Search

PDF file - 22K, Changes in shed Met do not correlate with changes in cHGF over the duration of the first cycle of onartuzumab administration in the Phase 1A study.

PDF file - 24K, cHGF levels are stable over time and are not variable between screening (any time during visit -24 hours) and predose (Day 0).

The objective of this study was to evaluate circulating hepatocyte growth factor (cHGF) as a pharmacodynamic biomarker of Met inhibition for onartuzumab (MetMAb, OA5D5v2) in a phase I trial in patients with advanced cancers and a phase II trial in non–small cell lung cancer (NSCLC). The phase I study was a dose escalation trial with onartuzumab administered i.v. once every three weeks. The phase II study was a randomized two-arm trial in which onartuzumab or placebo was administered in combination with erlotinib in 137 patients with second and third line (2/3L) NSCLC. cHGF levels were evaluated by ELISA at multiple time points over the treatment period. Onartuzumab administration resulted in an acute and sustained rise in cHGF in both the phase I and phase II studies. Elevation in cHGF was independent of dose or drug exposure and was restricted to onartuzumab treatment. Neither higher baseline nor elevated change in cHGF levels upon treatment could simply be attributed to tumor burden or number of liver metastasis. We have shown that elevated cHGF can consistently and reproducibly be measured as a pharmacodynamic biomarker of onartuzumab activity. The elevation in cHGF is independent of tumor type, dose administered, or dose duration. Although these studies were not powered to directly address the contribution of cHGF as a predictive, on-treatment, circulating biomarker, these data suggest that measurement of cHGF in future expanded studies is warranted. Mol Cancer Ther; 12(6); 1122–30. ©2013 AACR.

The RAS/RAF/MEK pathway is activated in more than 30% of human cancers, most commonly via mutation in the K-ras oncogene and also via mutations in BRAF. Several allosteric mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitors, aimed at treating tumors with RAS/RAF pathway alterations, are in clinical development. However, acquired resistance to these inhibitors has been documented both in preclinical and clinical samples. To identify strategies to overcome this resistance, we have derived three independent MEK inhibitor–resistant cell lines. Resistance to allosteric MEK inhibitors in these cell lines was consistently linked to acquired mutations in the allosteric binding pocket of MEK. In one cell line, concurrent amplification of mutant K-ras was observed in conjunction with MEK allosteric pocket mutations. Clonal analysis showed that both resistance mechanisms occur in the same cell and contribute to enhanced resistance. Importantly, in all cases the MEK-resistant cell lines retained their addiction to the mitogen-activated protein kinase (MAPK) pathway, as evidenced by their sensitivity to a selective inhibitor of the ERK1/2 kinases. These data suggest that tumors with acquired MEK inhibitor resistance remain dependent on the MAPK pathway and are therefore sensitive to inhibitors that act downstream of the mutated MEK target. Importantly, we show that dual inhibition of MEK and ERK by small molecule inhibitors was synergistic and acted to both inhibit the emergence of resistance, as well as to overcome acquired resistance to MEK inhibitors. Therefore, our data provide a rationale for cotargeting multiple nodes within the MAPK signaling cascade in K-ras mutant tumors to maximize therapeutic benefit for patients. Mol Cancer Ther; 11(5); 1143–54. ©2012 AACR.

PDF file - 451K

PDF file - 158K

PDF file - 309K

Purpose: Elevated levels or increases in circulating tumor cells (CTC) portend poor prognosis in patients with epithelial cancers. Less is known about CTCs as surrogate endpoints or their use for predictive biomarker evaluation. This study investigated the utility of CTC enumeration and characterization using the CellSearch platform, as well as mutation detection in circulating tumor DNA (ctDNA), in patients with advanced non–small cell lung cancer (NSCLC).Experimental Design: Forty-one patients were enrolled in a single-arm phase II clinical trial of erlotinib and pertuzumab. Peripheral blood was analyzed for CTC enumeration, EGFR expression in CTCs, and detection of oncogenic mutations in CTCs and ctDNA. Changes in CTC levels were correlated with 2[18F]fluoro-2-deoxy-d-glucose–positron emission tomographic (FDG-PET) and computed tomographic (CT) imaging and survival endpoints.Results: CTCs were detected (≥1 CTC) at baseline in 78% of patients. Greater sensitivity for mutation detection was observed in ctDNA than in CTCs and detected mutations were strongly concordant with mutation status in matched tumor. Higher baseline CTC counts were associated with response to treatment by Response Evaluation Criteria in Solid Tumors (RECIST, P = 0.009) and decreased CTC counts upon treatment were associated with FDG-PET and RECIST response (P = 0.014 and P = 0.019) and longer progression-free survival (P = 0.050).Conclusion: These data provide evidence of a correlation between decreases in CTC counts and radiographic response by either FDG-PET or RECIST in patients with advanced NSCLC. These findings require prospective validation but suggest a potential role for using CTC decreases as an early indication of response to therapy and ctDNA for real-time assessment of mutation status from blood. Clin Cancer Res; 18(8); 2391–401. ©2012 AACR.

