Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy

Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by... Clinical Studies British Journal of Cancer (2007) 96, 1166 – 1169 & 2007 Cancer Research UK All rights reserved 0007 – 0920/07 $30.00 www.bjcancer.com Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy ,1 3 2 2,3 3 4 5 2 F Di Fiore , F Blanchard , F Charbonnier , F Le Pessot , A Lamy , MP Galais , L Bastit , A Killian , 2 2,6 7 1 2,3 2,6 1 2 R Sesbou¨e´ , JJ Tuech , AM Queuniet , B Paillot , JC Sabourin , F Michot , P Michel and T Frebourg Digestive Oncology Unit, Department of Hepato-Gastroenterology, Rouen University Hospital, Northwest Canceropole, France and Inserm U614, Faculty 2 3 of Medicine, Northwest Canceropole, Rouen, France; Inserm U614, Faculty of Medicine, Northwest Canceropole, Rouen, France; Department of Pathology, Rouen University Hospital, Northwest Canceropole, France; Digestive Oncology Unit, Department of Hepato-Gastroenterology, Caen University Hospital and Francois Baclesse Centre, Caen, Northwest Canceropole, France; Oncology Unit, St Hilaire Medical Centre, Rouen, France; 6 7 Department of Surgery, Rouen University Hospital, Northwest Canceropole, France; Digestive Oncology Unit, Department of Hepato-Gastroenterology, Elbeuf Hospital, France The predictive value of KRAS mutation in metastatic colorectal cancer (MCRC) patients treated with cetuximab plus chemotherapy has recently been suggested. In our study, 59 patients with a chemotherapy-refractory MCRC treated with cetuximab plus chemotherapy were included and clinical response was evaluated according to response evaluation criteria in solid tumours (RECIST). Tumours were screened for KRAS mutations using first direct sequencing, then two sensitive methods based on SNaPshot and PCR- ligase chain reaction (LCR) assays. Clinical response was evaluated according to gene mutations using the Fisher exact test. Times to progression (TTP) were calculated using the Kaplan–Meier method and compared with log-rank test. A KRAS mutation was detected in 22 out of 59 tumours and, in six cases, was missed by sequencing analysis but detected using the SNaPshot and PCR-LCR assays. Remarkably, no KRAS mutation was found in the 12 patients with clinical response. KRAS mutation was associated with disease progression (P ¼ 0.0005) and TTP was significantly decreased in mutated KRAS patients (3 vs 5.5 months, P ¼ 0.015). Our study confirms that KRAS mutation is highly predictive of a non-response to cetuximab plus chemotherapy in MCRC and highlights the need to use sensitive molecular methods, such as SNaPshot or PCR-LCR assays, to ensure an efficient mutation detection. British Journal of Cancer (2007) 96, 1166–1169. doi:10.1038/sj.bjc.6603685 www.bjcancer.com Published online 20 March 2007 & 2007 Cancer Research UK Keywords: colorectal cancer; EGFR; KRAS; molecular markers; mutation Colorectal cancer is one of the most common tumours and a major in MCRC. The first study, performed in 31 MCRC patients, showed cause of cancer death worldwide. The median overall survival of that an EGFR gene copy number increase was associated with a patients with a metastatic colorectal cancer (MCRC) has increased clinical response to anti-EGFR agents and that mutation of KRAS, from 12 months to approximately 20 months in the past decade downstream of EGF signaling, did not correlate with treatment (Douillard et al, 2003; Meyerhardt and Mayer, 2005; Saunders and sensitivity (Moroni et al, 2005). In contrast, the second study Iveson, 2006). This dramatic improvement was mainly due to the including 30 MCRC patients reported that KRAS mutation was introduction of both active new chemotherapeutic agents and highly predictive of tumour resistance to cetuximab (Lievre et al, novel targeted drugs. The rationale of targeted therapies is to 2006). inhibit biological pathways and key molecules involved in tumour The aim of the present study was to determine the clinical growth and progression. In CRC, the novel therapies that are relevance of KRAS mutation detection in MCRC patients treated currently used target the vascular endothelial growth factor and with cetuximab. epidermal growth factor (EGF) signaling pathways (Kabbinavar et al, 2003; Hurwitz et al, 2004; Saltz et al, 2004). The variability of the MCRC clinical response to anti-EGFR agents has highlighted MATERIALS AND METHODS the urgent need to identify reliable markers with a predictive value Patients to select the appropriate patients who can benefit from these treatments. Essential in this context are two recent studies (Moroni s Patients with an MCRC treated with cetuximab (Erbitux , Merck, et al, 2005; Lievre et al, 2006) reporting the characterization of Lyon, France) between April 2004 and December 2005 and for molecular markers predictive of anti-EGFR antibodies sensitivity whom tumour DNA was available were included. All patients had previously received at least one chemotherapy regimen for MCRC. *Correspondence: Dr F Di Fiore; E-mail: frederic.di-fiore@chu-rouen.fr Cetuximab regimen was associated either with irinotecan or with Received 23 October 2006; revised 19 February 2007; accepted 19 oxaliplatin. Tumour response was evaluated according to the February 2007; published online 20 March 2007 response evaluation criteria in solid tumours (Therasse et al, KRAS mutational status and clinical response to