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First Published Online December 4, 2008
The Oncologist, Vol. 13, No. 12, 1270-1275, December 2008; doi:10.1634/theoncologist.2008-0181
© 2008 AlphaMed Press

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Gastrointestinal Cancer

High Concordance of KRAS Status Between Primary Colorectal Tumors and Related Metastatic Sites: Implications for Clinical Practice

Daniele Santinia, Fotios Loupakisb,c, Bruno Vincenzia, Irene Florianid, Irene Stasib, Emanuele Canestrarie, Eliana Rullid, Paolo Enrico Malteseb, Francesca Andreonie, Gianluca Masib, Francesco Grazianof, Giacomo Giulio Baldib, Lisa Salvatoreb, Antonio Russog, Giuseppe Perroneh, Maria Rosa Tommasinoi, Mauro Magnanie, Alfredo Falconeb,c, Giuseppe Toninia, Annamaria Ruzzoe

aDepartment of Oncology and hSurgical Pathology, University Campus Bio-Medico, Rome, Italy; bDepartment of Oncology, Azienda USL6 Livorno, Istituto Toscano Tumori, Italy; cDepartment of Oncology, Transplants and New Technologies in Medicine, University of Pisa, Pisa, Italy; dOncology Department, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; eDipartimento di Scienze Biomolecolari, University of Urbino, Urbino, Italy; fDivision of Medical Oncology, Azienda Ospedaliera San Salvatore, Pesaro, Italy; gSezione di Oncologia Medica, Dipartimento di Discipline Chirurugiche ed Oncologiche, and iDipartimento di Patologia Umana, Università di Palermo, Palermo, Italy

Key Words. Colorectal cancer • KRAS mutations • Concordance • Metastatic sites • Primary tumors

Correspondence: Daniele Santini, M.D., Ph.D., Medical Oncology, University Campus Bio-Medico, Via Alvaro del Portillo, 200, 00128 Rome, Italy. Telephone: 0039-06-225411206; Fax: 0039-06-225411934; e-mail: d.santini{at}unicampus.it

Received August 13, 2008; accepted for publication October 28, 2008; first published online in THE ONCOLOGIST Express on December 4, 2008.

Disclosure: The content of this article has been reviewed by independent peer reviewers to ensure that it is balanced, objective, and free from commercial bias. No financial relationships relevant to the content of this article have been disclosed by the authors, planners, independent peer reviewers, or staff managers.


   Learning Objectives
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 Learning Objectives
Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 Author Contributions
 References
 
After completing this course, the reader should be able to:

  1. Describe the importance of KRAS mutations in CRC patients.
  2. Explain the relevance to cancer treatment of concordance of KRAS status between primary tumors and metastases in CRC patients.
  3. Discuss the impact of KRAS mutations as a predictive/prognostic factor in CRC patients.

This article is available for continuing medical education credit at CME.TheOncologist.com


   ABSTRACT
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 Learning Objectives
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 Author Contributions
 References
 
Purpose. Several studies have suggested that KRAS somatic mutations may predict resistance to cetuximab- and panitumumab-based treatments in metastatic colorectal cancer (CRC) patients. Nevertheless, most experiences were conducted on samples from primaries. The aim of this study was to evaluate the grade of concordance in terms of KRAS status between primaries and related metastases.

Patients and Methods. We analyzed KRAS codon 12 and 13 mutations from formalin-fixed sections of 107 CRC primaries and related metastases. Eight pairs were excluded from the analysis because of the low amount of tumor tissue in the available samples. The main characteristics were: 50 men, 49 women; median age at diagnosis, 71 years (range, 41–84). The metastatic sites analyzed were the liver in 80 patients (80.8%), lung in seven patients (7.1%), and other sites in 12 patients (12.1%).

Results. A KRAS mutation was found in 38 (38.4%) primary tumors and in 36 (36.4%) related metastases. The rate of concordance was 96.0% (95% confidence interval, 90.0%–98.9%). Discordance was observed in only four (4%) patients.

Conclusions. Our results indicate that the detection of KRAS mutations in either primary or metastatic tumors from patients with CRC is concordant and this assessment could be used to predict response to targeted therapies such as cetuximab and panitumumab.


