Annals of Oncology Advance Access originally published online on July 31, 2008
Annals of Oncology 2009 20(1):84-90; doi:10.1093/annonc/mdn541
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gastrointestinal tumours |
PI3KCA/PTEN deregulation contributes to impaired responses to cetuximab in metastatic colorectal cancer patients
1 Experimental Molecular Pathology, Department of Pathology
2 Medical Oncology Unit 2, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
3 Laboratory of Molecular Diagnostics, Institute of Pathology, Locarno, Switzerland
4 Preventive-Predictive Medicine Unit
5 Colorectal Surgery Unit
6 Scientific Management, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
* Correspondence to: Dr S. Pilotti MD, Department of Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Venezian 1, 20133 Milan, Italy. Tel: +39-02-2390-2293; Fax: +39-02-2390-2198; E-mail: silvana.pilotti{at}istitutotumori.mi.it
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Abstract |
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Background: It has been reported that KRAS mutations (and to a lesser extent KRAS mutations with the BRAF V600E mutation) negatively affect response to anti-epidermal growth factor receptor (EGFR) mAbs in metastatic colorectal cancer (mCRC) patients, while the biological impact of the EGFR pathway represented by PI3K/PTEN/AKT on anti-EGFR treatment is still not clear.
Patients and methods: We analysed formalin-fixed samples from a cohort of 32 mCRC patients treated with cetuximab by means of EGFR immunohistochemistry, EGFR and PTEN FISH analysis, and KRAS, BRAF, PI3KCA, and PTEN genomic sequencing.
Results: Ten (31%) of 32 patients showed a partial response to cetuximab and 22 (69%) did not [nonresponder (NR)]. EGFR immunophenotype and FISH-based gene status did not predict an anti-EGFR mAb response, whereas KRAS mutations (24%) and PI3K pathway activation, by means of PI3KCA mutations (13%) or PTEN mutation (10%)/loss (13%), were significantly restricted to, respectively, 41% and 37% of NRs.
Conclusion: These findings suggested that KRAS mutations and PI3KCA/PTEN deregulation significantly correlate with resistance to cetuximab. In line with this, patients carrying KRAS mutations or with activated PI3K profiles can benefit from targeted treatments only by switching off molecules belonging to the downstream signalling of activated EGFR, such as mammalian target of rapamycin.
Key words: cetuximab, KRAS, metastatic colorectal cancer, PI3KCA, PTEN
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introduction |
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TopAbstractintroductionpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
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Recently published reports of clinical trials indicate that cetuximab treatment is associated with stable disease (SD) according to the RECIST criteria or a response to therapy in 39% of patients with advanced colorectal cancer (CRC) [1]. The CRC patients in all these studies were selected on the basis of the expression of epidermal growth factor receptor (EGFR) in accordance with the Food and Drug Administration guidelines, but simple immunohistochemical evidence of EGFR expression is no longer considered clinically useful as the intensity of tumour staining correlates poorly with the response to cetuximab [2, 3]; furthermore, there have been reports of a response in patients with EGFR-negative tumours [4, 5].
Recent FISH data have shown a positive correlation between a gain in EGFR gene copy number and the response to cetuximab [2, 6–8] or panitumumab [9]. However, Italiano et al. [10] have reported that EGFR gene status alone is insufficient to predict the response to anti-EGFR mAbs because a fraction of the tumours with an increased EGFR gene copy number did not respond and some without did. The same finding has been observed in a few individual cases in treated CRC series despite the statistically significant association between a high EGFR gene copy number and response to anti-EGFR mAbs observed in the series as a whole [2, 6, 8, 9].
Although not fully validated, molecular markers associated with disease control following cetuximab treatment are high levels of messenger RNA for the EGFR ligands epiregulin and amphiregulin as quantified by gene expression profiling [11].
Other factors involved in response/resistance to cetuximab have been investigated in metastatic colorectal cancer (mCRC) patients, including KRAS mutations [2, 3, 7, 12–14], RAS/RAF signalling pathway activation [15], and the loss of PTEN expression [2]. As expected, the results of these investigations indicate that all of these alterations negatively affect the response to anti-EGFR mAbs.
