Maintenance and consolidation therapy in patients with unresectable stage III/IV non-small cell lung cancer.

Globally, lung cancer is the leading cause of cancer-related mortality. Current chemotherapy combinations for the first-line treatment of advanced disease (stage IIIB with malignant pleural effusion/stage IV) and chemoradiotherapy regimens for the treatment of unresectable locally advanced disease (stage IIIA and IIIB without malignant pleural effusion) appear to have reached an efficacy plateau. The addition of new compounds including targeted agents to standard first-line cytotoxic doublets, administered concurrently and/or as maintenance therapy in patients who have not experienced disease progression after such treatment, has been shown to improve efficacy beyond this plateau in patients with advanced disease. However, to date, such approaches have been less successful in the treatment of patients with unresectable locally advanced stage III disease. The purpose of this review is to summarize the data from recent randomized phase III studies involving agents administered as maintenance or consolidation therapy in the treatment of unresectable stage III/IV non-small cell lung cancer (NSCLC). A possible alternative approach to the use of cytotoxic or molecularly targeted agents in this setting is the administration of therapeutic anticancer vaccines, which are designed to stimulate a host immunological response against the tumor. Current data in relation to the potential of vaccine therapy for NSCLC are therefore also reviewed, with a particular focus on belagenpumatucel-L and L-BLP25 vaccines, which are currently undergoing phase III evaluation as maintenance therapies in patients with unresectable stage III/IV NSCLC who have tumor control following first-line therapy.

Aurora kinases as anticancer drug targets.

The human aurora family of serine-threonine kinases comprises three members, which act in concert with many other proteins to control chromosome assembly and segregation during mitosis. Aurora dysfunction can cause aneuploidy, mitotic arrest, and cell death. Aurora kinases are strongly expressed in a broad range of cancer types. Aurora A expression in tumors is often associated with gene amplification, genetic instability, poor histologic differentiation, and poor prognosis. Aurora B is frequently expressed at high levels in a variety of tumors, often coincidently with aurora A, and expression level has also been associated with increased genetic instability and clinical outcome. Further, aurora kinase gene polymorphisms are associated with increased risk or early onset of cancer. The expression of aurora C in cancer is less well studied. In recent years, several small-molecule aurora kinase inhibitors have been developed that exhibit preclinical activity against a wide range of solid tumors. Preliminary clinical data from phase I trials have largely been consistent with cytostatic effects, with disease stabilization as the best response achieved in solid tumors. Objective responses have been noted in leukemia patients, although this might conceivably be due to inhibition of the Abl kinase. Current challenges include the optimization of drug administration, the identification of potential biomarkers of tumor sensitivity, and combination studies with cytotoxic drugs. Here, we summarize the most recent preclinical and clinical data and discuss new directions in the development of aurora kinase inhibitors as antineoplastic agents.

Cyclin D1 in non-small cell lung cancer: a key driver of malignant transformation.

PURPOSE: To review the evidence implicating the deregulation of cyclin D1 in the pathogenesis of non-small cell lung cancer (NSCLC), and to discuss the opportunities for targeted clinical intervention. METHODS: Data published until June 2006 are summarized, and previously unpublished results from our own research are included. RESULTS: In normal cells, cyclin D1 complexes with and activates cyclin-dependent kinases (CDK) and acts as a transcriptional regulator. The protein is frequently overexpressed in a wide range of cancers, sometimes coincident with CCND1 (cyclin D1) gene amplification (5-20% of tumours). A low level of somatic mutations have been seen in certain tumours. CCND1 is amplified in NSCLC and cyclin D1 is frequently overexpressed in tumours and pre-invasive bronchial lesions, generally from one parental allele. Mutation analyses revealed a frequent CCND1 gene polymorphism (A870G) that modulates alternative splicing and allows expression of an alternative cyclin D1 transcript (transcript cyclin D1b). The encoded cyclin D1b protein lacks a specific phosphorylation site required for nuclear export. Genotype has been correlated with the risk and/or severity of disease or drug response across a range of malignancies, including lung cancer. Together, these findings suggest a strong pathological role for cyclin D1 deregulation in bronchial neoplasia. CONCLUSION: Current data indicate that cyclin D1 overexpression is not a consequence of, but rather a pivotal element in the process of malignant transformation in the lung and other tissues. This understanding may open new avenues for lung cancer diagnosis, treatment and prevention.

