NCCN: Continuing Education
Accreditation Statement
This activity has been designated to meet the educational needs of physicians and nurses involved in the management of patients with cancer. There is no fee for this article. No commercial support was received for this article. The National Comprehensive Cancer Network (NCCN) is accredited by the ACCME to provide continuing medical education for physicians.
NCCN designates this journal-based CME activity for a maximum of 1.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only the credit commensurate with the extent of their participation in the activity.
NCCN is accredited as a provider of continuing nursing education by the American Nurses Credentialing Center‘s Commission on Accreditation.
This activity is accredited for 1.0 contact hour. Accreditation as a provider refers to recognition of educational activities only; accredited status does not imply endorsement by NCCN or ANCC of any commercial products discussed/displayed in conjunction with the educational activity. Kristina M. Gregory, RN, MSN, OCN, is our nurse planner for this educational activity.
All clinicians completing this activity will be issued a certificate of participation. To participate in this journal CE activity: 1) review the learning objectives and author disclosures; 2) study the education content; 3) take the posttest with a 66% minimum passing score and complete the evaluation at http://education.nccn.org/node/54141; and 4) view/print certificate.
Release date: October 13, 2014; Expiration date: October 13, 2015
Learning Objectives
Upon completion of this activity, participants will be able to:
Summarize emerging data on non-exon 2 KRAS mutations, NRAS mutations, and BRAF mutations, and how these data may impact clinical decision-making
Identify established predictive markers for cetuximab and panitumumab
Discuss the clinical implications of data regarding potential new biomarkers for anti-EGFR therapy
Colorectal cancer (CRC) is one of the most commonly diagnosed malignancies. In 2014, an estimated 136,830 new cases will be diagnosed and 50,310 deaths will be attributed to CRC in the United States alone.1 With the introduction of more active chemotherapeutic agents and targeted therapies against both vascular endothelial growth factor and epidermal growth factor receptor (EGFR), survival has improved. However, outcomes remain highly variable because CRC is a heterogeneous multipathway disease.2,3 It is becoming increasingly important to individualize treatment based on specific tumor characteristics, preferably driver mutations, to avoid unnecessary toxicity and cost for patients whose disease is unlikely to respond.4
After the introduction of anti-EGFR targeted therapy with cetuximab and panitumumab, only a subset of colorectal tumors were found to respond to these agents. KRAS exon 2-activating mutations in codons 12 and 13 were later found to be negative predictive markers for response.5,6 Anti-EGFR therapy has since been limited to patients lacking these mutations.7 However, a substantial number of patients with KRAS wild-type tumors failed to respond.5,6 This article reviews established predictive markers for cetuximab and panitumumab, and discusses the clinical implications of recently published data regarding new potential biomarkers for anti-EGFR therapy.
Background
EGFR is a receptor tyrosine kinase that plays a crucial role in cellular proliferation and oncogenesis. Binding of ligands such as epidermal growth factor and transforming growth factor alpha (TGF-α) to the extracellular domain activates 2 key intracellular signaling pathways: the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathways (Figure 1).8 On stimulation, RAS proteins, which constitute the first part of the MAPK pathway, exchange GDP for GTP and sequentially activate RAF, followed by MEK, and then MAPK. Alternatively, ligand-bound EGFR can translocate PI3K to the cell membrane and activate AKT and other downstream targets.9 These two seemingly separate cascades have significant cross-talk.10 Stimulation of both pathways results in cellular proliferation, differentiation, angiogenesis, survival, and migration.9,11,12
Under normal circumstances, the EGFR pathway is very tightly regulated. Deregulation leads to uncontrolled growth and oncogenesis.12 Tumor cells can exploit and upregulate the EGFR pathway through various mechanisms, including overexpression of EGFR on the cell surface, gene amplification, activating mutations of the receptor or any downstream protooncogenes (eg, RAS, RAF), and inactivating mutations of tumor suppressor genes (eg, PTEN), all of which result in constitutive activation of downstream signaling.13 Conversely, blockade of this pathway inhibits tumor growth and proliferation.14,15
