Systematic review of pharmacogenetic testing for predicting clinical benefit to anti-EGFR therapy in metastatic colorectal cancer

Jennifers Lin; Elizabeth M Webber; Caitlyn A Senger; Rebecca S Holmes; Evelyn P Whitlock

. 2011 May 6;1(5):650–662.

Systematic review of pharmacogenetic testing for predicting clinical benefit to anti-EGFR therapy in metastatic colorectal cancer

Jennifers Lin ¹, Elizabeth M Webber ¹, Caitlyn A Senger ¹, Rebecca S Holmes ¹, Evelyn P Whitlock ¹

PMCID: PMC3139487 NIHMSID: NIHMS306369 PMID: 21779535

Abstract

Pharmacogenetic testing can help identify patients with metastatic colorectal cancer more likely to respond to anti-EGFR therapy. We systematically reviewed the benefits and harms of EGFR-related pharmacogenetic testing of molecular targets downstream to KRAS in the treatment of metastatic colorectal cancer. We searched five electronic databases from January 2000 through November 2010, and conducted separate grey literature and conference abstracts searches. Two reviewers independently assessed all articles for relevance and quality. We identified 27 studies, primarily fair- to marginal-quality, small retrospective, and single-arm cohort studies with significant overlap in patient populations. We identified seven studies that studied BRAF in independent patient populations, one that studied NRAS, four that studied PIK3CA, eight that studied PTEN expression, and five that studied AKT expression. The best evidence for BRAF, NRAS, and PIK3CA comes from the largest retrospective study (n=649) of chemorefractory patients from seven European countries. In this study, BRAF mutation was present in 6.5% of KRAS wild-type tumors. Only 8.3% of persons with BRAF mutations, compared to 38% of persons without BRAF mutations (p=0.0012), responded to chemotherapy with cetuximab. Clinical sensitivity and the false positive fraction (1- specificity) were estimated at 9.8% (95% CI 6.3, 14.5) and 1.6% (95% CI 0.2, 5.6), respectively. BRAF mutation was also associated with worse median progression-free survival (absolute difference 18 weeks, p<0.0001), and overall survival (absolute difference 28 weeks, p<0.0001). In the only study comparing outcomes in persons who did (n=227) and did not (n=332) receive cetuximab with combination chemotherapy, those with BRAF mutation had worse survival outcomes regardless of whether or not they received cetuximab. Although NRAS and PIK3CA exon 20 mutations were also associated with worse outcomes compared to persons without these mutations, evidence is based on a small number of identified mutations. Evidence for protein expression of PTEN and AKT is more sparse and limited by variable methods for assessing protein expression. Low-quality evidence addressing clinical validity of pharmacogenetic testing in metastatic colorectal cancer patients suggests that BRAF mutations are associated with poorer treatment response and survival outcomes, although this association may be independent of treatment with EGFR inhibitors.

Keywords: BRAF, NRAS, PTEN, AKT, metastatic colorectal cancer, anti-EGFR monoclonal antibodies, cetuximab, pani-tumumab, pharmacogenetic test, systematic review

Introduction

Colorectal cancer is the third leading cause of cancer-related death in the United States [1]. Despite improvements in chemotherapy for metastatic colorectal cancer, overall five-year survival remains poor at just 11% for patients with metastatic disease [1]. Currently, cetuximab (Erbitux®, ImClone Systems) and panitumumab (Vectibix®, Amgen) are approved by the U.S. Food and Drug Administration (FDA) for the treatment of metastatic colorectal cancer in the refractory disease setting [2,3]. These monoclonal antibodies bind to the EGFR, preventing binding and activation of the downstream signaling pathways, which are important for cancer cell proliferation, invasion, metastasis, and ne-ovascularization [4]. Tumors with molecular alterations in the EGFR signaling pathway (e.g., mutations in KRAS, NRAS, BRAF, PIK3CA, loss of PTEN protein expression, or AKT over expression), however, may lead to a constitutively activated pathway not responsive to EGFR-targeted treatment.

Pharmacogenetic testing for KRAS has already entered clinical practice, such that persons with metastatic colorectal cancer whose tumors have KRAS mutations are not treated with EGFR monoclonal antibodies. In April 2009, ASCO issued a Provisional Clinical Opinion stating that all patients with metastatic colorectal cancer who are candidates for EGFR antibody therapy should have their tumor tested for KRAS mutations, and that persons with a KRAS mutation in codon 12 or 13 should not receive EGFR antibody as part of their treatment [5]. In July 2009, the FDA revised the label for cetuximab and panitumumab to advise against use of these agents in persons with colorectal cancer positive for KRAS mutations [6]. Even among patients with wild-type KRAS, however, the response rate to EGFR monoclonal antibodies is less than 20% [7]. Primary research suggests that molecular alterations in the EGFR signaling pathway downstream to KRAS may also predict non-response to EGFR monoclonal antibodies. These alterations are less frequently occurring than KRAS (Figure 1), but testing for additional molecular alterations in those without KRAS mutations has the potential to identify other patients not likely to respond to anti-EGFR therapy before treatment begins, therefore preventing unnecessary treatment and associated harms and costs [8,7].

Mutation frequencies and loss of protein expression in the EGFR signaling pathway.

We systematically reviewed the evidence for the clinical benefit and harms of EGFR-related pharmacogenetic testing (downstream to KRAS) in predicting non-response to treatment with anti-EGFR therapy. We asked four key questions (KQ) (Figure 2):

Analytic framework for clinical benefit and harms of EGFR-related pharmacogenetic testing of molecular targets in the treatment of metastatic colorectal cancer.

Clinical utility

KQ 1: In patients with mCRC, can other EGFR-related testing improve (or lead to non-inferior) patient outcomes or decision making compared to not using additional testing?

Clinical validity

KQ 2: How well do each of these tests predict treatment effectiveness?

KQ 3: How well do each of these tests predict important health outcomes?

Harms

KQ 4: What are the potential harms to patients in using these tests to guide treatment decisions?

Methods

Studies were identified by searching electronic databases, conference abstracts, regulatory documents, and trial registries. MEDLINE was searched from January 2000 to November 2010 for English language abstracts. This search was adapted for four additional databases (Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials, Database of Abstracts of Reviews of Effects, and Health Technology Assessments Database) and limited to publications between 2000 and 2010, with no language restrictions. We also searched Conference Papers Index (via CSA) from 2009 to 2010 and hand searched selected scientific conferences from 2009 to November 2010. Relevant studies were also identified by searching clinicaltrials.gov, NIH RePORTER, Current Controlled Trials (International Standard Randomized Controlled Trial Number Register), WHO International Clinical Trials Registry Platform, and FDA regulatory documents via Drugs@FDA. The last search for all databases was performed on November 24, 2010. Search details are provided in Search Strategies document (Supplementary Table 1).

Two investigators independently reviewed 3,365 abstracts and 191 articles against a priori specified inclusion criteria (Figure 3). For all key questions, we considered studies that included persons with metastatic colorectal cancer being treated with cetuximab or panitumumab, either alone or in combination with other chemotherapeutic agents. Studies that only included patients with locally advanced disease were excluded. Testing included assays for mutations in BRAF, NRAS, PIK3CA, and protein expression for PTEN and AKT. We also included studies examining PTEN and AKT mutations and gene copy number. We excluded testing for EGFR protein expression or gene copy number; upstream molecular drivers (i.e., EGFR ligands epiregulin and amphiregulin); and molecular targets not directly part of the EGFR signaling cascade, but mediators in adjacent pathways. We considered any study reporting one or more of the following outcomes: overall survival, clinical response to treatment (e.g., progression free survival, time to progression), health-related quality of life, radiologic evidence of tumor progression, or potential adverse effects (e.g., incorrect genotype assignment leading to incorrect treatment assignment, delayed treatment, negative psychological effects, and ethical, legal, and social issues/concerns). We did not exclude studies based on study design or study quality. Excluded studies are listed in Supplementary Table 2.

Two investigators independently assessed the quality of each study using the quality criteria proposed by the EGAPP working group [9], supplemented by the Newcastle Ottawa Scale developed for observational studies [10], and reporting standards checklist (REMARK) developed for prognostic and predictive studies [11]. Articles were rated good-, fair-, or marginal-quality. Good-quality studies were those that met the following criteria: prospective design; large, well-defined, and representative study population; genetic testing was described well; blinded assessment of genetic testing in relation to outcome; homogeneous treatment; low rate of missing data; sufficiently long follow-up; and well-described and well-conducted analysis of outcomes. Fair-quality studies did not meet all the criteria, but did not have any fatal flaws in study design. Marginal- or poor-quality studies had significant flaws or lack of reporting that implied bias affecting interpretation of study results. Disagreements about inclusion and quality were resolved by consensus with a third reviewer.

One investigator extracted all relevant data from the studies into evidence tables that included the following study details: critical features of study design and quality, funding source, patient characteristics (e.g., age, sex, race/ethnicity, Eastern Cooperative Oncology Group [ECOG] performance status, metastatic disease), treatment regimen and setting, genetic testing details (e.g., gene mutation(s)/protein expression, tumor sample, assay technique, scoring method), frequency of gene mutation or protein expression, a priori specified study outcomes (stratified by KRAS wild-type if available), and any outcome representing potential adverse effects. A second reviewer verified all extracted data.

