Israel Cañadas

Increased ALK Gene Copy Number and Amplification are Frequent in Non-small Cell Lung Cancer

Introduction: Translocation of the anaplastic lymphoma kinase (ALK) gene is involved in the tumorigenesis of a subset of non-small cell lung carcinomas (NSCLCs) and identifies patients sensitive to ALK inhibitors. ALK copy number changes and amplification, which plays an oncogenic role in tumors such as neuroblastoma, are poorly characterized in NSCLC. We aimed to study the prevalence of ALK copy number changes and their correlation to ALK protein expression, epidermal growth factor receptor (EGFR) status, and clinicopathological data in patients with NSCLC. Methods: ALK status was evaluated by fluorescence in situ hybridization (FISH). Specimens with ALK translocation were studied for echinoderm microtubule-associated protein-like 4 (EML4), KIF5B, and TFG status. ALK expression was assessed by immunohistochemistry. EGFR gene and protein status were evaluated in adenocarcinomas. Survival analysis was performed. Results: One hundred seven NSCLC cases were evaluated. There were two cases of EML4-ALK translocation and one with an atypical translocation of ALK. Both cases of EML4-ALK translocation had ALK protein expression, whereas in the rest, ALK was undetected. Eleven cases (10%) exhibited ALK amplification and 68 (63%) copy number gains. There was an association between ALK amplification and EGFR FISH positivity (p < 0.0001) but not with prognosis. In conclusion, EML4-ALK translocation is a rare event in NSCLC. Conclusion: The study reveals a significant frequency of ALK amplification and its association with EGFR FISH positivity in lung adenocarcinomas. Based on these findings, a potential role of ALK amplification in the response to ALK inhibitors alone or combined with EGFR inhibitors in NSCLC merits further studies.

Emergence of Multiple EGFR Extracellular Mutations during Cetuximab Treatment in Colorectal Cancer

Purpose: Patients with colorectal cancer who respond to the anti-EGFR antibody cetuximab often develop resistance within several months of initiating therapy. To design new lines of treatment, the molecular landscape of resistant tumors must be ascertained. We investigated the role of mutations in the EGFR signaling axis on the acquisition of resistance to cetuximab in patients and cellular models. Experimental Design: Tissue samples were obtained from 37 patients with colorectal cancer who became refractory to cetuximab. Colorectal cancer cells sensitive to cetuximab were treated until resistant derivatives emerged. Mutational profiling of biopsies and cell lines was performed. Structural modeling and functional analyses were performed to causally associate the alleles to resistance. Results: The genetic profile of tumor specimens obtained after cetuximab treatment revealed the emergence of a complex pattern of mutations in EGFR, KRAS, NRAS, BRAF, and PIK3CA genes, including two novel EGFR ectodomain mutations (R451C and K467T). Mutational profiling of cetuximab-resistant cells recapitulated the molecular landscape observed in clinical samples and revealed three additional EGFR alleles: S464L, G465R, and I491M. Structurally, these mutations are located in the cetuximab-binding region, except for the R451C mutant. Functionally, EGFR ectodomain mutations prevent binding to cetuximab but a subset is permissive for interaction with panitumumab. Conclusions: Colorectal tumors evade EGFR blockade by constitutive activation of downstream signaling effectors and through mutations affecting receptor–antibody binding. Both mechanisms of resistance may occur concomitantly. Our data have implications for designing additional lines of therapy for patients with colorectal cancer who relapse upon treatment with anti-EGFR antibodies. Clin Cancer Res; 21(9); 2157–66. ©2015 AACR.

Targeting Epithelial-to-Mesenchymal Transition with Met Inhibitors Reverts Chemoresistance in Small Cell Lung Cancer

Purpose: Met receptor phosphorylation is associated with poor prognosis in human small cell lung cancer (SCLC). The aim of our work was to investigate the effects of hepatocyte growth factor (HGF)/Met–mediated epithelial-to-mesenchymal transition (EMT) in SCLC and to evaluate the role of Met inhibition in mesenchymal/chemorefractory SCLC models. Experimental Design: SCLC models of HGF-induced EMT were evaluated in vitro and in vivo (subcutaneous xenografts in BALB/c nude mice) for chemosensitivity and response to Met inhibition with PF-2341066 (crizotinib). Human SCLC samples at diagnosis (N = 87) and relapse (N = 5) were evaluated by immunohistochemistry and immunofluorescence for EMT markers and Met status and these were correlated with patient outcome. Results: We identified that the activation of the Met receptor through HGF induced expression of mesenchymal markers, an aggressive phenotype, and chemoresistance. Blockade of this process with the Met inhibitor resensitized cells to chemotherapy in vitro and in vivo. Moreover, mesenchymal markers in human SCLC specimens were associated with Met activation, predicted worse survival, and were upregulated in chemorefractory disease. Conclusion: These results provide novel evidence on an important role of Met-dependent EMT in the adverse clinical behavior of SCLC and support clinical trials of Met inhibitors and chemotherapy in this fatal disease. Clin Cancer Res; 20(4); 938–50. ©2013 AACR.