Nanospan, an alternatively spliced isoform of sarcospan, localizes to the sarcoplasmic reticulum in skeletal muscle and is absent in limb girdle muscular dystrophy 2F.

BACKGROUND: Sarcospan (SSPN) is a transmembrane protein that interacts with the sarcoglycans (SGs) to form a tight subcomplex within the dystrophin-glycoprotein complex that spans the sarcolemma and interacts with laminin in the extracellular matrix. Overexpression of SSPN ameliorates Duchenne muscular dystrophy in murine models. METHODS: Standard cloning approaches were used to identify nanospan, and nanospan-specific polyclonal antibodies were generated and validated. Biochemical isolation of skeletal muscle membranes and two-photon laser scanning microscopy were used to analyze nanospan localization in muscle from multiple murine models. Duchenne muscular dystrophy biopsies were analyzed by immunoblot analysis of protein lysates as well as indirect immunofluorescence analysis of muscle cryosections. RESULTS: Nanospan is an alternatively spliced isoform of sarcospan. While SSPN has four transmembrane domains and is a core component of the sarcolemmal dystrophin-glycoprotein complex, nanospan is a type II transmembrane protein that does not associate with the dystrophin-glycoprotein complex. We demonstrate that nanospan is enriched in the sarcoplasmic reticulum (SR) fractions and is not present in the T-tubules. SR fractions contain membranes from three distinct structural regions: a region flanking the T-tubules (triadic SR), a SR region across the Z-line (ZSR), and a longitudinal SR region across the M-line (LSR). Analysis of isolated murine muscles reveals that nanospan is mostly associated with the ZSR and triadic SR, and only minimally with the LSR. Furthermore, nanospan is absent from the SR of δ-SG-null (Sgcd-/-) skeletal muscle, a murine model for limb girdle muscular dystrophy 2F. Analysis of skeletal muscle biopsies from Duchenne muscular dystrophy patients reveals that nanospan is preferentially expressed in type I (slow) fibers in both control and Duchenne samples. Furthermore, nanospan is significantly reduced in Duchenne biopsies. CONCLUSIONS: Alternative splicing of proteins from the SG-SSPN complex produces δ-SG3, microspan, and nanospan that localize to the ZSR and the triadic SR, where they may play a role in regulating resting calcium levels as supported by previous studies (Estrada et al., Biochem Biophys Res Commun 340:865-71, 2006). Thus, alternative splicing of SSPN mRNA generates three protein isoforms (SSPN, microspan, and nanospan) that differ in the number of transmembrane domains affecting subcellular membrane association into distinct protein complexes.

Cyclin D1 (CCND1) A870G gene polymorphism modulates smoking-induced lung cancer risk and response to platinum-based chemotherapy in non-small cell lung cancer (NSCLC) patients.

PURPOSE: The cyclin D1 (CCND1) A870G gene polymorphism is linked to the outcome in patients with resectable non-small cell lung cancer (NSCLC). Here, we investigated the impact of this polymorphism on smoking-induced cancer risk and clinical outcome in patients with NSCLC stages I-IV. METHODS: CCND1 A870G genotype was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis (RFLP) of DNA extracted from blood. The study included 244 NSCLC patients and 187 healthy control subjects. RESULTS: Patient characteristics were: 70% male, 77% smokers, 43% adenocarcinoma, and 27% squamous cell carcinoma. Eighty-one percent of the patients had stages III-IV disease. Median age at diagnosis was 60 years and median survival was 13 months. Genotype frequencies of patients and controls both conformed to the Hardy Weinberg equilibrium. The GG genotype significantly correlated with a history of heavy smoking (>or=40 py, P=0.02), and patients with this genotype had a significantly higher cigarette consumption than patients with AA/AG genotypes (P=0.007). The GG genotype also significantly correlated with tumor response or stabilization after a platinum-based first-line chemotherapy (P=0.04). Survival analysis revealed no significant differences among the genotypes. CONCLUSION: Evidence was obtained that the CCND1 A870G gene polymorphism modulates smoking-induced lung cancer risk. Further studies are required to explore the underlying molecular mechanisms and to test the value of this gene polymorphism as a predictor for platinum-sensitivity in NSCLC patients.

