Abstract
Initial experience with the epidermal growth factor receptor monoclonal antibodies (EGFR MoAb) in unselected patients with metastatic colorectal cancer (mCRC) showed that most of the treated patients did not derive therapeutic benefit. This outcome has driven the search for biomarkers for this population. Recent advances have further shown the heterogeneous nature of this disease with multiple interlinked pathways being implicated. Two such pathways downstream to the EGFR, mitogen-activated protein kinase (MAPK) and (phosphoinositide 3-kinase) PI3K, have gained increasing attention and become targets for development of novel biomarkers and therapeutic agents. Here, we highlight recent progress. Clin Cancer Res; 16(15); 3811–8. ©2010 AACR.
Background
Epidermal growth factor receptor (EGFR) is a 170-KDa transmembrane growth factor receptor, which, after being bound by 1 of 10 different ligands, undergoes homo- or heterodimerization and triggers a series of signaling events via a receptor-linked tyrosine kinase, mainly through the RAS/RAF/MEK and the phosphoinositide 3-kinase (PI3K)/AKT pathways (1, 2). Currently, two EGFR monoclonal antibodies (MoAb) are U.S. Food and Drug Administration (FDA) approved for metastatic colorectal cancer (mCRC), and several are in development. Cetuximab is a chimeric mouse-human immunoglobulin G1 (IgG1) MoAb, whereas panitumumab is a fully human monoclonal IgG2 antibody. Initial EGFR MoAb trials that preselected patients on the basis of EGFR immunohistochemistry (IHC) failed to show a correlation with response to EGFR MoAb, and lead to an intense search for other predictive biomarkers downstream to EGFR (3).
KRAS is a GTPase that encodes the human cellular homolog of the transforming gene Kirsten rat sarcoma-2 virus immediately downstream to EGFR. Activating mutations of KRAS are an early component of colon cancer carcinogenesis, occurring in up to 58% of polyps > 1 cm (4–6). KRAS mutations in CRC occur with a prevalence of 30 to 40% with the most common mutations being in codons 12 and 13 of exon 2 (7–9). A randomized phase III study, evaluating panitumumab as third line therapy versus best supportive care in mCRC (n = 463, 184 with KRAS mutation), was the first large study confirming the negative predictive value of KRAS mutations. The response rate (RR) in KRAS wild-type (WT) versus mutant (MT) was 10% versus 0%, and progression free survival (PFS) 12.3 versus 7.4 weeks (10). These results were extended by the National Cancer Institute (NCIC) CO.17 trial evaluating third line cetuximab versus best supportive care in 394 patients (42% patients with KRAS mutations; ref. 11).
Several recently reported clinical trials have evaluated EGFR MoAbs in the first and second line settings in combination with cytotoxic chemotherapy. The CRYSTAL trial (540 out of 1,198 tested for KRAS mutation) evaluated FOLFIRI ± cetuximab, and the OPUS trial (233 out of 337 tested for KRAS mutation) evaluated FOLFOX ± cetuximab (12, 13). Both these trials in the first line setting confirmed the negative predictive value of KRAS mutation for benefit with EGFR MoAb. A recently updated meta-analysis of KRAS WT patients from these two studies further confirmed these findings with significant improvements in RR [odds ratio (OR) 2.16, P < 0.0001), PFS [hazard ratio (HR) 0.66, P < 0.0001], and overall survival [(OS); HR 0.81, P = 0.006; ref. 14]. In contrast, the large COIN trial evaluating first line fluoropyrimidine-based chemotherapy and oxaliplatin ± cetuximab did not show any benefit with the addition of cetuximab in the metastatic setting even in KRAS WT patients (15).
With regards to panitumumab, the PRIME trial evaluating FOLFOX4 ± panitumumab showed improvement in PFS (9.6 versus 8 months, P = 0.02) in the first line setting in the KRAS WT population (16). A similar reduction in PFS was seen in KRAS MT patients treated with panitumumab. The 20050181 (“181”) study examined panitumumab in the second line setting (FOLFIRI ± panitumumab), and also showed improvement in PFS (5.9 versus 3.9 months, P = 0.004) with addition of panitumumab in KRAS WT patients (17). Overall, except for the COIN study, these studies showed improved outcomes in the KRAS WT patients with addition of EGF MoAbs to cytotoxic chemotherapy. The reasons for the contradictory findings of the COIN study, and the shorter median survival compared with other recent trials, are unclear.