We identified 27 studies (reported in 34 articles); however there was a significant overlap of populations studied. Our evidence synthesis focuses on studies with independent patient populations and, when possible, results in persons with metastatic colorectal cancer that have KRAS wild-type tumors. We focus on three primary outcomes— tumor response (or disease control if tumor response is not reported) based on radiographic findings, progression-free survival (PFS), and overall survival (OS). Studies either used RECIST or World Health Organization (WHO) criteria (based on radiographic findings) to assess tumor response or disease control. Most individual studies reported tumor response rates with or without an odds ratio (OR). For tumor response, we also calculated the true positive fraction (TPF or clinical sensitivity) and false positive fraction (FPF or 1-specificity) if sufficient data were reported in persons with KRAS wild-type tumors [12]. For continuous outcomes (survival), in addition to reporting hazard ratios (HR), we also report absolute differences between groups in weeks or months of median progression-free or overall survival.

We summarize results qualitatively and provide these results in tables for easy comparison across studies representing unique populations. For tumor response based on imaging, we attempted quantitative synthesis (meta-analyses) for sensitivity, specificity, and odds ratios to evaluate the predictive value for each genetic test with sufficient data. Due to overlapping populations and lack of outcome reporting for individuals with KRAS wild-type tumors, only 3 studies could be included in the meta-analyses. We attempted bivariate analyses for sensitivity and specificity (of BRAF and PTEN testing) simultaneously [13], as well as univariate meta-analyses for sensitivity, specificity, and diagnostic odds ratios, separately using random effects models [14,15]. However, the small number of studies and clinical heterogeneity among studies prohibited us from producing meaningful combined estimates. We instead focused on the best available evidence (e.g., single large, well-reported study) to provide the best estimate of clinical validity. We also considered how additional studies with independent and overlapping populations confirmed, disagreed, and/or contributed additional information to the best evidence detailed.

In addition to a summary of evidence table, we also provide a summary table focusing on the strength of the body of evidence, based on the GRADE (Grading of Recommendations Assessment, Development and Evaluations) approach [16]. The following four domains were assessed: risk of bias, consistency, directness, and precision. The overall strength of evidence was graded as high, moderate, low, or very low (insufficient).

Results

Key Question 1 to 3: Benefits of pharmacogenetic testing

We found no studies that directly assessed whether pharmacogenetic testing improves (or leads to non-inferior) important patient health outcomes (e.g., morbidity, mortality, health related quality of life) in metastatic colorectal cancer patients who received pharmacogenetic testing to guide EGFR monoclonal antibody treatment decisions, compared to those who did not receive pharmacogenetic testing (Key Question 1).

We found a total of 27 fair- to marginal-quality studies that evaluated pharmacogenetic testing for EGFR molecular targets downstream to KRAS and their association with tumor response (Key Question 2) or survival outcomes (Key Question 3) in patients with metastatic colorectal cancer treated with cetuximab or panitumumab [17-39]. Most trials had very limited reporting on important patient characteristics (Table 3) and were conducted in European countries. Patients in the few trials or centers that reported race/ethnicity were overwhelmingly white. Only two small studies included patients from the US [21,20], one of which included 23% non-white participants [21]. One small study was conducted in South Korea [40]. In studies that provided baseline patient characteristics, the age of patients ranged from 22 to 94 years, with the mean age ranging from 57 to 67 years. The cohorts of patients studied were 46 to 71% male.

Most studies evaluated response to cetuximab, either as monotherapy or in combination with other chemotherapy. Two studies included patients who received either cetuximab or panitumumab [27,24], and only one study included patients who exclusively received panitumumab [21]. All studies, except for one, included a majority of patients who had received prior chemotherapy, or in some cases included exclusively patients identified as chemorefractory. This study by Tol and colleagues, a retrospective evaluation of the Dutch RCT CAIRO2, evaluated the addition of cetuximab to combination chemotherapy (capecitabine, oxaliplatin, bevacizumab) as first-line treatment in patients with metastatic colorectal cancer [18]. Only eight studies reported the patients' performance score, and the majority of patients in these cohorts had no significant activity impairments (ECOG performance status of 0-1) [37,30,21,22,41, 28,40,18]. Among all patients studied, BRAF mutations ranged from 0 to 17%, NRAS 3%, PIK3CA 3 to 18%, loss of PTEN expression ranged from 12 to 42%, and loss of AKT expression ranged from 33 to 60%.

We found no good-quality studies. Most studies were retrospective single-arm evaluations of mutations or protein expression in cohorts of patients who received chemotherapy with cetuximab or panitumumab, comparing outcomes for persons with and without identified mutations or protein expression. Of the two studies that included both persons treated with and without cetuximab [18,20], only one reported outcomes comparing those receiving cetuximab versus those who did not [18]. Most of the studies were small, with less than 100 patients included in the analysis. Therefore, these studies may not have been adequately powered to examine the predictive ability of less frequently occurring tumor mutations. Most of the studies evaluated cetuximab in various treatment settings, and many studies combined patients receiving different combinations of chemotherapy, with differing histories of prior chemotherapy. Some studies combined persons who received either cetuximab or panitumumab. All but one study were retrospective and stated a priori for inclusion that “sufficient” tumor sample had to be available. Some studies specified that tumor samples had to be EGFR expression positive. Details about how patients were selected were not reported, or were very minimal, in about one-third of the studies. In the single prospective study (n=110), only 73% (80 of 110) of the tumor samples were assessed for BRAF [22]. No BRAF mutations were identified in this cohort. The level of reporting in the remaining studies was frequently inadequate to determine the proportion of missing data (from all persons eligible for the study). Information about unanalyzable or missing data (i.e., the number of and reasons for) was only reported in a few studies. Most studies did not provide sufficient data to determine if patients in the retrospectively-identified cohort were similar in terms of prognostic risk. Only a few of the studies performed analyses to determine if any important patient and treatment setting characteristics influenced the mutations' association with tumor response or survival. Studies rarely reported duration of study follow-up.

In addition to individual study quality concerns, included studies had significant overlap in populations studied. Of the 27 included studies, only seven studies evaluating BRAF (n=1,224) [17-22,40], one evaluating NRAS (n=649) [17], four evaluating PIK3CA (n=1,030) [17,18,20, 21], eight evaluating PTEN (n=742) [7,18,23, 25,26,28,29,31], and five evaluating AKT (n=249) [23-25,28,30] included independent patient populations (Figure 4). The largest study, conducted by DeRoock and colleagues, was a retrospective analysis of chemorefractory metastatic colorectal cancer patients who received cetuximab (n=649). This study provides the best evidence for the predictive value of BRAF, NRAS and PIK3CA [17]. The best evidence for PTEN protein expression comes from a smaller retrospective study (n=173) [26]. No studies of AKT expression reported outcomes stratified by KRAS wild-type.

Included studies illustrating overlapping populations.

BRAF (codon 600, V600E)

Overall, we identified seven studies (n=1224) that reported tumor response outcomes [17-22,40], and three studies (n=968) [17,18,20] that reported survival outcomes, in independent patient populations. The best evidence for BRAF pharmacogenetic testing comes from one large, fair-quality retrospective analysis of a consortium of European patients with chemorefractory metastatic colorectal cancer who received cetuximab (Supplementary Tables 3 and 4) [17]. This study by DeRoock and colleagues (n=649) included chemorefractory patients with sufficient primary tumor sample available from 11 centers in seven European countries who were treated with cetuximab in combination with other chemotherapy from 2001 to 2008. This cohort was mostly men (58%) with an average age of 61 years. Mutations from primary tumor samples were assessed using MassARRAY multiplex polymerase chain reaction (PCR), with a subset of samples independently validated using direct sequencing or allele-specific PCR. Ninety-five percent (350/370) of KRAS wild-type samples had BRAF mutation status assigned and outcome data available. BRAF mutation (V600E) was present in 6.5% (24/350) of KRAS wild-type. Only 8.3% of persons with BRAF mutations responded to chemotherapy with cetuximab (p= 0.0012), compared to 38% of persons with BRAF and KRAS wild-type tumors. Calculated true positive fraction (clinical sensitivity) and false positive fraction (1 -specificity) were estimated at 9.8% (95% CI 6.3 -14.5) and 1.6% (95% CI 0.2-5.6), respectively. BRAF mutation was also associated with worse progression-free survival (absolute difference 18 weeks, p< 0.0001), and overall survival (absolute difference 28 weeks, p< 0.0001). Odds ratios for tumor response (Supplementary Table 4) and hazard ratios for progression-free and overall survival (Supplementary Table 5) were adjusted for age, sex, previous chemotherapy, and treatment center. Findings from other studies with [42,30,7,34,35,32,37,24,26, 38, 29,41,20,39,18] and without [22,19,40, 20,18] overlapping populations were either not informative (because of low or no mutations identified, limitations in outcome reporting) or consistent with findings from the study by DeRoock and colleagues.