cetuximab F Di Fiore et al 0 0 2000). Patient’s tumour response to cetuximab was classified as specific upstream primer 5 -TTGTGGTAGTTGGAGCTGA-3 (c.35 0 0 complete response (CR), partial response (PR), stable disease (SD) G4A; p.G12D), or 5 -GTTGTGGTAGTTGGAGCTGC-3 (c.35 0 0 or progressive disease (PD). G4C; p.G12A), or 5 -TTGTGGTAGTTGGAGCTGT-3 (c.35G4T; p.G12V) and the common downstream primer 5 -TGGCGTAGGC AAGAGTGCC-3 ; the c.38 nucleotide using the specific upstream DNA extraction 0 0 primer 5 -TGGTAGTTGGAGCTGGTGA-3 (c.38 G4A; p.G13D) 0 0 DNA was extracted from paraffin-embedded tumour tissue and the downstream primer 5 -CGTAGGCAAGAGTGCCTTGAC-3 . samples using the DNA extraction kits from Takara (Madison, Upstream primers contain at their 5 end the M13F additional 0 0 WI, USA) or Ambion (Huntingdon, Cambridgeshire, UK), sequence (5 -ACTGTAAAACGACGGCCAGTGT-3 ) and down- according to the manufacturer’s instructions. Among the 59 stream primers at their 3 end the M13R additional sequence 0 0 patients analysed, DNA was extracted from the primary tumour (5 -TGGTCATAGCTGTTTCCTGCA-3 ). Upstream primers were 0 0 in 53 cases and from metastases in the six remaining cases. 5 -6FAM labelled and the downstream primers were 5 phosphory- lated. Ligase chain reaction reactions were performed in a final volume of 12.5ml containing 2 U of Pfu DNA ligase (Stratagene, la Sequencing analysis Jolla, CA, USA), and 1.25mM of each primer. After denaturation at KRAS exon 2 was PCR-amplified from tumour DNA using the 951C for 20 s, 40 two-steps cycles of 941C for 10 s alternating with following sense and antisense primers: 5 -AAGGCCTGCTGAAAA 651C for 2 min were performed. Ligation products were analysed 0 0 0 TGACTG-3 and 5 -CAAAGAATGGTCCTGCACCAG-3 . After on an ABI Prism 3100 DNA sequencer and the Gene Scan V3.7.1 purification using the gel extraction kit from Qiagen (Courtaboeuf, (Applied Biosystems). For each sample analysed, PCR-LCR was France), PCR products were sequenced using the Big Dye V3.1 performed twice. Terminator Kit (Applied Biosystems, Foster City, CA, USA) and an ABI Prism 377 or 3100 DNA sequencer (Applied Biosystems). Statistical analysis Considering the presence of non malignant cells in tumour samples, the presence of an heterozygous KRAS mutation in the Response to treatment according the mutational status was tumour was defined as the appearance of a mutant peak with an evaluated using the Fisher exact test. Patients with CR or PR or height of at least one-third of that of the wild type. All sequencing SD were considered as patients with controlled disease (CD). A analyses were performed at least twice on two independent PCRs. P-value equal or o0.05 was considered to indicate statistical significance. The time to progression (TTP) was calculated as the period from the beginning of treatment to the first SNaPshot multiplex assay observation of disease progression or to death. The TTP were After purification using gel extraction kit, PCR-amplified KRAS estimated using the Kaplan–Meier method and compared with exon 2 was analysed for the presence of KRAS mutations at the log-rank test. nucleotides c.34, c.35, c.37 and c.38, using the ABI PRISM SNaPshot Multiplex kit (Applied Biosystems, Foster City, CA, RESULTS USA) and four primers including at their 5 end, an additional tail allowing their simultaneous detection. The sequences of the sense A total of 59 patients were assessed in the present study. primers allowing the extension at nucleotides c.34, c.35, c.37 and After 3 months of treatment, 31 patients (52.5%) had a CD, 12 0 0 0 c.38 were, respectively, 5 -AACTTGTGGTAGTTGGAGCT-3 ,5 -N patients (20.3%) had a CR or PR (2 and 10 patients, respectively) 0 0 ACTTGTGGTAGTTGGAGCTG-3,5 -N TTGTGGTAGTTGGAG and 19 (32.2%) had an SD. The TTP in patients with CD was 6 0 0 0 CTGGT-3 and 5 -N TGTGGTAGTTGGAGCTGGTG-3 (N months, as compared to 3 months, in patients with PD indicating the additional nucleotides). The multiplex SNaPshot (Po0.0001). reaction was performed in a final volume of 10ml, containing one- fifth of the PCR reaction, 2.5ml of the SNaPshot Multiplex Ready Reaction Mix, 1ml of sequencing buffer from the Big Dye V3.1 KRAS mutation and response to treatment with cetuximab Terminator Kit and SNaPshot primers at a concentration of 0.02– plus chemotherapy 0.05mM. Cycling conditions were 25 cycles of rapid thermal ramp We detected a KRAS mutation by sequencing analysis of DNA to 961C, 961C for 10 s; rapid thermal ramp to 501C, 501C for 5 s; extracted from tumour sample in 16 out of 59 (27%) patients and rapid thermal ramp to 601C and 601C for 30 s. SNaPshot (Table 1). Among the 16 patients harbouring a somatic KRAS products were then treated 1 h at 371C with 3 U of shrimp alkaline mutation, 13 had a PD and three had an SD. Remarkably, no KRAS phosphatase (Amersham Biosciences/GE Healthcare Europe mutation was found in the 12 patients with CR or PR. Considering GmbH, Saclay, France). After heat inactivation of the alkaline that the genetic heterogeneity of tumours may hamper the phosphatase 15 min at 751C, labelled products were separated using a 25 min run on an ABI Prism 3130 DNA sequencer and data were analysed using the GeneMapper Analysis Software version 4.0 (Applied Biosystems). Table 1 KRAS mutations and response status to cetuximab-based chemotherapy in 59 MCRC patients PCR-LCR KRAS exon 2 was PCR-amplified using the sense primer 5 - Controlled disease AAGGTACTGGTGGAGTATTTGATAGTG-3 and the antisense 0 0 Complete Partial Stable Progressive primer 5 -TGTTGGATCATATTCGTCCACAAAA-3 . Ligase chain reaction (LCR) was then performed, as described by Shi et al response response disease disease (2004), on PCR-amplified exon 2 of KRAS, after purification using KRAS mutation the Qiagen gel extraction kit. The c.34 nucleotide was explored 0 Present 0 0 3 (5) 13 (17) using the specific upstream primer 5 -AACTTGTGGTAGTTGGA Absent 2 (2) 10 (10) 16 (14) 15 (11) 0 0 GATA-3 (c.34 G4A, p.G12S), or 5 -ACTTGTGGTAGTTGGAG ATT-3 (c.34 G4T, p.G12C) and the common downstream primer Numbers in brackets correspond to the corrected numbers of patients when 0 0 sequencing analysis was completed by SNaPshot and PCR – LCR assays. 