The EGFR has three domains: an extracellular-binding domain, a transmembrane domain, and a catalytic domain. It is activated as a dimer by binding to various types of ligands, which leads to the phosphorylation of both molecules and the switching on of several pathways, including RAS/RAF/mitogen-activated protein kinase (MAPK), PI3KCA/AKT/mammalian target of rapamycin (mTOR), and JAK-STAT. EGFR may therefore be targeted by inhibiting its extracellular domain using mAbs that block its interactions with natural ligands, by inhibiting the catalytic domain using small molecules that compete with intracellular ATP, or by inhibiting the downstream effectors such as MAPK or mTOR, which are triggered by activated EGFR.
We studied a cohort of 32 cetuximab-treated mCRC patients in order to investigate the mechanisms thought to favour or impair responses to the drug by means of immunophenotype, molecular, and cytogenetic analyses. The results confirmed the negative predictive role of KRAS mutations and indicated that activation of the EGFR signalling pathway as result of the deregulation of PI3KCA/PTEN could contribute to the failure of cetuximab, a finding that may have considerable implications for the treatment of mCRC patients.
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patients and methods |
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patients and treatment
We analysed 32 irinotecan-refractory mCRC patients (20 males and 12 females with a median age of 57 years) who received cetuximab (C225, Erbitux®, Merck-Serono, Darmstadt, Germany) plus irinotecan at Medical Oncology Unit 2 of the Fondazione IRCSS Istituto Nazionale dei Tumori (Milan, Italy) (Table 1). They were considered refractory to irinotecan because they had experienced disease progression during treatment or within 3 months of the end of the last received cycle. Irinotecan was given in the form of an infusion at a dose of 300 mg/m2 every 3 weeks and cetuximab at 400 mg/m2 followed by a weekly dose of 250 mg/m2. Seventeen patients received the combination after two treatment lines and 15 after three or more. All the patients had previously received 5-fluorouracil or oral fluoropyrimidines, oxaliplatin, and irinotecan for metastatic disease.
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The response to the cetuximab/irinotecan was evaluated every 9 weeks and, in the case of an objective response, the therapy was continued for up to 27 weeks; in the case of SD, the treatment was discontinued after 18 weeks. Responses were assessed by means of computed tomography according to the Response Evaluation Criteria in Solid Tumors and classified as partial response (PR), SD, and progressive disease (PD). For this analysis, the patients showing a partial response were classified as partial responders (PRs) and those with SD or PD as nonresponders (NRs).
All the patients gave their informed consent to participate in the study.
samples
The analyses were made using 44 formalin-fixed, paraffin-embedded (FFPE) surgical specimens or biopsies of primary tumour and synchronous (s) or metachronous (m) metastases from 12 patients, primary tumour alone in 12 and metastases alone in eight (Table 2). All the metastases were extracolonic: 17 hepatic, two pulmonary, and one uterine. Seven samples were referred and 37 were institutional.
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immunohistochemistry
The immunohistochemistry analyses were made using 2-µm thick sections of FFPE primary tumour and/or metastasis specimens.
MLH1, MSH2, CDX2.
The tissue sections were incubated in a 3% H2O2 solution to inhibit endogenous peroxidases and were then treated with 5 mM citrate buffer (pH 6) in a steamer for 6–15 min at 95°C to promote antigen retrieval. To limit nonspecific reactions, the sections were incubated with Ultra V Block (Lab Vision) for 10 minutes at room temperature and then with antibodies against: hMLH1 (G168-15, Santa Cruz, diluted 1 : 10), hMSH2 (NA27-100 µg, Oncogene, diluted 1 : 50), and CDX2 (MU392-UC, BioGenex, diluted 1 : 100) for 1 h. Surgical CRC samples from hereditary nonpolyposis CRC patients carrying MLH1 or MSH2 germline mutations were used as positive controls.
EGFR.
EGFR immunoreactivity was detected and scored using the EGFR PharmDx Kit (Dako Cytomation, Glostrup, Denmark) as previously described [16].