Currently, it is recommended that mCRC patients being considered for EGFR MoAb therapy be tested for mutations in codons 12 and 13 in exon 2 of the KRAS gene (18). Although KRAS mutation testing seems to have a 100% negative predictive value for response to EGFR MoAb therapy, there is only an incremental improvement in RR, PFS, and (in some studies) OS with this selection. Thus, a significant portion of the KRAS WT patients still derives no benefit. Furthermore, most studies also indicate that KRAS is not a prognostic marker (3, 8, 19–22). Overall, these studies show a need for the search for better biomarkers for EGFR MoAb therapy.
On the Horizon
Although prior studies suggest that more than 95% of all KRAS mutations are limited to codons 12 and 13, a recent study revealed that 9 of 106 colon cancer samples (8.4%) had mutations outside of these hotspots, with 7 in codon 146. Another 2% had KRAS gene amplification. Furthermore, these genetic changes had a differential phenotypic expression in vitro with a mutation in codon 164 actually being equivalent to KRAS WT (23). Although these new findings may contribute to our understanding of EGFR MoAb resistance in KRAS WT patients, the majority seem to have aberrant signaling through other components of the MAPK pathway such as BRAF, MEK, and/or other pathways downstream to EGFR, especially PI3K (24). Recent advances provide considerable information on these pathways, which is being used to generate new predictive and/or prognostic information (Table 1), and novel therapeutic options (Table 2).
Predictive biomarkers for EGFR MoAb treatment in mCRC
Summary of selected drugs in clinical trials targeting MAPK and/or PI3K pathways
BRAF is a serine-threonine kinase belonging to the RAF family of proteins (BRAF, CRAF, and ARAF). KRAS activates BRAF, which in turn activates MEK (see Fig. 1; refs. 25, 26). Large population-based studies indicate that activating mutations of BRAF occur in 10 to 20% of CRC (27–29), and all these mutations are in the P-loop (exon 11) or activation segment (exon 15) of the kinase domain of the gene. The mutation in the kinase domain is a single substitution (V600E) accounting for nearly 90% of all the mutations (30). Interestingly, with very rare exceptions, KRAS and BRAF mutations are mutually exclusive, and BRAF mutations are far more frequent in colon than rectal cancers (9). BRAF mutations also seem to be associated with right-sided tumors with sporadic microsatellite instability (MSI) and CpG island methylator phenotype (9, 31, 32). A recent retrospective study showed high concordance in the mutational status of BRAF and KRAS between primary and metastatic tumor biopsies, with less than 5% of the tumors acquiring a new mutation at metastasis (33). This finding suggests that KRAS and BRAF mutations can be analyzed with fairly high accuracy by sequencing specific hot spots from either the primary or the metastatic site.
EGFR signaling pathways. Ligand binding to EGFR leads to receptor dimerization. Subsequently, downstream pathways, including the MAPK pathway (through RAS) and the PI3K pathway (through p110 and subsequently PIP3), are activated. In the PI3K pathway, PIP3 activates AKT by facilitating its phosphorylation by PDPK1 and PDPK2 (mtorC2). AKT, in turn, activates mTOR. PTEN regulates activity of the PI3K pathway by converting PIP3 back to PIP2. In the MAPK pathway, RAS, RAF, MEK, and ERK are activated by sequential kinase activity. Both the pathways regulate multiple cellular processes vital for the malignant cell. Ligand binding and, therefore, downstream activation are blocked by EGFR MoAb. The most common aberrations leading to inappropriate activation of the pathways in cancer cells are also depicted.
