Keynote Comment

EGFR FISH in colorectal cancer: what is the current reality?

Colon cancer (CC) therapies have improved patient outcomes significantly over the last decades in both the adjuvant and metastatic settings. With the introduction of a number of novel agents, both traditional chemotherapies and biologically targeted agents, the need to identify subgroups that are likely and not likely to respond to a particular treatment regimen is paramount. This will allow patients who are likely to benefit to receive optimal care, while sparing those unlikely to benefit from unnecessary toxicity and cost. With the identification of several novel biomarkers and a variety of technologies to interrogate the genome, we already are able to rapidly study patient tumor or blood samples and normal tissues to generate a large dataset of aberrations within the cancer. How to digest this complex information to obtain accurate, reliable, and meaningful results that will allow us to provide truly personalized care for CC patients is just starting to be addressed. In this article, we briefly review the history of CC treatment, with an emphasis on current clinical standards that incorporate a “personalized medicine” approach. We then review strategies that will potentially improve our ability to individualize therapy in the future.

KRAS and BRAF mutations predict the resistance of colorectal carcinomas to therapy targeted to the epidermal growth factor receptor, but their detection can be challenging because of high testing volume, frequently low tumor content, and the spectrum of rarer mutations in these genes. To address these issues, we evaluated a locked nucleic acid (LNA)-PCR sequencing assay to detect low levels of mutant DNA, and we also evaluated a mass spectrometry genotyping assay (Sequenom, San Diego, CA) that is suitable for broad mutation screening. Clinical cases (n = 308) previously tested for KRAS and BRAF by standard sequencing were retested by LNA-PCR sequencing incorporating an LNA oligonucleotide to suppress amplification of nonmutant DNA, and by a Sequenom assay panel targeting common mutations in both genes. Standard sequencing detected 121 KRAS (39%) and 10 BRAF mutations; retesting with the LNA-based method and the Sequenom assay detected 19 (140/308, 45%) and 6 (127/308, 41%) additional KRAS mutants, respectively. One additional BRAF mutant was detected by the Sequenom assay. The analytical sensitivities were 0.3% for both KRAS and BRAF by LNA-PCR and from 1% to 10% for the Sequenom assays, depending on the specific mutation. Given these results, standard sequencing is suboptimal for mutation detection in metastatic and treated lesions even with predissection for tumor enrichment. High-sensitivity LNA-PCR sequencing detects significantly more mutations, whereas the Sequenom platform shows intermediate sensitivity but offers significant advantages for broader mutation screening.

Anti-EGFR monoclonal antibodies in metastatic colorectal cancer (mCRC) treatment are only effective in patients with KRAS wild type tumours. Here we assess the predictive value of other potential relevant markers involved in the epidermal growth factor receptor (EGFR) signalling pathways for response to cetuximab-based treatment.

Formalin-fixed paraffin-embedded colorectal cancer tissue of the primary tumour was obtained from 559 mCRC patients treated with chemotherapy and bevacizumab with or without cetuximab (phase III CAIRO2 study). DNA was isolated for mutation analysis of BRAF (V600E), KRAS (codon 12 and 13) and PIK3CA (exon 9 and 20). Tissue microarray’s (TMA’s) were constructed for the assessment of EGFR and HER2 gene copy number (GCN), and EGFR and PTEN protein expression. The results of these markers, individually or in combination, were correlated with progression-free survival (PFS) and overall survival (OS) in the subgroup of patients with a KRAS wild type tumour treated in the cetuximab-arm. KRAS wild type patients treated without cetuximab were used as a control group.

A total of 208 tumours (39.4%) contained a KRAS mutation, 8.7% a BRAF mutation and 9.9% a PIK3CA mutation. Loss of PTEN expression and the presence EGFR protein expression were observed in 42.0% and 61.7% of the samples, respectively. An increased EGFR GCN was observed in 15.3% of the samples, and 11.5% of the evaluable samples contained an increased HER2 GCN. In KRAS wild type patients treated with cetuximab a BRAF mutation was significantly and independently associated with PFS and OS. In patients treated without cetuximab the PFS and OS were also associated with the BRAF genotype. No prognostic or predictive value was observed for any of the other markers when tested individually or in combination.