We identified only one retrospective evaluation of pharmacogenetic testing in an RCT of persons with metastatic colorectal cancer receiving chemotherapy with (n=227) or without cetuximab (n=332) as first-line treatment [18]. In this study by Tol and colleagues, BRAF mutation was identified in 8.7% (45 of 518) of all persons. Outcomes based on imaging criteria were not reported in the subset of those with KRAS wild-type. For all patients, disease control was not statistically significantly different between persons with BRAF mutations versus BRAF wild-type, whether or not patients received cetuximab (Supplementary Table 4). For persons treated with cetuximab, median PFS was shorter for persons with BRAF mutation versus KRAS and BRAF wild-type, 6.5 months versus 11.4 months, respectively (absolute difference 4.9 months, p<0.0001). Results were similar for persons who received combination chemotherapy without cetuximab; median PFS was also shorter for persons with BRAF mutation versus wild-type, 5.7 months versus 10.8 months, respectively (absolute difference 5.1 months, p<0.0001). Overall survival, a secondary outcome in this study, showed a pattern consistent with that of PFS. Differences by BRAF mutation status for PFS and OS were essentially the same for both treatment groups (with or without cetuximab). PFS and OS were noticeably better for patients in this study (receiving first-line chemotherapy) compared with the chemorefractory patients in the two other retrospective analyses that reported survival outcomes, which suggests important clinical heterogeneity among studies [20,17].

NRAS (codons 12, 13, and 61) and PIK3CA (exons 9 and 20)

Pharmacogenetic testing for mutations other than BRAF have less evidence. We found only one study for NRAS (n=649) [17] and four studies for PIK3CA (n=1030) [17,18,20,21]. Again, the best evidence comes from the largest multi-center retrospective analysis (n=649) by DeRoock and colleagues in chemorefractory metastatic colorectal cancer [17]. In this study, only 82% (302/370) of KRAS wild-type tumors had NRAS status and outcomes. Four percent (13/302) of KRAS wild-type tumors had NRAS mutations and 13% (49/370) had PIK3CA mutations (exon 9 and 20). Although NRAS and PIK3CA exon 20 (not exon 9) mutations were associated with poorer outcomes, this evidence is based on a very small number of mutations from one study (Supplementary Tables 3 and 4). Overall, there was no statistically significant difference in PFS or OS between persons with tumors that had PIK3CA mutations versus wild-type (Supplementary Table 5). Authors conducted subgroup analyses (presumed a priori) for mutations in exon 9 versus mutations in exon 20 because of different proposed biological effects for domains encoded by these two exons. Compared with PIK3CA wild-type, PIK3CA exon 20 mutations, but not mutations in exon 9, appeared to predict poor tumor response and survival outcomes (Tables 3 and 4). The remaining two studies with independent patient populations did not report results by KRAS wild-type [20,21] (Supplementary Table 4). No other studies examining PIK3CA, with [18,20,33,7,37,38,24,29] or without [21] overlapping populations, report results for PIK3CA exon 9 and 20 separately.

Protein expression of PTEN and AKT

Most studies focusing on molecular alterations in PTEN and AKT studied protein expression instead of mutations or gene copy number. Studies used immunohistochemistry (IHC) to examine protein expression of PTEN and AKT, but used different antibodies and scoring systems. We found eight studies for PTEN (n=742) [7,18,23,25,26,28,29,31], six of which (n=652) reported survival outcomes [7,18,23,26,28,29] (Supplementary Tables 3 and 4). There was some evidence to suggest that PTEN loss may be associated with non-response, though results are conflicting between studies. The best evidence comes from a retrospective cohort (n=173) by Laurent-Puig and colleagues [26]. In this study, about 20% of the KRAS wild-type tumors had loss of PTEN protein expression. Loss of protein expression was not associated with tumor response or progression-free survival, but was associated with slightly worse overall survival (Supplementary Tables 3 and 4). Based on a small number of fair- to marginal-quality studies with differences in assay methodologies, PTEN expression does not appear to have clinically robust ability to predict survival response to cetuximab or panitumumab. Only five small studies (n=294) studied AKT protein expression [23-25,28,30], only two of which (n=194) reported survival outcomes [23,28] (Supplementary Tables 3 and 4) and none of which reported results for AKT loss in KRAS wild -type. None of the five studies showed a statistically significant association between AKT expression and tumor response or survival. Based on one study, PTEN and AKT protein expression are only concordant in 60% and 68% respectively, of primary and metastatic tumors [28].

Key Question 4: Harms of pharmacogenetic testing

We did not hypothesize any clinically significant harms to testing other than incorrect genotype assignment leading to incorrect treatment assignment (i.e., leading to subsequent withholding of potentially effective therapy, or giving therapy that has significant adverse effects and cost with little to no benefit). None of the included studies reported harms of testing, and we found no studies that explicitly addressed harms or that addressed psychological, ethical, legal, or social implications of testing.

The best evidence to estimate harms associated with incorrect treatment assignment based on testing for mutations in BRAF, NRAS, and PIK3CA comes from the largest retrospective study of chemorefractory patients with metastatic colorectal cancer by DeRoock and colleagues [17]. Overall, the specificity of mutations in EGRF-related genes was very high, and therefore the false positive fraction (1-specificity) was low. These false positives are those few patients who would respond to treatment despite a mutation identified through genetic testing, from whom potentially effective treatment is withheld. Point estimates of false positive fractions for BRAF, NRAS, and PIK3CA exon 20 are 1.6%, 0.9%, and 0.0% of responders, respectively (Supplementary Table 4).

Discussion

Of the studied molecular targets downstream from KRAS, the evidence is most promising for BRAF mutation as a negative predictor of response to EGFR monoclonal antibodies, and is most robust for persons with chemorefractory metastatic disease receiving cetuximab in combination chemotherapy (Supplementary Table 6). BRAF mutation is less common than KRAS mutations, approximately 5 to 20% versus 30 to 45%, respectively [43,44,45,46]. In the largest study, which was exclusively in chemorefractory patients, BRAF mutation was present in 6.5% of KRAS wild-type [17]. The calculated true positive fraction (possible benefit), was 9.8% (95% CI 6.3-14.5), which meant that an additional (after KRAS testing) 9.8% of persons who did not respond to treatment were identified with BRAF testing. The calculated false positive fraction (possible harm) was 1.6% (95% CI 0.2-5.6), which meant that of those who responded to treatment, the proportion with BRAF mutation was small. BRAF mutation also was associated with worse median progression-free survival (absolute difference of 18 weeks) and overall survival (absolute difference of 28 weeks) in chemorefractory persons. One retrospective evaluation of an RCT that compared persons receiving cetuximab and combination chemotherapy to those receiving combination chemotherapy without cetuximab as first-line chemotherapy showed that persons with BRAF mutations had shorter progression-free and overall survival regardless of cetuximab, suggesting prognostic ability independent of treatment with cetuximab [18]. While the overall magnitude of association (odds ratio) of BRAF mutation on tumor response and survival is similar to the association of KRAS mutation on tumor response and survival, the clinical sensitivity is much lower. In a recent good quality systematic review of KRAS testing in this clinical scenario, the sensitivity was 49% (95% CI 44-54) [47]. In addition, the body of evidence for BRAF testing is much smaller and primarily comprises single-arm retrospective studies with poorly-characterized cohorts of patients (Supplementary Table 7). Similar to KRAS mutations, two studies that currently represent the best evidence for BRAF mutations showed association is greater in chemorefractory patients than in patients receiving cetuximab in combination with other chemotherapy as first-line therapy [17,18]. However, unlike KRAS, BRAF mutation appears to have prognostic ability independent of predicting response to cetuximab. Additional retrospective evaluations of RCTs comparing persons who received chemotherapy with and without EGFR monoclonal antibodies would help clarify the extent to which BRAF mutation predicts poor response to anti-EGFR therapy, or predicts poor prognosis independent of treatment effect. We identified conference abstracts, without full publication of results, of retrospective analyses of RCTs evaluating the addition of cetuximab to first-line therapy that also suggest that BRAF mutation in persons with metastatic colorectal cancer was a strong negative prognostic factor (independent of treatment effect) [47,48]. When these and other studies are fully reported, it would be important to attempt to clarify the prognostic significance of BRAF mutations in metastatic colorectal cancer, and separate out whether there is any additional pharma-cogenetic treatment selection role for BRAF mutation testing for anti-EGFR therapy in first-line versus second-line or higher treatment of metastatic colorectal cancer.

Important details that may the affect test accuracy and reproducibility of assays are not routinely reported in studies addressing clinical validity. The analytic validity of BRAF testing in colorectal cancer was not part of this systematic review. In general, we found that the published literature on analytic validity was sparse, and does not reflect the technology of the assays used in the studies. The analytic validity of BRAF testing in colorectal cancer is likely good, based on the one study by DeRoock and colleagues that independently validated assay results in a subset of patients using allele-specific PCR [17]. Most applicable and least biased analytic validity evidence for BRAF testing should be available from proficiency testing programs, although the proficiency testing data would not address important pre-analytic factors (that relate to tumor specimen and dissection of tissue) that may also influence test performance.

Evidence for EGFR-related pharmacogenetic testing, other than KRAS testing, in metastatic colorectal cancer to guide the use of anti-EGFR chemotherapy is still very limited (Supplementary Table 8). Evidence for these tests comes almost exclusively from fair- to marginal-quality retrospective studies without a comparison cohort who did not receive EGFR monoclonal antibodies. In general, if the gene mutation is not uncommon, prospective studies are clearly preferred. If prospective evaluation studies are not available or feasible, then better retrospective studies are needed. These studies should be based on well described cohorts, either nested in trials or clinical settings with high quality of information (patient and outcome assessment and documentation), with good descriptions of patient and setting characteristics (in terms of prognostic factors and treatment), and good follow-up and measurement of patient outcomes.