   INTRODUCTION
Top
 Learning Objectives
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 Author Contributions
 References
 
Colorectal cancer (CRC) is one of the leading causes of cancer-related death in western countries [1]. Recent therapeutic strategies are based on targeting the epidermal growth factor receptor (EGFR), which is overexpressed in CRC [2]. Cetuximab and panitumumab are monoclonal antibodies (mAbs) directed against EGFR. Blockade of EGFR disrupts downstream signaling pathways such as the phosphatidylinositol 3' kinase/Akt, Ras/Raf/mitogen-activated protein kinase (MAPK), and signal transducer and activator of transcription pathways, all of which are crucial in the regulation of cell growth, proliferation, apoptosis, invasion, migration, and angiogenesis [1]. On the basis of the Bowel Oncology with Cetuximab Antibody (BOND) trial, cetuximab was approved [3] in European countries in combination with irinotecan for the treatment of patients with EGFR+ metastatic CRC who progressed on a prior irinotecan-based chemotherapy regimen. In subsequent retrospective analyses, no correlation was found between EGFR expression assessed by immunohistochemistry (IHC) and clinical outcome [3, 4]. The need for alternative predictive factors has become imperative in order to avoid unnecessary toxicities and waste of resources.

KRAS mutations occur in about 40% of primary CRCs [5, 6], and such mutations have been demonstrated to be predictors of resistance to anti-EGFR mAbs. In the presence of specific mutations of the KRAS gene, the Ras protein, which has intrinsic GTPase activity, is constitutively activated, and subsequent signaling events are unregulated and independent from EGFR control [7]. Several studies [8–13] in patients with advanced or metastatic CRC have indicated that the presence of KRAS mutations (KRAS point mutations in codons 12 and 13) is associated with a lack of response to cetuximab.

More recently, in patients with KRAS mutations, panitumumab was demonstrated to be ineffective [14], and therefore this drug was granted approval for the treatment of patients with wild-type KRAS who have chemotherapy-refractory CRC. In all the cited studies [8–13], the mutational analysis was conducted almost exclusively on primary tumors.

It has been shown that primary colorectal tumors may differ from their corresponding metastases in terms of EGFR [15, 16], Akt, and MAPK protein expression assessed by IHC [17]. While it is well known that KRAS mutations occur in the first steps of colorectal carcinoma progression [18], additional data have demonstrated that the frequency of KRAS mutations in lymph node metastases is higher than in the related primary CRCs [19]. Because mutations in downstream targets of EGFR have been shown to correlate with a lesser response to the EGFR agent cetuximab, assessing this intracellular molecular target might help to predict response to such treatments. Therefore, it may be of primary importance to verify the correlation between primaries and related metastases with regard to KRAS status.

The primary endpoint of this study was to verify the concordance in terms of KRAS status between primary CRCs and paired metastatic sites.


   PATIENTS AND METHODS
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 Learning Objectives
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 Author Contributions
 References
 
Patient Selection
Patients were selected from a pathology database of CRC cases undergoing surgical resection of the primary tumor and surgical resection or diagnostic biopsy of the synchronous or metachronous corresponding metastatic site and collected in the pathology departments of the Azienda USL6 of Livorno, Università degli Studi di Pisa, Università Campus Bio-Medico, and Università degli Studi di Palermo between 1998 and 2007. All these patients had not been, at the time of specimen collection, treated with any targeted agents such as cetuximab and panitumumab.

KRAS Mutational Status
We searched for KRAS point mutations in codons 12 and 13, two hotspots that include >95% of mutations in this gene, as already reported [20].

Polymerase Chain Reaction Amplification
DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) CRC specimens using the Qiamp DNA FFPE tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. In total, 25–50 ng of DNA was added to a volume of 25 µl with 150 µM deoxynucleotide triphosphates, 6.25 pmol of each primer, 1 U of Hot-Rescue DNA Polymerase (Diatheva, Fano, Italy), 5% dimethylsulfoxide, 2 mM MgCl2, and 2.5 µl of 10x polymerase chain reaction (PCR) buffer. Primers spanned codon 12 and 13 of the KRAS gene. Primers for the 214-bp amplicon that spanned exon 2 were 5'-GTGTGACATGTTCTAATATAGTCA-3' (forward) and 5'-GAATGGTCCTGCACCAGTAA-3' (reverse). PCR cycling was run according to the following conditions: one cycle of 95°C for 10 minutes; 40 cycles of 95°C for 45 seconds, 56°C for 45 seconds, and 72°C for 45 seconds; and one cycle of 72°C for 10 minutes.

DNA Sequencing
PCR products were column purified using the MinElute PCR Purification Kit (Qiagen) according to the manufacturer's instructions and eluted in a 15-µl volume. Concentrations were estimated with the ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE).

The cleaned PCR product (10–15 ng) was then used as a template in cycle sequencing with the BigDye Terminator v.1.1 CycleSequencing Kit (Applied Biosystems, Foster City, CA). The reaction mix consisted of 1 µl Terminator ready reaction mix, 1x sequencing buffer, and 3.2 pmol sequencing primer in a 20-µl total volume.

