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Supplementary Table 2 from Paxillin Is a Target for Somatic Mutations in Lung Cancer: Implications for Cell Growth and Invasion

Supplementary Figure 5 from Paxillin Is a Target for Somatic Mutations in Lung Cancer: Implications for Cell Growth and Invasion

Supplementary Figure Legends 1-2, Table Legend 1 from The MET Receptor Tyrosine Kinase Is a Potential Novel Therapeutic Target for Head and Neck Squamous Cell Carcinoma

Supplementary Figure 1 from Paxillin Is a Target for Somatic Mutations in Lung Cancer: Implications for Cell Growth and Invasion

Supplementary Figure 2 from Paxillin Is a Target for Somatic Mutations in Lung Cancer: Implications for Cell Growth and Invasion

Supplementary Table 3 from Paxillin Is a Target for Somatic Mutations in Lung Cancer: Implications for Cell Growth and Invasion

PDF file - 66K, This table contains the detailed immunohistochemical data scoring depicted in Figure A and B

Esophageal cancer incidence is increasing and has few treatment options. In studying receptor tyrosine kinases associated with esophageal cancers, we have identified EPHB4 to be robustly overexpressed in cell lines and primary tumor tissues. In total, 94 squamous cell carcinoma, 82 adenocarcinoma, 25 dysplasia, 13 Barrett esophagus, and 25 adjacent or unrelated normal esophageal tissues were evaluated by immunohistochemistry. EPHB4 expression was significantly higher in all the different histologic categories than in adjacent normal tissues. In 13 esophageal cancer cell lines, 3 of the 9 SCC cell lines and 2 of the 4 adenocarcinomas expressed very high levels of EPHB4. An increased gene copy number ranging from 4 to 20 copies was identified in a subset of the overexpressing patient samples and cell lines. We have developed a novel 4-nitroquinoline 1-oxide (4-NQO)-induced mouse model of esophageal cancer that recapitulates the EPHB4 expression in humans. A specific small-molecule inhibitor of EPHB4 decreased cell viability in a time- and dose-dependent manner in 3 of the 4 cell lines tested. The small-molecule inhibitor and an EPHB4 siRNA also decreased cell migration (12%–40% closure in treated vs. 60%–80% in untreated), with decreased phosphorylation of various tyrosyl-containing proteins, EphB4, and its downstream target p125FAK. Finally, in a xenograft tumor model, an EPHB4 inhibitor abrogated tumor growth by approximately 60% compared with untreated control. EphB4 is robustly expressed and potentially serves as a novel biomarker for targeted therapy in esophageal cancers. Cancer Res; 73(1); 184–94. ©2012 AACR.

Supplementary Table 4 from Paxillin Is a Target for Somatic Mutations in Lung Cancer: Implications for Cell Growth and Invasion

Supplementary Figure 1 from The MET Receptor Tyrosine Kinase Is a Potential Novel Therapeutic Target for Head and Neck Squamous Cell Carcinoma

Lung cancer is characterized by abnormal cell growth and invasion, and the actin cytoskeleton plays a major role in these processes. The focal adhesion protein paxillin is a target of a number of oncogenes involved in key signal transduction and important in cell motility and migration. In lung cancer tissues, we have found that paxillin was highly expressed (compared with normal lung), amplified (12.1%, 8 of 66) and correlated with increased MET and epidermal growth factor receptor (EGFR) gene copy numbers, or mutated (somatic mutation rate of 9.4%, 18 of 191). Paxillin mutations (19 of 21) were clustered between LD motifs 1 and 2 and the LIM domains. The most frequent point mutation (A127T) enhanced lung cancer cell growth, colony formation, focal adhesion formation, and colocalized with Bcl-2 in vitro. Gene silencing from RNA interference of mutant paxillin led to reduction of cell viability. A murine in vivo xenograft model of A127T paxillin showed an increase in tumor growth, cell proliferation, and invasion. These results establish an important role for paxillin in lung cancer. [Cancer Res 2007;68(1):132–42]