Low GRADE retrospective observational evidence suggests that BRAF testing in metastatic colorectal cancer patients is a negative predictor of response and survival in those treated with cetuximab (Supplementary Table 8). However, it is unclear if the association of BRAF mutation with worse tumor response and survival is due to predicting response to treatment with cetuximab or prognosis independent of treatment. Evidence for NRAS and PIK3CA exon 20 is thus far based on a very limited number of tumors with identified mutations and needs to be replicated in other populations and treatment settings. Evidence for PTEN loss of expression is conflicting, and may be due to clinical heterogeneity or variation in analytic and pre-analytic factors. IHC assays (the antibodies and scoring system) for protein expression need to be validated and standardized. The evidence is rapidly evolving, with numerous relevant conference abstracts presented in 2009 and 2010, without full publication of results, which most certainly will add to the knowledge base about the clinical validity of these tests (Supplementary Table 9). Improved reporting of important patient characteristics, treatment setting, and details of assays and tumor sample will help inform the applicability and implementation of clinically valid pharmacogenetic tests into practice.

Acknowledgments

The authors thank Smyth Lai, MLS for conducting the literature searches, Rongwei (Rochelle) Fu, PhD for statistical consultation, and Kevin Lutz, MFA for his editorial support. They also thank the following reviewers for their contribution to this evidence review: B. Nedrow (Ned) Calonge, MD, MPH; W. David Dotson, PhD; Stephanie Melillo, MPH; C. Sue Richards, PhD, FACMG; Matthew Thompson, MD, MPH, D.PHIL; and Katrina Goddard, PhD. This review was conducted as part of the Comparative Effectiveness Research in Genetics in Colorectal Cancer, NCI RC 2-CA148471.

Competing interests

The authors declare that they have no competing interests.

Supplementary Tables

Supplementary Table 1.

Search Strategies

Published Literature
Source	Dates	Search Details
OvidMEDLINE	OvidMedline 1996-November Week 3 2010	1 Colorectal Neoplasms/
		2 Colonic Neoplasms/
		3 Sigmoid Neoplasms/
		4 Rectal Neoplasms/
	Medline In Process November 26, 2010	5 Anus Neoplasms/
		6 Anal Gland Neoplasms/
		7 CRC.ti.ab.
		8 mCRC.ti.ab.
	Daily Update November 26, 2010	9 ((colon or colorectal or colonic or sigmoid or rectal or rectum) adj3 (cancer* or neoplasm* or neoplasia* or tumor* or tumour* or carcinoma* or adenocarcinoma* or metastatic or metastasis or metastases)).ti,ab.
		10 or/1-9
		11 “Proto-Oncogene Proteins B-raf”/
		12 “Proto-Oncogene Proteins C-raf”/
		13 BRAF protein, human.nm.
		14 KRAS protein, human.nm.
		15 Genes, ras/
		16 ras Proteins.sh.nm.
		17 PTEN Phosphohydrolase.sh.nm.
		18 PTEN protein, human.nm.
		19 1-Phosphatidylinositol 3-Kinase/
		20 PIK3CA protein, human.nm.
		21 p13k.ti,ab.
		22 PIK3CA.ti,ab.
		23 PTEN.ti.ab.
		24 “neuroblastoma RAS”.ti,ab.
		25 (k-ras or b-raf or n-ras or h-ras).ti,ab.
		26 (kras or braf or nras or hras).ti,ab.
		27 “V-Ki-ras2”.ti,ab.
		28 “Ki-ras*”.ti,ab.
		29 “Kirsten rat sarcoma”.ti,ab.
		30 “V-raf murine sarcoma”.ti,ab.
		31 V600E.ti,ab.
		32 ((ras or raf) adj3 (family or mutation* or gene* or pathway* or signal)).ti,ab.
		33 or/11-32
		34 Cetuximab.nm.
		35 Panitumumab.nm.
		36 Pharmacogenetics/
		37 pharmacogen*.ti,ab.
		38 erbitux.ti.ab.
		39 vectibix.ti.ab.
		40 “abx-egf”.ti,ab.
		41 or/34-40
		42 Antibodies, Monoclonal.sh.nm.
		43 “monoclonal antibod*”.ti,ab.
		44 or/42-43
		45 Receptor, Epidermal Growth Factor.sh.nm.
		46 EGFR protein, human.nm.
		47 “epidermal growth factor”.ti,ab.
		48 egfr.ti.ab.
		49 or/45-48
		50 44 and 49
		51 33 or 41 or 50
		52 10 and 51
		53 limit 52 to yr=“2000 -Current”
		54 limit 53 to english language
		55 remove duplicates from 54

CDSR, CENTRAL, DARE, HTA - searched simultaneously in Cochrane Library via Wiley Online Library	2000-2010	#1 (colon or colorectal or colonic or sigmoid or rectal or anus or anal) :ti,ab,kw
		#2 (cancer or cancers or neoplasm or neoplasms or carcinoma or carcinomas or adenocarcinoma or adenocarcinomas or metastatic or metastasis or metastases):ti,ab,kw
		#3 (#1 AND #2)
		#4 (crc or mcrc):ti,ab,kw
		#5 (kras or braf or nras or pten or p13k or pik3ca):ti,ab,kw
		#6 (ras or raf):ti,ab,kw and (family or mutation or mutations or gene or genes or pathway or pathways or signal):ti,ab,kw
		#7 (k-ras or b-raf or n-ras or pten or p13k or pik3ca):ti,ab,kw
		#8 (egfr or “epidermal growth factor”):ti,ab,kw
		#9 (“monoclonal antibodies” or cetuximab or panitumumab or erbitux or vectibix or pharmacogenetics or pharmacogenomics or pharmacogenetic or pharmacogenomic or oncogene or oncogenes):ti,ab,kw
		#10 (#3 OR #4)
		#11 (#5 OR #6 OR #7 OR #8 OR #9)
		#12 (#10 AND #11), from 2000 to 2010

Grey Literature

*Conference Papers Index - via CSA*	2009-2010	(KW=(kras or braf or nras or hras or pten or plk3ca or p13k or vraf)
		AND
		(KW=(colon or colonic or colorectal or rectal or rectum)
		AND
		(KW=(cancer or cancers or neoplasm or neoplasms or carcinoma or carcinomas or tumor or tumour or metastatic)
		Limited to: English Only

*DRUGS@FDA*	No Limits	Cetuximab
		OR
		Panitumumab
		No action dates or other limits imposed.
		Medical, Statistical, Chemistry, Pharmacology, and Clinical Pharmacology Biopharmaceutics Reviews pdfs downloaded for each drug.

*FDA.gov*	2008-2010	Food & Drug Administration Oncologic Drugs Advisory Committee [FDA ODAC] Meetings 2008-current, hand-searched

*clinicaltrials.gov*	No Limits	(braf OR kras OR nras OR hras OR PTEN OR PIK3CA OR P13K OR vraf)
		AND
		(colon OR colonic OR colorectal)
		AND
		(cancer OR cancers OR neoplasm OR neoplasms OR carcinoma OR carcinomas OR tumor OR tumour OR tumors OR tumours OR metastatic)

*NIH RePORTER*	No Limits	“Term search” field used for each statement
		(braf AND colon) OR (kras AND colon) OR (nras AND colon) OR (hras AND colon) OR (PTEN AND colon) OR (PIK3CA AND colon) OR (P13K AND colon) OR (vraf AND colon)

*Current Controlled Trials (ISRCTN)*	No Limits	kras OR braf OR nras OR hras OR pten OR pik3ca OR p1k3 OR vraf

*WHO ICTRP Search Portal*	No Limits	All searches were limited to: Condition: colorectal cancer Recruitment status: all
		Title: kras OR Title: braf OR Title: nras OR Title: hras OR Title: PTEN OR Title: PIK3CA OR Title: P13K OR Title: vraf

*Hand searched conference abstracts*	2009 to November 2010	American Society of Clinical Oncology (ASCO) General Meeting; Gastrointestinal Cancers Symposium; American Association for Cancer Research (AACR) Annual Meeting; College of American Pathologists (CAP); American Society for Therapeutic Radiology and Oncology (ASTRO) Annual Meeting; American College of Gastroenterology (ACG) Annual Meeting; Digestive Disease Week; European Crohn's and Colitis Organization (ECCO); International Symposium on Targeted Anticancer Therapies and World Congress on Gastrointestinal Cancer

Reference	Reason for exclusion
Albitar M, Yeh C, Ma W, Albitar M, Yeh C, Ma W. K-ras mutations and cetuximab in colorectal cancer. N Engl J Med. 2009;360:834-836.	No relevant outcomes

Al-Kuraya KS, Al-Kuraya KS. KRAS and TP53 mutations in colorectal carcinoma. Saudi Journal of Gastroenterology. 2009;15:217-219.	Not original research

Bibeau F, Lopez-Crapez E, Di FF et al. Impact of Fc{gamma}Rlla-Fc{gamma}Rllla polymorphisms and KRAS mutations on the clinical outcome of patients with metastatic colorectal cancer treated with cetuximab plus irinotecan. J Clin Oncol. 2009;27:1122-1129.	KRAS clinical validity only

