 12, 13 and 61 mutational status was analyzed in KRAS/BRAF wt patients. We collected pathological and clinical features in the overall population and outcome data in a subset of NRAS mutated chemo-refractory patients treated with anti-EGFR MoAbs in advanced lines. NRAS was mutated in 47/786 (6%) mCRCs. NRAS and KRAS mutated tumors did not show significant differences in terms of clinical and pathological characteristics, except for a lower prevalence of mucinous histology (p 5 0.012) and lung metastases (p 5 0.012) among NRAS mutated tumors. In the uni- and multivariate model, NRAS mutations were associated with shorter overall survival (OS) compared to all wt patients (median OS 25.6 vs 42.7 months; univ: HR 5 1.91, 95% CI 1.39–3.86, p 5 0.0013; multiv: HR 5 1.75, 95% CI 1.1.3–2.72, p 5 0.013). None of the chemo-refractory NRAS mutated patients evaluable for response to anti-EGFRs achieved response. In conclusion, NRAS mutations have a relevant incidence in patients with mCRC and showed an association with specific clinical and pathological features. NRAS mutations affect mCRC patients’ prognosis and predict lack of response to anti-EGFRs.

RAS are GTPase proteins involved in signal transduction, cellular growth, differentiation, proliferation and survival1,2 and are encoded by RAS family members genes. KRAS and HRAS were firstly identified from studies of Kirsten sarcoma3 and Harvey sarcoma virus4 and then in human cancer cells.5 The discovery of NRAS came some years later in human neuroblastoma cells.6 Mutations of RAS lead to a defective GTPase activity that causes a hyperactivation of the signalling casKey words: metastatic colorectal cancer, NRAS, BRAF, KRAS, antiEGFR Abbreviations: EGFR: epidermal growth factor receptor; mCRC: metastatic colorectal cancer; MoAbs: monoclonal antibodies; OS: overall survival; PFS: progression free survival; PS: performance status; WT: wild-type; Vs: versus Additional Supporting Information may be found in the online version of this article. M.S. and C.C. contributed equally to this work. Grant sponsor: ARCO No-profit Foundation DOI: 10.1002/ijc.28955 History: Received 7 Feb 2014; Accepted 25 Apr 2014; Online 8 May 2014 Correspondence to: Fotios Loupakis, MD, PhD, Unit of Medical Oncology 2, University Hospital of Pisa, Via Roma, 67 56126 Pisa, Italy, Fax: 139050992069, E-mail: [email protected]

C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V

cade,7 determining an uncontrolled activity of the downstream effectors such as RAF proteins and MAP-Kinases. RAS mutations occur in about 20–25% of human cancers8; in colorectal cancers (CRC) KRAS mutations are found in about 40% of cases and more frequently affect codon 12 (79%) and codon 13 (17)%,9 HRAS mutations are negligible events and NRAS mutations are found in about 3–5% of CRC, more frequently located in codon 61 (60%).2,10 In the last years, KRAS mutations have been widely investigated in patients with metastatic CRC (mCRC) and are now recognized as determinants of resistance to antiepidermal growth factor receptor (EGFR) monoclonal antibodies (MoAbs).11–18 Moreover KRAS mutated tumors have been associated with specific clinical traits such as the occurrence of lung and brain metastases or right-sided primary tumors.19–21 In terms of clinical outcome, the effect of KRAS mutations, both in the adjuvant and in the metastatic setting, is still a matter of debate despite having been advocated as a mild negative prognostic marker in many studies.12,22–26 Also RAF has been well characterized as a GTP-ase protein involved in signal transduction, cellular proliferation and cancerogenesis.27,28 Activating mutations of BRAF, mainly affecting codon 600, are found in about 8% of CRCs tumors.29 In mCRC, BRAF mutation has a well-known negative prognostic impact24,30–32 and is associated with a peculiar

Cancer Genetics

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NRAS mutations as prognostic and predictive markers in mCRC

Cancer Genetics

What’s new? The RAS family of genes plays a key role in human cancers. While mutations in KRAS and BRAF have been widely studied. The clinico-pathological features and the prognostic impact of NRAS mutations are less known. Here the authors identify NRAS mutations in 6% of metastatic colorectal cancers. Clinical and pathological characteristics of NRAS-mutant tumors were similar to those with KRAS mutations. Moreover NRAS mutations functioned as reliable predictors of resistance to anti-EGFR monoclonal antibody therapy. This study highlights the importance of including analysis of NRAS mutations into the molecular characterization of metastatic colorectal tumors.