Supplementary Table 1 from Paxillin Is a Target for Somatic Mutations in Lung Cancer: Implications for Cell Growth and Invasion

Supplementary Figure 3 from Paxillin Is a Target for Somatic Mutations in Lung Cancer: Implications for Cell Growth and Invasion

Supplementary Figure 4 from Paxillin Is a Target for Somatic Mutations in Lung Cancer: Implications for Cell Growth and Invasion

Recurrent/metastatic head and neck cancer remains a devastating disease with insufficient treatment options. We investigated the MET receptor tyrosine kinase as a novel target for the treatment of head and neck squamous cell carcinoma (HNSCC). MET/phosphorylated MET and HGF expression was analyzed in 121 tissues (HNSCC/normal) by immunohistochemistry, and in 20 HNSCC cell lines by immunoblotting. The effects of MET inhibition using small interfering RNA/two small-molecule inhibitors (SU11274/PF-2341066) on signaling, migration, viability, and angiogenesis were determined. The complete MET gene was sequenced in 66 head and neck cancer tissue samples and eight cell lines. MET gene copy number was determined in 14 cell lines and 23 tumor tissues. Drug combinations of SU11274 with cisplatin or erlotinib were tested in SCC35/HN5 cell lines. Eighty-four percent of the HNSCC samples showed MET overexpression, whereas 18 of 20 HNSCC cell lines (90%) expressed MET. HGF overexpression was present in 45% of HNSCC. MET inhibition with SU11274/PF-2341066 abrogated MET signaling, cell viability, motility/migration in vitro, and tumor angiogenesis in vivo. Mutational analysis of 66 tumor tissues and 8 cell lines identified novel mutations in the semaphorin (T230M/E168D/N375S), juxtamembrane (T1010I/R988C), and tyrosine kinase (T1275I/V1333I) domains (incidence: 13.5%). Increased MET gene copy number was present with >10 copies in 3 of 23 (13%) tumor tissues. A greater-than-additive inhibition of cell growth was observed when combining a MET inhibitor with cisplatin or erlotinib and synergy may be mediated via erbB3/AKT signaling. MET is functionally important in HNSCC with prominent overexpression, increased gene copy number, and mutations. MET inhibition abrogated MET functions, including proliferation, migration/motility, and angiogenesis. MET is a promising, novel target for HNSCC and combination approaches with cisplatin or EGFR inhibitors should be explored. [Cancer Res 2009;69(7):3021–31]

Supplementary Table 1 from The MET Receptor Tyrosine Kinase Is a Potential Novel Therapeutic Target for Head and Neck Squamous Cell Carcinoma

Recurrent/metastatic head and neck cancer remains a devastating disease with insufficient treatment options. We investigated the MET receptor tyrosine kinase as a novel target for the treatment of head and neck squamous cell carcinoma (HNSCC). MET/phosphorylated MET and HGF expression was analyzed in 121 tissues (HNSCC/normal) by immunohistochemistry, and in 20 HNSCC cell lines by immunoblotting. The effects of MET inhibition using small interfering RNA/two small-molecule inhibitors (SU11274/PF-2341066) on signaling, migration, viability, and angiogenesis were determined. The complete MET gene was sequenced in 66 head and neck cancer tissue samples and eight cell lines. MET gene copy number was determined in 14 cell lines and 23 tumor tissues. Drug combinations of SU11274 with cisplatin or erlotinib were tested in SCC35/HN5 cell lines. Eighty-four percent of the HNSCC samples showed MET overexpression, whereas 18 of 20 HNSCC cell lines (90%) expressed MET. HGF overexpression was present in 45% of HNSCC. MET inhibition with SU11274/PF-2341066 abrogated MET signaling, cell viability, motility/migration in vitro, and tumor angiogenesis in vivo. Mutational analysis of 66 tumor tissues and 8 cell lines identified novel mutations in the semaphorin (T230M/E168D/N375S), juxtamembrane (T1010I/R988C), and tyrosine kinase (T1275I/V1333I) domains (incidence: 13.5%). Increased MET gene copy number was present with >10 copies in 3 of 23 (13%) tumor tissues. A greater-than-additive inhibition of cell growth was observed when combining a MET inhibitor with cisplatin or erlotinib and synergy may be mediated via erbB3/AKT signaling. MET is functionally important in HNSCC with prominent overexpression, increased gene copy number, and mutations. MET inhibition abrogated MET functions, including proliferation, migration/motility, and angiogenesis. MET is a promising, novel target for HNSCC and combination approaches with cisplatin or EGFR inhibitors should be explored. [Cancer Res 2009;69(7):3021–31]