Maspin - the most commonly-expressed gene of the 18q21.3 serpin cluster in lung cancer - is strongly expressed in preneoplastic bronchial lesions.

Maspin, SCCA1/2 and hurpin were identified by cDNA microarray analyses as dramatically differentially expressed transcripts in primary non-small cell lung cancer (NSCLC). These sequences are located within a 10-gene serpin cluster on 18q21.3. Using comparative RT-PCR, we have investigated the expression of each of these serpins, including their flanking loci, in an independent NSCLC series. Whereas six of the genes (maspin, SCCA1, SCCA2, hurpin, megsin and pAI-2) were commonly differentially expressed in primary lesions, each significantly more often in squamous cell tumours, maspin was identified as the most frequently over-represented sequence in both squamous cell carcinoma and adenocarcinoma. Using a well-characterized monoclonal antibody, we have shown strong maspin expression in tumour protein extracts, detected multiple isoforms of the 42 kDa protein and shown that maspin is localized specifically to the tumour cells within neoplastic lesions. In contrast, most cells in non-neoplastic lung tissue appear not to express the gene, with the exception of the multipotent basal epithelial cells that line the bronchial airway. These reserve cells generally show strong predominantly nuclear localization of maspin. Strong nuclear expression of maspin within primary tumour cells is correlated with increased survival (P=0.05) and a longer remission duration (P=0.02) in resectable-staged patients. However, within the airways of cancer patients and somewhat in contrast to this observation, such expression was more frequently detected in the superficial cells of preneoplastic over non-neoplastic epithelia (P<0.0001), consistent with a role for the protein in early neoplasia.

Expression profiling of primary non-small cell lung cancer for target identification.

Using a panel of cDNA microarrays comprising 47 650 transcript elements, we have carried out a dual-channel analysis of gene expression in 39 resected primary human non-small cell lung tumours versus normal lung tissue. Whilst approximately 11 000 elements were scored as differentially expressed at least twofold in at least one sample, 96 transcripts were scored as over-represented fourfold or more in at least seven out of 39 tumours and 30 sequences 16-fold in at least two out of 39 tumours, including 24 transcripts in common. Transcripts (178) were found under-represented fourfold in at least seven out of 39 tumours, 31 of which are under-represented 16-fold in at least two out of 39 lesions. The relative expression levels of representative genes from these lists were analysed by comparative multiplex RT-PCR and found to be broadly consistent with the microarray data. Two dramatically over-represented genes, previously designated as potential tumour suppressors in breast (maspin) and lung and breast (S100A2) cancers, were analysed more extensively and demonstrate the effectiveness of this approach in identifying potential lung cancer diagnostic or therapeutic targets. Whilst it has been reported that S100A2 is downregulated in NSCLC at an early stage, our microarray, cmRT-PCR, Western and immunohistochemistry data indicate that it is strongly expressed in the majority of tumours.

FOLFOX4 with cetuximab vs. UFOX with cetuximab as first-line therapy in metastatic colorectal cancer: The randomized phase II FUTURE study.

