David M. Jablons

Randomized Phase II Trial Comparing Bevacizumab Plus Carboplatin and Paclitaxel With Carboplatin and Paclitaxel Alone in Previously Untreated Locally Advanced or Metastatic Non-Small-Cell Lung Cancer

PURPOSE To investigate the efficacy and safety of bevacizumab plus carboplatin and paclitaxel in patients with advanced or recurrent non-small-cell lung cancer. PATIENTS AND METHODS In a phase II trial, 99 patients were randomly assigned to bevacizumab 7.5 (n = 32) or 15 mg/kg (n = 35) plus carboplatin (area under the curve = 6) and paclitaxel (200 mg/m(2)) every 3 weeks or carboplatin and paclitaxel alone (n = 32). Primary efficacy end points were time to disease progression and best confirmed response rate. On disease progression, patients in the control arm had the option to receive single-agent bevacizumab 15 mg/kg every 3 weeks. RESULTS Compared with the control arm, treatment with carboplatin and paclitaxel plus bevacizumab (15 mg/kg) resulted in a higher response rate (31.5% v 18.8%), longer median time to progression (7.4 v 4.2 months) and a modest increase in survival (17.7 v 14.9 months). Of the 19 control patients that crossed over to single-agent bevacizumab, five experienced stable disease, and 1-year survival was 47%. Bleeding was the most prominent adverse event and was manifested in two distinct clinical patterns; minor mucocutaneous hemorrhage and major hemoptysis. Major hemoptysis was associated with squamous cell histology, tumor necrosis and cavitation, and disease location close to major blood vessels. CONCLUSION Bevacizumab in combination with carboplatin and paclitaxel improved overall response and time to progression in patients with advanced or recurrent non-small-cell lung cancer. Patients with nonsquamous cell histology appear to be a subpopulation with improved outcome and acceptable safety risks.

Activating Mutations in the Tyrosine Kinase Domain of the Epidermal Growth Factor Receptor Are Associated with Improved Survival in Gefitinib-Treated Chemorefractory Lung Adenocarcinomas

Purpose: Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) confer a strong sensitivity to gefitinib, a selective tyrosine kinase inhibitor of EGFR. Experimental Design: We examined EGFR mutations at exons 18, 19, and 21 in tumor tissue from 68 gefitinib-treated, chemorefractory, advanced non–small cell lung cancer patients from the United States, Europe, and Asia and in a highly gefitinib-sensitive non–small cell lung cancer cell line and correlated their presence with response and survival. In addition, in a subgroup of 28 patients for whom the remaining tumor tissue was available, we examined the relationship among EGFR mutations, CA repeats in intron 1 of EGFR, EGFR and caveolin-1 mRNA levels, and increased EGFR gene copy numbers. Results: Seventeen patients had EGFR mutations, all of which were in lung adenocarcinomas. Radiographic response was observed in 16 of 17 (94.1%) patients harboring EGFR mutations, in contrast with 6 of 51 (12.6%) with wild-type EGFR (P < 0.0001). Probability of response increased significantly in never smokers, patients receiving a greater number of prior chemotherapy regimens, Asians, and younger patients. Median survival was not reached for patients with EGFR mutations and was 9.9 months for those with wild-type EGFR (P = 0.001). EGFR mutations tended to be associated with increased numbers of CA repeats and increased EGFR gene copy numbers but not with EGFR and caveolin-1 mRNA overexpression (P = not significant). Conclusions: The presence of EGFR mutations is a major determinant of gefitinib response, and targeting EGFR should be considered in preference to chemotherapy as first-line treatment in lung adenocarcinomas that have demonstrable EGFR mutations.

SOCS-3 is frequently silenced by hypermethylation and suppresses cell growth in human lung cancer

Lung cancer is the leading cause of cancer death in the world, but the molecular mechanisms for its development have not been well characterized. The suppressors of cytokine signaling (SOCS) are inhibitors of cytokine signaling that function via the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. Eight SOCS proteins with similar structures have been identified so far. SOCS family members, however, have distinct mechanisms of inhibition of JAK/STAT signaling. Abnormalities of the JAK/STAT pathway are associated with cancer. Inhibition of signaling results in growth suppression in various cell types. Recently, the involvement of SOCS-1 in carcinogenesis has been reported. Here, we report identification of frequent hypermethylation in CpG islands of the functional SOCS-3 promoter that correlates with its transcription silencing in cell lines (lung cancer, breast cancer, and mesothelioma) and primary lung cancer tissue samples. Restoration of SOCS-3 in lung cancer cells where SOCS-3 was methylation-silenced resulted in the down-regulation of active STAT3, induction of apoptosis, and growth suppression. Our results suggest that methylation silencing of SOCS-3 is one of the important mechanisms of constitutive activation of the JAK/STAT pathway in cancer pathogenesis. The data also suggest that SOCS-3 therapy may be useful in the treatment of cancer.