PAX5 is a nuclear transcription factor required for B cell development, and its expression was evaluated in upper aerodigestive malignancies and pancreatic cancer by immunoblotting. The PAX5 protein expression was relatively strong in small-cell lung cancer (SCLC, 11/12); however, its expression was not detected in non-SCLC (NSCLC, n=13), mesothelioma (n=7), pancreatic (n=6), esophageal (n=6) and head and neck cancer cell lines (n=12). In comparison, PAX8 and PAX3 expressions were absent or non-detectable in SCLC cell lines; however, PAX8 was expressed in most of the tested NSCLC cell lines (13/13) and also frequently in all the other cell lines. We also detected frequent expressions of PAX2 and PAX9 protein in the various cell lines. Utilizing neuroendocrine tumor samples, we found that the frequency as well as the average intensity of the expression of PAX5 increased from pulmonary carcinoid (9%, moderate and strong PAX5 expression, n=44), to large-cell neuroendocrine carcinoma (LCNC, 27%, n=11) to SCLC (33%, n=76). FISH analysis revealed no translocations of the PAX5 gene, but polyploidy in some SCLC tumor tissues (6/37). We determined that PAX5 could regulate the transcription of c-Met using luciferase-coupled reporter and chromatin immunoprecipitation analysis. In addition, the phospho-c-Met (active form) and PAX5 were both localized to the same intra-nuclear compartment in hepatocyte growth factor treated SCLC cells and interacted with each other. Finally, we determined the therapeutic translational potential of PAX5 using PAX5 knockdown SCLC cells in conjunction with Topoisomerase 1 (SN38) and c-Met (SU11274) inhibitors. Loss of endogenous PAX5 significantly decreased the viability of SCLC cells, especially when combined with SN38 or SU11274, and maximum effect was seen when both inhibitors were used. Therefore, we propose that PAX5 could be an important regulator of c-Met transcription and a potential target for therapy in SCLC.

The c-MET receptor can be overexpressed, amplified, or mutated in solid tumours including small cell lung cancer (SCLC). In c-MET-overexpressing SCLC cell line NCI-H69, hepatocyte growth factor (HGF) dramatically induced c-MET phosphorylation at phosphoepitopes pY1230/1234/1235 (catalytic tyrosine kinase), pY1003 (juxtamembrane), and also of paxillin at pY31 (CRKL-binding site). We utilised a global proteomics phosphoantibody array approach to identify further c-MET/HGF signal transduction intermediates in SCLC. Strong HGF induction of specific phosphorylation sites in phosphoproteins involved in c-MET/HGF signal transduction was detected, namely adducin-alpha [S724], adducin-gamma [S662], CREB [S133], ERK1 [T185/Y187], ERK1/2 [T202/Y204], ERK2 [T185/Y187], MAPKK (MEK) 1/2 [S221/S225], MAPKK (MEK) 3/6 [S189/S207], RB [S612], RB1 [S780], JNK [T183/Y185], STAT3 [S727], focal adhesion kinase (FAK) [Y576/S722/S910], p38alpha-MAPK [T180/Y182], and AKT1[S473] and [T308]. Conversely, inhibition of phosphorylation by HGF in protein kinase C (PKC), protein kinase R (PKR), and also CDK1 was identified. Phosphoantibody-based immunohistochemical analysis of SCLC tumour tissue and microarray established the role of c-MET in SCLC biology. This supports a role of c-MET activation in tumour invasive front in the tumour progression and invasion involving FAK and AKT downstream. The c-MET serves as an attractive therapeutic target in SCLC, as shown through small interfering RNA (siRNA) and selective prototype c-MET inhibitor SU11274, inhibiting the phosphorylation of c-MET itself and its downstream molecules such as AKT, S6 kinase, and ERK1/2. Investigation of mechanisms of invasion and, ultimately, metastasis in SCLC would be very useful with these signal transduction molecules.

