In the present study, the first conducted in a mCRC population with known EGFR status, we showed that KRAS mutation is significantly associated with cetuximab failure in EGFR FISH+ patients, even if it does not preclude response. Although preclinical data supported the negative impact of MET, IGF1R, and PI3KCA on cetuximab activity, in our study none of them demonstrated a clinical relevance as predictors for primary resistance, irrespective of the EGFR FISH result.
Previous studies demonstrated that KRAS
mutations represent the most relevant mechanism responsible for anti-EGFR
strategy failure (Lièvre et al, 2006
; Di Fiore et al, 2007
; Khambata-Ford et al, 2007
; Amado et al, 2008
; De Roock et al, 2008
). Nevertheless, in CRC no data existed whether KRAS
mutations drove cetuximab resistance in a patient population potentially sensitive to anti-EGFR
agents, neither whether such events occurred in EGFR
FISH+ individuals. In the present study, we observed that the incidence of KRAS
mutation was similar in EGFR
FISH+ and FISH− subgroups, indicating that these phenomena are not mutually exclusive. It is noteworthy that no difference in outcome was observed in the group of patients EGFR
FISH− irrespective of KRAS
status, while the difference in response was significant only in EGFR
FISH+. These results suggest that the drug maybe ineffective in absence of the target, while KRAS
activation is able to drive drug resistance even if EGFR
is present. Interestingly, in our population of KRAS
mutated patients, four individuals responded to the therapy, and all were EGFR
FISH positive. This finding raises the possibility that, in presence of an overexpressed target, occasionally tumour shrinkage could occur also in individuals harbouring the mutation.
mutations were identified in 66% of melanomas, and in smaller percentage in other human cancers including colorectal (Davies et al, 2002
). Our study confirmed that such mutation is not frequent in CRC and could represent an additional mechanism responsible for cetuximab failure. None of the patients carrying BRAF
mutation responded to the therapy, with a trend for shorter TTP and survival, differences not significant probably because of the small number of patients.
A recent study showed that colorectal cancer cell lines with activating PI3KCA mutations or with loss of PTEN expression were more resistant to cetuximab therapy than PI3KCA wild type/PTEN expressing cell lines (Jhawer et al, 2008). Importantly, simultaneous mutations of KRAS and PI3KCA conferred maximal resistance to cetuximab. In our study, combination of multiple mutation tests (KRAS or BRAF±PI3KCA) did not provide additional information over a single mutation test (data not shown).
Previous studies have investigated MET
in CRC with semiquantitative techniques such as immunoblotting or IHC (Di Renzo et al, 1995
; Hiscox et al, 1997
; Fujita and Sugano, 1997
; Fukuura et al, 1998
). Our study represents the first report in CRC assessing MET
at the genomic level using FISH. We report here that MET
amplification is a rare event in CRC, occurring in about 2% of cases. Only two patients had MET
amplification and both responded to cetuximab therapy. Although the number of patients was too low for any conclusion, the level of MET
gene gain observed in our study population was lower than reported in a previous study conducted on cell lines and patients with acquired resistance to anti EGFR agents (Engelman et al, 2007
). Although obtained in tissues collected before starting cetuximab therapy, these findings suggest that only high level of MET
gene gain could be responsible for resistance, levels probably occurring only under therapeutic pressure and rarely present in a general population of patients unexposed to anti-EGFR agents.
IGF1R is overexpressed in 50–90% of CRC (Weber et al, 2002
; Koda et al, 2004
), and preclinical studies suggested that this target could be responsible for resistance to anti-EGFR
strategies (Chakravarti et al, 2002
). In our study, IGF1R expression was not associated with cetuximab resistance, probably because the IGF1R pathway did not affect the antiproliferative activity of cetuximab, as recently observed in a lung cancer model (Morgillo et al, 2007
). In the study conducted by Morgillo et al (2007
) only the treatment with gefitinib, an EGFR
-TKI, but not cetuximab, induced EGFR
-IGF1R heterodimerisation and activation of IGF1R and its downstream signalling mediators, resulting in increased survivin expression in NSCLC cell lines with high levels of IGF1R expression. Interestingly, IGF1R-expressing patients had longer survival than IGF1R negative. A similar pattern has been observed in other malignancies such as non-small cell lung cancer or soft tissue sarcomas (Ahlén et al, 2005
; Cappuzzo et al, 2006
), whereas the opposite situation was found in other malignancies such as uveal melanoma and breast cancer (Turner et al, 1997
; All-Ericsson et al, 2002
). The significant association with survival is of particular relevance and merits further investigations, considering the number of strategies interfering with the IGF1R pathway under evaluation in solid cancers, including CRC. In our study, no patient had IGF1R
gene amplification and the level of IGF1R
gene gain was very low in the whole analysed population, suggesting that such event is not involved in primary resistance. Moreover, no association was found between IGF1R
gene and protein expression, suggesting post-transcriptional events could also interfere with the gene function.
To conclude, the present study showed that presence of KRAS mutations represents the strongest predictor for cetuximab failure in EGFR FISH-positive CRC patients. The rarity of MET and IGF1R gene amplification suggest that such biological events play a limited role in primary resistance to anti-EGFR agents. The impact of BRAF mutation on cetuximab resistance as well as the potential prognostic implications of IGF1R expression requires further investigation.