C-MET as a new therapeutic target for the development of novel anticancer drugs

MET is a tyrosine kinase receptor that, upon binding of its natural ligand, the hepatocyte growth factor (HGF), is phosphorylated and subsequently activates different signalling pathways involved in proliferation, motility, migration and invasion. MET has been found to be aberrantly activated in human cancer via mutation, amplification or protein overexpression. MET expression and activation have been associated with prognosis in a number of tumour types and predict response to MET inhibitors in preclinical models. Here we review the HGF/MET signalling pathway, its role in human cancer and the different inhibitory strategies that have been developed for therapeutic use.

MET phosphorylation predicts poor outcome in small cell lung carcinoma and its inhibition blocks HGF-induced effects in MET mutant cell lines

Background:Small cell lung carcinoma (SCLC) has poor prognosis and remains orphan from targeted therapy. MET is activated in several tumour types and may be a promising therapeutic target.Methods:To evaluate the role of MET in SCLC, MET gene status and protein expression were evaluated in a panel of SCLC cell lines. The MET inhibitor PHA-665752 was used to study effects of pathway inhibition in basal and hepatocyte growth factor (HGF)-stimulated conditions. Immunohistochemistry for MET and p-MET was performed in human SCLC samples and association with outcome was assessed.Results:In MET mutant SCLC cells, HGF induced MET phosphorylation, increased proliferation, invasiveness and clonogenic growth. PHA-665752 blocked MET phosphorylation and counteracted HGF-induced effects. In clinical samples, total MET and p-MET overexpression were detected in 54% and 43% SCLC tumours (n=77), respectively. MET phosphorylation was associated with poor median overall survival (132 days) vs p-MET negative cases (287 days)(P<0.001). Phospho-MET retained its prognostic value in a multivariate analysis.Conclusions:MET activation resulted in a more aggressive phenotype in MET mutant SCLC cells and its inhibition by PHA-665752 reversed this phenotype. In patients with SCLC, MET activation was associated with worse prognosis, suggesting a role in the adverse clinical behaviour in this disease.

The first-in-class anti-EGFR antibody mixture Sym004 overcomes cetuximab-resistance mediated by EGFR extracellular domain mutations in colorectal cancer

Purpose: Approved anti-EGFR antibodies cetuximab and panitumumab provide significant clinical benefit in patients with metastatic colorectal cancer (MCRC). However, patients ultimately develop disease progression, often driven by acquisition of mutations in the extracellular domain (ECD) of EGFR. Sym004 is a novel 1:1 mixture of two nonoverlapping anti-EGFR mAbs that recently showed promising clinical activity in a phase I trial in MCRC. Our aim was to determine the efficacy of Sym004 to circumvent cetuximab resistance driven by EGFR ECD mutations. Experimental Design: Functional studies were performed to assess drug–receptor binding as well as ligand-dependent activation of individual EGFR mutants in the presence of cetuximab, panitumumab, and Sym004. Cell viability and molecular effects of the drugs were assayed in cetuximab-resistant cell lines and in tumor xenograft models. Efficacy of Sym004 was evaluated in patients progressing to cetuximab that harbored EGFR mutation in the post-cetuximab tumor sample. Results: Contrary to cetuximab and panitumumab, Sym004 effectively bound and abrogated ligand-induced phosphorylation of all individual EGFR mutants. Cells resistant to cetuximab harboring mutations in EGFR maintained sensitivity to Sym004, which was consistent with an effective suppression of EGFR downstream signaling, translating into profound and sustained tumor regression in the xenograft model. As proof-of-principle, a patient with a tumor harboring an EGFR mutation (G465R) following cetuximab therapy benefited from Sym004 therapy. Conclusions: Sym004 is an active drug in MCRC resistant to cetuximab/panitumumab mediated by EGFR mutations. EGFR mutations are potential biomarkers of response to Sym004 to be evaluated in ongoing large clinical trials. Clin Cancer Res; 22(13); 3260–7. ©2016 AACR.