The EGFR pathway is a logical therapeutic target in CRC, because 60% to 80% of tumors express EGFR on the cell surface.16-18 With the development of the anti-EGFR monoclonal antibodies cetuximab and panitumumab, several investigators reported activity in CRC. In a phase II trial of cetuximab-alone in chemotherapy-refractory tumors expressing EGFR, partial responses were seen in 9% patients and stable disease in 37%.19 A larger randomized trial showed comparable response (8%) and stable disease (31%) rates, but improved overall survival (OS) with cetuximab versus best supportive care (BSC) (6.1 vs 4.6 months, respectively).20 Cunningham et al21 reported higher response rates and progression-free survival (PFS) in irinotecan-refractory patients given cetuximab plus irinotecan versus cetuximab alone, suggesting synergy between chemotherapy and anti-EGFR agents. Similarly, panitumumab improved PFS compared with BSC in previously treated patients.22 In the first-line setting, the CRYSTAL (Cetuximab Combined With Irinotecan in First-Line Therapy for Metastatic Colorectal Cancer) study randomized patients with metastatic CRC (mCRC) to FOLFIRI (leucovorin, infusional 5-FU, and irinotecan)/cetuximab versus FOLFIRI and showed higher response rates and PFS with cetuximab.23
Simplified schematic representation of the epidermal growth factor receptor (EGFR) pathway. Ligand binding causes homodimerization or heterodimerization of the EGFR and subsequent autophosphorylation of the intracellular domain. The MAPK or PI3K/AKT pathways are activated, which promote cellular growth, proliferation, and survival. In reality, these pathways have significant crosstalk; however, they have been shown as separate pathways for simplification. Anti-EGFR monoclonal antibodies inhibit the EGFR pathway by blocking the extracellular domain of the receptor but are ineffective if downstream messengers (eg, RAS) are mutated.
Citation: Journal of the National Comprehensive Cancer Network J Natl Compr Canc Netw 12, 10; 10.6004/jnccn.2014.0140
Outcomes of Selected Phase II/III Studies of Anti-EGFR Therapy With Regard to KRAS Mutation
Early trials evaluating anti-EGFR agents exclusively enrolled tumors expressing EGFR by immunohistochemistry (IHC), yet only a subset of patients experienced response. Although the degree of skin rash correlated with improved survival, no relationship between the level of EGFR expression by IHC and response rates was seen.19,21 Furthermore, responses were noted in tumors lacking IHC detectable EGFR.24,25 Thus, EGFR expression could not be used as a meaningful predictor of response and the quest for more precise biomarkers continued. Levels of EGFR ligand expression and EGFR gene copy number were also investigated; however, studies were equivocal and neither is currently used in clinical practice.26-28
KRAS
With an improved understanding of the EGFR pathway, Lievre et al29 hypothesized that mutations in downstream effectors of EGFR could modulate response to targeted therapies. Tumors from 30 patients treated with cetuximab were analyzed. No responses were seen in 13 patients harboring mutations in KRAS. Conversely, none of the 11 responders had KRAS mutations.29 This and several other uncontrolled studies suggested that KRAS mutations predicted for a lack of response to EGFR inhibitors.30,31 These findings were subsequently confirmed in multiple large trials when tumor samples were retrospectively analyzed for KRAS exon 2 (codons 12 and 13) mutations (Table 1).5,6,32
HRAS, KRAS, and NRAS belong to the RAS group of proteins that lie downstream of EGFR. The most common RAS mutations, seen in almost 40% of patients with mCRC, are activating mutations of KRAS exon 2 at codons 12 and 13.5,6 They lead to constitutive activation of downstream signaling and thus resistance to EGFR inhibitors. Based on increasing clinical data, in 2009 ASCO released provisional guidelines recommending that all patients with advanced CRC have KRAS exon 2 mutation testing before the initiation of anti-EGFR therapy.7 Subsequently, the FDA indications for both cetuximab and panitumumab were modified to reflect this change.33 Hence, KRAS exon 2 mutations became the first clinically useful negative predictive markers for anti-EGFR therapy.