gene expression signature.33 Moreover, BRAF mutated tumors share some histopathological and clinical characteristics: they occur more frequently in elderly, females, are right-sided, mucinous, microsatellite instable and show a high rate of nodal and peritoneal metastases.19,30,34,35 Finally, several evidences underlined the lack of efficacy of anti-EGFR MoAbs in BRAF mutated tumors.36–38 Due to the possible role of BRAF mutation as a driver signal in tumor progression, many targeted strategies are under investigation for BRAF mutated tumors.39 In this complex and intriguing scenario, NRAS mutated tumors might be considered as a neglected issue, since up today they have never been characterized from a histological and clinical point of view, as well as with regard to mCRC patients’ prognosis. At the time the present study was started the major report on NRAS mutations in mCRC was a retrospective series suggesting the potential role of such mutations as predictors of resistance to anti-EGFR MoAbs in later lines of treatment in a relatively small cohort of mutated patients.36 Additional data were presented from a large (N 5 1,630) phase 3 randomized study of first-line chemotherapy plus or minus cetuximab.40 Interestingly, overall survival (OS) outcome for 38 NRAS mutated patients suggested a negative prognostic impact similar to that of KRAS mutations. Nevertheless, no details on clinical and pathological features were reported since NRAS mutations were mainly tested with the purpose of verifying their predictive role to anti-EGFR MoAbs. Therefore, we planned and conducted the present analysis in order to address the following issues concerning NRAS mutated tumors: 1. incidence in a large dataset of patients with mCRC; 2. clinical and pathological features; 3. correlation with survival and 4. correlation with response to anti-EGFR MoABs in a subset of chemo-refractory patients with mCRC.

Material and Methods Patients selection and data collection

Patients with available KRAS, BRAF and NRAS mutational status were included: KRAS codon 12, 13, 61 and BRAF codon 600 mutational status were evaluated in tissue specimens of patients with mCRC analyzed at our pathology

department from 2009 to 2012. NRAS codons 12, 13 and 61 mutational status was analyzed only in KRAS and BRAF wild-type (wt) patients. Based on their mutational status, we categorized patients as: NRAS mutated, KRAS mutated, BRAF mutated and all wt (NRAS, KRAS, BRAF wt). The following clinical and pathological features were collected: sex, ECOG performance status (PS), age at the time of diagnosis of metastatic disease, site of primary tumor (right versus (vs) left vs rectum), mucinous histology (yes vs no), depth of tumoral penetration (pT0–4), pathological involvement of locoregional lymph nodes (pN0–2), resected primary tumor (yes vs no), time to metastases (synchronous vs metachronous), metastatic sites at diagnosis. Data about first line systemic treatment were also collected. Among NRAS mutated patients, those who had received a salvage treatment with an anti-EGFR MoAb were identified and were defined as “evaluable for response” those meeting the following criteria:  irinotecan-refractory (whose disease had progressed during or within 3 months after the treatment with an irinotecanbased regimen),  evaluable disease according to RECIST,  adequate clinical re-evaluation by means of 8 weekly CT scan. Mutational status analyses

Mutational analyses were performed on primary and/or metastatic samples. KRAS codons 12, 13 (exon 2) and 61 (exon 3) and BRAF codon 600 (exon 15) were analyzed by pyrosequencing as previously reported.41 The results were analyzed with PyroMark Q24 1.0.9 software and independently evaluated by two investigators. NRAS codons 12, 13 (exon 2) and 61 (exon 3) were evaluated by means of PCR amplification and direct-sequencing in KRAS and BRAF wt samples. PCR were performed used HotStarTaq Master Mix Kit (Qiagen, Hilden, Germany) as previously reported.41 All reactions were done in duplicate. The primers used are as follows: NRAS codons 12/13: (sense) 50 -GATGTGGCTCGCCAAT TAAC-30 ; (antisense) 50 -CACTGGGGCTCACCTCTATG-30 ; the amplicon size was 183 bp. NRAS codon 61: (sense) 50 -CACCCCCAGGATTCTTAC AG-30 ; (antisense) 50 -CCCCATAAAGATTCAGAACACA-30 ; the amplicon size was 247 bp. C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V

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The samples were purified with a QIAquick PCR Purification Kit (Qiagen) and submitted to cycle sequencing with BigDye Terminator (version 3.1) Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol and with the same primers used in PCR. The sequencing products were ethanol-precipitated before running on a 3130 Genetic Analyzer (Applied Biosystems). The sequencing data were visualized by use of Sequencing Analysis (Applied Biosystems), and were independently evaluated by two investigators. Statistical analyses

Results Mutations’ frequencies

Seven hundred eighty-six patients with mCRC were included in the study population. KRAS and BRAF mutations were found in 393 (50%) and 72 (9%) patients respectively. KRAS mutations affected codon 12, 13 and 61 in 308 (78%), 70 (18%) and 16 (4%) cases, respectively. NRAS was found mutated in 47 (15%) out of 321 KRAS and BRAF wt patients. In the study population NRAS mutation rate was 6%. NRAS mutations affected codon 12, 13 and 61 in 14 (30%), 6 (13%) and 27 (57%) cases, respectively. Two hundred seventy-four (35%) patients were KRAS, BRAF and NRAS wt and therefore defined as all wt (Supporting Information Fig. 1). Association of clinical and pathological features with mutational status

Clinical characteristics according to mutational status are displayed in Table 1. A significant difference among mutational groups was identified for the following categories: age (p 5 0.02), sex (p 5 0.02), ECOG PS (p 5 0.03), primary C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V

Figure 1. OS according to mutational status.