5 -GTGGCGTAGGCAAGAGTGC-3 ; the c.35 nucleotide using the & 2007 Cancer Research UK British Journal of Cancer (2007) 96(8), 1166 – 1169 Clinical Studies Clinical Studies KRAS mutational status and clinical response to cetuximab F Di Fiore et al relationship between KRAS status and sensitivity to anti-EGFR AB c.34G monoclonal antibodies had not been found previously by Moroni et al (2005) in a series of 31 patients. This discrepancy might c.35G probably be explained, at least in part, by the limited number of c.37G c.38G patients in these latter series. Direct sequencing allowed us to detect a KRAS mutation in 16 out of 59 patients (27%) with MCRC, c.35T and among the 43 patients without detectable KRAS mutations, Control DNA 15 presented a PD. We hypothesized that we missed some KRAS c.35 mutations by direct sequencing of tumour DNA, since malignant tumours are genetically heterogeneous. Furthermore, it is im- portant to highlight that our study was based on paraffin- embedded tumours from which it is more difficult to obtain c.35C high-quality DNA. Using two independent sensitive methods, respectively, based on SNaPshot and PCR-LCR assays specifically Tumour 12292 designed to detect KRAS mutation, we detected additional c.35T mutations in two patients with SD, four with PD but none in 11 with CR/PR. This demonstrates the need to use highly sensitive molecular techniques to ensure detection in tumours of mutations Figure 1 Detection by SNaPShot and PCR-LCR assays of KRAS conferring resistance to treatments. Considering the heterogeneity mutations not detected by direct sequencing. Direct sequencing of KRAS of tumour cells, sampling tissue is particularly important. We exon 2 from control DNA and tumour 12292. The black arrow indicates determined using SnaPshot assay the KRAS status in two to three the nucleotide c.35 (A). SNaPShot detection of the c.35G4T (p.G12V) different areas of three tumours including two with KRAS mutation in tumour 12292. Each peak corresponds to a specific extended mutation. These analyses showed that the results did not differ primer. The red arrows indicates the peak specific of the c.35G4T according to the site of the analysis. The absence of KRAS mutation (B). PCR-LCR analysis of tumour 12292, using a dye-labelled mutation in 11 out of 28 patients with PD in our series is probably primer specific for the mutant c.35G4C (p.G12A) or c.35G4T (p.G12V) KRAS allele. The arrow indicates the peak specific of the c.35G4T explained by the fact that KRAS mutation is not the only genetic mutation (C). Note that the c.35G4T mutation detected by both the alteration conferring resistance to anti-EGFR antibodies. Indeed SNaPShot and PCR-LCR assays cannot be clearly detected by sequencing somatic alterations hitting other downstream effectors of the EGFR analysis alone. transduction cascade, such as RAF, MEK or ERK, may have a similar effect. In conclusion, these results should prompt further studies on larger MCRC series to definitely establish the clinical relevance detection by direct sequencing of heterozygous mutations present of KRAS mutation detection in anti-EGFR antibodies based on in a small fraction of tumour cells, we screened the tumours chemotherapy. They also highlight the need to use sensitive without detectable KRAS mutations, using two sensitive methods molecular methods to detect mutations conferring resistance and able to detect specifically KRAS exon 2 mutations. We developed a the two assays presented in this study should facilitate the multiplex SNaPshot assay based on primer extension able to detect detection of KRAS mutations in CRC, on a routine basis. A major simultaneously in a single tube the different KRAS mutations and a criticism that should be made to all studies on predictive markers fluorescent PCR-LCR assay. These two analyses were performed of clinical response to anti-EGFR agents in MCRC, including ours, in 11 out of 12 CR/PR patients, in 15 out of 16 patients with SD and is that the clinical response is evaluated on the metastatic disease in the 15 PD patients, in whom direct sequencing from tumour whereas the presence of the molecular marker is assessed from the DNA had revealed no mutation. Five additional KRAS mutations primary tumour. In the present study we had the opportunity to were detected by both methods (Figure 1) and one mutation was compare the KRAS mutational status between primary tumour and detected only by PCR-LCR assay. These six additional mutations metastases in five patients whom samples were available. In these were found in two SD and four PD patients (Table 