Two sequencing reactions were performed for each template with the nested primer 5'-GCCTGCTGAAAATGACTGAA-3' (forward) and 5'-TGAATTAGCTGTATCGTCAAGGCACT-3' (reverse).

Reactions were run according to the following protocol: one cycle of 96°C for 2 minutes and 40 cycles of 96°C for 10 seconds, 55°C for 5 seconds, and 60°C for 2 minutes. Sequencing reactions were purified with the DyeEx 96 Kit (Qiagen) and run on an ABI PRISM 310 Genetic Analyser (Applied Biosystems). The sense and antisense strands were aligned and analyzed with Sequence Navigator Software (Applied Biosystems).

Statistical Analysis
Considering the primary endpoint, the level of concordance of KRAS status between primaries and related metastatic samples was stated at a minimum desirable of 90%, while it was not acceptable at a level <80%. Using the design proposed by A'Hern for the binomial distribution [21], setting the probability of erroneously concluding that the concordance is >80% at 5% (one-sided {alpha} = .05) and the probability of correctly concluding that the concordance is at least 90% at 85% (β = 0.15), it was necessary to perform both evaluations in at least 94 patients. The minimum number of concordant samples was set at 82 of 94, because this result is associated with the lower limit of the 90% confidence interval (CI) of concordance of 80.1%.


   RESULTS
Top
 Learning Objectives
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 Author Contributions
 References
 
We retrospectively analyzed KRAS codon 12 and 13 mutations in primary tumors and related metastatic sites from 107 CRC patients. Eight pairs were excluded from the analysis because of the low amount of tumor tissue in the available samples. Ninety-nine patients were available for the final analysis (Table 1)—50 men (50.5%) and 49 women (49.5%); median age at diagnosis, 71 years (range, 41–84 years). Thirty-three were affected by right CRC, 46 by left CRC (including intraperitoneal rectal adenocarcinoma), and 20 by extraperitoneal rectal cancer. CRC adenocarcinoma was the unique histotype, and in 10 cases a mucinous differentiation was detected. Histologic grade 1–2 and grade 3 were described in 63 (63.6%) and 36 (36.4%) tumors, respectively.


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  		Table 1. Patient characteristics

 
In total, 99 pathologic samples from metastatic sites were analyzed. The most common site of origin of metastatic tissue was the liver, which was noted in 80 (80.8%) cases (synchronous in 75 cases and metachronous in the remaining 24 cases), and the lung, which was noted in seven cases (metachronous in five cases). The others organs of tumor origin in the remaining cases are shown in Table 2.


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  		Table 2. Sites of metastasis

 
A KRAS mutation was found in 38 (38.4%) primary tumors and in 36 (36.4%) related metastatic sites. The grade of concordance between primaries and metastatic sites was 96.0% (95% CI, 90.0%–98.9%). Details regarding the frequencies of the different codon 12 and 13 KRAS mutations in this population are reported in Table 3. All patients, with the exception of one, with wild-type KRAS primaries also showed wild-type KRAS metastases. Discordance was observed in only four (4%) patients. In particular, in one patient KRAS was wild-type in the primary tumor and mutated in the metastatic site (peritoneum, metachronous); in three patients KRAS was mutated in the primary tumor and wild-type in the metastatic site (liver in all three cases, two synchronous and one metachronous).


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  		Table 3. Codon distribution of specific KRAS mutations

 

   DISCUSSION
Top
 Learning Objectives
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
Author Contributions
 References
 
Metastatic CRC represents a major global health problem, but the introduction of a novel class of targeted antineoplastic agents, mAbs, directed against the EGFR has significantly changed the therapeutic options available for these patients. As EGFR targeting provides real advantages only in a small subgroup of patients, several attempts have been made to identify predictive factors of treatment benefit.

Positive IHC staining for EGFR has been used as a criterion for patient selection. All the reported studies, however, have failed to demonstrate any correlation between the efficacy of cetuximab and EGFR IHC staining [3, 4]. Nowadays, several retrospective studies have clearly demonstrated the high predictive value of KRAS mutations in metastatic CRC patients treated with anti-EGFR mAb-based therapy [8–13]. Because identification of the mutational status of KRAS could help to select patients who have a high probability of benefiting from anti-EGFR antibodies [14], it may be of primary importance to verify the degree of correlation between primaries and related metastases with regard to KRAS status.