Background A 70-year-old Japanese–American woman who had never smoked was diagnosed with stage IV non-small-cell lung cancer with rib metastases. She had previously been well and she had no family history of malignancy. While receiving treatment with erlotinib, an epidermal growth factor receptor small-molecule inhibitor, she progressed and developed new brain metastases. She failed further chemotherapy treatments and subsequently developed extensive symptomatic leptomeningeal carcinomatosis associated with diplopia, hemiparesis, weight loss, and incontinence. Investigations Chest X-ray, head and chest CT scan, R2 lymph-node biopsy, histopathology, immunohistochemistry, MRI of head and spine, lumbar puncture, laser microdissection and EGFR genomic DNA sequencing of the R2 lymph node and cerebrospinal fluid tumor cells. Diagnosis Erlotinib-refractory stage IV lung adenocarcinoma and end-stage symptomatic leptomeningeal metastases with a novel double L858R + E884K somatic mutation of the EGFR. Management Carboplatin, paclitaxel and erlotinib, whole-brain radiotherapy, temozolomide with and without irinotecan, and gefitinib.

Esophageal cancer incidence is increasing and has few treatment options. In studying receptor tyrosine kinases associated with esophageal cancers, we have identified EPHB4 to be robustly overexpressed in cell lines and primary tumor tissues. In total, 94 squamous cell carcinoma, 82 adenocarcinoma, 25 dysplasia, 13 Barrett esophagus, and 25 adjacent or unrelated normal esophageal tissues were evaluated by immunohistochemistry. EPHB4 expression was significantly higher in all the different histologic categories than in adjacent normal tissues. In 13 esophageal cancer cell lines, 3 of the 9 SCC cell lines and 2 of the 4 adenocarcinomas expressed very high levels of EPHB4. An increased gene copy number ranging from 4 to 20 copies was identified in a subset of the overexpressing patient samples and cell lines. We have developed a novel 4-nitroquinoline 1-oxide (4-NQO)-induced mouse model of esophageal cancer that recapitulates the EPHB4 expression in humans. A specific small-molecule inhibitor of EPHB4 decreased cell viability in a time- and dose-dependent manner in 3 of the 4 cell lines tested. The small-molecule inhibitor and an EPHB4 siRNA also decreased cell migration (12%–40% closure in treated vs. 60%–80% in untreated), with decreased phosphorylation of various tyrosyl-containing proteins, EphB4, and its downstream target p125FAK. Finally, in a xenograft tumor model, an EPHB4 inhibitor abrogated tumor growth by approximately 60% compared with untreated control. EphB4 is robustly expressed and potentially serves as a novel biomarker for targeted therapy in esophageal cancers. Cancer Res; 73(1); 184–94. ©2012 AACR.

Lung cancer is characterized by abnormal cell growth and invasion, and the actin cytoskeleton plays a major role in these processes. The focal adhesion protein paxillin is a target of a number of oncogenes involved in key signal transduction and important in cell motility and migration. In lung cancer tissues, we have found that paxillin was highly expressed (compared with normal lung), amplified (12.1%, 8 of 66) and correlated with increased MET and epidermal growth factor receptor (EGFR) gene copy numbers, or mutated (somatic mutation rate of 9.4%, 18 of 191). Paxillin mutations (19 of 21) were clustered between LD motifs 1 and 2 and the LIM domains. The most frequent point mutation (A127T) enhanced lung cancer cell growth, colony formation, focal adhesion formation, and colocalized with Bcl-2 in vitro. Gene silencing from RNA interference of mutant paxillin led to reduction of cell viability. A murine in vivo xenograft model of A127T paxillin showed an increase in tumor growth, cell proliferation, and invasion. These results establish an important role for paxillin in lung cancer. [Cancer Res 2007;68(1):132–42]