Evidence suggests that BRAF has a significant negative prognostic value and possibly no predictive value for EGFR-targeting MoAb therapy. A retrospective analysis of mCRC patients who received therapy with EGFR MoAbs showed that 11 out of 113 (10%) tumors had a BRAF V600E mutation, and none of them responded to EGFR monotherapy (P = 0.029). Moreover, those with a mutation had significantly shorter PFS and OS (34). A retrospective analysis of 516 tumors from the phase III CAIRO2 trial, evaluating the role of two biological agents in the first line setting (CapOx + bevacizumab ± cetuximab), with 8.7% of the tumors having this mutation showed a negative prognostic value for BRAF V600E with significantly decreased OS (15.2 versus 21.5 months, P = 0.01), PFS (6.6 versus 10.4 months, P = 0.001), but no difference in rate of response to cetuximab (35). In another retrospective study of 173 refractory KRAS WT mCRC samples treated with cetuximab that analyzed for EGFR amplification, PTEN and BRAF status again showed that 5 out of 116 tumors had BRAF V600E, and this mutation was weakly associated with lack of response (P = 0.063), but strongly associated with shorter PFS and OS (P = 0.001; ref. 36). Data from Ogino and colleagues also showed that the 105 out of 649 patients (17%) who had BRAF V600E mutation had increased CRC-specific mortality (multivariate HR 1.97), confirming the negative prognostic value of BRAF mutation (32). A planned analysis of 1,404 patients from PETACC-3 [irinotecan versus irinotecan + infusional 5-fluorouracil (5-FU) in adjuvant setting for stages II and III CRC] showed that 7.9% of patients had BRAF mutations, and they had decreased OS [HR 1.66, 95% confidence interval (CI) 1.15-2.40, P = 0.0069]. This effect was more pronounced in patients with MSI-low and MSI-stable tumors (HR 2.19, 95% CI 1.43-3.37, P = 0.0034). Finally, a recent update on the CRYSTAL trial showed that 59 out of 625 evaluated patients (9%) had the BRAF V600E mutation. In this small group, no difference was found in OR, OS, and PFS when cetuximab was added to FOLFIRI. However, when compared with the patients with BRAF WT, these patients had lower median OS (10.3 versus 21.8 months on FOLFIRI and 14.1 versus 25.1 months on FOLFIRI + cetuximab) and median PFS (5.6 months versus 8.8 months on FOLFIRI and 8 months versus 10.9 months on FOLFIRI + cetuximab; ref. 19).
Apart from KRAS and BRAF, two other groups of molecules related to the EGFR pathway have also emerged as potential biomarker candidates: EGFR ligands and FcγR polymorphisms. EGFR is activated by a variety of ligands such as amphiregulin, epiregulin, EGF, and transforming growth factor-α. Using transcriptional profiling and reverse transcriptase-PCR results of 80 mCRC patients, Khambata-Fort and colleagues showed that elevated epiregulin and amphiregulin had a positive predictive value in determining response to cetuximab (37). Furthermore, patients with higher levels of these ligands had a significantly better PFS (103 and 115 versus 57 days for high versus low levels of epiregulin and amphiregulin, respectively). These results were validated in two other recent studies. A retrospective analysis of 385 patients enrolled in the NCIC CO.17 trial showed that patients with both high epiregulin and KRAS WT had a significantly better response to cetuximab (38). A second study evaluated 220 mCRC samples from patients treated with irinotecan + cetuximab and showed that patients with KRAS WT and higher levels of epiregulin and amphiregulin had significantly better PFS, OS, and response to treatment (39). Finally, another study evaluated 95 mCRC patients treated with cetuximab + irinotecan and similarly showed significant prognostic and predictive values to higher levels of epiregulin and amphiregulin (40).
Cetuximab, being an IgG1 antibody, should be capable of initiating antibody-dependent cell-mediated cytotoxicity (ADCC) mediated via the Fc-receptor (FcR). Data from other MoAbs (trastuzumab in breast cancer, rituximab in lymphoma) suggest that polymorphisms in two receptor genes, FcγRIIa-H131R and FcγRIIIa-V158F, are clinically relevant. These polymorphisms were evaluated in normal colonic tissue from 69 irinotecan-resistant mCRC patients treated with irinotecan + cetuximab. In patients with KRAS WT, the subset with genotypes FcγRIIIa-158V/V and/or FcγRIIa-131H/H were found to have better PFS and OS as compared with carriers of the polymorphisms (41). However, further analysis suggested that there is a high degree of linkage disequilibrium between these two polymorphisms in the Caucasian population and only FcγRIIIa-158V/V has predictive value (42). If true, this finding would suggest better response with antibodies that can initiate ADCC; however, there is no difference in RRs between cetuximab and panitumumab. Moreover, if this effect were clinically significant, KRAS mutant tumors should also have some benefit with cetuximab that has not been observed so far.
