A Phase II Trial of Alisertib (MLN8237) in Salvage Malignant Mesothelioma.

LESSONS LEARNED: Treatment with the Aurora kinase A inhibitor yields often durable disease control, but limited tumor regression, in heavily pretreated patients with unresectable malignant pleural or peritoneal mesothelioma. In a limited sample size, MYC copy-number gain or gene amplification, a candidate predictive biomarker for alisertib, did not correlate with improved response numbers or patient outcomes. BACKGROUND: Malignant mesothelioma is an aggressive disease for which few effective therapies are available. The Aurora family kinases are critical for mitotic fidelity and highly expressed in mesothelioma, wherein their inhibition leads to growth arrest in vitro. We evaluated the efficacy of alisertib, an Aurora A kinase inhibitor, in relapsed malignant mesothelioma. METHODS: Twenty-six patients with previously treated, unresectable pleural or peritoneal mesothelioma were enrolled on a single-arm, single-institution phase II trial of alisertib at a dosage of 50 mg twice daily for 7 of every 21 days. The primary endpoint was 4-month disease control rate. Secondary endpoints included overall response rate, progression free survival, overall survival, safety/toxicity, and correlation of endpoints with MYC copy number. RESULTS: Of the 25 evaluable patients treated on study, 8 (32%) experienced 4-month disease control, surpassing the futility endpoint. There were no confirmed partial or complete responses. Median progression-free and overall survival were 2.8 months and 6.3 months, respectively. No associations between MYC copy number and outcomes were observed. CONCLUSION: Alisertib has modest activity in this unselected malignant mesothelioma population. Several patients achieved durable disease control. Although the study did meet its prespecified futility endpoint, the sponsor elected to close the trial at the interim analysis.

Clinical outcomes and biomarker profiles of elderly pretreated NSCLC patients from the BATTLE trial.

BACKGROUND: Treating elderly non-small-cell lung cancer (NSCLC) patients in the salvage setting is challenging because of concerns of intolerance to therapy. Here we report outcomes (survival and toxicity) of elderly patients on the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) trial. METHODS: Two hundred and fifty-five chemorefractory NSCLC patients received tumor molecular analysis, and were randomized to erlotinib, erlotinib-bexarotene, vandetanib, or sorafenib. Retrospective subgroup analyses were conducted comparing outcomes among age groups (< 65 versus ≥ 65 years; < 70 versus ≥ 70 years; < 75 versus ≥ 75 years), treatments, and sex. RESULTS: Median age was 62 years (range, 26-84); 38% were aged 65 years or more. No significant differences among age groups were seen in rates of biopsy-related pneumothorax, treatment-related death, compliance, grade 3 to 4 hematologic toxicities, response rate, nor overall survival. However, older women aged 65 years or more had more grade 3 to 4 nonhematologic toxicities (p = 0.05). Elderly men aged 65 years or more (p = 0.008) had a higher disease-control rate at 8 weeks and a better progression-free survival (PFS) (p = 0.0068). Elderly women aged 70 years or more had a trend toward higher 8-week disease-control rate (p = 0.06). Older men aged 65 years or more treated with vandetanib had a better median PFS (p = 0.03) whereas PFS of older women aged 70 years or more was worse (p = 0.03) compared with younger patients. Elderly men aged 70 years or more treated with sorafenib had a higher overall survival compared with younger men (p = 0.04). Tumor tissue biomarkers show distinct differences by sex and age. CONCLUSION: Fit elderly NSCLC patients should be considered for salvage targeted therapy. In this subset of patients, older men seem to have significant clinical benefit from certain agents. Tumor biomarker analysis demonstrates sex and age variations, and is hypothesis-generating.

MicroRNA 96 is a post-transcriptional suppressor of anaplastic lymphoma kinase expression.