The c-Met receptor tyrosine kinase is emerging as a novel target in many solid tumors, including lung cancer. PHA-665752 was identified as a small molecule, ATP competitive inhibitor of the catalytic activity of the c-Met kinase. Here, we show that treatment with PHA665752 reduced NCI-H69 (small cell lung cancer) and NCI-H441 (non–small cell lung cancer) tumorigenicity in mouse xenografts by 99% and 75%, respectively. Reduction in tumor size was also observed by magnetic resonance imaging of tumors in mice. PHA665752 inhibited c-Met phosphorylation at the autophosphorylation and c-Cbl binding sites in mouse xenografts derived from non–small cell lung cancer cell lines (NCI-H441 and A549) and small cell lung cancer cell line (NCI-H69). PHA665752 also inhibited angiogenesis by >85% in all the abovementioned cell lines and caused an angiogenic switch which resulted in a decreased production of vascular endothelial growth factor and an increase in the production of the angiogenesis inhibitor thrombospondin-1. These studies show the feasibility of selectively targeting c-Met with ATP competitive small molecule inhibitors and suggest that PHA665752 may provide a novel therapeutic approach to lung cancer. [Cancer Res 2007;67(8):3529–34]

c-Met receptor tyrosine kinase (RTK) has not been extensively studied in malignant pleural mesothelioma (MPM). In this study, c-Met was overexpressed and activated in most of the mesothelioma cell lines tested. Expression in MPM tissues by immunohistochemistry was increased (82%) in MPM in general compared with normal. c-Met was internalized with its ligand hepatocyte growth factor (HGF) in H28 MPM cells, with robust expression of c-Met. Serum circulating HGF was twice as high in mesothelioma patients as in healthy controls. There was a differential growth response and activation of AKT and extracellular signal–regulated kinase 1/2 in response to HGF for the various cell lines. Dose-dependent inhibition (IC50 < 2.5 μmol/L) of cell growth in mesothelioma cell lines, but not in H2052, H2452, and nonmalignant MeT-5A (IC50 >10 μmol/L), was observed with the small-molecule c-Met inhibitor SU11274. Furthermore, migration of H28 cells was blocked with both SU11274 and c-Met small interfering RNA. Abrogation of HGF-induced c-Met and downstream signaling was seen in mesothelioma cells. Of the 43 MPM tissues and 7 cell lines, we have identified mutations within the semaphorin domain (N375S, M431V, and N454I), the juxtamembrane domain (T1010I and G1085X), and an alternative spliced product with deletion of the exon 10 of c-Met in some of the samples. Interestingly, we observed that the cell lines H513 and H2596 harboring the T1010I mutation exhibited the most dramatic reduction of cell growth with SU11274 when compared with wild-type H28 and nonmalignant MeT-5A cells. Ultimately, c-Met would be an important target for therapy against MPM. (Cancer Res 2006; 66(1): 352-61

Non–small cell lung cancer (NSCLC) is a difficult disease to treat. The c-Met receptor is an attractive potential target for novel therapeutic inhibition in human cancers. We provide strong evidence that c-Met is overexpressed, activated, and sometimes mutated in NSCLC cell lines and tumor tissues. Expression of c-Met was found in all (100%) of the NSCLC tumor tissues examined (n = 23) and most (89%) of the cell lines (n = 9). Sixty-one percent of tumor tissues strongly expressed total c-Met, especially adenocarcinoma (67%). Specific expression of phospho-Met (p-Met) [Y1003] and [Y1230/1234/1235] was seen by immunohistochemistry. p-Met expression was preferentially observed at the NSCLC tumor invasive fronts. c-Met alterations were identified within the semaphorin domain (E168D, L299F, S323G, and N375S) and the juxtamembrane domain (R988C, R988C + T1010I, S1058P, and alternative splice product skipping entire juxtamembrane domain) of a NSCLC cell line and adenocarcinoma tissues. We validated c-Met as potential therapeutic target using small interfering RNA down-regulation of the receptor expression by 50% to 60% in NSCLC cells. This led to inhibition of p-Met and phospho-AKT and up to 57.1 ± 7.2% cell viability inhibition at 72 hours. The selective small molecule inhibitor of c-Met SU11274 inhibited cell viability in c-Met-expressing NSCLC cells. SU11274 also abrogated hepatocyte growth factor–induced phosphorylation of c-Met and its downstream signaling. Here, we provide first direct evidence by small interfering RNA targeting and small molecule inhibitor that c-Met is important in NSCLC biology and biochemistry. These results indicate that c-Met inhibition will be an important therapeutic strategy against NSCLC to improve its clinical outcome.