Boccia RV, Cosgriff TM, Headley DL, Badarinath S, Dakhil SR. A phase II trial of FOLFOX6 and cetuximab in the first-line treatment of patients with metastatic colorectal cancer. Clinical Colorectal Cancer. 2010;9:102-107.	No relevant gene

Chung KY, Shia J, Kemeny NE et al. Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. J Clin Oncol. 2005;23:1803-1810.	No relevant gene

Daemen A, Gevaert O, De BT et al. Integrating microarray and proteomics data to predict the response on cetuximab in patients with rectal cancer. Pac Symp Biocomput. 2008; 166-177.	No relevant gene

Erben P, Strobel P, Horisberger K et al. KRAS and BRAF Mutations and PTEN Expression Do Not Predict Efficacy of Cetuximab-Based Chemoradiotherapy in Locally Advanced Rectal Cancer. Int J Radiat Oncol Biol Phys. 2010.	Genotyping not performed in tumor samples

Gebbia V, Del PS, Borsellino N et al. Efficacy and safety of cetuximab/irinotecan in chemotherapy-refractory metastatic colorectal adenocarcinomas: a clinical practice setting, multicenter experience. Clinical Colorectal Cancer. 2006;5:422-428.	No relevant gene

Goncalves A, Esteyries S, Taylor-Smedra B et al. A polymorphism of EGFR extracellular domain is associated with progression free-survival in metastatic colorectal cancer patients receiving cetuximab-based treatment. BMC Cancer. 2008;8:169.	KRAS clinical validity only

Graziano F, Canestrari E, Loupakis F et al. Genetic modulation of the Let-7 microRNA binding to KRAS 3'-untranslated region and survival of metastatic colorectal cancer patients treated with salvage cetuximab-irinotecan. Pharmacogenomics J. 2010.	No relevant gene

Graziano F, Ruzzo A, Loupakis F et al. Pharmacogenetic profiling for cetuximab plus irinotecan therapy in patients with refractory advanced colorectal cancer. J Clin Oncol. 2008;26:1427-1434.	No relevant gene

Hebbar M, Di FF, Conroy T et al. Assessment of baseline clinical predictive factors of response to cetuximab-irinotecan in patients with irinotecan-refractory metastatic colorectal cancer. Oncology. 2007;73:185-191.	No relevant gene

Hebbar M, Wacrenier A, Desauw C et al. Lack of usefulness of epidermal growth factor receptor expression determination for cetuximab therapy in patients with colorectal cancer. Anticancer Drugs. 2006; 17:855-857.	No relevant gene

Hoy SM, Wagstaff AJ, Hoy SM, Wagstaff AJ. Panitumumab in the treatment of metastatic colorectal cancer: profile report. Biodrugs. 2007;21:135-137.	Not original research

Irahara N, Baba Y, Nosho K et al. NRAS mutations are rare in colorectal cancer. Diagn Mol Pathol. 2010; 19:157-163.	No monoclonal antibodies

Italiano A, Follana P, Caroli FX et al. Cetuximab shows activity in colorectal cancer patients with tumors for which FISH analysis does not detect an increase in EGFR gene copy number. Ann Surg Oncol. 2008; 15:649-654.	No relevant gene

Jacobs B, De RW, Piessevaux H et al. Amphiregulin and epiregulin mRNA expression in primary tumors predicts outcome in metastatic colorectal cancer treated with cetuximab. J Clin Oncol. 2009;27:5068-5074.	KRAS clinical validity only

Jehan Z, Bavi P, Sultana M et al. Frequent PIK3CA gene amplification and its clinical significance in colorectal cancer. J Pathol. 2009;219:337-346.	No monoclonal antibodies

Jhawer M, Goel S, Wilson AJ et al. PIK3CA mutation/PTEN expression status predicts response of colon cancer cells to the epidermal growth factor receptor inhibitor cetuximab. Cancer Res. 2008;68:1953-1961.	Genotyping not performed in tumor samples

Katsios C, Ziogas DE, Roukos DH. Colorectal cancer: cetuximab, KRAS, BRAF, PIK3CA mutations and beyond. Expert review of gastroenterology & hepatology. 2010;4:525-529.	No relevant outcomes

Laurent-Puig P, Lievre A, Blons H, Laurent-Puig P, Lievre A, Blons H. Beyond the KRAS test. Eur J Cancer. 2009;45 Suppl 1:398-399.	Not original research

Lelli G, Cataldo S, Carandina I et al. The role of cetuximab in pre-treated refractory patients with metastatic colorectal cancer: outcome study in clinical practice. J Chemother. 2008;20:374-379.	No relevant gene

Lenz HJ, Van CE, Khambata-Ford S et al. Multicenter phase II and translational study of cetuximab in metastatic colorectal carcinoma refractory to irinotecan, oxaliplatin, and fluoropyrimidines. J Clin Oncol. 2006;24:4914-4921.	No relevant gene

Liao W, Liao Y, Zhou JX et al. Gene Mutations in Epidermal Growth Factor Receptor Signaling Network and Their Association With Survival in Chinese Patients With Metastatic Colorectal Cancers. Anat Rec (Hoboken). 2010.	No monoclonal antibodies

Lopez-Crapez E, Mineur L, Emptas H et al. KRAS status analysis and anti-EGFR therapies: is comprehensiveness a biologist's fancy or a clinical necessity? Br J Cancer. 2010; 102:1074-1075.	Not original research

Lurje G, Nagashima F, Zhang W et al. Polymorphisms in cyclooxygenase-2 and epidermal growth factor receptor are associated with progression-free survival independent of K-ras in metastatic colorectal cancer patients treated with single-agent cetuximab. Clin Cancer Res. 2008; 14:7884-7895.	No relevant gene

Mao C, Liao RY, Chen Q, Mao C, Liao RY, Chen Q. Loss of PTEN expression predicts resistance to EGFR-targeted monoclonal antibodies in patients with metastatic colorectal cancer. Br J Cancer. 2010;102:940.	Not original research

Moosmann N, Fischer-von WL, Vehling KU et al. Cetuximab plus XELIRI (capecitabine + irinotecan) versus cetuximab plus XELOX (capecitabine + oxaliplatin) for first-line treatment of metastatic colorectal cancer: a randomized trial of the AIO CRC Study Group. SO: Onkologie. 2006;29:141.	No relevant gene

Oden-Gangloff A, Di FF, Bibeau F et al. TP53 mutations predict disease control in metastatic colorectal cancer treated with cetuximab-based chemotherapy. Br J Cancer. 2009; 100:1330-1335.	No relevant gene

Pantaleo MA, Fanti S, Lollini PL et al. PET detection of epidermal growth factor receptor in colorectal cancer: a real predictor of response to cetuximab treatment? European Journal of Nuclear Medicine & Molecular Imaging. 2007;34:1510-1511.	Not original research

Paule B, Castagne V, Picard V et al. MDR1 polymorphism role in patients treated with cetuximab and irinotecan in irinotecan refractory colorectal cancer. Med Oncol. 2009.	No relevant gene

Personeni N, Fieuws S, Piessevaux H et al. Clinical usefulness of EGFR gene copy number as a predictive marker in colorectal cancer patients treated with cetuximab: a fluorescent in situ hybridization study. Clin Cancer Res. 2008;14:5869-5876.	No relevant gene

Personeni N, Hendlisz A, Gallez J et al. Correlation between the response to cetuximab alone or in combination with irinotecan and the activated/phosphorylated epidermal growth factor receptor in metastatic colorectal cancer. Semin Oncol. 2005;32:S59-S62.	No relevant gene

Reidy DL, Vakiani E, Fakih MG et al. Randomized, phase II study of the insulin-like growth factor-1 receptor inhibitor IMC-A12, with or without cetuximab, in patients with cetuximab- or panitumumab-refractory metastatic colorectal cancer. J Clin Oncol. 2010;28:4240-4246.	Not original research

Saltz LB. Can the addition of cetuximab to irinotecan improve outcome in colorectal cancer? SO: Nature clinical practice Oncology. 2005;2:20-21.	Not original research

Sartore BA, Moroni M, Veronese S et al. Epidermal growth factor receptor gene copy number and clinical outcome of metastatic colorectal cancer treated with panitumumab. SO: Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2007;25:3238-3245.	No relevant gene

Scartozzi M, Bearzi I, Mandolesi A et al. Epidermal Growth Factor Receptor (EGFR) gene copy number (GCN) correlates with clinical activity of irinotecan-cetuximab in K-RAS wild-type colorectal cancer: a fluorescence in situ (FISH) and chromogenic in situ hybridization (CISH) analysis. BMC Cancer. 2009;9:303.	No relevant gene

Scartozzi M, Bearzi I, Pierantoni C et al. Nuclear factor-kB tumor expression predicts response and survival in irinotecan-refractory metastatic colorectal cancer treated with cetuximab-irinotecan therapy. J Clin Oncol. 2007;25:3930-3935.	No relevant gene

Scartozzi M, Mandolesi A, Giampieri R et al. Insulin-like growth factor 1 expression correlates with clinical outcome in K-RAS wild type colorectal cancer patients treated with cetuximab and irinotecan. Int J Cancer. 2010.	No relevant gene