The negative predictive value of KRAS exon 2 mutations for anti-EGFR therapy was validated in multiple studies,5,6 but some evidence showed that not all mutations were equal, and responses varied depending on the specific mutation. Two large retrospective pooled subgroup analyses from previous major trials suggested that patients with KRAS G13D mutation experienced response to anti-EGFR therapy.34,35 However, these responses were not as robust as those seen in patients with KRAS wild-type tumors, and other analyses were unable to replicate these findings.36 Therefore, patients with KRAS G13D mutations are not routinely offered anti-EGFR therapy in clinical practice.36,37
The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Colon Cancer currently recommend that all patients with mCRC have either primary or metastatic tumor tissue genotyped for KRAS and NRAS (discussed later) mutations at diagnosis of stage IV disease (to view the most recent version of these guidelines, visit NCCN.org). Patients bearing these mutations should not be treated with anti-EGFR agents alone or in combination with chemotherapy. No specific test is recommended, but specimens should be analyzed at a Clinical Laboratory Improvement Amendments-certified laboratory.38
Other RAS Mutations
As seen in multiple trials,5,6 a significant number of patients with KRAS exon 2 wild-type mCRC experience no response to anti-EGFR therapy. The lack of KRAS exon 2 mutations does not necessarily predict response, and clearly other factors are involved. Increasing evidence suggests that less common RAS mutations outside the routinely tested “hotspots” on exon 2 may lead to constitutive activation of the MAPK pathway.39 Loupakis et al40 first showed that 8 patients with KRAS exon 3 (codon 61) and exon 4 (codon 146) mutations had resistance to cetuximab despite being KRAS exon 2 wild-type. De Roock et al41 performed a retrospective analysis of more than 700 chemotherapy-refractory patients who received cetuximab plus chemotherapy and analyzed tumor samples for NRAS, PIK3CA, BRAF, and additional KRAS somatic mutations based on data from COSMIC (Catalogue of Somatic Mutations in Cancer).41 Despite being KRAS exon 2 wild-type, patients with mutations had a lower response rate to cetuximab than those with the so-called quadruple-negative phenotype.
Douillard et al42 performed a prospective-retrospective analysis of the PRIME (Panitumumab Randomized Trial In Combination With Chemotherapy for Metastatic Colorectal Cancer to Determine Efficacy) study, which randomized patients to upfront panitumumab plus FOLFOX4 (infusional 5-FU, leucovorin, oxaliplatin) versus FOLFOX4 alone. Using Sanger sequencing and polymerase chain reaction, tumors of 620 patients with KRAS exon 2 wild-type mCRC were retrospectively analyzed for other mutations at KRAS (exons 3, 4) and NRAS (exons 2, 3, 4). Additional RAS mutations were detected in 108 (17%) patients with mCRC originally classified as KRAS exon 2 wild-type. These patients did not benefit from panitumumab, and in fact had inferior PFS and OS compared with those treated with FOLFOX alone. Those without any RAS mutation did significantly better with the addition of panitumumab. Notably, this was the first study to hint at a detrimental effect of panitumumab in patients bearing RAS mutations in addition to KRAS mutations in exon 2.
A recently reported phase II trial showed comparable findings.43 The PEAK (Panitumumab Efficacy in Combination With mFOLFOX6 Against Bevacizumab Plus mFOLFOX6 in mCRC Subjects With Wild-Type KRAS Tumors) study evaluated FOLFOX plus panitumumab or bevacizumab in untreated patients with KRAS exon 2 wild-type mCRC. In a prospective-retrospective manner, tumors were tested for mutations in KRAS exon 3 (codons 59, 61), exon 4 (codons 117, 146) and NRAS exon 2 (codons 12, 13), exon 3 (codons 59, 61), and exon 4 (codons 117, 146). Patients with all-RAS wild-type mCRC had improved PFS with panitumumab/FOLFOX compared with those treated with bevacizumab/FOLFOX (13.1 vs 9.5 months; P=.02), and although the survival data were immature, a trend toward improved survival with panitumumab was seen. Among the RAS-mutated KRAS exon 2 wild-type group, no difference was seen in PFS or OS between the treatment arms.
Using the novel technique of massively parallel multigene sequencing (also known as next-generation sequencing [NGS]), investigators analyzed tumor samples previously assessed for KRAS (codons 12, 13) from patients enrolled in the 20020408 trial for 9 different genes, including RAS-activating mutations (KRAS codon 61; NRAS codons 12, 13, 61).44 They reported improved PFS with panitumumab compared with BSC in patients with wild-type KRAS (codons 12, 13, 61) and NRAS (codons 12, 13, 61) tumors.22,44 The analysis was subsequently expanded to include mutations in exon 4 of KRAS and NRAS, and an additional 9 and 2 patients were identified, respectively. The all-RAS wild-type tumors had an overall response rate of 15% with panitumumab compared with 1% in the RAS-mutant group.45 Patients with exon 4 mutations did not benefit from panitumumab.