1). SNaPshot five patients, SnaPshot assay had indicated that a KRAS mutation and PCR-LCR assays confirmed the absence of KRAS mutations in was present in one case and absent in the remaining five cases. For the CR/PR patients. In this series of 59 MCRC, sequencing analysis these five patients, analysis of the corresponding metastatic site completed by SNaPshot multiplex and PCR-LCR assays led, showed that the KRAS mutation status was identical between the therefore, to the detection of a KRAS mutation in 22 samples primary tumour and metastases. Considering the genetic evolution (37%). The presence of KRAS mutation was in this series of metastases compared to primary tumour, we think nevertheless significantly associated with PD (P ¼ 0.0005). The predictive value that it will be important in the future to screen directly metastases of KRAS mutation for PD could be estimated to 77.2%, and the for the presence of alterations conferring either sensitivity or sensitivity and specificity of KRAS mutation for progression to resistance to these targeted therapies. treatment (CD vs PD) to 60.7 and 83.8%, respectively. The TTP was significantly decreased in patients harbouring KRAS mutation as compared to those without detectable mutation (3 vs 5.5 months, P ¼ 0.015). ACKNOWLEDGEMENTS We are indebted to Dr Scott, Dr De Puyfontaine, Dr Lafay, Dr DISCUSSION Jacob, Dr Elfadel, Dr Chasle, Dr Arsene, Dr Rousselot, Dr Piot, This study performed on 59 MCRC patients confirms that the Dr Damour, Dr Moran, Dr Bokanowski, Dr Ducastelle, Dr Anger, presence of KRAS mutation in tumour is highly predictive of a Dr Paoletti, Dr Petit, Dr Gallet, Dr El Amarti, Dr Patrier, Dr non-response to treatment based on cetuximab plus chemo- Roquet, Dr Albin, Dr Lepille, Dr Bonnafous, Dr Barhoum, Dr therapy, as shown previously in a series of 30 patients (Lievre Toulemonde, Dr Gomez, Dr Randrianarivelo, Dr Demmane and et al, 2006). It is important to highlight that, in our series, the Ms Delannoy for providing clinical information, and also Professor proportions of CR/PR, SD and PD patients were 20.3, 32.2 and Mario Tosi for critical review of the paper. This work was 47.5%, respectively, and this distribution is similar to that reported supported by a grant from the French National Institute of Cancer in the randomised cetuximab trial (Cunningham et al, 2004). The (INCA) to the North-West Canceropole. British Journal of Cancer (2007) 96(8), 1166 – 1169 & 2007 Cancer Research UK KRAS mutational status and clinical response to cetuximab F Di Fiore et al REFERENCES Cunningham D, Humblet Y, Siena S, Khayat D, Bleiberg H, Santoro A, Bets Meyerhardt JA, Mayer RJ (2005) Systemic therapy for colorectal cancer. D, Mueser M, Harstrick A, Verslype C, Chau I, Van Cutsem E (2004) N Engl J Med 352: 476–487 Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan- Moroni M, Veronese S, Benvenuti S, Artale S, Bardelli A, Siena S (2005) refractory metastatic colorectal cancer. N Engl J Med 351: 337–345 Gene copy number for epidermal growth factor receptor (EGFR) and Douillard JY, Sobrero A, Carnaghi C, Comella P, Diaz-Rubio E, Santoro A, clinical response to antiEGFR treatment in colorectal cancer: a cohort Van Cutsem E (2003) Metastatic colorectal cancer: integrating irinotecan study. Lancet Oncol 6: 279–286 into combination and sequential chemotherapy. Ann Oncol 14(Suppl 2): Saltz LB, Meropol NJ, Loehrer PJ, Needle MN, Kopit J, Mayer RJ (2004) ii7 –ii12 Phase II trial of cetuximab in patients with refractory colorectal cancer Hurwitz H, Fehrenbacher I, Novotny W, Cartwright T, Hainsworth J, Heim that expresses the epidermal growth factor receptor. J Clin Oncol 22: W, Berlin J, Baron A, Griffing S, Holmgren E, Ferrara N, Fyfe G, Rogers 1201– 1208 B, Ross R, Kabbinavar F (2004) Bevacizumab plus irinotecan, Saunders M, Iveson T (2006) Management of advanced colorectal cancer: fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J state of the art. Br J Cancer 95: 131–138 Med 350: 2335–2342 Shi C, Eshleman SH, Jones D, Fukushima N, Hua L, Parker AR, Yeo CJ, Kabbinavar F, Hurwitz HI, Fehrenbacher I, Meropol NJ, Novotny WF, Hruban RH, Goggins MG, Eshleman JR (2004) LigAmp for sensitive Lieberman G, Griffing S, Bergsland E (2003) Phase II, randomized trial detection of single-nucleotide differences. Nat Methods 1: 141–147 comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/ Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, LV alone in patients with metastatic colorectal cancer. J Clin Oncol 21: Rubinstein L, Veirweij J, Van Glabbeke M, van Oosterom AT, 60–65 Christian MC, Gwyther SG (2000) New guidelines to evaluate the Lievre A, Bachet JB, Le Corre D, Boige V, Landi B, Emile JF, Cote JF, response to treatment in solid tumours: European Organization for Tomasic G, Penna C, Ducreux M, Rougier P, Penault-Llorca F, Laurent- Research and Treatment of Cancer, National Cancer Institute of the Puig P (2006) KRAS mutation status is predictive of response to United States, National Cancer Institute of Canada. J Natl Cancer Inst 92: cetuximab therapy in colorectal cancer. Cancer Res 66: 3992 –3995 205–216 & 2007 Cancer Research UK British Journal of Cancer (2007) 96(8), 1166 – 1169 Clinical Studies http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png British Journal of Cancer Springer Journals

Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy

Loading next page...