FISH
EGFR and PTEN gene copy numbers were defined by FISH of 2-µm cut tissue sections as previously described [16]. Briefly, the LSI EGFR/CEP 7 dual colour probe (#32-191053 Vysis, Downers Grove, IL) was used for EGFR and the LSI PTEN SO/CEP 10 SG dual colour probe (Vysis) for PTEN. EGFR gene status was scored as the average number of EGFR red signal per nucleus [8, 9]. PTEN homozygous or hemizygous gene deletion was defined as the presence of only centromeric signals or the presence of one gene signal coupled with two centromeric signals in neoplastic cells.
DNA extraction
Microscopic dissection of 7-µm methylene blue-stained tissue sections (with >80% tumour cells) allowed the precise separation of neoplastic and normal tissue. Genomic DNA was extracted using the Qiamp DNA Kit (Qiagen, Chatsworth, CA) in accordance with the manufacturer's instructions.
mutational analyses
The KRAS, BRAF, PI3KCA, and PTEN mutational analyses were made by means of PCR using 100 ng of genomic DNA for each PCR reaction.
All the PCR products were directly sequenced using an ABI Prism 377 (Applied Biosystems, Foster City, CA) and were then evaluated by means of Sequence Navigator software (Applied Biosystems). Each sequence was carried out at least twice, starting from an independent amplification reaction.
KRAS.
Exon 1 of the KRAS gene was amplified in order to seek potential mutations on the two foremost codons (12 and 13), which have been reported as mutated in CRC. PCR amplification was carried out using a standard protocol and previously described primers and conditions [17].
BRAF.
Exons 11 and 15 of the BRAF gene, including the classical mutation V600E, were amplified using previously described conditions and primers [18].
PI3KCA.
Exons 9 and 20 of the alpha polypeptide (the catalytic subunit of the PI3K protein, PI3KCA), which are frequently mutated in CRC, were amplified using a standard PCR protocol and previously described primers [6].
PTEN.
In order to avoid amplifying the well-known PTEN pseudogene, the PTEN-coding sequences of exons 5, 6, 7, 8, and 9 were amplified using the following intronic primers designed by means of Primer3 software: exon 5, sense 5'-TGCAACATTTCTAAAGTTACCTACT-3' antisense 5'-GAGGAAAGGAAAAACATCAAAAA-3', 353 bp; exon 6, sense 5'-TTTTTCAATTTGGCTTCTCTTTTT-3' antisense 5'-TGTTCCAATACATGGAAGGATG-3', 220 bp; exon 7, sense 5'-CAGTTAAAGGCATTTCCTGTG-3' antisense 5'-TTTTGGATATTTCTCCCAATGAA-3', 256 bp; exon 8, sense 5'-TGTCATTTCATTTCTTTTTCTTTTC-3' antisense 5'-AAGTCAACAACCCCCACAAA-3', 305bp; and exon 9, sense 5'-TCATGGTGTTTTATCCCTCTTG-3' antisense 5'-TGAGTCATATTTGTGGGTTTTCA-3', 264 bp. The annealing temperature for all pairs of primers was 55°C.
statistical analysis
Receiver operating characteristic (ROC) curve analysis was used to determine a cut-off point for the EGFR gene copy number as a continuous variable [8, 9]: for each value, sensitivity and specificity were obtained as percentages, and the criterion value for which sensitivity and specificity were the highest was used as the best cut-off point. The samples showing a mean number of EGFR signals per nucleus of that was equal to or more than the cut-off point identified by ROC analysis were classified as FISH positive, and those showing a mean number of EGFR signals per nucleus that was less than the cut-off point were classified as FISH negative.
Fisher's one-tailed exact test was used to calculate the P values for the associations between gene alterations and the response to cetuximab. The level of significance was set at P = 0.05.
Progression-free survival (PFS) was calculated from the first day of cetuximab treatment to the date of tumour progression or death from any cause or the date of the last follow-up at which the patients were censored.
