Sensitivity of epidermal growth factor receptor and ErbB2 exon 20 insertion mutants to Hsp90 inhibition

The mature epidermal growth factor receptor (EGFR) neither associates with nor requires the molecular chaperone heat-shock protein 90 (Hsp90). Mutations in EGFR exons 18, 19, and 21 confer Hsp90 chaperone dependence. In non-small cell lung cancer (NSCLC), these mutations are associated with enhanced sensitivity to EGFR inhibitors in vitro and with clinical response in vivo. Although less prevalent, insertions in EGFR exon 20 have also been described in NSCLC. These mutations, however, confer resistance to EGFR inhibitors. In NSCLC, exon 20 insertions have also been identified in the EGFR family member ErbB2. Here, we examined the sensitivity of exon 20 insertion mutants to an Hsp90 inhibitor currently in the clinic. Our data demonstrate that both EGFR and ErbB2 exon 20 insertion mutants retain dependence on Hsp90 for stability and downstream-signalling capability, and remain highly sensitive to Hsp90 inhibition. Use of Hsp90 inhibitors should be considered in NSCLC harbouring exon 20 insertions in either EGFR or ErbB2

Mutational status of epidermal growth factor receptor is the only predictor of clinical response to gefitinib treatment in non-small cell lung cancer

The clinical profile of lung cancer in Hong Kong shows distinct characteristics. The incidences in both genders are comparable to those in Western populations with male (M):female (F) ratio of 2.4:1 in 2002. However, the majority of the women have never smoked and the smoker (SM):non-smoker (NS) ratio was 0.3:1, compared to 6.2:1 in male patients. Consistent with findings in various Asian populations, we have previously shown that mutations of the epidermal growth factor receptor (EGFR) in lung cancers are associated with adenocarcinomas, women and non-smokers. The purpose of this study was to examine the clinical correlates and EGFR mutational status (exons 18 to 21) of non-small cell lung cancer (NSCLC) in relation to clinical responses to the tyrosine kinase inhibitor gefitinib (Iressa). All patients who had resistant disease to conventional chemotherapy, had available clinical material (biopsy, fine needle aspiration, lavage or pleural effusion fluid) for mutation analysis, and who received gefitinib treatment in our institution were retrospectively evaluated. There were 27 F (26 NS, 1 SM) and 16 M (9 NS, 7 SM, p=0.002) aged 35-85 (median 64), and included 32 histologically confirmed adenocarcinomas (AD), 2 squamous and 9 NSCLC of advanced stage disease (IIIB and IV). Gefitinib (250mg/day) was given for 0.5 to 21.2 (median 3) months and patients were monitored for 0.5 to 26.4 (median 6.3) months. The median survival was 10.5 months (95% CI, 8.3-12.7) and one-year survival rate was 37.3%. There were 25 mutations (9 substitutions, 17 deletions) that resulted in amino acid changes from 20 F, 5 M (p=0.010), and 23 NS, 2 SM (p=0.052). One case showed both exon 19 deletion and exon 20 substitution. Common changes (exon 19 deletions, L858R) involved 88% of the mutants. 2 other cases showed silent mutations. Clinical response (CR) from gefitinib treatment was observed in 23 (53.5%) patients who showed either reduction of longest tumour diameter by ≥30%, improvement in performance status, and/or reduced serum carcinoembryonic antigen tumour marker. They included 17/27 (63%) female, 6/16 (37.5%) male, 19/35 (54.3%) NS, 4/8 (50%) SM, and 19/32 (59.4%) AD. Clinical response was correlated only with the EGFR mutation status (19/25 mutants, 4/18 wild types, p<0.001) but not patient gender, smoking history or tumor diagnosis. Survival analysis showed that patients with EGFR mutation had significantly better outcome than those without mutation (one-year survival rate 55.5% vs. 10.6%, p=0.013). Within the limits of the small patient number of this study, the data suggested that the EGFR mutational status is the only predictor of clinical response to gefitinib treatment for NSCLC in our population. (Supported by HKU CRCG 200507176201