Although attempts at developing drugs targeting KRAS (farnesyl inhibitors) have largely been unsuccessful, several BRAF inhibitors have been discovered. Sorafenib is an oral multikinase inhibitor that targets both WT BRAF and oncogenic BRAF V600E and has in vitro activity in CRC cell lines with this mutation (34, 43). However, sorafenib also inhibits multiple other tyrosine kinases, especially those involved in angiogenesis, and it is unclear how much effect sorafenib exerts through the MAPK pathway. Currently, several phase II-III clinical trials are evaluating the role of sorafenib in mCRC treatment in relation to KRAS and/or BRAF mutation status, which should help define the role of this agent in this setting. More selective RAF inhibitors are being studied and are in various stages of development (Table 2; refs. 44, 45). However, a series of recent studies has shown that BRAF V600E inhibitors may, in fact, cause paradoxical activation of the MAPK pathway in KRAS MT/BRAF WT or kinase dead tumors through formation of CRAF homo- or heterodimers (with BRAF), and subsequent interaction with the upstream mutated KRAS (46–48). The clinical implications of these findings in colorectal cancer treatment are yet unclear. Although BRAF is the best-studied kinase, at least five kinases phosphorylate MEK (ARAF, BRAF, CRAF, Tpl2, and Mos). This high level of redundancy at the level of activation of MEK would suggest that inhibition of this enzyme may be a more effective strategy to inhibit MAPK signaling than at KRAS or BRAF, and multiple agents have been developed toward this target (Table 2; ref. 45).
PI3K is an effector pathway of EGFR and multiple other receptors. This pathway has been reviewed extensively (49, 50), and will only be summarized briefly here. PI3Ks are divided into classes and subclasses of which class IA is the most extensively studied. The class IA PI3Ks are heterodimers consisting of a regulatory subunit, p85 that has three isoforms, p85α, p85β, and p55γ collectively called p85, and the p110 catalytic subunit with p110α, p110β (ubiquitous distribution), and p110γ (expressed mainly in leukocytes) isoforms. p85α has an inhibitory effect on the p110α catalytic subunit in the basal state, but upon activation by receptor tyrosine kinases or G-protein coupled receptors, it mediates localization of the enzyme to the membrane. The p110α catalytic subunit, upon activation, phosphorylates membrane phosphatidyl-inositol-, 4,5-triphosphate (PIP2) to phosphatidyl-inositol-3, 4,5-triphosphate (PIP3). PIP3 is an important lipid second messenger that provides docking sites for multiple downstream components, including a putative 3-phosphoinositide-dependent protein kinase 1 (PDPK 1) and AKT. The cellular levels of PIP3, and thus the activity of the PI3K pathway, are tightly regulated by the opposing lipid phosphatase activity of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN). AKT is a serine-threonine protein kinase expressed as three isoforms (AKT1, AKT2, and AKT3), and upon docking with PIP3, undergoes phosphorylation at two sites by PDPK1 and PDPK2 to be activated. Once activated, it activates multiple downstream proteins important in protein synthesis [mammalian target of rapamycin (mTOR), S6K], metabolism (glycogen synthase3), and cell survival and proliferation (FOXO1, MDM2, etc). mTOR is also a serine-threonine protein kinase belonging to the PI3K superclass, with significant structural homology to the PI3Ks. mTOR exists in two distinct complexes (mTORC1, mTORC2), each with multiple subunits. Upon activation, mTORC1 targets ribosomal and translational components important in protein synthesis, whereas mTORC2 is thought to be the primary PDPK2 phosphorylating AKT.
Recent studies have shown convincing evidence that many components of the PI3K pathway are frequently altered in numerous human cancers. Germline mutations in the PTEN gene cause syndromes with predisposition to hamartomatous CRC (Cowden's syndrome; ref. 51). Although somatic mutations in the PTEN gene are rare in CRC, several retrospective studies have shown by using IHC that 20 to 40% of CRC samples have loss of PTEN expression (52, 53). In contrast to the PTEN gene, the PIK3CA gene encoding the p110α subunit of class IA PI3K is frequently mutated in CRC. Most studies, including the large population-based study by Nosho and colleagues (54), have only evaluated exons 9 and 20 of this gene and showed a mutation rate of 10 to 15% (52–56). A retrospective study that sequenced all the coding exons of this gene, in contrast, revealed that 74 of the 234 (31.6%) evaluated colorectal cancer samples had a mutation in the PIK3CA gene. Although this study suggests that a significant proportion of the mutations may occur outside exons 9 and 20, this study, like others on this gene, is not population based and thus may have suffered from a selection bias (57). Overall, on the basis of current data, it seems that the PIK3CA gene mutation frequency in CRC is probably between 15 and 25%, and that these mutations show a more scattered pattern than those in the KRAS and BRAF genes. The PIK3R1 gene encoding p85α has also been reported to be mutated in CRC (58).These mutations cause constitutive activation of the PI3K pathway in preclinical models (59, 60).