Activation of the Wnt pathway in non small cell lung cancer: evidence of dishevelled overexpression

Non small cell lung cancer (NSCLC) is the leading cause of cancer deaths in the United States and worldwide. Unfortunately, standard therapies remain inadequate. An increased understanding of the molecular biology of lung cancer biology is required to develop more effective new therapies. In this report, we show that the Wnt pathway is activated through Dishevelled (Dvl) overexpression in NSCLC. Analysis of freshly resected tumors and lung cancer cell lines demonstrate that Dvl-3, a critical mediator of Wnt signaling, is overexpressed. Specifically, Dvl-3 was overexpressed significantly in 75% of fresh NSCLC microdissected samples compared to control paired matched normal lung samples. To evaluate the biological significance of Wnt signaling and, in particular, Dvl function in lung cancer, we transfected siRNA (designed to inhibit selectively human Dvl-1, -2, and -3), to the NSCLC cell line H1703, which is known to have β-catenin-mediated Tcf-dependent transcriptional activity. Here, we demonstrate that Dvl-specific siRNA treatment in H1703 decreases significantly Dvl and β-catenin expression, resulting in reduction of Tcf-dependent transcriptional activity, and, importantly, growth inhibition. Taken together, these data support the novel hypothesis that Dvl overexpression is critical to Wnt signaling activation and cell growth in NSCLC.

Wnt Inhibitory Factor-1 Is Silenced by Promoter Hypermethylation in Human Lung Cancer

Aberrant activation of the Wingless-type (Wnt) signaling pathway is associated with a variety of human cancers, and we recently reported the importance of aberrant Wnt signaling in lung cancer. On the other hand, inhibition of Wnt signaling suppresses growth in numerous cell types. Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist that can bind Wnt in the extracellular space and inhibit Wnt signaling. Recently, down-regulation of WIF-1 has been reported in several human cancers. To discover the mechanism of WIF-1 silencing in lung cancer, we first identified the human WIF-1 promoter and subsequently examined the methylation status in the CpG islands. By using methylation-specific PCR and sequence analysis after bisulfite treatment, we demonstrate here frequent CpG island hypermethylation in the functional WIF-1 promoter region. This hypermethylation correlates with its transcriptional silencing in human lung cancer cell lines. Moreover, treatment with 5-aza-2′-deoxycytidine restores WIF-1 expression. We then studied WIF-1 expression in 18 freshly resected lung cancers, and we show a down-regulation in 15 of them (83%). This silencing also correlates with WIF-1 promoter methylation. Our results suggest that methylation silencing of WIF-1 is a common and likely important mechanism of aberrant activation of the Wnt signaling pathway in lung cancer pathogenesis, raising its therapeutic interest.

Inhibition of Wnt-2-mediated signaling induces programmed cell death in non-small-cell lung cancer cells

In this report, we have demonstrated that Wnt-2 protein is overexpressed in freshly resected human non-small-cell lung cancer (NSCLC) tissues. We have also developed a monoclonal antibody against the N-terminus of human Wnt-2 protein. This monoclonal antibody induces apoptosis in human NSCLC cell lines that overexpress Wnt-2 protein. Incubation of this antibody with normal human airway cells lacking Wnt-2 expression does not induce apoptosis. Wnt-2 signaling blockade by the anti-Wnt-2 antibody is confirmed by downregulation of cytosolic β-catenin and reduction in TCF-dependent transcriptional activity (TOPFLASH assay). In addition, Wnt-2-specific small interfering RNA (siRNA) treatment in the NSCLC cell line A549 also downregulated cytosolic β-catenin and induced apoptosis. Moreover, downregulation of an inhibitor of apoptosis family protein, Survivin, was noticed both in the Wnt-2 antibody- and siRNA-treated NSCLC cells, suggesting that inhibition of Wnt-2-mediated signaling induces apoptosis through inactivating Survinin.

A Monoclonal Antibody against Wnt-1 Induces Apoptosis in Human Cancer Cells

Aberrant activation of the Wingless-type (Wnt)/beta-catenin signaling pathway is associated with a variety of human cancers. Little is known regarding the role that Wnt ligands play in human carcinogenesis. To test whether a Wnt-1 signal is a survival factor in human cancer cells and thus may serve as a potential cancer therapeutic target, we investigated the effect of inhibition of Wnt-1 signaling in a variety of human cancer cell lines, including non small cell lung cancer, breast cancer, mesothelioma, and sarcoma. Both monoclonal antibody and RNA interference (RNAi) were used to inhibit Wnt-1 signaling. We found that incubation of a monoclonal anti-Wnt-1 antibody induced apoptosis and caused downstream protein changes in cancer cells overexpressing Wnt-1. In contrast, apoptosis was not detected in cells lacking or having minimal Wnt-1 expression after the antibody incubation. RNAi targeting of Wnt-1 in cancer cells overexpressing Wnt-1 demonstrated similar downstream protein changes and induction of apoptosis. The antibody also suppressed tumor growth in vivo. Our results indicate that both monoclonal anti-Wnt-1 antibody and Wnt-1 siRNA inhibit Wnt-1 signaling and can induce apoptosis in human cancer cells. These findings hold promise as a novel therapeutic strategy for cancer.