INTRODUCTION: The purpose of this study was to assess the efficacy and safety of FOLFOX4, comprising infusional 5-fluorouracil (5-FU)/leucovorin (LV) and oxaliplatin, with cetuximab compared with UFOX, comprising UFT, an oral prodrug of 5-FU, LV, and oxaliplatin, with cetuximab as first-line treatment for mCRC. PATIENTS AND METHODS: Patients, unselected by tumor KRAS status, were randomized 1:1 to FOLFOX4 with cetuximab or UFOX with cetuximab. Treatment was continued until disease progression or unacceptable toxicity. The primary end point, assessed in the intention-to-treat population, was progression-free survival (PFS). Secondary end points included tumor response, overall survival, and safety. Outcome according to KRAS mutation status was investigated. RESULTS: Recruitment was curtailed at 302 patients after reporting of the importance of tumor KRAS mutation status for cetuximab activity. Baseline characteristics were balanced between treatment groups. PFS was significantly longer in the FOLFOX4 with cetuximab group compared with UFOX with cetuximab group (median 8.2 vs. 6.6 months; hazard ratio, 0.68; 95% confidence interval [CI], 0.52-0.89; P = .0048). The response rate was also significantly greater in the FOLFOX4 with cetuximab group (51.3% vs. 37.5%, respectively; odds ratio, 1.76; 95% CI, 1.11-2.78; P = .0160), although overall survival was comparable. In the KRAS wild type subgroup, efficacy outcomes were similar to those in the intention-to-treat population. Side effect profiles were manageable and consistent with expectations. CONCLUSION: In the first-line treatment of mCRC, UFOX with cetuximab had an acceptable safety profile but inferior activity compared with FOLFOX4 with cetuximab in relation to PFS and response. The regimens were comparable with regard to overall survival.

Meta-analysis of individual patient data from randomized trials of chemotherapy plus cetuximab as first-line treatment for advanced non-small cell lung cancer.

OBJECTIVES: Four randomized phase II/III trials investigated the addition of cetuximab to platinum-based, first-line chemotherapy in patients with advanced non-small cell lung cancer (NSCLC). A meta-analysis was performed to examine the benefit/risk ratio for the addition of cetuximab to chemotherapy. MATERIALS AND METHODS: The meta-analysis included individual patient efficacy data from 2018 patients and individual patient safety data from 1970 patients comprising respectively the combined intention-to-treat and safety populations of the four trials. The effect of adding cetuximab to chemotherapy was measured by hazard ratios (HRs) obtained using a Cox proportional hazards model and odds ratios calculated by logistic regression. Survival rates at 1 year were calculated. All applied models were stratified by trial. Tests on heterogeneity of treatment effects across the trials and sensitivity analyses were performed for all endpoints. RESULTS: The meta-analysis demonstrated that the addition of cetuximab to chemotherapy significantly improved overall survival (HR 0.88, p=0.009, median 10.3 vs 9.4 months), progression-free survival (HR 0.90, p=0.045, median 4.7 vs 4.5 months) and response (odds ratio 1.46, p<0.001, overall response rate 32.2% vs 24.4%) compared with chemotherapy alone. The safety profile of chemotherapy plus cetuximab in the meta-analysis population was confirmed as manageable. Neither trials nor patient subgroups defined by key baseline characteristics showed significant heterogeneity for any endpoint. CONCLUSION: The addition of cetuximab to platinum-based, first-line chemotherapy for advanced NSCLC significantly improved outcome for all efficacy endpoints with an acceptable safety profile, indicating a favorable benefit/risk ratio.

ERCC1 mRNA expression is not associated with response and survival after platinum-based chemotherapy regimens in advanced non-small cell lung cancer.

BACKGROUND: Platinum-based therapy is pivotal to the treatment of advanced non-small cell lung Cancer (NSCLC). Excision repair cross-complementation group 1 (ERCC1) is a key component of the platinum-DNA repair machinery responsible for nucleotide excision repair. We sought to determine the influence of ERCC1 mRNA expression in advanced NSCLC on chemotherapy response, toxicity, and survival after platinum-based chemotherapy. METHODS: Patients randomized to a phase III trial of platinum-based chemotherapy were eligible for inclusion. Formalin-fixed paraffin-embedded tumor biopsies were retrieved for mRNA extraction and purification before quantitative real-time polymerase chain reaction analysis using Taqman technology. Expression data were correlated with treatment response, toxicity, and overall survival. RESULTS: Sixty-six patients were enrolled. No statistically significant relationship existed between ERCC1 mRNA expression and response to chemotherapy (p = 0.794) or hematological toxicity. No statistically significant difference in median survival was demonstrated according to ERCC1 expression (high expression, 415 days, 95% confidence interval [95%CI]: 197-633 days; low expression, 327 days [95%CI: 211-433 days]; p = 0.801). High ERCC1 mRNA expression was associated with a hazard ratio for death of 0.96 (95% CI 0.919-1.004; p = 0.08). CONCLUSION: In contrast to recent publications, ERCC1 mRNA expression in our study did not favor a prognostically better outcome after platinum-based chemotherapy in advanced NSCLC. We explore potential reasons for this, including the need for cautious interpretation of mRNA expression data from archival materials and highlight the need for additional translational research linking gene expression with a promising ERCC1 polymorphism.