The data on PTEN status are more limited. Because PTEN can be mutated, deleted, or silenced, ascertainment of PTEN status is usually done at the protein level. A small retrospective study showed that none of 11 patients with PTEN loss had response to cetuximab, in contrast to 10 out of 16 with intact PTEN expression who had partial response (P < 0.001; ref. 61). Another retrospective study evaluated PTEN expression by IHC and KRAS mutation analysis by DNA sequencing in primary, metastatic, and paired (96, 59, and 53, respectively) samples from patients with irinotecan-resistant mCRC treated with cetuximab (53). The concordance rate between primary and metastatic tumors for PTEN was 60%, and PTEN loss in metastatic tumors significantly predicted lack of response to cetuximab (36% versus 5%, P = 0.007). Moreover, patients with PTEN null metastases had shorter PFS (3.3 versus 4.7 months, P = 0.005), which was even more significant in KRAS WT patients. However, in sharp contrast, the PTEN analysis of the primary tumor did not reveal any predictive or prognostic information. Although the relatively low concordance rate between primary and metastatic tumors for PTEN expression could be secondary to selection of clonal populations during metastasis, it could also be secondary to the subjective nature of IHC testing with significant method and observer variability. This consideration and the possible need to analyze PTEN from metastatic tumors may limit the role of PTEN as a biomarker in CRC. This study also analyzed AKT expression by IHC and failed to show any correlation with prognosis or response to cetuximab. Studies, however, have shown a role for mutations or amplification of AKT1 and amplification of AKT2 gene in aberrant activation of this pathway (62).
Another retrospective study analyzing KRAS, PTEN, and PIK3CA mutations in 110 mCRC patients treated with either of the EGFR MoAbs showed that none of the 15 patients with a PIK3CA mutation responded to treatment (P = 0.038). This effect was even stronger when limited to patients with KRAS WT (P = 0.016). Furthermore, patients with PIK3CA mutations had shorter PFS (P = 0.0035), which was again more significant when limited to KRAS WT tumors (P = 0.0021). PTEN loss was also associated with decreased OS (P = 0.0048; ref. 52). However, Prenen and colleagues, who retrospectively analyzed PIK3CA and KRAS mutation status in 200 mCRC patients treated with cetuximab ± irinotecan, provided conflicting data. No significant difference was found between PIK3CA WT and MT in response to cetuximab or prognosis in their study (56).
PIK3CA mutations were also analyzed as a prognostic marker in 450 patients with resected nonmetastatic CRC. This study showed that upon multivariate analysis controlling for other risk factors for recurrence, PIK3CA mutations predicted increased CRC specific mortality (multivariate HR 2.23; 95% CI, 1.21-4.11) However, this effect seemed to be limited to patients with KRAS WT (55). PIK3CA mutations were also shown to be associated with increased risk of local recurrence upon multivariate analysis in 19 out of 240 patients with resected rectal cancer who did not receive radiation (HR 3.4; 95% CI 1.2-9.2, P = 0.017; ref. 63).
Given the key role of the PI3K pathway in many cell processes vital for tumorigenesis, this pathway has been targeted recently for drug development. The first generation PI3K inhibitors (Wortmannin and LY294002) were nonselective for the different classes and had poor pharmacological properties (49, 50). Chemotypes more specific to PI3K class I, and further, more specific to different isoforms, are in various stages of development (Table 2). One of the p110α specific compounds, PI-103, was found to have the ability to inhibit mTOR as well, which is not surprising given the structural homology between PI3Ks and mTOR. This finding has led to the development of a new class of compounds with dual effects at two different sites in the pathway, with the prospect of more potent inhibition (49, 50).