Seruca R, Velho S, Oliveira C, Leite M, Matos P, Jordan P. Unmasking the role of KRAS and BRAF pathways in MSI colorectal tumors. Expert review of gastroenterology & hepatology. 2009;3:5-9.	Not original research

Survival Data in FDA Approval for ERBITUX((R)) (CETUXIMAB) Supports Use as a Single Agent in Patients with Advanced Colorectal Cancer. Cancer Biol Ther. 2007;6:1671-1673.	No relevant gene

Tappenden P, Jones R, Paisley S, Carroll C. Systematic review and economic evaluation of bevacizumab and cetuximab for the treatment of metastatic colorectal cancer (Provisional abstract). SO: Health Technology Assessment. 2007;11:1-146.	No relevant gene

Tie J, Gibbs P, Lipton L et al. Optimizing targeted therapeutic development: Analysis of a colorectal cancer patient population with the BRAFV600E mutation. Int J Cancer. 2010.	No monoclonal antibodies

Tol J, Koopman M, Rodenburg CJ et al. A randomised phase III study on capecitabine, oxaliplatin and bevacizumab with or without cetuximab in first-line advanced colorectal cancer, the CAIRO2 study of the Dutch Colorectal Cancer Group (DCCG). An interim analysis of toxicity. Ann Oncol. 2008; 19:734-738.	No relevant gene

Tsuchihashi Z, Khambata-Ford S, Hanna N et al. Responsiveness to cetuximab without mutations in EGFR. N Engl J Med. 2005;353:208-209.	No relevant gene

Vallbohmer D, Zhang W, Gordon M et al. Molecular determinants of cetuximab efficacy. J Clin Oncol. 2005;23:3536-3544.	No relevant gene

Vincenzi B, Santini D, Tonini G, Vincenzi B, Santini D, Tonini G. Lack of response of cetuximab plus oxaliplatin in advanced colorectal cancer patients resistant to both oxaliplatin and cetuximab plus irinotecan. Ann Oncol. 2006;17:527-528.	No relevant gene

Wainberg Z, Hecht JR. A phase III randomized, open-label, controlled trial of chemotherapy and bevacizumab with or without panitumumab in the first-line treatment of patients with metastatic colorectal cancer. Clinical Colorectal Cancer. 2006;5:363-367.	Not original research

Winder T, Zhang W, Yang D et al. Germline Polymorphisms in Genes Involved in the IGF1 Pathway Predict Efficacy of Cetuximab in Wild-type KRAS mCRC Patients. Clin Cancer Res. 2010; 16:5591-5602.	Genotyping not performed in tumor samples

Windsor AC, Cohen R, Jiao LR et al. Cetuximab in the first-line therapy of metastatic colorectal carcinoma: not so CRYSTAL clear. Future Oncology. 2008;4:741-744.	Not original research

Wong R, Cunningham D, Wong R, Cunningham D. Using predictive biomarkers to select patients with advanced colorectal cancer for treatment with epidermal growth factor receptor antibodies.[Erratum appears in J Clin Oncol. 2009 Jun 20;27(18):3070]. J Clin Oncol. 2008;26:5668-5670.	Not original research

Zhang W, Azuma M, Lurje G et al. Molecular predictors of combination targeted therapies (cetuximab, bevacizumab) in irinotecan-refractory colorectal cancer (BOND-2 study). Anticancer Res. 2010;30:4209-4217.	No relevant gene

Zhang W, Gordon M, Press OA et al. Cyclin D1 and epidermal growth factor polymorphisms associated with survival in patients with advanced colorectal cancer treated with Cetuximab. Pharmacogenetics & Genomics. 2006;16:475-483.	Genotyping not performed in tumor samples

Zlobec I, Molinari F, Kovac M et al. Prognostic and predictive value of TOPK stratified by KRAS and BRAF gene alterations in sporadic, hereditary and metastatic colorectal cancer patients. Br J Cancer. 2010;102:151-161.	No relevant outcomes

Author, Year	% Mutation		% Negative protein expression		N	Median Age, years	% Male	% White	Rx	Prior chemo	Perf score	Length of follow-up	Rx efficacy	Survival outcomes
DeRoock 2010[17]	BRAF NRAS PIK3CA	6.5 4.1 13.0	NR		649	61	58.1	NR	C-mab	Yes	NR	NR	RECIST or WHO	PFS OS

Prenen 2009[33]^*	PIK3CA	11.6^§	NR		200	61	60	NR	C-mab	Yes	NR	NR	RECIST	PFS OS

DeRoock 2008[42]^*	BRAF NRAS	5.6^§ 4.4^§	NR		113	60	61.9	NR	C-mab	Yes	NR	NR	RECIST and WHO	NR

Cappuzzo 2008[30] ^*	BRAF PIK3CA	5.1^§ 17.7^§	AKT	NR^∥	82	63	64	NR	C-mab	Yes	Yes	18 months	RECIST	OS

Sartore-Bianchi 2009[27]^*	PIK3CA exon 9 exon 20	5.2 14.3	PTEN	8.3^§	81	64	65	NR	C-mab	Yes	NR	NR	RECIST	PFS OS

Sartore-Bianchi 2009[7]^*	BRAF	11.6	PTEN	36^§	114	64	65	NR	Both	Yes	NR	NR	RECIST	NR

Molinari 2009[34] ^*	BRAF	28.5^¶	PTEN	14.3	12	67	63	NR	Both	Yes	NR	NR	RECIST	NR

DiNicolantonio 2008[35]^*	BRAF	13.9^§	NR		79	63^‡	71	NR	Both	Yes	NR	NR	RECIST	PFS OS

Frattini 2007[36]^*	NR		PTEN	40.1^§	27	66^‡	67	NR	C-mab	Yes	NR	NR	RECIST	NR

Benvenuti 2007[32]^*	BRAF	12.8^§	NR		47	62^‡	63	NR	Both	Yes	NR	60 weeks	RECIST	NR

Moroni 2005[37]^*	BRAF PIK3CA	3.3^§ 9.7^§	NR		30	66^‡	71	NR	Both	Yes	Yes	48 weeks	RECIST	NR

Perkins 2010[24]^*	BRAF PIK3CA	4.3 8.7	AKT	NR	42	61.8^‡	57	NR	Both	Yes	NR	10 months	RECIST	NR

Laurent-Puig 2009[26]^*	BRAF	2.9^§	PTEN	19.8^§	162	NR	NR	NR	C-mab	Yes	NR	NR	RECIST	PFS OS

Lievre 2006[38] ^*	BRAF PIK3CA	0^§ 6.7^§	NR		30	62^‡	63	NR	C-mab	Yes	NR	NR	RECIST	OS

Perrone 2009[29]^*	BRAF PIK3CA	9.7^§ 12.9^§	PTEN	NR^∥	30	57^‡	63	NR	C-mab	Yes	NR	27 weeks	RECIST	PFS

Loupakis 2009[28]^*	NR		PTEN AKT	48.9 59.8^§	102	62	59	NR	C-mab	Yes	Yes	21 months	RECIST	PFS OS

Loupakis 2009[41]^*	BRAF	14.9	NR		87	66	60	NR	C-mab	Yes	Yes	NR	RECIST	PFS OS

Souglakos 2009[20]	BRAF PIK3CA	12.3 17.9	NR		92	59	52	NR	C-mab	Yes	NR	NR	NR	PFS

Saridaki 2010[39]^†	BRAF	8.3^§	NR		48	64	57	NR	C-mab	Yes	NR	NR	NR	PFS OS

Tol 2010[18]	BRAF PIK3CA	8.7 9.9^§	PTEN	42^§	227	62	60	NR	C-mab	No	Yes	35 months	WHO	PFS OS

Negri 2010[23]	NR		PTEN AKT	11.6^§ NR	50	NR	NR	NR	C-mab	NR	NR	23 months	NR	PFS OS

Montagut 2010[19]	BRAF	6.3	NR		48	NR	65	NR	C-mab	Yes	NR	NR	RECIST	NR

Barbier 2010[25]	NR		PTEN AKT	40.7^§ 28.6^§	18	64	57	NR	C-mab	Yes	NR	NR	RECIST	NR

Sohn 2009[40]	BRAF	0	NR		66	58	61	NR	C-mab	Yes	Yes	NR	RECIST	NR

Razis 2008[31]	NR		PTEN	13.9^§	72	60	56	NR	C-mab	Yes	NR	17 months	NR	NR

Freeman 2008[21]	BRAF PIK3CA	6.5 3.2^§	NR		62	62	60	77	P-mab	Yes	Yes	NR	RECIST and WHO	NR

Khambata-Ford 2007[22]	BRAF	0	NR		110	61	46	NR	C-mab	Yes	Yes	NR	WHO	NR

Study details		Population details		Intervention details	Outcomes: NRAS (codons 12, 13, 61) (In patients with KRAS wild-type tumor, unless otherwise specified)

Author, year Country Quality	Design Selection criteria	N (patients) Median Age Sex	Treatment	Genetic test	Mutation frequency	Objective tumor response^†	Calculated TPF (95%CI)^‡ FPF (95%CI)^§	Odds ratio for tumor response^∥ (95%CI)
DeRoock 2010[17]	Retrospective cohort	N= 649	C-mab combination therapy	MassArray genotyping, subset independently validated by direct sequencing or allele-specific PCR	All patients : 2.6% (17/644)	Mut: 7.7% (1/13)	TPF : 0.063 (0.033, 0.107)	OR : 0.14 (0.01, 0.70)
7 European countries	Chemotherapy refractory patients from 11 centers, 2001-2008	61 yrs [28-86]			In KRAS WT : 4.1% (13/315)	WT: 38.1% (110/289)^* p= 0.013	FPF : 0.009 (0.0002, 0.49)	Adj OR : 0.087 (0.004, 0.511)
Fair		58% men						Adjusted for age sex, number of previous chemotherapy, center
								OR defined with mutation predicting response

Number and Design of Studies	Major Limitations	Validity of Evidence	Summary of Findings
**KQ1. Clinical Utility: In patients with mCRC, does EGFR-related genetic testing (downstream to KRAS) reduce morbidity and/or prolong survival compared to not testing for these markers?**

None	Not applicable	Not Applicable	Currently, there are no studies designed to assess if testing for EGFR-related markers, other than KRAS, in patients with metastatic colorectal cancer directly reduces morbidity and or prolongs survival, compared to patients without genetic testing.