BRAF genotype is correlated with PFS and OS in KRAS wild type mCRC patients, which is independent of cetuximab treatment. PIK3CA mutation, loss of PTEN expression, EGFR GCN and HER2 GCN have no predictive value for response to treatment with cetuximab, neither individually nor in combination with other markers.

The aims of this study were first, to systematically assess the inter-observer reproducibility of mean Epidermal Growth Factor Receptor (EGFR) gene copy number (MCN) in histological and cytological specimens from lung and non-lung cancers, second to compare the performance of this quantitative approach to the current Colorado criteria for the assessment of fluorescence in situ hybridization (FISH) positivity and third to develop a model to convert cytology into histology MCN. EGFR FISH analysis was performed on 170 histological and 153 cytological specimens. The MCN and Colorado criteria were assessed by two independent observers and agreement evaluated using Bland–Altman plots and kappa values. Conversion of cytology into histology MCN was tested on two randomized subgroups of specimens. Applying the Colorado criteria, agreement between observers was moderate with histology (k = 0.5) and excellent with cytology (k = 0.94). Positivity in histology versus cytology led to fair agreement (k = 0.33). MCN was significantly greater in cytology compared to histology (p < 0.001) with excellent inter-observer agreement in both sample types (r = 0.99 and 0.89, respectively). The average difference in MCN between observers was −0.003 (95%CI: −0.05 to 0.05) and 0.008 (95%CI: −0.09 to 0.11) for cytology and histology, respectively. A reliable conversion model with an R²-value of 0.91 in the validation subgroup (p < 0.001) was obtained. The MCN is a reproducible scoring method for evaluating EGFR FISH in samples from lung and non-lung cancers. This quantitative scoring system may constitute an alternative to current methods of gene copy number assessment and will further help to optimize patient selection for targeted therapies.

The treatment of metastatic colorectal cancer has undergone major advances yielding significant improvements in survival over the past decade. These advances have evolved due to the benefits of combination chemotherapy and the incorporation of biologic therapy. However, as we struggle to provide optimum care while sparing patients ineffective therapy and undue cost, the importance of tailored therapy to maximize benefit will become increasingly important. This article reviews the major advances in the treatment of patients with metastatic colorectal cancer and the burgeoning developments in individualized therapy.

Epidermal growth factor receptor (EGFR) is a therapeutic target for colorectal cancer (CRC). However, technical challenges have limited in vivo imaging of EGFR in CRCs. Confocal laser endomicroscopy (CLE) enables accurate microscopic visualization of CRC in patients during endoscopy. We evaluated the ability to use CLE in vivo for instantaneous molecular imaging of EGFR in CRC models.

Tumors were grown in mice (n = 68) from human CRC cell lines that expressed high (SW480 cells) or low (SW620 cells) levels of EGFR. Tumors were visualized in vivo with a handheld CLE probe after injection of fluorescently labeled EGFR antibodies. EGFR-specific fluorescence was graded from 0 to 3+. Neoplastic and non-neoplastic specimens from human colorectal mucosa were examined. In vivo findings were correlated with histopathology, immunohistochemistry, and fluorescence microscopy analyses.

CLE analysis of cell cultures confirmed the different expression levels of EGFR between cell lines. In living animals, CLE differentiated EGFR expression levels between tumor cell limes (mean fluorescence, 1.92 ± 0.22 [SW480] and 0.59 ± 0.21 [SW620], P = .0004). CLE analysis of EGFR expression in human specimens allowed distinction of neoplastic from non-neoplastic tissues (mean fluorescence, 2.0 ± 0.37 vs 0.25 ± 0.16, respectively, P = .0035).

CLE can be used for in vivo, molecular analysis of CRC and to differentiate EGFR expression patterns in xenograft tumors and human tissue samples. Because CLE can be performed during endoscopy, in vivo molecular imaging might be used in diagnosis of CRC and to predict response to targeted therapies.