In the present observational analysis, we clearly reported a very high concordance (96.0%; 95% CI, 90.0%–98.9%) between primaries and related metastases in terms of KRAS mutational status. A similar grade of concordance was reported by Molinari et al. [22], who analyzed KRAS mutational status in a small sample of 30 consecutive patients with CRC with synchronous or metachronous metastasis. The same mutational pattern between primaries and corresponding metastases was observed in 26 cases. In three cases the mutation was restricted to the primary, and in one case it was restricted to the metastatic lesion, with a total grade of concordance of 86.6%.

We believe that the data reported in this paper have to be considered to be of strong clinical impact. Notably, we included a very large sample of patients (99 patients) with the same clinical and pathological characteristics of those included in the clinical trials investigating cetuximab- and panitumumab-based therapies. Furthermore, the prevalence (36.4%) of KRAS mutations in our sample is similar to that reported in the literature (about 40%) [5, 6]. We excluded from the analysis patients with a low amount of available tumor tissue to avoid false-negative cases derived from the analysis of the mutational status of normal tissue. In addition, the mutational status of KRAS was analyzed in a double-blinded fashion for each primary and metastatic sample. In the few cases in which the mutational status was uncertain, this was interpreted by a third molecular biologist.

The correlation analysis between the primary cancer and corresponding metastatic site was discordant in only four cases. In particular, in one patient KRAS was wild-type in the primary tumor and mutated in the metastatic site; in three patients KRAS was mutated in the primary tumor and wild-type in metastatic site. This high grade of concordance confirms that KRAS mutation represents a very early mutational step in CRC pathogenesis and plays a central role in tumor progression. Indeed, KRAS mutations are commonly found in patients with CRC and in the near normal mucosa and, in general, this mutation might represent an early, stable genetic event that precedes the appearance of histologically detectable aberrations in colonic epithelial cells [23].

Recent findings underline the importance of a true outcome predictor for patients with metastatic CRC who are treated with an anti-EGFR mAb. An improvement in terms of the response rate and progression-free survival time for the combination of cetuximab plus chemotherapy versus chemotherapy alone in the first-line treatment of metastatic CRC was recently demonstrated in a phase III randomized trial [24]. In this setting, the vascular endothelial growth factor (VEGF) inhibitor bevacizumab has already been approved [25] in combination with irinotecan, fluorouracil, and leucovorin for the treatment of metastatic CRC. In the near future, the selection of patients who really benefit from anti-EGFR mAbs will be even more imperative, especially for those with metastatic lesions, who are potential candidates for secondary resection in which tumor shrinkage is the major goal of treatment. For these reasons, future prospective studies may be aimed at evaluating the role of KRAS mutations in directing the choice of which biologic (anti-EGFR, anti-VEGF, or both) could be the best partner for upfront chemotherapy. At the same time, KRAS analysis may help to avoid unnecessary toxicities and to optimize the allocation of resources for treating patients in the second- or third-line settings. To our knowledge, this is the first report indicating that the analysis of any available neoplastic tissue (primary or metastatic) for KRAS status is to be considered adequate and reliable in the vast majority of patients and this will be of great importance in routine clinical practice.


   AUTHOR CONTRIBUTIONS
Top
 Learning Objectives
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 Author Contributions
References
 
Conception/design: Daniele Santini, Fotios Loupakis, Francesco Graziano, Giacomo Giulio Baldi, Alfredo Falcone, Giuseppe Tonini

Financial support: Daniele Santini

Provision of study materials: Fotios Loupakis, Eliana Rulli, Paolo Enrico Maltese, Francesca Andreoni, Gianluca Masi, Lisa Salvatore, Antonio Russo, Giuseppe Perrone, Maria Rosa Tommasino, Mauro Magnani, Alfredo Falcone, Giuseppe Tonini, Annamaria Ruzzo

Collection/assembly of data: Bruno Vincenzi, Irene Stasi, Emanuele Canestrari, Eliana Rulli, Paolo Enrico Maltese, Francesca Andreoni, Gianluca Masi, Giacomo Giulio Baldi, Lisa Salvatore, Antonio Russo, Giuseppe Perrone, Maria Rosa Tommasino, Mauro Magnani, Alfredo Falcone, Giuseppe Tonini, Annamaria Ruzzo

Data analysis: Bruno Vincenzi, Irene Floriani, Francesco Graziano

Manuscript writing: Fotios Loupakis, Bruno Vincenzi, Irene Floriani, Francesco Graziano

Final approval of manuscript: Daniele Santini, Irene Stasi, Emanuele Canestrari, Gianluca Masi, Francesco Graziano, Antonio Russo, Maria Rosa Tommasino, Mauro Magnani, Alfredo Falcone, Giuseppe Tonini, Annamaria Ruzzo


   REFERENCES
Top
 Learning Objectives
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 Author Contributions
 References
 

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