Mitogen-activated protein kinase phosphatase-1 (MKP-1) impairs the response to anti-epidermal growth factor receptor (EGFR) antibody cetuximab in metastatic colorectal cancer patients

Background:The validation of KRAS mutations as a negative marker of response to anti-epidermal growth factor receptor (EGFR) antibodies has meant a seminal advance towards treatment individualisation of colorectal cancer (CRC) patients. However, as a KRAS wild-type status does not guarantee a response to anti-EGFR antibodies, a current challenge is the identification of other biomarkers of response. On the basis of pre-clinical evidence, we hypothesised that mitogen-activated protein kinase phosphatase-1 (MKP-1), a phosphatase that inactivates MAPKs, could be a mediator of resistance to anti-EGFR antibodies.Methods:Tumour specimens from 48 metastatic CRC patients treated with cetuximab-based chemotherapy were evaluated for KRAS and BRAF mutational status and MKP-1 expression as assessed by immunohistochemistry.Results:As expected, clinical benefit was confined to wild-type KRAS and BRAF patients. Mitogen-activated protein kinase phosphatase-1 was overexpressed in 16 patients (33%) and was not associated with patient baseline clinicopathological characteristics and KRAS mutational status. All patients with BRAF mutations (n=3) had MKP-1 overexpression. Among KRAS wild-type patients, MKP-1 overexpressors had a 7% response rate (RR), whereas patients not overexpressing MKP-1 had a 44% RR (P=0.03). Moreover, median time to progression was significantly longer in MKP-1 non-overexpressing patients (32 vs 13 weeks, P=0.009).Conclusion:These results support the concept of MKP-1 as a promising negative marker of response to cetuximab-based treatment in CRC patients with wild-type KRAS.

Cytogenetic characterization of NCI-H69 and NCI-H69AR small cell lung cancer cell lines by spectral karyotyping

Small cell lung cancer (SCLC) shows an excellent sensitivity to chemotherapy, but commonly develops resistance after a few months. An early identification of a genomic marker in drug discovery may help to select patients who would respond to treatment in clinical trials. Herein, we characterized the parental NCI-H69 (sensitive) and NCI-H69AR (anthracycline-resistant) cell lines by G-banding and spectral karyotyping (SKY). In the H69 cell line, SKY allows us to redefine three alterations that are not well characterized by G-banding and to confirm seven. For H69AR, SKY redefined 10 chromosomal alterations and confirmed four observed by G-banding. Fluorescence in situ hybridization confirmed the amplification of the MYCN gene (dmin or hsr) in these two cell lines, although only the H69AR cell line showed MYCN amplification in the form of homogeneously staining regions. It should be noted that a new derivative chromosome appears in the H69AR cell line, a der(16)t(3;16;18;5;18), characterized by SKY as showing 18q amplification. Amplification of genes located in this region may correlate with resistance to anticancer therapies. We suggest that the 18q marker may have a broader application in SCLC. In conclusion, SKY provides a useful complementary technique to routine cytogenetics for the accurate characterization of SCLC cell lines and could provide some relevant information concerning regions involved in chemoresistance.

Angiopoietin-2 is a negative prognostic marker in small cell lung cancer

BACKGROUND Small cell lung cancer (SCLC) is a highly lethal disease due to its chemorefractory nature after initial treatment. Angiogenesis plays an important role in tumor growth, metastasis and chemoresistance. We hypothesized that angiogenesis could predict chemoresistance in SCLC patients and be potentially a therapeutic target in this disease. METHODS Serum samples from forty-three SCLC patients were prospectively obtained at diagnosis, response evaluation and progression. Angiogenesis-related cytokines (Angiopoietin-2, VEGF-A, C and D) were simultaneously quantified by Luminex Technology. Clinical data were prospectively recorder. RESULTS Significantly higher concentration of angiogenesis-related cytokines were found in SCLC patients at diagnosis compared to healthy volunteers. High baseline serum concentration of Angiopoietin-2 (sAngiopoietin-2) were associated with a worse overall survival (p=0.006) and remained independently associated with survival in the multivariate analysis (p=0.008). In addition, sAngiopoietin-2 significantly increased at progression when compared to baseline. CONCLUSION These data provide novel evidence on a role of sAngiopoietin-2 in the adverse clinical behavior of SCLC and could be a potential therapeutic target in this disease.

ALK status in a primary lung tumour and metachronous metastases.

Sir: The anaplastic lymphoma kinase (ALK) protein has recently become a promising target in the treatment of small but significant subset of patients with non-small cell lung cancer (NSCLC). This is due to the high response rates obtained with the ALK inhibitor, Crizotinib, in ALK gene translocated NSCLC patients. ALK translocations are present in approximately 3–5% of patients with NSCLC and are predictive of response to therapy with the ALK inhibitor, Crizotinib. There is little information available about the correlation of ALK status between the primary tumour and metastatic sites. Here we present a case of a patient with an ALKpositive lung primary tumour with a metachronous metastasis where ALK was analysed. A 75-year-old man, former smoker (35 packs ⁄ year) was diagnosed after complete clinical staging [computerized tomography (CT), positron emission tomography (PET), bronchoscopy and cytology by