Anaplastic lymphoma kinase (ALK) constitutes a part of the oncogenic fusion proteins nucleophosmin-ALK and echinoderm microtubule-associated protein like 4-ALK, which are aberrantly expressed in a subset of T-cell anaplastic large-cell lymphoma and non-small-cell lung cancer, respectively. The expression of mutated, constitutively active ALK also occurs in a subset of neuroblastoma tumors. ALK is believed to play an important role in promoting tumor survival. Nevertheless, the mechanisms underlying the expression of ALK in cancer cells are not completely known. MicroRNA (miR) has been implicated in the regulation of the expression of both oncogenes and tumor suppressor genes. We tested the hypothesis that the expression of ALK could be regulated by miR. Three Internet-based algorithms identified miR-96 to potentially bind with the ALK 3'-untranslated region. Notably, miR-96 levels were markedly decreased in ALK-expressing cancer cell lines and primary human tumors compared with their normal cellular and tissue counterparts. Transfection of the cell lines with miR-96 decreased levels of the different forms of ALK protein, without significant effects on ALK mRNA. Furthermore, miR-96 decreased the phosphorylation of ALK target proteins, including Akt, STAT3, JNK, and type I insulin-like growth factor receptor, and it down-regulated JunB. These effects were associated with reduced proliferation, colony formation, and migration of ALK-expressing cancer cells. These data provide novel evidence that decreases in miR-96 could represent a mechanism underlying the aberrant expression of ALK in cancer cells.

Phase II study of dasatinib in patients with advanced non-small-cell lung cancer.

PURPOSE: Src family kinases (SFKs) promote cancer progression and are commonly expressed in non-small-cell lung cancer (NSCLC), but the clinical effects of SFK inhibition in NSCLC are unknown. We conducted a phase II trial of the SFK inhibitor dasatinib for advanced NSCLC. We tested the hypotheses that the activation of epidermal growth factor receptor (EGFR) or SFK or modulation of serum cytokines may predict a response to dasatinib. PATIENTS AND METHODS: Patients received dasatinib as first-line therapy. Response was measured by tumor size on computed tomography scans and by metabolic activity on positron emission tomography scans. Tissue samples taken before patients received dasatinib were tested for EGFR and Kras mutation and phosphorylated SFK expression. RESULTS: Thirty-four patients were enrolled. The overall disease control rate (partial responses plus stable disease) for dasatinib was 43%. One patient had a partial response to therapy. Eleven patients (32%) had a metabolic response to dasatinib. SFK activation and EGFR and Kras mutations in tumor tissue did not predict response to dasatinib. Significant toxicities included fatigue and dyspnea. The presence of a pleural effusion before dasatanib therapy predicted the development of a clinically significant effusion during therapy. CONCLUSION: Dasatinib as a single agent had modest clinical activity that was lower than that generally observed in patients with NSCLC who receive chemotherapy. Pleural effusion was an expected and problematic toxicity that was successfully treated with steroids, diuretics, and dose interruptions. Marked activity in one patient and prolonged stable disease in four others suggested a potential subpopulation of patients with dasatinib-sensitive NSCLC.

Phase II randomized, placebo-controlled trial of green tea extract in patients with high-risk oral premalignant lesions.

Epidemiologic and preclinical data support the oral cancer prevention potential of green tea extract (GTE). We randomly assigned patients with high-risk oral premalignant lesions (OPL) to receive GTE at 500, 750, or 1,000 mg/m(2) or placebo thrice daily for 12 weeks, evaluating biomarkers in baseline and 12-week biopsies. The OPL clinical response rate was higher in all GTE arms (n = 28; 50%) versus placebo (n = 11; 18.2%; P = 0.09) but did not reach statistical significance. However, the two higher-dose GTE arms [58.8% (750 and 1,000 mg/m(2)), 36.4% (500 mg/m(2)), and 18.2% (placebo); P = 0.03] had higher responses, suggesting a dose-response effect. GTE treatment also improved histology (21.4% versus 9.1%; P = 0.65), although not statistically significant. GTE was well tolerated, although higher doses increased insomnia/nervousness but produced no grade 4 toxicity. Higher mean baseline stromal vascular endothelial growth factor (VEGF) correlated with a clinical (P = 0.04) but not histologic response. Baseline scores of other biomarkers (epithelial VEGF, p53, Ki-67, cyclin D1, and p16 promoter methylation) were not associated with a response or survival. Baseline p16 promoter methylation (n = 5) was associated with a shorter cancer-free survival. Stromal VEGF and cyclin D1 expression were downregulated in clinically responsive GTE patients and upregulated in nonresponsive patients at 12 weeks (versus at baseline). An extended (median, 27.5 months) follow-up showed a median time to oral cancer of 46.4 months. GTE may suppress OPLs, in part through reducing angiogenic stimulus (stromal VEGF). Higher doses of GTE may improve short-term (12-week) OPL outcome. The present results support longer-term clinical testing of GTE for oral cancer prevention.