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patients
After a median treatment duration of 25 weeks, 10 of 32 (31%) patients partially responded to cetuximab (PRs; median PFS 8.5 months) (Table 1); the remaining 22 (13 with SD and nine with PD) did not gain any clinical benefit from the mAb regimen and were considered NRs (median PFS 4.5 months).
microsatellite status
All the 44 samples (24 primary tumour and 20 metastases specimens) from the 32 unselected mCRC patients showed nuclear immunostaining for CDX2, MLH1, and MSH2 and were therefore considered positive for microsatellite stability [19].
EGFR analyses
IHC.
Intermediate or high EGFR immunoreactivity was observed in 14 of the 32 cases (44%); the remaining cases showed low EGFR expression (Table 2). The primary (4/7 = 57%) and metastatic tumour samples (3/6 = 50%) of the subjects responding to cetuximab had higher expression scores than the primary (4/16 = 25%) and metastatic tumour samples (5/13 = 38%) of the NRs, but this difference was not statistically significant. The patients with SD and PD showed similar EGFR expression (38% versus 44%).
FISH.
FISH analysis revealed EGFR gene amplification in 2 of 31 cases (6%) (Table 2); all the other cases showed balanced EGFR and CEP7 copy numbers. ROC analysis identified a mean of 2.79 EGFR copies/nucleus with 80.9% sensitivity and 62.5% specificity (Figure 1). Using this cut-off point, 23 patients (74%) were classified as EGFR FISH positive and eight (26%) as FISH negative (Table 2). Five of 23 FISH-positive patients (22%) and four of eight FISH-negative patients (50%) responded to cetuximab, and so this cut-off point did not discriminate PRs from NRs.
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The paired primary and metastatic tumour samples had the same EGFR gene score (Table 2).
correlations between EGFR immunophenotype and gene status
As previously observed in other tumours [16], there was no correlation between EGFR expression and EGFR gene status (Table 2).
KRAS mutational profile
KRAS point mutations were detected in seven of 29 successfully analysed patients (24%), and all involved codons 12 (70%) and 13 (30%). There was no significant difference in the frequency of KRAS mutations between the primary (18%) and metastatic tumour samples (23%). None of the patients harbouring a somatic KRAS mutation responded to cetuximab therapy (7/19 = 37%), whereas no mutations were observed in the tumours or metastases of the 10 PR patients (P = 0.03) (Table 2). The frequency of KRAS mutations was similar in the patients with SD or PD (4/12 = 33% versus 3/7 = 43%).
BRAF mutational profile
Three of 31 patients (10%) showed BRAF point mutations (Table 2): two were the classical V600E transition and one the previously described activating substitution K601E [20]. There was no difference in mutation occurrence between the primary and metastatic tumour samples, although the BRAF mutation in one case (#22) was only detected in the metastasic sample and, interestingly, the paired primary tumour sample carried the G13D KRAS mutation (Table 2). Two of three patients with BRAF point mutations were PRs (20%) and one a NR with SD (5%).
PI3KCA mutational profile
Four of 31 successfully analysed samples (13%) carried a PI3KCA point mutation. The missense mutations were found in the hot spots located in exon 9 of the PI3KCA gene (E542K, E545A, E545K) (Table 2); one unpublished point mutation was observed in an NR (#29), and involved a GCA
GtA transition in codon 1020 of exon 20 inducing the substitution of an alanine by a valine (Figure 2A). In one case (#25), the PI3KCA point mutation detected in the KRAS wild-type primary tumour was absent in the paired metastatic sample which, however, showed KRAS mutation (Table 2). It is worth noting that all four mutations of the PI3KCA gene were found in NR patients (19%), but the difference between PR and NR cases was not statistically significant. Moreover, the frequency of PI3KCA mutations was similar in the patients with SD (2/13 = 15%) or PD (2/8 = 25%).
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PTEN profile
FISH.
Thirty-one cases were cytogenetically analysed successfully, four (13%) of which showed a decreased PTEN gene copy number accounting for three hemizygous deletions (10%) (Figure 2B) and one monosomy of chromosome 10 (3%). Among the remaining cases, 15 (48%) were disomic and 11 (35%) showed an increased PTEN copy number, accounting for nine cases of low polysomy (29%) and two of high polysomy of chromosome 10 (6%) (Table 2).