Given the central role of AKT in the PI3K pathway and its potential to activate multiple downstream effector proteins, it promises to be an attractive anticancer therapeutic target and many such compounds are either in the preclinical or early phase trials. Perifosine is a synthetic alkylphospholipid similar to miltefosine, but without the latter's severe gastrointestinal side effects, and has been shown to have strong antineoplastic effects in preclinical and early clinical studies (64, 65). It is thought that this compound inhibits signaling through multiple pathways, including the MAPK, JNK, and PI3K (via AKT) pathways. Perifosine has been shown to have activity in combination with capecitabine (P-CAP) in heavily treated mCRC, especially in patients previously refractory to 5-FU in a small phase II trial that compared this combination to capecitabine with placebo (CAP). The overall median OS for P-CAP versus CAP was 24.3 versus 16.3 months (P = 0.1348), and the median time to progression and OS for refractory 5-FU patients (P-CAP versus CAP) were 18 versus 10.3 weeks (P = 0.0188) and 24.3 versus 11.7 months (P = 0.0346), respectively (66). In contrast to AKT inhibitors, mTOR inhibitors have been in clinical use for a considerable time, first as antifungals, later as immunosuppressives (sirolimus), and, finally, as antineoplastic agents in renal cell carcinoma (sirolimus, temsirolimus, and everolimus). However, mTOR inhibitors may have alternate effects by inhibiting either mTORC1 (protein synthesis), mTORC2 (PDPK2 activation of AKT), or both (49, 50). Multiple mTOR inhibitors are currently in development (Table 2). Preclinical studies have suggested a role for mTOR inhibitors in treatment of CRC, and this is being explored in phase I-II clinical trials with everolimus. A recent retrospective study suggested that the PI3K pathway could have a particularly high rate of derangement in hereditary nonpolyposis CRC (HNPCC), with 51 out of 58 (88%) of the analyzed tumors having alteration in at least one pathway component, raising the intriguing possibility of targeting this pathway in this small, but significant, subset of CRC (67). Finally, preclinical evidence suggests that the PI3K and MAPK pathways are strongly interlinked, and that the combination of MEK and PI3K inhibitors act synergistically to inhibit tumor cells with RAS mutations (68).
In summary, both MAPK and PI3K pathways are stimulated by EGFR, with important implications for EGFR MoAb therapy and future drug development. Current American Society of Clinical Oncology (ASCO) guidelines recommend testing only for KRAS mutations in codons 12 and 13, in patients being considered for EGFR MoAb therapy. Although the data reviewed here are mostly from retrospective studies and need to be confirmed before routine clinical use, it appears that BRAF has a negative prognostic value, and possibly little predictive value. In the case of the PI3K pathway, its activation by either loss of PTEN or mutation of PIK3CA appears to have significant negative predictive and prognostic effects that are more pronounced in KRAS WT tumors. The subjective nature of PTEN assessment, however, is a significant challenge. Finally, early data suggest that elevated levels of EGFR ligands appear to hold promise as positive predictive biomarkers. New drugs are being developed against numerous targets in these pathways, and many are in early clinical stages.
Several important issues need to be addressed during this process of developing novel drugs targeting these pathways. Treatment of patients with EGFR MoAb without KRAS preselection cost the U.S. health care system billions of dollars, and also caused toxicities in thousands of patients with no chance of benefit from 2004 to 2009. It would be far more economical to collect tumor samples and/or design biomarker-driven clinical trials during development of these targeted agents, prior to widespread use, without the knowledge gained from such studies. It is notable that the majority of biomarker studies with EGFR MoAbs originated outside of the United States, where, ironically, biomarker studies have been chronically underfunded despite massive sums spent on unselected patient populations. Finally, the ability of the cancer cell to develop drug resistance via new mutations or alternate signaling pathways also needs to be addressed by combination therapy, and, if possible, analysis of tumor tissue upon progression. Given the frequency of alterations in the PI3K and MAPK pathways in colorectal cancer, these agents represent a promising avenue of investigation.
Disclosure of Potential Conflicts of Interest
W. Messersmith, unpaid advisory board, AstraZeneca, Genentech, commercial research grant, Pfizer, Roche. A. Dasari disclosed no potential conflict of interest.
- Received February 1, 2010.
- Revision received May 13, 2010.
- Accepted May 18, 2010.
References
Volume 16, Issue 15