Expression of epidermal growth factor (EGF)/transforming growth factor-alpha by human lung cancer cells determines their response to EGF receptor tyrosine kinase inhibition in the lungs of mice.

Epidermal growth factor receptor (EGFR) has been extensively targeted in the treatment of non-small cell lung cancer, producing responses in a small number of patients. To study the role of ligand expression in mediating response to EGFR antagonism, we injected NCI-H441 [EGFR and EGF/transforming growth factor-alpha (TGF-alpha) positive] or PC14-PE6 (EGFR positive and EGF/TGF-alpha negative) human lung adenocarcinoma cells into the lungs of nude mice. We randomized the mice to receive treatment with the EGFR tyrosine kinase inhibitors gefitinib or AEE788 or vehicle. Treatment of mice bearing NCI-H441 but not PC14-PE6 lung tumors resulted in a significant reduction in primary tumor growth, pleural effusion, and lymph node metastasis. Immunohistochemical analyses revealed that NCI-H441 and PC14-PE6 cells expressed EGFR but that the expression of EGF/TGF-alpha was high in NCI-H441 cells and very low in PC14-PE6 cells. Consequently, EGFR was activated in both tumor and tumor-associated endothelial cells in the NCI-H441 tumors but not in the PC14-PE6 tumors. Antagonism of EGFR signaling by treatment of mice with AEE788 decreased proliferation and increased apoptosis of both tumor cells and tumor-associated endothelial cells in NCI-H441 tumors but not in PC14-PE6 tumors. However, after transfection of PC14-PE6 cells with TGF-alpha, lung tumors derived from the transfected cells expressed and activated EGFR in both tumor and tumor-associated endothelial cells and tumors responded to treatment with AEE788. Collectively, these results strongly suggest that the response of human lung cancers growing orthotopically in mice to the inhibition of EGFR signaling is determined by ligand (EGF/TGF-alpha) expression by tumor cells. Our findings provide an additional explanation for the susceptibility of lung cancers to treatment with EGFR tyrosine kinase inhibitors.

Clinicopathologic characteristics of the EGFR gene mutation in non-small cell lung cancer.

BACKGROUND: The authors sought to define clinicopathologic features associated with mutations of the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC). METHODS: The authors evaluated surgically resected NSCLC tumors for EGFR (exons 18-21) and KRAS (codons 12-13) mutations and immunohistochemistry (EGFR, phosphorylated-EGFR, and HER2/Neu), and correlated results with clinical outcome and patient and disease features. After their analysis on 159 patients was completed, they selected a second cohort of Asian patients (n = 22) and compared EGFR mutation results to place of birth and immigration to the United States. RESULTS: Of 159 patients, 14 had EGFR mutations and 18 had KRAS mutations. EGFR mutations were associated with adenocarcinoma (p = 0.002), female gender (p = 0.02), never-smoking (p < 0.0001), Asian ethnicity (p = 0.005), air bronchograms (p = 0.004), and multiple wedge resections (p = 0.03). Although statistical significance was not reached, a higher incidence of synchronous primary cancers (36% versus 17%; p = 0.09) and a smaller median tumor size (11.8 cm versus 24.0 cm; p = 0.24) were seen. There was no difference in disease-free survival; however, median overall survival in patients with EGFR mutations was shorter (3.49 versus 4.29 years; p = 0.85). EGFR mutation did not correlate with immunohistochemistry. In the second cohort of 22 Asian patients, 12 (55%) had the mutation. Of interest, there was no geographic difference in incidence of EGFR mutation. Asian women with the EGFR mutation developed adenocarcinoma at an earlier age than other lung cancer patients. CONCLUSION: There is a distinct clinical profile for NSCLC patients with the EGFR mutation. However, this mutation does not alter disease-free survival and is likely attributable to an inherited susceptibility instead of an environmental effect.

Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis is inhibited by Bcl-2 but restored by the small molecule Bcl-2 inhibitor, HA 14-1, in human colon cancer cells.

PURPOSE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that induces apoptosis in multiple tumor cell types while sparing most normal cells. We determined the effect of ectopic Bcl-2 expression on TRAIL-induced apoptosis and whether the small molecule Bcl-2 inhibitor, HA14-1, could increase TRAIL sensitivity. EXPERIMENTAL DESIGN: SW480 human colon cancer cells were stably transfected with the PC3-Bcl-2 plasmid or vector alone. Cells were incubated with recombinant human TRAIL +/- HA14-1 or caspase-9 inhibitor (Z-LEHD-FMK). Apoptosis was analyzed by Annexin V-fluorescein isothiocyanate labeling and DNA fragmentation factor 45 (DFF45) cleavage. Clonigenic survival was also studied. Caspase activation was determined by immunoblotting or colorimetric assay. The cytosolic expression of Bid, Bax, and XIAP and release of cytochrome c and Smac/DIABLO were determined by immunoblotting. RESULTS: Bcl-2 overexpression partially protected SW480 cells from a dose-dependent induction of apoptosis by TRAIL, as did a caspase-9 inhibitor, and increased their clonogenic survival. Bcl-2 overexpression attenuated TRAIL-induced cleavage of caspase-8, indicating its activation upstream and downstream of mitochondria, as well as cleavage of Bid and caspase-3. Bcl-2 inhibited TRAIL-induced Bax translocation, cytosolic release of cytochrome c and Smac/DIABLO, and the downstream cleavage of XIAP and DFF45. Coadministration of HA14-1 and TRAIL increased apoptosis in SW480/Bcl-2 cells by restoring Bax redistribution and cytochrome c release. CONCLUSIONS: Bcl-2 confers apoptosis resistance to TRAIL by inhibiting a mitochondrial amplification step and by inactivating downstream XIAP in SW480 cells. HA14-1 reversed Bcl-2-mediated TRAIL resistance, suggesting a novel strategy for increasing TRAIL sensitivity in Bcl-2-overexpressing colon cancers.

Cyclooxygenase-2 overexpression reduces apoptotic susceptibility by inhibiting the cytochrome c-dependent apoptotic pathway in human colon cancer cells.

The cyclooxygenase-2 (COX-2) gene encodes an inducible enzyme that converts arachidonic acid to prostaglandins and is up-regulated in colorectal neoplasms. Evidence indicates that COX-2 may regulate apoptosis and can influence the malignant phenotype. Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX enzymes and induce apoptosis in colorectal cancer cell lines, which may contribute to their antitumor effects. To determine whether forced COX-2 expression modulates susceptibility to drug-induced apoptosis, HCT-15 colon carcinoma cells were stably transfected with the COX-2 cDNA, and two clones overexpressing COX-2 were isolated. Selective COX-2 (NS398) and nonselective (sulindac sulfide) COX inhibitors, as well as 5-fluorouracil (5-FU), induced apoptosis (terminal deoxynucleotidyl transferase-mediated nick end labeling in a dosage-dependent manner. Forced COX-2 expression significantly attenuated induction of apoptosis by all three of the drugs compared with parental HCT-15 cells. NSAIDs and 5-FU induced the mitochondrial release of cytochrome c as well as caspase-3 and -9 activation, and to a much lesser extent, caspase-8. COX-2-overexpressing cells showed reduced cytochrome c and caspase activation, relative to parental cells. A specific inhibitor of caspase-3 restored cell survival after drug treatment. COX-2 transfectants were found to overexpress the antiapoptotic Bcl-2 mRNA and protein relative to parental cells. In conclusion, forced COX-2 expression significantly attenuates apoptosis induction by NSAIDs and 5-FU through predominant inhibition of the cytochrome c-dependent apoptotic pathway. COX-2-mediated up-regulation of Bcl-2 suggests a potential mechanism for reduced apoptotic susceptibility.