All the patients with a decreased PTEN gene copy number were NRs (19%) (three SD and one PD). Increased PTEN gene copy numbers were more frequent among PRs than NRs (50% versus 28%), but this difference was not statistically significant. The same trend was observed in both primary (3/7 = 43% versus 4/15 = 27%) and metastatic tumour samples (3/6 = 50% versus 4/12 = 33%).
PTEN mutations
PTEN mutations were observed in 3 of 30 cases (10%), with no differences between the primary and metastatic tumour samples. One case (#19) carried two different mutations (the missense P103S and the nonsense E99stop) coupled with a PTEN hemizygous deletion (Figure 2B) and, in another one (#11), the PTEN mutation was coupled with a PI3KCA mutation. Two cases had silent point mutations (Table 2).
All the patients with PTEN mutations were NRs to cetuximab therapy (3/21 = 14%; 2 SD and 1 PD) (Table 2).
deregulation of EGFR signalling pathways in PRs and NRs
Activation of the KRAS/BRAF signalling pathway was found in PRs (20%) and NRs (37%), but the KRAS mutations were limited to the NRs (P = 0.03) and the BRAF mutations occurred in both. Furthermore, evidence of the activation of the PI3KCA/PTEN signalling pathway was significantly restricted to the NRs (41%) (P = 0.02) (Table 2). In particular, PI3KCA and PTEN mutations occurred in 6 of 22 NRs (27%), and with a similar frequency among the patients with SD (38%) or PD (44%).
The NRs as a whole showed alterations in the KRAS/BRAF and/or PI3KCA/PTEN signalling pathway significantly (P = 0.02) more frequently (14/22 = 64%) than the PRs (2/10 = 20%), in whom only BRAF mutations were found.
Five of 12 patients for whom paired primary and metastatic tumour specimens were available had deregulated gene profiles, which were functionally similar in three cases (#15, #22, and #25) (Table 2).
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discussion |
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TopAbstractintroductionpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
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The results of our combined immunohistochemical, molecular, and cytogenetic investigations of a cohort of 32 mCRC patients confirm the statistically significant value of KRAS mutations in predicting resistance to cetuximab therapy (P = 0.03) and extend the spectrum of deregulated gene products belonging to the EGFR signalling pathways that negatively affect such a response to PI3KCA and PTEN genes. We found that PI3KCA/PTEN gene deregulation (due to direct PI3KCA mutations or an indirect result of the loss of PTEN gene function/expression due to gene mutations or allele loss) significantly correlated with an impaired response to cetuximab (P = 0.02).
Cumulatively, we found evidence of the activation of the KRAS/RAF and PI3K pathways in, respectively, 37% and 41% of the NR patients. The correlation between KRAS mutations and resistance to anti-EGFR treatments has been well validated in vitro and in vivo [2, 3, 7, 12–14], but the predictive role of PI3KCA/PTEN deregulation (reported here for the first time in a clinical setting) should be further investigated in other independent series of treated mCRC patients, although in vitro studies of PI3KCA/PTEN expression/mutation status have found a significant difference in cetuximab response, with PI3KCA-mutant/PTEN-null colon cancer cell lines being consistently more resistant than wild-type lines [21].
As KRAS and PI3KCA/PTEN mutations, respectively, affected 37% and 27% of the NRs and evoked the activation of MAPK/AKT/mTOR, our findings open up new avenues to the treatment of mCRC patients with mTOR inhibitors or their analogues. As we used FFPE material, we could not provide evidence of the activation of the MAPK, AKT, or mTOR or its downstream effectors, whose phosphorylation status could be revealed by a biochemical analysis of frozen specimens.
The occurrence of a PI3KCA mutation in the primary tumour and a KRAS mutation in its synchronous metastasis in one NR (#25) not only reconfirms the validity of the principle of exclusiveness (as each mutation belongs to one of the two pathways) but also underlines the clonal heterogeneity of CRC, which was also observed in another NR (#22) in whom the primary and paired synchronous metastatic tumour samples carried KRAS and BRAF mutations belonging to the same pathway. PI3KCA mutations have been previously reported in two NR CRC patients treated with cetuximab by Lièvre et al. [7] but, as they were associated with KRAS mutations, they were not informative in relation to the role of PI3KCA itself. The same authors pointed out that no correlation has yet been reported between PI3KCA mutations and a response to cetuximab [3], which is in line with the findings of Moroni et al. [6].