Double EGFR mutants containing rare EGFR mutant types show reduced in vitro response to gefitinib compared with common activating missense mutations

Epidermal growth factor receptor (EGFR) mutations are common in lung adenocarcinomas, especially from nonsmoking women of Asian descent. We have previously shown EGFR mutations occur in >70% of lung adenocarcinoma from nonsmokers in our population with a complex mutational profile, including 13% of EGFR double mutations. In this study, we investigated the in vitro gefitinib response of four EGFR double mutants identified in untreated patients, including Q787R+L858R, E709A+G719C, T790M+L858R, and H870R+L858R. The phosphorylation profiles of EGFR and downstream effectors AKT, STAT3/5, and ERK1/2 were compared by immunoblot analyses among the single and double mutants transfected into H358 cells. Results showed that mutants responded to in vitro gefitinib treatment with different sensitivities. The G719C and L858R single mutants showed the highest gefitinib sensitivity compared with the corresponding coexisting single mutants E709A, Q787R, H870R, and T790M. The double mutants E709A+G719C, Q787R+L858R, and H870R+L858R showed attenuated responses to gefitinib in the EGFR and downstream effector phosphorylation profiles compared with G719C or L858R alone. T790M+L858R showed strong resistance to gefitinib. Clinically, the patient whose tumor contained H870R+L858R showed tumor stabilization by 250 mg oral gefitinib daily but cerebral metastasis developed 6 months later. Correlation with the in vitro phosphorylation profile of H870R+L858R suggested that treatment failure was probably due to inadequate suppression of EGFR signaling by the drug level attainable in the cerebrospinal fluid at the given oral dosage. Overall, the findings suggested that rare types of EGFR substitution mutations could confer relative gefitinib resistance when combined with the common activating mutants. Copyright © 2009 American Association for Cancer Research.link_to_subscribed_fulltex

Src promotes survival and invasion of lung cancers with epidermal growth factor receptor abnormalities and is a potential candidate for molecular-targeted therapy

Molecular-targeted therapy using tyrosine kinase inhibitors against epidermal growth factor receptor (EGFR) is an effective therapy for non-small cell lung cancer that harbor EGFR mutations. This study aimed to investigate the role of Src, a close EGFR associator, as a drug target in NSCLC cells with different EGFR genomic statuses. Src inhibition was achieved using 4-(4′-Phenoxyanilino)-6,7-dimethoxyquinazolinee (SKI-1) and the specificity of action was verified by RNA interference. The results showed that SKI-1 induced significant apoptosis in a dose-dependent manner in cancer cells with high basal Src activation. Activation of FAK and p130Cas was involved in Src-mediated invasion in SKI-1-sensitive cells. SKI-1 inhibited phosphorylation of EGFR as well as EGFR downstream effectors, such as signal transducers and activators of transcription 3/5, extracellular signal-regulated kinase 1/2 and AKT in the mutant cells but not the wild-type cells. This inhibition profile of EGFR implicates that induction of apoptosis and sensitivity of mutant cells to SKI treatment is mediated by EGFR and EGFR downstream pathways. Cotreatment with SKI-1 and gefitinib enhanced apoptosis in cancer cells that contained EGFR mutation and/or amplification. SKI-1 treatment alone induced significant apoptosis in H1975 cells known to be resistant to gefitinib. Src phosphorylation was shown by immunohistochemistry in around 30% of primary lung carcinomas. In 152 adenocarcinomas studied, p-Src was associated with EGFR mutations (P = 0.029). Overall, the findings indicated that Src could be a useful target for treatment of non-small cell lung cancer. Besides EGFR genomic mutations, other forms of EGFR and related family member abnormalities such as EGFR amplification might enhance SKI sensitivity. Copyright © 2009 American Association for Cancer Research.link_to_subscribed_fulltex