Findings from the panitumumab studies have been upheld by trials evaluating cetuximab. The recently presented phase III FIRE-3 trial underscores the importance of other RAS mutations in selecting patients for treatment with cetuximab.46 Untreated patients with KRAS exon 2 wild-type mCRC were randomized to FOLFIRI with either cetuximab or bevacizumab. As part of a preplanned analysis, the effect of mutations at KRAS (exons 3, 4), NRAS (exons 2, 3, 4), and BRAF (V600E) on outcomes was assessed. Sequencing of all RAS mutations was possible in 455 of 592 enrolled patients; 65 harbored another RAS mutation, whereas 48 had mutated BRAF. Patients lacking any RAS mutations showed a higher overall response rate with FOLFIRI/cetuximab compared with FOLFIRI/bevacizumab (76.0% vs 65.2%; P=.044). No statistically significant improvement in PFS was noted, but improved OS was observed in the patients with all-RAS wild-type tumors treated with cetuximab (33.1 vs 25.9 months; P=.010).
Distribution of RAS Mutations Across Recent Studies
Updated results of the phase II OPUS (Oxaliplatin and Cetuximab in First-Line Treatment of mCRC) trial, which randomized patients to first-line FOLFOX with or without cetuximab, were also recently presented.47 Using the highly sensitive BEAMing (Beads, Emulsions, Amplification, and Magnetics) technology, KRAS exon 2 wild-type tumors were retrospectively evaluated for mutations in KRAS (exons 3, 4) and NRAS (exons 2, 3, 4). Of the 118 evaluable tumors, mutations at other loci were detected in 36 (31%). The all-RAS wild-type population had significantly improved PFS and response rates with FOLFOX/cetuximab, whereas PFS was better with FOLFOX-alone in patients harboring any RAS mutation. Cetuximab nonsignificantly improved OS in the wild-type population but seemed to be detrimental in the RAS-mutated group.
Also using BEAMing technology, retrospective tissue analysis of the larger phase III CRYSTAL study (FOLFIRI with or without cetuximab) is currently underway.48 Additional KRAS (exons 3, 4) and NRAS (exons 2, 3, 4) mutations are being examined in patients with KRAS exon 2 wild-type tumors. These studies are summarized in Tables 2 and 3.
BRAF
Up to 9% of colorectal tumors harbor the BRAF V600E mutation, which leads to constitutive activation of the EGFR pathway.49,50 Because RAF lies downstream of RAS and activates many of the same intracellular pathways, it is plausible that BRAF mutation status may also have predictive value for anti-EGFR therapy.40,51 Early studies supported such a role; however, pooled data from the CRYSTAL and OPUS trials showed no relationship of BRAF mutation to cetuximab response in the first-line setting.49,51,52 Similarly, the PRIME study reported that BRAF mutation was a negative prognostic factor but did not predict response to panitumumab.42 In contrast, the PICCOLO (Panitumumab, Irinotecan & Ciclosporin in Colorectal Cancer Therapy; irinotecan with or without panitumumab) study suggested that panitumumab had a detrimental effect on OS in patients with BRAF-mutated, fluoropyrimidine-refractory mCRC.53
Currently, the role of BRAF as a negative prognostic marker is well-established in mCRC, but its predictive value for anti-EGFR therapy remains controversial.52,54-56 The NCCN Guidelines for Colon Cancer uphold that although BRAF V600E mutation is associated with poor prognosis, insufficient data exist to use BRAF mutation status to guide anti-EGFR therapy. Testing for BRAF mutation in patients with RAS wild-type mCRC should only be used for prognostic purposes.38
Outcomes of Selected First-Line Phase II/III Studies With Extended RAS Mutation Testing
Others
Other potential biomarkers, such as PIK3CA mutations and loss of PTEN, have also been studied with conflicting results (Table 4); therefore, routine analysis is not recommended.55,57,58 A recent meta-analysis suggests a possible negative predictive value for these biomarkers, but further studies are needed and a detailed discussion is beyond the scope of this review.59
Discussion
Until recently, only KRAS exon 2 mutation testing was routinely performed in patients with mCRC who were candidates for anti-EGFR therapy. Significant work has been done to further delineate the group of patients likely to respond to anti-EGFR therapy, and newly presented data clearly show that genetic profiling should be expanded to include other RAS mutations. Improved outcomes with anti-EGFR agents in all-RAS wild-type tumors have now been seen in several studies. By the same token, worse outcomes have been documented in patients with non-exon 2 KRAS or NRAS mutations treated with anti-EGFR therapy, indicating that treatment toxicity should be avoided in these patients. Thus far, extended RAS mutations seem to be mutually exclusive of KRAS exon 2 mutations.60