In one of three NRs carrying disabling PTEN mutations (#19), the observed association between a nonsense mutation and a hemizygous PTEN deletion indicated a loss of PTEN function. The other two (#11 and #31) showed the same previously unreported PTEN missense mutation involving codon 85 which, by clustering in the phosphatase domain (exon 5), could inactivate or reduce the phosphatase activity of PTEN which, as demonstrated for most missense mutations within this domain [22], is critical for its tumour suppressor function. Interestingly, as PI3KCA mutations or the loss of PTEN function can independently activate the PI3K pathway [23], the double mutation of PTEN and PI3KCA in one of these two cases (#11) may have an additive effect on PI3K activation. Furthermore, the biological effect of the decreased PTEN gene copy number, which was represented in four NRs by a hemizygous PTEN deletion or monosomy of chromosome 10, could be related to the functional haploinsufficiency of this tumour suppressor gene, which is critical for tumour progression [24].
Two of our PR patients carried BRAF mutations. Both were characterised by a stable immunophenotype profile [19] and microsatellite stability as assessed by BAT25 and BAT26 microsatellite loci (data not shown) and lacked the morphological patterns of serrated polyp neoplasia [25]. These BRAF mutations are in line with the lack of correlation between the mutational status of the BRAF gene alone and the cetuximab response/resistance reported in the literature [7, 15] and with the 50-fold less transforming activity of V600EBRAF in comparison with KRAS mutations [26]. However, given recent preclinical data indicating that the inhibition of both EGFR and RAS/RAF downstream signalling effectors may be more effective [15], and recent clinical data indicating that cetuximab is active in patients carrying a disomic EGFR pattern [10], the inhibition of both EGFR and MAPK could well be considered for patients bearing BRAF or KRAS mutations.
We observed a positive correlation between high/intermediate EGFR expression and a response to cetuximab in primary (57%) and metastatic tumours (50%), but this was probably attributable to the fact that the scoring system, which considered both the percentage of positive cells and the intensity of staining, closely mirrored the intratumoral heterogeneity of EGFR expression. However, the lack of a significant correlation between cetuximab response and EGFR expression [2, 4, 5] is not unexpected as the current immunohistochemical detection system cannot distinguish high- and low-affinity EGFRs, and low-affinity EGFRs may be over-represented in this system. This finding is in keeping with cell line data showing that low-affinity EGFRs make up >95% of all EGFRs [27].
As our ROC analysis produced a mean EGFR gene copy number/nucleus of 2.79 that did not discriminate sensitive patients and NRs, our FISH data support the idea that analysing EGFR gene status by means of FISH alone is insufficient to predict the efficacy of anti-EGFR therapies [10]. It should therefore be combined with the analysis of downstream EGFR pathways, including the JAK-STAT pathway, which is directly stimulated after EGFR activation and has not yet been investigated in mCRC patients receiving anti-EGFR treatment. However, the small number of our cases could be critical in driving this conclusion.
In conclusion, our results obtained from a cohort of mCRC patients treated at the same institution confirm and expand the number of activated downstream effectors belonging to the EGFR pathways that may negatively affect the response to cetuximab by adding the PI3KCA and PTEN genes. More importantly from the clinical point of view, they show a two-fold increase in NRs whose CRC molecular profiles otherwise suggest the benefit of targeted treatments inhibiting MAPK or mTOR, alone or in combination with cetuximab. However, the nondefinitive role of EGFR gene copy number as a biomarker capable of identifying cetuximab-responsive patients implies that patients without any signs of EGFR signalling pathway activation (50% of our cohort) should not be denied access to anti-EGFR treatment because they may respond to cetuximab [2, 4, 5].