 
/lp/springer-journals/clinical-relevance-of-kras-mutation-detection-in-metastatic-colorectal-ubjDWizsDQ

References (24)

Publisher
Springer Journals
Copyright
Copyright © 2007 by The Author(s)
Subject
Biomedicine; Biomedicine, general; Cancer Research; Epidemiology; Molecular Medicine; Oncology; Drug Resistance
ISSN
0007-0920
eISSN
1532-1827
DOI
10.1038/sj.bjc.6603685
Publisher site
See Article on Publisher Site

Abstract

Clinical Studies British Journal of Cancer (2007) 96, 1166 – 1169 & 2007 Cancer Research UK All rights reserved 0007 – 0920/07 $30.00 www.bjcancer.com Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy ,1 3 2 2,3 3 4 5 2 F Di Fiore , F Blanchard , F Charbonnier , F Le Pessot , A Lamy , MP Galais , L Bastit , A Killian , 2 2,6 7 1 2,3 2,6 1 2 R Sesbou¨e´ , JJ Tuech , AM Queuniet , B Paillot , JC Sabourin , F Michot , P Michel and T Frebourg Digestive Oncology Unit, Department of Hepato-Gastroenterology, Rouen University Hospital, Northwest Canceropole, France and Inserm U614, Faculty 2 3 of Medicine, Northwest Canceropole, Rouen, France; Inserm U614, Faculty of Medicine, Northwest Canceropole, Rouen, France; Department of Pathology, Rouen University Hospital, Northwest Canceropole, France; Digestive Oncology Unit, Department of Hepato-Gastroenterology, Caen University Hospital and Francois Baclesse Centre, Caen, Northwest Canceropole, France; Oncology Unit, St Hilaire Medical Centre, Rouen, France; 6 7 Department of Surgery, Rouen University Hospital, Northwest Canceropole, France; Digestive Oncology Unit, Department of Hepato-Gastroenterology, Elbeuf Hospital, France The predictive value of KRAS mutation in metastatic colorectal cancer (MCRC) patients treated with cetuximab plus chemotherapy has recently been suggested. In our study, 59 patients with a chemotherapy-refractory MCRC treated with cetuximab plus chemotherapy were included and clinical response was evaluated according to response evaluation criteria in solid tumours (RECIST). Tumours were screened for KRAS mutations using first direct sequencing, then two sensitive methods based on SNaPshot and PCR- ligase chain reaction (LCR) assays. Clinical response was evaluated according to gene mutations using the Fisher exact test. Times to progression (TTP) were calculated using the Kaplan–Meier method and compared with log-rank test. A KRAS mutation was detected in 22 out of 59 tumours and, in six cases, was missed by sequencing analysis but detected using the SNaPshot and PCR-LCR assays. Remarkably, no KRAS mutation was found in the 12 patients with clinical response. KRAS mutation was associated with disease progression (P ¼ 0.0005) and TTP was significantly decreased in mutated KRAS patients (3 vs 5.5 months, P ¼ 0.015). Our study confirms that KRAS mutation is highly predictive of a non-response to cetuximab plus chemotherapy in MCRC and highlights the need to use sensitive molecular methods, such as SNaPshot or PCR-LCR assays, to ensure an efficient mutation detection. British Journal of Cancer (2007) 96, 1166–1169. doi:10.1038/sj.bjc.6603685 www.bjcancer.com Published online 20 March 2007 & 2007 Cancer Research UK Keywords: colorectal cancer; EGFR; KRAS; molecular markers; mutation Colorectal cancer is one of the most common tumours and a major in MCRC. The first study, performed in 31 MCRC patients, showed cause of cancer death worldwide. The median overall survival of that an EGFR gene copy number increase was associated with a patients with a metastatic colorectal cancer (MCRC) has increased clinical response to anti-EGFR agents and that mutation of KRAS, from 12 months to approximately 20 months in the past decade downstream of EGF signaling, did not correlate with treatment (Douillard et al, 2003; Meyerhardt and Mayer, 2005; Saunders and sensitivity (Moroni et al, 2005). In contrast, the second study Iveson, 2006). This dramatic improvement was mainly due to the including 30 MCRC patients reported that KRAS mutation was introduction of both active new chemotherapeutic agents and highly predictive of tumour resistance to cetuximab (Lievre et al, novel targeted drugs. The rationale of targeted therapies is to 2006). inhibit biological pathways and key molecules involved in tumour The aim of the present study was to determine the clinical growth and progression. In CRC, the novel therapies that are relevance of KRAS mutation detection in MCRC patients treated currently used target the vascular endothelial growth factor and with cetuximab. epidermal growth factor (EGF) signaling pathways (Kabbinavar et al, 2003; Hurwitz et al, 2004; Saltz et al, 2004). The variability of the MCRC clinical response to anti-EGFR agents has highlighted MATERIALS AND METHODS the urgent need to identify reliable markers with a predictive value Patients to select the appropriate patients who can benefit from these treatments. Essential in this context are two recent studies (Moroni s Patients with an MCRC treated with cetuximab (Erbitux , Merck, et al, 2005; Lievre et al, 2006) reporting the characterization of Lyon, France) between April 2004 and December 2005 and for molecular markers predictive of anti-EGFR antibodies sensitivity whom tumour DNA was available were included. All patients had previously received at least one chemotherapy regimen for MCRC. *Correspondence: Dr F Di Fiore; E-mail: frederic.di-fiore@chu-rouen.fr Cetuximab regimen was associated either with irinotecan or with Received 23 October 2006; revised 