Based on this new evidence, the most recent NCCN Guidelines for Colon Cancer strongly recommend KRAS and NRAS genotyping for all patients with mCRC.38 Patients with known KRAS and NRAS mutations should not be treated with anti-EGFR therapy alone or in combination with chemotherapy. Although the European Medicines Agency indications for cetuximab and panitumumab have been updated to include only patients with RAS wild-type (KRAS/NRAS) mutations,61,62 the FDA indications for cetuximab and panitumumab do not yet reflect the newer data.63,64
The question arises whether these results need to be confirmed by prospective studies before expanded KRAS and NRAS mutation testing is incorporated into routine clinical practice? The authors categorically believe that randomized prospective clinical trials to evaluate biomarkers remain the gold standard. However, these trials are expensive and may not be feasible because of the sheer number of patients needed for statistical significance. In the authors’ opinion, prospective-retrospective studies should be considered acceptable if certain criteria are fulfilled, such as adequate tissue samples, validated testing methodologies, and reproducibility across multiple studies.65 All of the clinical studies described had sufficient tumor tissue that was tested with validated biomarker assays and follow-up was adequate. Furthermore, results from archival specimens have now been validated by several studies. In addition, KRAS exon 2 mutation testing was accepted as standard of care based on similar prospective-retrospective studies.
Summary of PFS and OS Based on PIK3CA Mutation Status
With regard to validated biomarker assays, no FDA-approved tests currently exist for extended RAS mutational analysis. Testing methodologies such as pyrosequencing, single nucleotide polymorphism genotyping followed by mass spectrometry analysis, and targeted exome sequencing have been used.66-68 NGS is becoming increasingly popular, and may help in obtaining more information per sample and potentially identifying new predictive biomarkers in the future. NGS results include information about allele frequency that can identify small clonal populations. BEAMing is also a very sensitive upcoming method that can be used to detect small tumor populations.69 In the future, these approaches may help guide therapeutic strategies for an individual patient in the third- and fourth-line settings as disease clones evolve with treatment.
Conclusions
In conclusion, standard testing for KRAS exon 2 mutations is not sufficient to identify patients with mCRC who are likely to benefit from anti-EGFR therapies. Evidence now suggests that expanded genetic profiling, including extended KRAS and NRAS mutation analysis, may be necessary not only to enable better patient selection but also to avoid potential harm caused by therapy. Data on other biomarkers, such as EGFR, PTEN, PIK3CA, and BRAF, are immature and should not yet be incorporated into routine practice to inform clinical decisions regarding use of anti-EGFR therapy. Expanded RAS mutation analysis will further narrow the subset of patients likely to benefit from anti-EGFR therapy, but more studies are needed to identify other predictive biomarkers. Mutation testing needs to be standardized and made more cost-effective to allow for widespread implementation into clinical practice. NGS and other newer techniques may enable rapid and sensitive identification of current and novel predictive biomarkers in the future.
Drs. Ashraf and Kothari have disclosed that they have no financial interests, arrangements, affiliations, or commercial interests with the manufacturers of any products discussed in this article or their competitors Dr. Kim has disclosed that he has received honoraria from Bristol-Myers Squibb Company and Roche; grant and/or research support from Bayer AG and Novartis Pharmaceuticals; and is a consultant for Eli Lilly and Company.
CE AUTHORS
Deborah J. Moonan, RN, BSN, Director, Continuing Education & Grants, has disclosed that she has no relevant financial relationships.
Ann Gianola, MA, Manager, Continuing Education & Grants, has disclosed that she has no relevant financial relationships.
Kristina M. Gregory, RN, MSN, OCN, Vice President, Clinical Information Operations, has disclosed that she has no relevant financial relationships.
Rashmi Kumar, PhD, Senior Manager, Clinical Content, has disclosed that she has no relevant financial relationships.
Deborah A. Freedman-Cass, PhD, Oncology Scientist/Senior Medical Writer, has disclosed that she has no relevant financial relationships.
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