MET is a receptor tyrosine kinase that plays a critical role in proliferation, angiogenesis, invasion, and metastasis. It has been shown to be dysregulated in a number of malignancies, including non-small cell lung cancer (NSCLC). Despite reports of alterations in MET, and its ligand, HGF, being highly associated with advanced pathological stage and worse outcome in patients with NSCLC, the value of MET as a prognostic biomarker remains unclear. The primary objective of this study is to determine the correlation between MET alterations and expression with stage and overall survival in adenocarcinoma (AC) patients in a large cohort of patients. The secondary objectives are to determine the correlation between overall survival and the following: 1) epithelial mesenchymal transition (EMT), 2) EGFR mutations & expression, 3) KRAS mutations, 4) TP53 mutations, and 5) CBL mutations. In addition, sera levels of circulating MET and HGF will be evaluated in order to determine their potential as prognostic factors. MET, EGFR exons 18–21, TP53 exons 4–10, KRAS exon 2, and CBL exons 2–16 were sequenced using standard PCR and sequencing techniques. Standard immunohistochemistry (IHC) techniques were used to evaluate MET, phosphorylated (pMET Y1003 and Y1230/34/35) p53, HGF, EGFR, and E-cadherin expression. A total of 280 patients will be included in this study, and the interim analysis reported herein evaluated 100 patients. The intensity of cytoplasmic or membranous staining was scored on fourpoint scale: (0, no staining; 1+, weak; 2+, moderate; 3+, strong staining). The extent of staining was scored on a similar scale (0, negative; 1+, 1–10%; 2+, 11–50%; 3+, > 50%). The product of the intensity and extent of staining yielded final scores between 0 and 9. The mean expressions were: MET 3.6 (±0.3); pY1230/34/35 MET 2.0 (±0.2); pY1003MET 4.6 (±0.3); HGF 4.4 (±0.3); EGFR 4.3 (±0.3); TP53 3.7 (±0.3); and E-cadherin 5.5 (±0.3). In ten patient samples, six non-synonymous (NS) mutations were detected in MET (SEMA domain: E168D, M362T, N375S, and Q318K; JM domain: T992I and R970C). In EGFR, the NS mutation L858R was detected in two patients. We detected 12 NS mutations in TP53 (exon 4: E68*, P72R; exon 5: V157F, R175H, I162F, H193Y, Y163D; exon 8: R273L, R273C, V274L, A276F, and G266*). Four NS mutations were detected in exon 2 of KRAS (G12C, G12V, G12D, and G12S). Three mutations were found in the proline-rich region of CBL (L676P, A677S and A678S). ELISAs were utilized to determine soluble MET and HGF levels in pre- and postoperative sera samples. Soluble MET serum levels were significantly increased (p