19 February 2007; accepted 19 oxaliplatin. Tumour response was evaluated according to the February 2007; published online 20 March 2007 response evaluation criteria in solid tumours (Therasse et al, KRAS mutational status and clinical response to cetuximab F Di Fiore et al 0 0 2000). Patient’s tumour response to cetuximab was classified as specific upstream primer 5 -TTGTGGTAGTTGGAGCTGA-3 (c.35 0 0 complete response (CR), partial response (PR), stable disease (SD) G4A; p.G12D), or 5 -GTTGTGGTAGTTGGAGCTGC-3 (c.35 0 0 or progressive disease (PD). G4C; p.G12A), or 5 -TTGTGGTAGTTGGAGCTGT-3 (c.35G4T; p.G12V) and the common downstream primer 5 -TGGCGTAGGC AAGAGTGCC-3 ; the c.38 nucleotide using the specific upstream DNA extraction 0 0 primer 5 -TGGTAGTTGGAGCTGGTGA-3 (c.38 G4A; p.G13D) 0 0 DNA was extracted from paraffin-embedded tumour tissue and the downstream primer 5 -CGTAGGCAAGAGTGCCTTGAC-3 . samples using the DNA extraction kits from Takara (Madison, Upstream primers contain at their 5 end the M13F additional 0 0 WI, USA) or Ambion (Huntingdon, Cambridgeshire, UK), sequence (5 -ACTGTAAAACGACGGCCAGTGT-3 ) and down- according to the manufacturer’s instructions. Among the 59 stream primers at their 3 end the M13R additional sequence 0 0 patients analysed, DNA was extracted from the primary tumour (5 -TGGTCATAGCTGTTTCCTGCA-3 ). Upstream primers were 0 0 in 53 cases and from metastases in the six remaining cases. 5 -6FAM labelled and the downstream primers were 5 phosphory- lated. Ligase chain reaction reactions were performed in a final volume of 12.5ml containing 2 U of Pfu DNA ligase (Stratagene, la Sequencing analysis Jolla, CA, USA), and 1.25mM of each primer. After denaturation at KRAS exon 2 was PCR-amplified from tumour DNA using the 951C for 20 s, 40 two-steps cycles of 941C for 10 s alternating with following sense and antisense primers: 5 -AAGGCCTGCTGAAAA 651C for 2 min were performed. Ligation products were analysed 0 0 0 TGACTG-3 and 5 -CAAAGAATGGTCCTGCACCAG-3 . After on an ABI Prism 3100 DNA sequencer and the Gene Scan V3.7.1 purification using the gel extraction kit from Qiagen (Courtaboeuf, (Applied Biosystems). For each sample analysed, PCR-LCR was France), PCR products were sequenced using the Big Dye V3.1 performed twice. Terminator Kit (Applied Biosystems, Foster City, CA, USA) and an ABI Prism 377 or 3100 DNA sequencer (Applied Biosystems). Statistical analysis Considering the presence of non malignant cells in tumour samples, the presence of an heterozygous KRAS mutation in the Response to treatment according the mutational status was tumour was defined as the appearance of a mutant peak with an evaluated using the Fisher exact test. Patients with CR or PR or height of at least one-third of that of the wild type. All sequencing SD were considered as patients with controlled disease (CD). A analyses were performed at least twice on two independent PCRs. P-value equal or o0.05 was considered to indicate statistical significance. The time to progression (TTP) was calculated as the period from the beginning of treatment to the first SNaPshot multiplex assay observation of disease progression or to death. The TTP were After purification using gel extraction kit, PCR-amplified KRAS estimated using the Kaplan–Meier method and compared with exon 2 was analysed for the presence of KRAS mutations at the log-rank test. nucleotides c.34, c.35, c.37 and c.38, using the ABI PRISM SNaPshot Multiplex kit (Applied Biosystems, Foster City, CA, RESULTS USA) and four primers including at their 5 end, an additional tail allowing their simultaneous detection. The sequences of the sense A total of 59 patients were assessed in the present study. primers allowing the extension at nucleotides c.34, c.35, c.37 and After 3 months of treatment, 31 patients (52.5%) had a CD, 12 0 0 0 c.38 were, respectively, 5 -AACTTGTGGTAGTTGGAGCT-3 ,5 -N patients (20.3%) had a CR or PR (2 and 10 patients, respectively) 0 0 ACTTGTGGTAGTTGGAGCTG-3,5 -N TTGTGGTAGTTGGAG and 19 (32.2%) had an SD. The TTP in patients with CD was 6 0 0 0 CTGGT-3 and 5 -N TGTGGTAGTTGGAGCTGGTG-3 (N months, as compared to 3 months, in patients with PD indicating the additional nucleotides). The multiplex SNaPshot (Po0.0001). reaction was performed in a final volume of 10ml, containing one- fifth of the PCR reaction, 2.5ml of the SNaPshot Multiplex Ready Reaction Mix, 1ml of sequencing buffer from the Big Dye V3.1 KRAS mutation and response to treatment with cetuximab Terminator Kit and SNaPshot primers at a concentration of 0.02– plus chemotherapy 0.05mM. Cycling conditions were 25 cycles of rapid thermal ramp We detected a KRAS mutation by sequencing analysis of DNA to 961C, 961C for 10 s; rapid thermal ramp to 501C, 501C for 5 s; extracted from tumour sample in 16 out of 59 (27%) patients and rapid thermal ramp to 601C and 601C for 30 s. SNaPshot (Table 1). Among the 16 patients harbouring a somatic KRAS products were then treated 1 h at 371C with 3 U of shrimp alkaline mutation, 13 had a PD and three had an SD. Remarkably, no KRAS phosphatase (Amersham Biosciences/GE Healthcare Europe mutation was found in the 12 patients with CR or PR. Considering GmbH, Saclay, France). After heat inactivation of the alkaline that the genetic heterogeneity of tumours may hamper the phosphatase 15 min at 751C, labelled products were separated using a 25 min run on an ABI Prism 3130 DNA sequencer and data were analysed using the GeneMapper