Glutathione-S-transferase P1 isoenzyme polymorphisms, platinum-based chemotherapy, and non-small cell lung cancer.

BACKGROUND: Polymorphisms within the P1 isoenzyme of GST (GSTP1) are associated with alterations in enzyme activity and may change sensitivity to platinum-based chemotherapy. We investigated the relationship between exon 5 and exon 6 GSTP1 gene polymorphisms and treatment response, hematological, and nonhematological toxicity and overall survival for patients receiving platinum-based chemotherapy for advanced non-small cell lung cancer (NSCLC). METHODS: Between 2001 and 2002, 108 patients with chemotherapy-naïve advanced NSCLC were recruited. Associations between the GSTP1 polymorphisms (Ile105Val, Thr110Ser, Ala114Val, and Asp 147Tyr) and GSTP1*A, *B, and *C haplotypes and treatment response and toxicity were evaluated using the Pearson chi and Kruskal-Wallis tests, respectively. Associations with survival were compared using Kaplan-Meier survival curves and Cox proportional hazard ratios. RESULTS: No significant associations were noted between GSTP1 polymorphisms and treatment response or survival. Significantly less neutropenic toxicity was demonstrated for patients possessing the 105Val allele (p = 0.020) or the GSTP1*B haplotype (p = 0.038). However, the variant allele GSTP1 105Val, and patients possessing a GSTP1*B allele demonstrated notable trends toward inferior response and survival. CONCLUSIONS: GSTP1 haplotype can be used to stratify hematological toxicity after platinum-based chemotherapy, but the lack of significant associations with response or survival suggests that GSTP1 polymorphisms may not be strong pharmacogenomic markers in this population. Additional large prospective studies incorporating the GSTP1 haplotype may clarify the reported discrepancies.

Xeroderma pigmentosum group D haplotype predicts for response, survival, and toxicity after platinum-based chemotherapy in advanced nonsmall cell lung cancer.

BACKGROUND: The treatment of lung cancer has reached a therapeutic plateau. Several mechanisms of platinum resistance have been described, including the removal of platinum-DNA adduct by nucleotide excision repair (NER). Polymorphisms within the Xeroderma pigmentosum Group D protein (XPD), a member of the NER pathway, are associated with alterations in enzyme activity and may change sensitivity to platinum-based chemotherapy. The authors investigated the relation between XPD polymorphisms and treatment response, toxicity, and overall survival in patients who received platinum-based chemotherapy for advanced nonsmall cell lung cancer (NSCLC). METHODS: Between 2001 and 2002, 108 patients with chemotherapy-naive, advanced NSCLC were recruited. Associations between XPD312/751 polymorphisms and XPD haplotype and treatment response, toxicity. and survival were evaluated. RESULTS: Significant correlations were observed between XPD haplotype and Grade 4 neutropenia and overall survival together with a greater response to platinum-based chemotherapy for the XPD *A haplotype. CONCLUSIONS: The XPD haplotype may represent a useful pharmacogenomic marker of platinum-based chemotherapy in patients with advanced NSCLC and requires prospective validation.

Over-expression of Microspan, a novel component of the sarcoplasmic reticulum, causes severe muscle pathology with triad abnormalities.