EGFR mutations are prevalent and independent from K-RAS mutations in lung adenocarcinomas from non-smokers

Lung cancer is common in Hong Kong with an unusually high incidence in female non-smokers (NS). Lung cancers in NS are likely to involve different carcinogenic processes from those in smokers, but the molecular targets are unclear. Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a crucial role in cell proliferation, survival and differentiation. It has been observed that non-small cell lung cancer (NSCLC) patients showing improved clinical response to tyrosine kinase inhibitor (TKI) treatment harbour somatic mutations in EGFR tyrosine kinase domain. Activating point mutations in the RAS oncogene has been recognized in adenocarcinomas of different organs including the lung. In order to find out the prevalence and patterns of EGFR and RAS mutations, as well as their relation with clinicopathological profiles of primary NSCLC in Hong Kong, 228 surgically resected primary lung cancers were analyzed. The tumors included 205 adenocarcinomas (AD), 14 squamous cell carcinomas (SCC) and 9 lymphoepithelioma-like carcinomas (LELC) from 97 men (55 current smokers (SM), 17 NS, 24 ex-smokers (EX) and 1 passive smokers (PS)), and 131 women (7 SM, 92 NS, 13 EX and 19 PS) were studied. PCR amplified products spanning the EGFR mutation hot spots (exons 18-21) were sequenced. An overall mutation rate of 49.1% (112/228) was observed, including 52 in-frame deletions, 5 in-frame insertions and 55 amino acid substitutions. Statistically significant associations were found between EGFR mutations and a) NS (79/109) compared with patients with any level of current or previous tobacco exposure (33/119 SM, PS, EX) or with SM only (12/62), P<0.0001 for both, b) female (83/131) compared to male (29/97), P<0.0001 and c) AD (112/205) compared to non-AD (0/23), P<0.0001. No association was found with patients’ age and pathological tumor stage. The findings suggest that EGFR mutation mediates important non-tobacco induced carcinogenic pathways in AD from NS, in contrast to SCC that are prevalent in smokers or the Epstein Barr virus-related LELC. The frequency of K-RAS codon 12 mutations was analyzed by dot-blot and sequencing which demonstrated 18/228 (7.9%) mutations. The mutations were significantly associated with history of any tobacco exposure (15/119, P=0.006) or SM only (10/62, P=0.002). Furthermore, all the K-RAS mutants were found in tumours with wild type EGFR (P<0.0001). In conclusion, EGFR mutations are prevalent and independent from K-RAS mutations in the carcinogenic process in NS with AD. EGFR mutation status of these patients is useful for predicting potential response to TKI therapy. (Supported by HKSAR RGC grants 7310/01M, 7486/03M, 7468/04M

Identification of discriminatory gene expression of EBV-associated primary lymphoepithelioma-like carcinoma of lung by oligonucleotide microarray analysis