Paxillin is a 68 kDa focal adhesion protein that provides multiple docking sites at the plasma membrane for an array of signaling molecules and helps form a structural link between the extracellular matrix and the actin cytoskeleton. It is essential in actin filament assembly and focal adhesion formation. Focal adhesion complexes are the drivers of cell spreading and migration, adhesion to the extracellular matrix and matrix remodeling by fibrillar adhesions in which paxillin plays a major role. It has been shown that the modulation of tyrosine phosphorylation of paxillin regulates both the assembly and turnover of adhesion sites and phosphorylated paxillin enhances lamellipodial protrusions whereas non-phosphorylated paxillin is essential for fibrillar adhesion formation. We have previously reported that in lung cancer, paxillin was over expressed, amplified and mutated in a significant number of patient samples and the protein upregulated in higher stages of lung cancer compared with lower stages (Jagadeeswaran et al, 2008). We recently showed paxillin gene to be amplified in some pre-neoplastic lung lesions. Among the mutations we described, A127T enhanced cell proliferation, focal adhesion formation and colocalization with Bcl-2 in lung cancer cells. We have now analyzed 50 epithelioid, 16 sarcomatoid, and one mixed malignant pleural mesothelioma tissue, and compared to 40 normal adjacent lung parenchyma (pneumocytes and endothelial cells) by immunohistochemistry. Automated cellular imaging was used to calculate average staining intensity as an integrated optical density (IOD). Whereas normal lung had IODs of 45 for paxillin, epithelioid MPM had IODs of 268 and sarcomatoid 331. This demonstrates that paxillin is highly upregulated in both epithelioid as well as sarcomatoid. To further study the effects of activating mutations of paxillin, we cloned the most commonly occurring paxillin mutations in a GFP tagged vector and transiently transfect HEK-293 cells. Utilizing live-cell imaging to systematically study the effects of wild-type paxillin versus mutants, we created a mathematical model that recapitulates the salient features of the measured dynamics, and conclude that compared to wild-type, some mutant clones confer a) enhanced focal adhesion formation, b) increased filopodia and lamellipodia formation c) increased mobility and d) increased cell displacement in transiently transfected HEK-293 cells. We believe that paxillin is an important molecule in thoracic cancer including lung and malignant mesothelioma, and its therapeutic potential needs to be explored further. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2341. doi:10.1158/1538-7445.AM2011-2341

Although various mutations of the epidermal growth factor receptor ( EGFR ) gene, most commonly L858R (exon 21) and short exon 19 deletions, have been identified to confer sensitivity toward EGFR tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, it is not known if there are mutations that may result in differential activities of the two inhibitors. We describe a 70-year-old Japanese American woman diagnosed with stage IV non-small-cell lung cancer (NSCLC) with rib metastasis. While receiving treatment with the EGFR small molecule TKI erlotinib, she progressed and developed new brain metastases. She failed further chemotherapy treatments and subsequently developed symptomatic extensive leptomeningeal carcinomatosis associated with diplopia, hemiparesis, weight loss, and incontinence. Monotherapy gefitinib 250 mg daily was initiated, and she showed striking response both clinically and radiographically within the first few weeks. Using laser microdissection (LMD), we performed genomic DNA extraction and EGFR gene sequencing from the enriched tumor cells in her pretreatment tumor biopsy specimen and tumor cells found in her cerebrospinal fluid. Two heterozygous somatic EGFR mutations, L858R (exon 21) and E884K (exon 22), were identified in both specimens. In vitro transfection and biochemical studies revealed that the novel E884K mutation confers opposite effects in sensitivity to the two EGFR inhibitors. EGFR E884K and EGFR L858R+E884K enhanced the sensitivity of the mutated receptor to gefitinib inhibition. Conversely, the E884K mutation resulted in decreased responsiveness of the receptor to erlotinib, and it significantly abrogated the drug sensitivity conferred by L858R (EGFR L858R+E884K ). This study demonstrates that it is possible to have differential response to alternative EGFR TKIs. This also represents the first report of a response of leptomeningeal metastases to EGFR inhibition by small molecule inhibitor gefitinib alone in NSCLC. Further structural studies of the mutant EGFR are warranted to improve individualized targeted therapy and small molecule inhibitors9 design in lung cancer in the future.

9539 Background: c-MET is a potential therapeutic target with its aberrant signaling mediating invasive cellular program in cancers. Novel therapies are needed for lung cancer, mesothelioma and squ...