Sarcospan (SSPN) is a core component of the dystrophin-glycoprotein complex (DGC). Multiple SSPN transcripts are ubiquitously expressed and SSPN splicing is disrupted in many lung tumors, suggesting the importance of SSPN-related mRNAs. We describe the isolation of an alternatively spliced isoform of SSPN, which we designate 'microspan' based on its small size relative to SSPN. Microspan has two transmembrane domains and a novel C-terminus. We demonstrate that microspan is not an integral component of the DGC and is not perturbed by the loss of dystrophin. Microspan protein is detected at the sarcoplasmic reticulum (SR) using indirect immunofluorescence and immunoelectron microscopy. Furthermore, microspan purifies with skeletal muscle SR membranes and not transverse tubules. Mice engineered to over-express microspan display severe kyphosis and die at approximately 8 weeks of age. Levels of ryanodine receptor, dihydropyridine receptor, and SERCA-1 are greatly reduced in microspan transgenic muscle. Furthermore, electron microscopy reveals that microspan over-expression causes a dramatic perturbation in triad structure. Our findings suggest that microspan is an important component of the SR and may contribute to excitation-contraction coupling.

Induced expression of human CCND1 alternative transcripts in mouse Cyl-1 knockout fibroblasts highlights functional differences.

Splicing of human cyclin D1 (CCND1) mRNA producing transcripts a and b is modulated by a common polymorphism (A --> G) located in a conserved splice donor region at nucleotide 870. CCND1 A/G(870) genotype is associated with tumour progression and clinical outcome in a variety of cancers. Although in vitro expression of cyclin D1 transcript a (CCND1(tra)) has been widely investigated, few studies have examined the expression of CCND1 transcript b (CCND1(trb)). We have studied the effects of inducible expression of human CCND1(trb) in comparison with human CCND1(tra) in a mouse fibroblast knock-out for cyclin D1 (MEF(Cyl-1-/-)). Inducible expression was in stable clones isolated from MEF(Cyl-1-/-) transfectants. Induction of CCND1(tra) produced a 36-kDa protein, which led to a significant increase in the proportion of cells in S-phase, as detected by BrdU incorporation after 32 hr, compared to non-induced cells (p = 0.012). Clones induced to express CCND1(tra) exhibited a significantly increased ability to grow in serum depleted (2% FCS) medium compared to non-induced clones (p = 0.0004). Induced expression of CCND1(trb) in MEF(Cyl-1-/-) transfectants produced a 31-kDa protein and resulted in no significant difference in DNA synthesis, neither did the cells acquire the ability to grow in serum-depleted conditions compared to non-induced cells. Induction of CCND1(trb) significantly enhanced the ability of MEF(Cyl-1-/-) transfectants to form colonies in soft agar, (average 30-fold increase) compared to non-induced clones or those induced to express CCND1(tra). Our data supports the emerging view that CCND1 alternate transcripts encode proteins with differing independent biological functions. We suggest that CCND1(tra) encodes a protein involved in regulating mitogen responsive, anchorage-dependent G(1) progression, whereas CCND1(trb) modulates the ability of the cell to grow in an anchorage-independent manner.

Quantitative trait locus analysis reveals two intragenic sites that influence O6-alkylguanine-DNA alkyltransferase activity in peripheral blood mononuclear cells.

The repair of specific types of DNA alkylation damage by O6-alkylguanine-DNA alkyltransferase (MGMT) is a major mechanism of resistance to the carcinogenic and chemotherapeutic effects of certain alkylating agents. MGMT expression levels vary widely between individuals but the underlying causes of this variability are not known. To address this, we used an expressed single nucleotide polymorphism (SNP) and demonstrated that the MGMT alleles are frequently expressed at different levels in peripheral blood mononuclear cells (PBMC). This suggests that there is a genetic component of inter-allelic variation of MGMT levels that maps close to or within the MGMT locus. We then used quantitative trait locus (QTL) analysis using intragenic SNPs and found that there are at least two sites influencing inter-individual variation in PBMC MGMT activity. One is characterized by an SNP at the 3' end of the first intron and the second by two SNPs in the last exon. The latter are in perfect disequilibrium and both result in amino acid substitutions-one of them, Ile143Val, affecting an amino acid close to the Cys145 residue at the active site of MGMT. Using in vitro assays, we further showed that while the Val143 variant did not affect the activity of the protein on methylated DNA substrate, it was more resistant to inactivation by the MGMT pseudosubstrate, O6-(4-bromothenyl)guanine. These findings suggest that further investigations of the potential epidemiological and clinical significance of inherited differences in MGMT expression and activity are warranted.