Lymphoepithelioma-like carcinomas (LELC) of the lung is a distinct type of primary lung cancer constituting 5-9% of non-small cell lung cancers. LELC occurring in oriental ethnic groups are etiologically related to EBV infection and demonstrate clonal episomal EBV genomes as well as EBV marker expression in tumor cells. There is no relation with tobacco exposure but a close morphological resemblance to nasopharyngeal undifferentiated carcinomas, which is also a common EBV-related tumor in Chinese, is striking. The molecular pathways involved in lung LELC are incompletely understood. In order to identify signature molecules of LELC, we compared the expression profiles of 8 untreated, EBV-positive primary lung LELC with 9 normal lung tissues by oligonucleotide microarray analysis (HGU133A chips, Affymetrix). Verification of the differential expression of selective genes was performed using quantitative PCR (tagman probes) and immunohistochemical evaluation on a tissue microarray. The data were evaluated with the method of feature partitioning for single and pair-wise gene expression (Yap et al, 2004) which identified a discriminatory gene set of 1480 genes capable of distinguishing LELC from the normal lung tissue expression profiles with 100% accuracy. The identified gene set included a higher proportion of genes related to active cellular proliferation, mitosis, cell cycle, DNA replication and nuclear division compared to the arrayed probe set. Examples of tumor-related genes in the discriminatory gene set included those involved in EBV viral gene interaction (BS69, EBI3, EIF5A, KPNA2, MYB, p100, RECK, TAL1), cell proliferation or growth (EZH2, HMGA1, PTTG1, STK6), cell adhesion or extracellular matrix modification (EPHB2, GPI, HMMR, MMP9, MMP12, TIMP3, TNXB), apoptosis or cell survival (AVEN, BCL2, BIRC5, GG2-1), tumor suppression (DAPK1, GADD45B, PLAGL1, PPARG, RTN4), cell cycle checkpoint (CDKN1C, CGR19, WFDC1), anti-angiogenesis (ADAMTS1) and negative regulation of cell growth (CTNNAL1, DLC1, DUSP6, TGFBR2, TGFBR3, TU3A). A change of cytoskeletal expression profile was also observed with underexpression of KRT 7, SCEL and overexpression of the keratins KRT5, 6A and B, 8, 13, 15, 17, 19 as well as some basal cell or cell junction markers such as BPAG1 and KIND1. In addition, several genes related to potential resistance to drug treatment showed elevated expression (ABCE1, CTPS, RRM2, TYMS). The identified genes could contribute to a better understanding of EBV- induced carcinogenesis as well as signify new molecular targets of therapy for EBV-related tumors of the lung and other body regions. (Supported by HKSAR RGC grants 7486/03M, 7468/04M

The EML4-ALK fusion gene is involved in various histologic types of lung cancers from nonsmokers with wild-type EGFR and KRAS

Background: The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4- ALK) fusion gene resulting from the chromosome inversion inv(2)(p21;p23) recently was identified in non- small cell lung cancer (NSCLC). The authors of this study investigated the frequency, genetic and clinicopathologic profiles of EML4-ALK in Chinese patients with NSCLC. Methods: EML4-ALK was investigated in 266 resected primary NSCLC, including adenocarcinomas (AD), lymphoepithelioma-like carcinomas, squamous cell carcinomas, mucoepidermoid carcinomas, and adenosquamous carcinomas, by reverse transcriptase-polymerase chain reaction and was verified by sequencing. EML4-ALK protein expression was studied by immunohistochemistry. Results: Thirteen tumors (4.9%) had EML4-ALK comprising 4 fusion transcript variants with fusion of the variable segments from 5' EML4 to 3' ALK and with preservation of the ALK kinase domain. The most common variant consisted of 8 tumors with variant 3 that involved EML4 exon 6. The others included 2 tumors with variant 1 (exon 13), 2 tumors with variant 2 (exon 20), and 1 tumor with the novel variant 5 (exon 18). There were 11 ADs and 2 unusual carcinomas with mixed squamous and glandular components. Immunohistochemistry demonstrated diffuse ALK fusion proteins in the tumor cell cytoplasm. EML4-ALK was associated with nonsmokers (P = .009). Tumors with the fusion gene had the wild-type epidermal growth factor receptor (EGFR)(P = .001) and v-Ki-ras2/Kirsten rat sarcoma viral oncogene homolog (KRAS) genes. Patients who had EML4-ALK-positive AD had a younger median age (P = .018) compared with patients who did not have the fusion gene. Conclusions: The EML4-ALK fusion gene was present in various histologic types of NSCLC. It occurred in mutual exclusion to EGFR and KRAS mutations and was associated with nonsmokers. The authors concluded that EML4-ALK may be useful for predicting the potential response to ALK inhibitors as a therapeutic option for patients with lung cancer. © 2009 American Cancer Society.link_to_subscribed_fulltex