tumor site (p < 0.0001), mucinous histology, (p 5 0.0003) and pathologic nodes’ involvement (p 5 0.043). NRAS mutated tumors were right-, left- sided or rectal in 33%, 36% and 31% of cases, respectively. The distribution of primary tumor site in NRAS mutated was not different from KRAS mutated patients, while a lower incidence of rightsided primaries was observed compared to BRAF mutated tumor (p 5 0.002). With regard to other clinical and pathological characteristics, NRAS and KRAS mutated tumors did not show significant differences except for a lower prevalence of mucinous histology in NRAS mutated tumors (NRAS vs KRAS 4% vs 26%, p 5 0.012). Mucinous histology was less frequent in NRAS mutated tumors also compared to BRAF mutated (50%, p < 0.0001) and all wt (24%, p 5 0.002). BRAF mutated tumors were more likely to occur in females in comparison to all wt (66% vs 36%, p 5 0.003) and KRAS mutated tumors (66% vs 41%, p 5 0.028) and in patients with ECOG PS 1–2 in comparison to all wt (34 vs 19%, p 5 0.015) and KRAS mutated patients (34% vs 22%, p 5 0.049). BRAF mutated tumors were more frequently right sided (64% right, 13% left, 23% rectal) also in comparison to KRAS mutated (p < 0.0001) and all wt (p < 0.0001) tumors. They were more frequently associated with mucinous histology in comparison to KRAS mutated (p 5 0.004) and all wt (p 5 0.005) tumors and showed a higher pathological involvement of locoregional lymph nodes in comparison to all wt (pN1–2: BRAF mutated 87% vs all wt 70%, p 5 0.012). Median age of all wt patients (62 years) was lower in comparison to KRAS mutated (63 years, p 5 0.005) and BRAF mutated (64 years, p 5 0.03). Patterns of metastatic spread according to mutational status are described in Table 2. NRAS and KRAS mutated patients showed rather similar metastatic sites with the exception of a higher incidence of lung metastases observed in KRAS mutated patients (NRAS vs KRAS 30% vs 36%, p 5 0.012). Moreover, lung metastases were more frequent in KRAS mutated patients compared to BRAF mutated (p 5 0.005) and all wt (p 5 0.005). BRAF mutated patients presented a higher incidence of peritoneal metastases in comparison to all wt (28% vs 13%, p 5 0.005).

Cancer Genetics

Results of KRAS, BRAF and NRAS mutational analyses were used as categorical variables (presence or absence of the mutation). Fisher’s exact test or v2 test was used when appropriate to compare clinical and biological features according to mutational status. OS was defined as the time from the diagnosis of metastatic disease to death due to any cause. In “evaluable for response” patients, progression free survival (PFS) was defined as the time from the beginning of the treatment with an anti-EGFR MoAb to disease progression or death due to any cause. OS and PFS analyses were determined according to the Kaplan–Meier method and survival curves were compared using the log-rank test. Statistical significance was set at p 5 0.05 for a bilateral test. The correlation of mutational status and clinical and pathological characteristics with survival was assessed in univariate analyses. Cox proportional hazard model was adopted in the multivariate analysis, including as covariates variables significantly correlated with survival in the univariate analyses.

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NRAS mutations as prognostic and predictive markers in mCRC

Table 1. Patients’ characteristics according to mutational status All wt (N 5 274) N (%)

Characteristics Sex

Age

ECOG PS

Primary tumor site

Mucinous histology

pT

Cancer Genetics

pN

Time to mts

Number of mts

KRAS mut (N 5 393) N (%)

NRAS mut (N 5 47) N (%)

BRAF mut (N 5 72) N (%)

p 0.020

Female

98 (36)

162 (41)

21 (45)

40 (66)

Male

176 (64)

231 (59)

26 (55)

32 (34)

Median

62

64

63

63

Range

25–88

25–88

32–84

42–86

0

222 (81)

306 (78)

31 (67)

47 (66)

1–2

52 (19)

87 (22)

16 (33)

25 (34)

Right colon

51 (19)

135 (35)

15 (33)

45 (64)

Left colon

129 (48)

161 (42)

16 (36)

9 (13)

Rectum

89 (33)

86 (23)

14 (31)

16 (23)

Na

5

11

2

2

Yes

27 (24)

44 (26)

1 (4)

20 (50)

No

84 (76)

127 (74)

26 (96)

20 (50)

Na

163

222

20

32

1–2

26 (12)

32 (10)

4 (11)

2 (3)

3–4

188 (88)

282 (90)

32 (89)

59 (97)

Tx

60

79

11

11

0

63 (30)

72 (23)

11 (31)

8 (13)

1–2

149 (70)

238 (77)

24 (69)

52 (87)

Na

62

83

12

12

Synchronous

192 (70)

286 (73)

35 (68)

55 (76)

Metachronous

82 (30)

107 (27)

15 (32)

17 (24)

1

141 (54)

166 (46)

21 (48)

32 (46)

>1

119 (46)

194 (54)

23 (52)

37 (54)

Na

14

33

3

3

0.020

0.030

Role of NRAS mutations as prognostic and predictive markers in metastatic colorectal cancer.

NRAS mutations occur in 3-5% of colorectal cancer. Differently from KRAS and BRAF mutations, the role of NRAS mutations as prognostic and predictive m...
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