Analysis Software version 4.0 (Applied Biosystems). Table 1 KRAS mutations and response status to cetuximab-based chemotherapy in 59 MCRC patients PCR-LCR KRAS exon 2 was PCR-amplified using the sense primer 5 - Controlled disease AAGGTACTGGTGGAGTATTTGATAGTG-3 and the antisense 0 0 Complete Partial Stable Progressive primer 5 -TGTTGGATCATATTCGTCCACAAAA-3 . Ligase chain reaction (LCR) was then performed, as described by Shi et al response response disease disease (2004), on PCR-amplified exon 2 of KRAS, after purification using KRAS mutation the Qiagen gel extraction kit. The c.34 nucleotide was explored 0 Present 0 0 3 (5) 13 (17) using the specific upstream primer 5 -AACTTGTGGTAGTTGGA Absent 2 (2) 10 (10) 16 (14) 15 (11) 0 0 GATA-3 (c.34 G4A, p.G12S), or 5 -ACTTGTGGTAGTTGGAG ATT-3 (c.34 G4T, p.G12C) and the common downstream primer Numbers in brackets correspond to the corrected numbers of patients when 0 0 sequencing analysis was completed by SNaPshot and PCR – LCR assays. 5 -GTGGCGTAGGCAAGAGTGC-3 ; the c.35 nucleotide using the & 2007 Cancer Research UK British Journal of Cancer (2007) 96(8), 1166 – 1169 Clinical Studies Clinical Studies KRAS mutational status and clinical response to cetuximab F Di Fiore et al relationship between KRAS status and sensitivity to anti-EGFR AB c.34G monoclonal antibodies had not been found previously by Moroni et al (2005) in a series of 31 patients. This discrepancy might c.35G probably be explained, at least in part, by the limited number of c.37G c.38G patients in these latter series. Direct sequencing allowed us to detect a KRAS mutation in 16 out of 59 patients (27%) with MCRC, c.35T and among the 43 patients without detectable KRAS mutations, Control DNA 15 presented a PD. We hypothesized that we missed some KRAS c.35 mutations by direct sequencing of tumour DNA, since malignant tumours are genetically heterogeneous. Furthermore, it is im- portant to highlight that our study was based on paraffin- embedded tumours from which it is more difficult to obtain c.35C high-quality DNA. Using two independent sensitive methods, respectively, based on SNaPshot and PCR-LCR assays specifically Tumour 12292 designed to detect KRAS mutation, we detected additional c.35T mutations in two patients with SD, four with PD but none in 11 with CR/PR. This demonstrates the need to use highly sensitive molecular techniques to ensure detection in tumours of mutations Figure 1 Detection by SNaPShot and PCR-LCR assays of KRAS conferring resistance to treatments. Considering the heterogeneity mutations not detected by direct sequencing. Direct sequencing of KRAS of tumour cells, sampling tissue is particularly important. We exon 2 from control DNA and tumour 12292. The black arrow indicates determined using SnaPshot assay the KRAS status in two to three the nucleotide c.35 (A). SNaPShot detection of the c.35G4T (p.G12V) different areas of three tumours including two with KRAS mutation in tumour 12292. Each peak corresponds to a specific extended mutation. These analyses showed that the results did not differ primer. The red arrows indicates the peak specific of the c.35G4T according to the site of the analysis. The absence of KRAS mutation (B). PCR-LCR analysis of tumour 12292, using a dye-labelled mutation in 11 out of 28 patients with PD in our series is probably primer specific for the mutant c.35G4C (p.G12A) or c.35G4T (p.G12V) KRAS allele. The arrow indicates the peak specific of the c.35G4T explained by the fact that KRAS mutation is not the only genetic mutation (C). Note that the c.35G4T mutation detected by both the alteration conferring resistance to anti-EGFR antibodies. Indeed SNaPShot and PCR-LCR assays cannot be clearly detected by sequencing somatic alterations hitting other downstream effectors of the EGFR analysis alone. transduction cascade, such as RAF, MEK or ERK, may have a similar effect. In conclusion, these results should prompt further studies on larger MCRC series to definitely establish the clinical relevance detection by direct sequencing of heterozygous mutations present of KRAS mutation detection in anti-EGFR antibodies based on in a small fraction of tumour cells, we screened the tumours chemotherapy. They also highlight the need to use sensitive without detectable KRAS mutations, using two sensitive methods molecular methods to detect mutations conferring resistance and able to detect specifically KRAS exon 2 mutations. We developed a the two assays presented in this study should facilitate the multiplex SNaPshot assay based on primer extension able to detect detection of KRAS mutations in CRC, on a routine basis. A major simultaneously in a single tube the different KRAS mutations and a criticism that should be made to all studies on predictive markers fluorescent PCR-LCR assay. These two analyses were performed of clinical response to anti-EGFR agents in MCRC, including ours, in 11 out of 12 CR/PR patients, in 15 out of 16 patients with SD and is that the clinical response is evaluated on the metastatic disease in the 15 PD patients, in whom direct sequencing from tumour whereas the presence of the molecular marker is assessed from the DNA had revealed no mutation. Five additional KRAS mutations primary tumour. In the present study we had the opportunity to were detected by both methods (Figure 1) and one mutation was compare the KRAS mutational status between primary tumour and detected only by PCR-LCR assay. These six additional