Analysis of KRAS/BRAF and PI3KCA/PTEN genes in the paired primary and metastatic tumour samples (particular when synchronous) underlined the clonal heterogeneity of CRC within each pathway. However, in the five cases with deregulated gene profiles, it seemed to be sufficiently informative to restrict the analysis to the primary tumour, a promising finding for targeted treatment decision making.
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Associazione Italiana per la Ricerca sul Cancro to Dr. Bertario Lucio.
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Acknowledgements |
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TopAbstractintroductionpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
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We would like to thank the following pathologists who kindly contributed case material: G. Angeli (Presidio ospedaliero S. Andrea, Vercelli), G. Coggi (Laboratorio Fleming, Milan), F. Crivelli (Presido ospedaliero S. Antonio Abate, Gallarate), C. Galli (ASL, Provincia di Lodi), G. Sangalli (Ospedale di Circolo, Busto Arsizio), and G. Viale (Istituto Europeo di Oncologia, Milan).
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Footnotes |
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Both authors contributed equally to this work. 
Received for publication March 7, 2008. Revision received July 3, 2008. Accepted for publication July 4, 2008.
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TopAbstractintroductionpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
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S. D. Richman, M. T. Seymour, P. Chambers, F. Elliott, C. L. Daly, A. M. Meade, G. Taylor, J. H. Barrett, and P. Quirke KRAS and BRAF Mutations in Advanced Colorectal Cancer Are Associated With Poor Prognosis but Do Not Preclude Benefit From Oxaliplatin or Irinotecan: Results From the MRC FOCUS Trial J. Clin. Oncol., December 10, 2009; 27(35): 5931 - 5937. [Abstract] [Full Text] [PDF]
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F. Loupakis, L. Pollina, I. Stasi, A. Ruzzo, M. Scartozzi, D. Santini, G. Masi, F. Graziano, C. Cremolini, E. Rulli, et al. PTEN Expression and KRAS Mutations on Primary Tumors and Metastases in the Prediction of Benefit From Cetuximab Plus Irinotecan for Patients With Metastatic Colorectal Cancer J. Clin. Oncol., June 1, 2009; 27(16): 2622 - 2629. [Abstract] [Full Text] [PDF]
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H. Prenen, J. De Schutter, B. Jacobs, W. De Roock, B. Biesmans, B. Claes, D. Lambrechts, E. Van Cutsem, and S. Tejpar PIK3CA Mutations Are Not a Major Determinant of Resistance to the Epidermal Growth Factor Receptor Inhibitor Cetuximab in Metastatic Colorectal Cancer Clin. Cancer Res., May 1, 2009; 15(9): 3184 - 3188. [Abstract] [Full Text] [PDF]
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C. J. Allegra, J. M. Jessup, M. R. Somerfield, S. R. Hamilton, E. H. Hammond, D. F. Hayes, P. K. McAllister, R. F. Morton, and R. L. Schilsky American Society of Clinical Oncology Provisional Clinical Opinion: Testing for KRAS Gene Mutations in Patients With Metastatic Colorectal Carcinoma to Predict Response to Anti-Epidermal Growth Factor Receptor Monoclonal Antibody Therapy J. Clin. Oncol., April 20, 2009; 27(12): 2091 - 2096. [Abstract] [Full Text] [PDF]
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P. Laurent-Puig, A. Lievre, and H. Blons Mutations and Response to Epidermal Growth Factor Receptor Inhibitors Clin. Cancer Res., February 15, 2009; 15(4): 1133 - 1139. [Abstract] [Full Text] [PDF]
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T. Takano, S. Ota, A. Hori, N. Seki, and K. Eguchi Can Epidermal Growth Factor Receptor-Fluorescent in Situ Hybridization Predict Clinical Benefit From Cetuximab Treatment in Patients With Non-Small-Cell Lung Cancer? J. Clin. Oncol., January 20, 2009; 27(3): 464 - 465. [Full Text] [PDF]
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R. Wong and D. Cunningham Using Predictive Biomarkers to Select Patients With Advanced Colorectal Cancer for Treatment With Epidermal Growth Factor Receptor Antibodies J. Clin. Oncol., December 10, 2008; 26(35): 5668 - 5670. [Full Text] [PDF]
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