NASOPHARYNGEAL CARCINOMA—REVIEW OF THE MOLECULAR MECHANISMS OF TUMORIGENESIS

Nasopharyngeal carcinoma (NPC) is a head and neck cancer rare throughout most of the world but common in certain geographic areas, such as southern Asia. While environmental factors and genetic susceptibility play important roles in NPC pathogenesis, the Epstein–Barr virus in particular has been implicated in the molecular abnormalities leading to NPC. There is upregulation of cellular proliferation pathways such as the Akt pathway, mitogen‐activated protein kinases, and the Wnt pathway. Cell adhesion is compromised due to abnormal E‐cadherin and β‐catenin function. Aberrations in cell cycle are due to dysregulation of factors such as p16, cyclin D1, and cyclin E. Anti‐apoptotic mechanisms are also upregulated. There are multiple abnormalities unique to NPC that are potential targets for novel treatments.

A practical molecular assay to predict survival in resected non-squamous, non-small-cell lung cancer: development and international validation studies.

BACKGROUND The frequent recurrence of early-stage non-small-cell lung cancer (NSCLC) is generally attributable to metastatic disease undetected at complete resection. Management of such patients depends on prognostic staging to identify the individuals most likely to have occult disease. We aimed to develop and validate a practical, reliable assay that improves risk stratification compared with conventional staging. METHODS A 14-gene expression assay that uses quantitative PCR, runs on formalin-fixed paraffin-embedded tissue samples, and differentiates patients with heterogeneous statistical prognoses was developed in a cohort of 361 patients with non-squamous NSCLC resected at the University of California, San Francisco. The assay was then independently validated by the Kaiser Permanente Division of Research in a masked cohort of 433 patients with stage I non-squamous NSCLC resected at Kaiser Permanente Northern California hospitals, and on a cohort of 1006 patients with stage I-III non-squamous NSCLC resected in several leading Chinese cancer centres that are part of the China Clinical Trials Consortium (CCTC). FINDINGS Kaplan-Meier analysis of the Kaiser validation cohort showed 5 year overall survival of 71·4% (95% CI 60·5-80·0) in low-risk, 58·3% (48·9-66·6) in intermediate-risk, and 49·2% (42·2-55·8) in high-risk patients (p(trend)=0·0003). Similar analysis of the CCTC cohort indicated 5 year overall survivals of 74·1% (66·0-80·6) in low-risk, 57·4% (48·3-65·5) in intermediate-risk, and 44·6% (40·2-48·9) in high-risk patients (p(trend)<0·0001). Multivariate analysis in both cohorts indicated that no standard clinical risk factors could account for, or provide, the prognostic information derived from tumour gene expression. The assay improved prognostic accuracy beyond National Comprehensive Cancer Network criteria for stage I high-risk tumours (p<0·0001), and differentiated low-risk, intermediate-risk, and high-risk patients within all disease stages. INTERPRETATION Our practical, quantitative-PCR-based assay reliably identified patients with early-stage non-squamous NSCLC at high risk for mortality after surgical resection. FUNDING UCSF Thoracic Oncology Laboratory and Pinpoint Genomics.

An Anti-Wnt-2 Monoclonal Antibody Induces Apoptosis in Malignant Melanoma Cells and Inhibits Tumor Growth

Activation of the Wnt/β-catenin signaling pathway has been associated with human cancers. To test whether Wnt-2 signal is a survival factor in human melanoma cells and thus represents a potential therapeutic target, we investigated the effects of inhibition of Wnt-2 signaling in human melanoma cell lines. We have developed a novel monoclonal antibody against the NH2 terminus of the human Wnt-2 ligand that induces apoptosis in human melanoma cells overexpressing Wnt-2. Whereas incubation of this antibody with normal cells lacking Wnt-2 expression does not induce apoptosis, Wnt-2 signaling blockade by the ligand-binding antibody is confirmed by down-regulation of Dishevelled and β-catenin. Wnt-2 small interfering RNA treatment in these cells yielded similar apoptotic effects and downstream changes. Down-regulation of an inhibitor of apoptosis family protein, survivin, was observed in both the Wnt-2 antibody-treated and small interfering RNA-treated melanoma cell lines, suggesting that the antibody induces apoptosis by inactivating survivin. In an in vivo study, this monoclonal anti-Wnt-2 antibody suppresses tumor growth in a xenograft model. These findings suggest that the anti-Wnt-2 monoclonal antibody may be useful for the treatment of patients with malignant melanoma.