KQ2. Clinical validity: In patients with mCRC, how well does EGFR-related genetic testing (i.e., BRAF, NRAS, PIK3CA, PTEN, AKT) predict response to anti-EGFR antibody treatment, as measured by tumor response/disease progression?

27 studies total - 25 retrospective single arm cohort - 1 retrospective analysis of RCT - 1 prospective single arm cohort Studies with unique populations: -7 BRAF (n=1,224) - 1 NRAS (n=649) -4P/K3CA(n=1,030) - 8 PTEN (n=742) - 5 AKT (n=294)	Overlapping populations. Retrospective and many small (n<100) studies. Limited reporting of important patient/setting characteristics. Very little evidence for panitumumab. Limited reporting of outcomes by KRAS wild-types (PIK3CA, PTEN, AKT). Only one study compared outcomes with persons who did not receive anti-EGFR therapy; this study reported outcomes by disease control (versus tumor response) and included persons eligible for combination chemotherapy as first line treatment. Limitations in reporting at the individual study level did not allow for meaningful pooled analyses.	Quality: Fair to poor Applicability: mostly European patients, poor descriptions of patient characteristics and setting of treatment; findings strongest for chemorefractory patients getting cetuximab; gene mutation testing on primary tumor sample, but may be of issue for protein expression; mutations primarily assessed or confirmed with direct sequencing	Best evidence for **BRAF, NRAS, and PIK3CA comes from the largest retrospective study (n=649) of chemorefractory patients from a European consortium. In this study, BRAF** mutation (V600E) was present in 6.5% of KRAS wild-type. Only 8.3% of persons with BRAF mutations, compared to 38% of persons without BRAF mutations responded to chemotherapy with cetuximab (p= 0.0012). Clinical sensitivity and specificity were estimated at 9.8% (95% CI 6.3-14.5) and 1.6% (95% CI 0.2-5.6), respectively. Four percent of KRAS wild-type tumors had *NRAS* mutations and 13% had *PIK3CA* mutations (exon 9 and 20). Although NRAS and PIK3CA exon 20 mutations are associated with poorer outcomes, confidence intervals are wide given low number of mutations. Findings from other studies with and without overlapping populations were either not informative (because low or no mutations identified, limitations in outcome reporting) or consistent with findings from the study by DeRoock and colleagues. No other studies report results for PIK3CA exon 9 and 20 separately. Studies evaluating protein expression of PTEN and AKT generally used IHC, but used different antibodies and scoring systems. There is some evidence to suggest that PTEN loss may be associated with non-response, though results are conflicting between studies. Limitations at the individual study level prevent more definitive conclusions. No studies reported results for AKT loss in KRAS wild-type. Based on one study, PTEN and AKT protein expression are only concordant in 60% and 68% respectively, of primary and metastatic tumors

KQ3: Clinical validity or utility: In patients with mCRC, do treatment choices based on genetic testing (i.e., BRAF, NRAS, PIK3CA, PTEN, AKT) lead to improvement in survival (overall survival, progression-free survival), improvement in quality of life, or reduction in morbidity?

14 studies total -13 retrospective single arm cohort - 1 retrospective analysis of RCT Studies with unique populations: - 3 BRAF (n =968) - 1 NRAS (n=649) - 3 PIK3CA (n =968) -6 PTEN(n=652) -2 AKT(n = 194)	Overlapping populations. Retrospective and many small (n<100) studies. Limited reporting of important patient/setting characteristics. Very little evidence for panitumumab. Sparse reporting of survival outcomes. Possible differing definitions of PFS. No quality of life outcomes. Limited reporting of outcomes by KRAS wild-types (PIK3CA, PTEN, AKT). Only one study compared outcomes with persons who did not receive anti-EGFR therapy.	Quality: Fair to poor Applicability: mostly European patients, poor descriptions of patient characteristics and setting of treatment; findings strongest for chemorefractory patients getting cetuximab; gene mutation testing on primary tumor sample, but may be of issue for protein expression; mutations primarily assessed or confirmed with direct sequencing	Best evidence for **BRAF, NRAS, and PIK3CA comes from the largest retrospective study (n=649) of a European consortium of patients. For mutation frequencies, see Key Question 2. In this study, BRAF mutation was also associated with worse progression-free survival (absolute difference 18 weeks, p< 0.0001), and overall survival (absolute difference 28 weeks, p< 0.0001). Although NRAS and PIK3CA exon 20 mutations are associated with poorer survival outcomes, confidence intervals are wide given low number of mutations. In another study comparing disease control in persons who did (n=227) and did not (n=332) receive cetuximab with combination chemotherapy, differences in PFS and OS by BRAF mutation were essentially the same for both treatment arms. Survival was noticeably better in this study, as compared to other studies, which suggests important clinical heterogeneity. Findings from other studies with and without overlapping populations were either not informative (because low or no mutations identified, limitations in outcome reporting) or consistent with findings from the study by DeRoock and colleagues. Studies used different thresholds to dichotomize protein expression positivity or loss. Based on limited evidence, PTEN expression does not appear to have clinically robust ability to predict survival response to cetuximab or panitumumab. No studies reported results for AKT** loss in KRAS wild-type.

KQ4: Harms: What are the potential harms to patients of performing these genetic tests related to anti-EGFR antibody treatment decisions, including incorrect genotype assignment leading to incorrect treatment assignment, ELSI risks, and other risks associated with this testing?

No additional studies identified.	No clinically significant harms of testing hypothesized (other than incorrect genotype assignment leading to incorrect treatment assignment). Limitations in same as for KQ2.	Quality: Fair to poor Applicability: applicability of test performance same as for KQ2.	Best evidence for **BRAF, NRAS, and PIK3CA** comes from the largest retrospective study (n=649) of a European consortium of patients. Calculated point estimates of false positives (from whom potentially effective treatment is withheld) for BRAF, NRAS, and PIK3CA exon 20 are estimated below 2.0%.

		KRAS (codons 12, 13)^*		BRAF (V600E)

Purpose of study	Mutation frequency	∼30 to 45%		∼5 to 20%

	Evidence^†	24 studies, n=2242^‡		7 studies, n=1224^§

	Indication	Chemorefractory persons with mCRC		Chemorefractory persons with mCRC, with KRAS WT tumor

	Description of study design^∥	^#Studies in ≥second-line tx	^#Studies in first-line tx	^#Studies in ≥second-line tx	^#Studies in first-line tx

Clinical utility or clinical validity	RCT of PGX testing vs. no testing strategies	None		None

	Prospective cohort study of PGX testing vs. no testing strategies	None		None

	Prospective evaluation of PGX test from RCT of tx vs. no tx	1	1	None

	Retrospective evaluation of PGX test from RCT of tx vs. no tx	1	4	None	1

Clinical validity (association only)	Prospective evaluation of PGX test from cohort who received tx	1	1	1^¶	None

	Retrospective evaluation of PGX test from cohort who received tx	13	1	5^¶^**	None

	Well done case-control study (only if outcomes of interest are rare)	Not applicable

Analytic validity	Not systematically reviewed	Multiple mutations in multiple codons, clinical performance may depend on analytic considerations of assay		Single mutation in single codon, assay(s) likely with good accuracy and reproducibility

			Factors that may decrease quality of evidence

Outcome (key question)	No. studies (n pts)	Study design	Risk of bias	Indirectness	Inconsistency	Imprecision	Publication bias	Overall quality of evidence
Rx efficacy: Tumor response (KQ2)	7 BRAF (n=1224)	Almost exclusively retrospective evaluation of single arm cohort of patients getting EGFR inhibitors	Serious	No, but mainly applicable to c-mab combo tx in chemorefractory	No	Serious	No known publication bias	Low
	1 NRAS (n=649)		Serious		Only one study	Very serious		Very low
	4 PIK3CA (n=1030)		Serious		Serious	Very serious
	8 PTEN (n=742)		Very serious		Very serious	Very serious
	5 AKT (n=294)		Very serious		No	Not applicable

Survival: PFS or OS (KQ3)	3 BRAF (n=968)	Almost exclusively retrospective evaluation of single arm cohort of patients getting EGFR inhibitors	Serious	No, but mainly applicable to c-mab combo tx in chemorefractory	No	Serious	No known publication bias, possible selective reporting bias	Low
	1 NRAS (n=649)		Serious		Only one study	Very serious		Very low
	3 PIK3CA (n=968)		Serious		Serious	Very serious
	6 PTEN (n=652)		Very serious		No	Very serious
	2 AKT (n=194)		Very serious		No	Not applicable