Cyclooxygenase-2 expression in human breast cancers and adjacent ductal carcinoma in situ.

Cyclooxygenase-2 (COX-2) is an inducible enzyme that converts arachidonic acid to prostaglandins. Overexpression of the COX-2 gene in mammary glands of transgenic mice was sufficient to induce tumorigenesis. We analyzed COX-2 expression in human breast cancers (and breast cancer cell lines) and adjacent ductal carcinoma in situ (DCIS) as well as its association with HER2/neu and clinicopathological variables. Archival primary breast carcinomas (n = 57), adjacent DCIS (n = 14) and DCIS alone (n = 2) were analyzed for COX-2 and HER2 expression by immunohistochemistry using specific monoclonal antibodies. An immunohistochemical scoring system was used. HER2 gene amplification had been analyzed previously by fluorescence in situ hybridization (n = 20). Histology of carcinomas included infiltrating ductal (n = 44), lobular (n = 2), and other (n = 7). Frozen breast cancers and adjacent normal tissue pairs (n = 9) were analyzed for COX-2 mRNA by reverse transcription-PCR. COX-2 and HER2 expression were also analyzed in human breast cancer cell lines (MCF-7, MCF-7/HER2, SK-BR-3, and MDA-MB-231) by immunoblotting. Cytoplasmic COX-2 expression was detected at an intermediate or high level in epithelial cells in 18 of 42 (43%) invasive breast cancers and in 10 of 16 (63%) cases of DCIS. Normal-appearing breast epithelia adjacent to cancer expressed COX-2 in 81% of cases and was generally focal and of similar or decreased intensity relative to adjacent neoplastic epithelia. COX-2 mRNA was detected in all samples analyzed by reverse transcription-PCR and was increased in eight of nine breast cancers relative to paired normal tissue. In archival tumors, no significant correlation was found between COX-2 and HER2 expression/amplification and clinicopathological variables. COX-2 expression was induced in MCF-7 cells stably transfected with HER2, in contrast to parental MCF-7 cells, and was detected in MDA-MB-231, but not SK-BR-3 cells. COX-2 is frequently overexpressed in invasive breast cancers and in adjacent DCIS and, thus, may be an early event in mammary tumorigenesis. Forced HER2 expression in MCF-7 cells was shown to up-regulate COX-2, although no association was found in human tumors. Our results suggest that nonsteroidal anti-inflammatory drugs and selective COX-2 inhibitors may be useful in the chemoprevention and therapy of human breast cancer.

In situ hybridization assay of androgen receptor gene in hepatocarcinogenesis.

AIM:To determine the correlation between expression of androgen receptor (AR) gene and hepatocarcinogenesis.METHODS:Male SD rats were used as experimental animals and the animal model of experimental hepatocarcinoma was established by means of 3'-me-DAB administration. Androgen receptor mRNA was detected by a non-radioactive in situ hybridization assay in neoplastic and non-neoplastic liver tissues.RESULTS:The expression of androgen receptor mRNA was observed only in neoplastic cells and some atypical hyperplastic cells. In the liver tissue of control animal and the remaining normal liver cells adjacent to the carcinoma tissue, no positive signal was seen.CONCLUSION:Androgen has an important correlation with hepatocarcinogenesis and the expression of androgen receptor gene might be a mark event during hepatocarcinogenesis.