mutations metastases in five patients whom samples were available. In these were found in two SD and four PD patients (Table 1). SNaPshot five patients, SnaPshot assay had indicated that a KRAS mutation and PCR-LCR assays confirmed the absence of KRAS mutations in was present in one case and absent in the remaining five cases. For the CR/PR patients. In this series of 59 MCRC, sequencing analysis these five patients, analysis of the corresponding metastatic site completed by SNaPshot multiplex and PCR-LCR assays led, showed that the KRAS mutation status was identical between the therefore, to the detection of a KRAS mutation in 22 samples primary tumour and metastases. Considering the genetic evolution (37%). The presence of KRAS mutation was in this series of metastases compared to primary tumour, we think nevertheless significantly associated with PD (P ¼ 0.0005). The predictive value that it will be important in the future to screen directly metastases of KRAS mutation for PD could be estimated to 77.2%, and the for the presence of alterations conferring either sensitivity or sensitivity and specificity of KRAS mutation for progression to resistance to these targeted therapies. treatment (CD vs PD) to 60.7 and 83.8%, respectively. The TTP was significantly decreased in patients harbouring KRAS mutation as compared to those without detectable mutation (3 vs 5.5 months, P ¼ 0.015). ACKNOWLEDGEMENTS We are indebted to Dr Scott, Dr De Puyfontaine, Dr Lafay, Dr DISCUSSION Jacob, Dr Elfadel, Dr Chasle, Dr Arsene, Dr Rousselot, Dr Piot, This study performed on 59 MCRC patients confirms that the Dr Damour, Dr Moran, Dr Bokanowski, Dr Ducastelle, Dr Anger, presence of KRAS mutation in tumour is highly predictive of a Dr Paoletti, Dr Petit, Dr Gallet, Dr El Amarti, Dr Patrier, Dr non-response to treatment based on cetuximab plus chemo- Roquet, Dr Albin, Dr Lepille, Dr Bonnafous, Dr Barhoum, Dr therapy, as shown previously in a series of 30 patients (Lievre Toulemonde, Dr Gomez, Dr Randrianarivelo, Dr Demmane and et al, 2006). It is important to highlight that, in our series, the Ms Delannoy for providing clinical information, and also Professor proportions of CR/PR, SD and PD patients were 20.3, 32.2 and Mario Tosi for critical review of the paper. This work was 47.5%, respectively, and this distribution is similar to that reported supported by a grant from the French National Institute of Cancer in the randomised cetuximab trial (Cunningham et al, 2004). The (INCA) to the North-West Canceropole. British Journal of Cancer (2007) 96(8), 1166 – 1169 & 2007 Cancer Research UK KRAS mutational status and clinical response to cetuximab F Di Fiore et al REFERENCES Cunningham D, Humblet Y, Siena S, Khayat D, Bleiberg H, Santoro A, Bets Meyerhardt JA, Mayer RJ (2005) Systemic therapy for colorectal cancer. D, Mueser M, Harstrick A, Verslype C, Chau I, Van Cutsem E (2004) N Engl J Med 352: 476–487 Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan- Moroni M, Veronese S, Benvenuti S, Artale S, Bardelli A, Siena S (2005) refractory metastatic colorectal cancer. N Engl J Med 351: 337–345 Gene copy number for epidermal growth factor receptor (EGFR) and Douillard JY, Sobrero A, Carnaghi C, Comella P, Diaz-Rubio E, Santoro A, clinical response to antiEGFR treatment in colorectal cancer: a cohort Van Cutsem E (2003) Metastatic colorectal cancer: integrating irinotecan study. Lancet Oncol 6: 279–286 into combination and sequential chemotherapy. Ann Oncol 14(Suppl 2): Saltz LB, Meropol NJ, Loehrer PJ, Needle MN, Kopit J, Mayer RJ (2004) ii7 –ii12 Phase II trial of cetuximab in patients with refractory colorectal cancer Hurwitz H, Fehrenbacher I, Novotny W, Cartwright T, Hainsworth J, Heim that expresses the epidermal growth factor receptor. J Clin Oncol 22: W, Berlin J, Baron A, Griffing S, Holmgren E, Ferrara N, Fyfe G, Rogers 1201– 1208 B, Ross R, Kabbinavar F (2004) Bevacizumab plus irinotecan, Saunders M, Iveson T (2006) Management of advanced colorectal cancer: fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J state of the art. Br J Cancer 95: 131–138 Med 350: 2335–2342 Shi C, Eshleman SH, Jones D, Fukushima N, Hua L, Parker AR, Yeo CJ, Kabbinavar F, Hurwitz HI, Fehrenbacher I, Meropol NJ, Novotny WF, Hruban RH, Goggins MG, Eshleman JR (2004) LigAmp for sensitive Lieberman G, Griffing S, Bergsland E (2003) Phase II, randomized trial detection of single-nucleotide differences. Nat Methods 1: 141–147 comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/ Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, LV alone in patients with metastatic colorectal cancer. J Clin Oncol 21: Rubinstein L, Veirweij J, Van Glabbeke M, van Oosterom AT, 60–65 Christian MC, Gwyther SG (2000) New guidelines to evaluate the Lievre A, Bachet JB, Le Corre D, Boige V, Landi B, Emile JF, Cote JF, response to treatment in solid tumours: European Organization for Tomasic G, Penna C, Ducreux M, Rougier P, Penault-Llorca F, Laurent- Research and Treatment of Cancer, National Cancer Institute of the Puig P (2006) KRAS mutation status is predictive of response to United States, National Cancer Institute of Canada. J Natl Cancer Inst 92: cetuximab therapy in colorectal cancer. Cancer Res 66: 3992 –3995 205–216 & 2007 Cancer Research UK British Journal of Cancer (2007) 96(8), 1166 – 1169 Clinical Studies

Journal

British Journal of CancerSpringer Journals

Published: Mar 20, 2007

There are no references for this article.