Quality of life (KQ3)	None	Not applicable

Harms (KQ4)	No additional studies	Retrospective evaluation of cohort studies	Serious	Serious	No	Serious	Not applicable	Low to very low

Source	Study ID or First Author	Title	Details
clinicaltrials.gov	NCT01086267	Safety and Efficacy Study of BMS-908662 Alone or in Combination With Cetuximab in Subjects With K-RAS or B-RAF Mutation Positive Advanced or Metastatic Colorectal Cancer	Recruiting Completion Date: July 2012

clinicaltrials.gov	NCT00975897	Study of Tumor Tissue Testing in Selecting Treatment for Patients With Metastatic or Locally Advanced Colorectal Cancer	Recruiting Completion Date: NR

clinicaltrials.gov	NCT00655499	Panitumumab and Irinotecan as Third-Line Therapy in Treating Patients With Metastatic Colorectal Cancer	Recruiting Completion Date: NR

clinicaltrials.gov	NCT01243372	Biomarkers in Predicting Response to Cetuximab in Patients with Advanced Colorectal Cancer	Not yet recruiting Completion Date: NR

clinicaltrials.gov	NCT01126450	Lenalidomide and Cetuximab in Treating Patients with Metastatic Colorectal Cancer	Recruiting Completion Date: NR

clinicaltrials.gov	NCT00202787	Safety and Efficacy of Combination Therapy with Cetuximab and FOLFOX4 in Patients with Colorectal Cancer	Completed February 2009 No published article found

ISRCTN	ISRCTN83171665	FOCUS 3: the feasibility of molecular selection of therapy using KRAS, BRAF, and topo-1 in patients with metastatic or locally advanced colorectal cancer	Recruiting Completion Date: NR, assigned July 2009 Sponsored by the Medical Research Council (UK)

ESMO GI 2009	Saletti, P.	RAF kinase inhibitor protein (RKIP) is not a predictive factor in patients with K-RAS wild-type metastatic colorectal cancer (MCRC) treated with cetuximab or pantiumuab	Conference abstract only No published article found

ECCO15-ESMO34	Spindler, K.	Triple mutational testing for response to EGFR inhibitor treatment with cetuximab and irinotecan in metastatic colorectal cancer	Conference abstract only No published article found

ECCO15-ESMO34	van Cutsem, E.	A meta-analysis of the CRYSTAL and OPUS studies combining cetuximab with chemotherapy (CT) as 1st-line treatment for patients with metastatic colorectal cancer (mCRC): results according to KRAS and BRAF mutation status	Conference abstract only No published article found

AACR 2010	Peeters, M.	Use of massively parallel, next-generation sequencing to identify gene mutations beyond KRAS that predict response to panitumumab in a randomized, phase 3, monotherapy study of metastatic colorectal cancer (mCRC)	Conference abstract only No published article found

ASCO 2010	di Salvatore, M.	KRAS and BRAF mutational status and PTEN, cMET, and IGF1R expression as predictive markers of response to cetuximab plus chemotherapy in metastatic colorectal cancer	Conference abstract only No published article found

ASCO 2010	Yokota, T.	PTEN/p-AKT expression as predictive markers for cetuximab in colorectal cancer	Conference abstract only No published article found

ASCO 2010	Sood, A.	Beyond KRAS: the quest for novel genetic markers predictive for response to anti-epidermal growth factor receptor (EGFR) therapy in patients with metastatic colorectal cancer	Conference abstract only No published article found

ASCO 2010	Weickhardt, A.	DUX study: a phase II study of evaluating dual targeting of the EGFR using the combination of cetuximab and erlotinib in patients with chemotherapy refractory metastatic colorectal cancer	Conference abstract only No published article found

ASCO 2010	Maughan, T.	Identification of potentially responsive subsets when cetuximab is added to oxaliplatin-fluoropyrimidine chemotherapy (CT) in first-line advanced colorectal cancer (aCRC): mature results of the MRC COIN trial	Conference abstract only No published article found

ASCO 2010	Schwartzberg, L.	PEAK: a randomized phase II study to compare the efficacy of panitumumab plus mFOLFOX6 in patients (pts) with previously untreated, unresectable metastatic colorectal cancer expressing wild-type KRAS	Conference abstract only No published article found

ASCO 2010	Blank, P.	Cost-effectiveness of novel predictive tests in treatment of metastatic colorectal cancer: An analysis from a Swiss perspective	Conference abstract only No published article found

ASCO 2010	Garrido-Laguna, I.	Phosphatase and tensin homologue (PTEN) loss and response to phase I trials targeting PI3K/AKT/mTOR pathway in patients with advanced cancer	Conference abstract only No published article found

ASCO 2010	Janku, F.	PIK3CA, KRAS, and BRAF mutations in patients with advanced cancers treated with PI3K/AKT/mTOR axis inhibitors	Conference abstract only No published article found

ASCO 2009	Garcia, E.	Use of combined biomarkers analysis to predict response to chemotherapy in colorectal cancer: a single-institution feasibility study	Conference abstract only No published article found

ASCO 2009	Bengala, C.	EGFR gene copy number, KRAS and BRAF status, PTEN and AKT expression analysis in patients with metastatic colon cancer treated with anti-EGFR monoclonal antibodies +/- chemotherapy	Conference abstract only No published article found

ASCO 2009	Ruzzo, A.	Association of BRAF mutations and EGFR Intron-1 L/L genotype with resistance to cetuximab plus irinotecan treatment in KRAS wild-type metastatic colorectal cancer patients	Conference abstract only Portion of results have been published: Loupakis F, et al. KRAS codon 61, 146 and BRAF Mutations…Br J Cancer. 2009. 101(4):715-21.

ASCO Gl 2009	Norguet, E.	Use of nuclear PTEN expression status to predict survival of metastatic colorectal cancer patients treated with cetuximab	Conference abstract only No published article found

ASCO GI 2010	Kato, K.	Efficacy of cetuximab against KRAS wild-type colorectal cancer with BRAF or PIK3CA mutations	Conference abstract only No published article found

ASCO GI 2010	Xie, L.	Practical application of using predictive biomarkers to select patients with metastatic colorectal cancer for treatment with epidermal growth factor receptor inhibitors	Conference abstract only No published article found

ASCO GI 2010	Sood, A.	Use of PTEN expression in the primary tumor as a predictive marker for radiologic response to anti-epidermal growth factor receptor (EGFR) based therapy in patients with metastatic colorectal cancer (mCRC)	Conference abstract only No published article found

ASCO GI 2010	Linot, B.	Impact of PI3K, BRAF, and KRAS mutations on efficacy intensified FOLFIRI + cetuximab regimen in advanced colorectal cancer	Conference abstract only No published article found

ASCO GI 2010	Bokemeyer, C.	Biomarkers predictive for outcome in patients with metastatic colorectal cancer (mCRC) treated with first-line FOLFOX4 plus or minus cetuximab: updated data from the OPUS study	Conference abstract only No published article found

ASCO GI 2010	Kohne, C.	Cetuximab with chemotherapy (CT) as first-line treatment for metastatic colorectal cancer: a meta-analysis for the CRYSTAL and OPUS studies according to KRAS and BRAF mutation status	Conference abstract only No published article found Results presented at ECCO15-ESMO34, ASCO 2010, and ESMO GI 2009.

ESMO 2010	Stintzing, S.	Cetuximab plus xeliri versus cetuximab plus xelox as first-line treatment for patients with metastatic colorectal cancer (mCRC): analysis of the randomized trial of the German AIO CRC study group: KRK-0204	Conference abstract only No published article found

ESMO 2010	Smith, C.G.	High throughput somatic profiling of the RAS-RAF-MAP pathways in advanced colorectal cancer and correlations with response to cetuximab	Conference abstract only No published article found

ESMO 2010	Rebersek, M.	Correlation of BRAF status with clinical response to cetuximab in KRAS wild type (WT) metastatic colorectal (mCRC) patients-single institution experience	Conference abstract only No published article found

PERMALINK

Systematic review of pharmacogenetic testing for predicting clinical benefit to anti-EGFR therapy in metastatic colorectal cancer

Jennifers Lin

Elizabeth M Webber

Caitlyn A Senger

Rebecca S Holmes

Evelyn P Whitlock

Abstract

Introduction

Figure 1.

Figure 2.

Clinical utility

Clinical validity

Harms

Methods

Figure 3.

Results

Key Question 1 to 3: Benefits of pharmacogenetic testing

Figure 4.

BRAF (codon 600, V600E)

NRAS (codons 12, 13, and 61) and PIK3CA (exons 9 and 20)

Protein expression of PTEN and AKT

Key Question 4: Harms of pharmacogenetic testing

Discussion

Acknowledgments

Competing interests

Supplementary Tables

Supplementary Table 1.

Supplementary Table 2.

Supplementary Table 3.

Table 4.

B.

C.

D.

E.

Table 5.

B.

C.

D.

E.

Supplementary Table 6.

Table 7.

Table 8.

Table 9.

References

ACTIONS

PERMALINK

RESOURCES

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