Purpose: Class 1 phosphatidylinositol 3-kinase (PI3K) plays a major role in cell proliferation and survival in a wide variety of human cancers. Here, we investigated biomarker strategies for PI3K pathway inhibitors in non–small-cell lung cancer (NSCLC).Experimental Design: Molecular profiling for candidate PI3K predictive biomarkers was conducted on a collection of NSCLC tumor samples. Assays included comparative genomic hybridization, reverse-transcription polymerase chain reaction gene expression, mutation detection for PIK3CA and other oncogenes, PTEN immunohistochemistry, and FISH for PIK3CA copy number. In addition, a panel of NSCLC cell lines characterized for alterations in the PI3K pathway was screened with PI3K and dual PI3K/mTOR inhibitors to assess the preclinical predictive value of candidate biomarkers.Results: PIK3CA amplification was detected in 37% of squamous tumors and 5% of adenocarcinomas, whereas PIK3CA mutations were found in 9% of squamous and 0% of adenocarcinomas. Total loss of PTEN immunostaining was found in 21% of squamous tumors and 4% of adenocarcinomas. Cell lines harboring pathway alterations (receptor tyrosine kinase activation, PI3K mutation or amplification, and PTEN loss) were exquisitely sensitive to the PI3K inhibitor GDC-0941. A dual PI3K/mTOR inhibitor had broader activity across the cell line panel and in tumor xenografts. The combination of GDC-0941 with paclitaxel, erlotinib, or a mitogen-activated protein–extracellular signal-regulated kinase inhibitor had greater effects on cell viability than PI3K inhibition alone.Conclusions: Candidate biomarkers for PI3K inhibitors have predictive value in preclinical models and show histology-specific alterations in primary tumors, suggesting that distinct biomarker strategies may be required in squamous compared with nonsquamous NSCLC patient populations. Clin Cancer Res; 18(24); 6771–83. ©2012 AACR.

XLSX file - 602KB, Supplementary Table 5. Cancer cell lines analyzed in this study. Supplementary Table 6. Tumor tissues analyzed in this study. Supplementary Table 7. List of 340 well characterized hotspot mutations interrogated by the amplicon libraries in this study. Variants also assayed by asPCR are indicated. Supplementary Table 8. Variants detected in 66 cancer cell lines, after applying post-processing filters. Note that indels and predicted deleterious SNVs are reported for all interrogated genes and transcripts. Supplementary Table 9. Variants detected in 73 FFPE endometrial tumor tissues, after applying post-processing filters. Note that indels and predicted deleterious SNVs are reported for all interrogated genes and transcripts. Supplementary Table 10. For cell line data, concordance with COSMIC somatic mutation annotation.

PDF file - 337K

Purpose: Cancer of the ovary confers the worst prognosis among women with gynecologic malignancies, underscoring the need to develop new biomarkers for detection of early disease, particularly those that can be readily monitored in the blood.Experimental Design: We developed an algorithm to identify secreted proteins encoded among ∼22,500 genes on commercial oligonucleotide arrays and applied it to gene expression profiles of 67 stage I to IV serous papillary carcinomas and 9 crudely enriched normal ovarian tissues, to identify putative diagnostic markers. ELISAs were used to validate increased levels of secreted proteins in patient sera encoded by genes with differentially high expression.Results: We identified 275 genes predicted to encode secreted proteins with increased/decreased expression in ovarian cancers (2-fold, P < 0.001). The serum levels of four of these proteins (matrix metalloproteinase-7, osteopontin, secretory leukoprotease inhibitor, and kallikrein 10) were significantly elevated in a series of 67 independent patients with serous ovarian carcinomas compared with 67 healthy controls (P < 0.001, Wilcoxon rank sum test). Optimized support vector machine classifiers with as few as two of these markers (osteopontin or kallikrein 10/matrix metalloproteinase-7) in combination with CA-125 yielded sensitivity and specificity values ranging from 96% to 98.7% and 99.7% to 100%, respectively, with the ability to discern early-stage disease from normal, healthy controls.Conclusions: Our data suggest that this assay combination warrants further investigation as a multi-analyte diagnostic test for serous ovarian adenocarcinoma.

PDF file - 110K

XLSX file - 17K, PI3K pathway alterations in NSCLC cell lines

XLSX file - 18K, PI3K pathway alterations in NSCLC FFPE tumor samples

PDF file - 731KB, Supplementary Figure 1. Schematic workflow of MMP-seq. Supplementary Figure 2. Examples of tiling and hotspot amplicon designs. Supplementary Figure 3. Consistent target enrichment and variant allele quantification in FF biological replicates. Supplementary Figure 4. Correlation between called variant frequencies in paired FF and FFPE samples. Supplementary Figure 5. UDG treatment improves sequencing specificity but has no impact on sensitivity. Supplementary Figure 6. PIK3R1 mutation profiles from TCGA endometrial cancer study. Supplementary Table 1. MMP-seq target genes. Supplementary Table 2. Latin Square Design. Supplementary Table 3. Latin square mutation read frequency detected when sequencing input cell lines for Latin square cross-dilutions. Supplementary Table 4. Pretreatment of FFPE DNA samples with uracil-DNA glycosylase (UDG) resulted in markedly reduction of false positives.

PDF file - 181K

Purpose: Non–small cell lung cancers (NSCLC) comprise multiple distinct biologic groups with different prognoses. For example, patients with epithelial-like tumors have a better prognosis and exhibit greater sensitivity to inhibitors of the epidermal growth factor receptor (EGFR) pathway than patients with mesenchymal-like tumors. Here, we test the hypothesis that epithelial-like NSCLCs can be distinguished from mesenchymal-like NSCLCs on the basis of global DNA methylation patterns.Experimental Design: To determine whether phenotypic subsets of NSCLCs can be defined on the basis of their DNA methylation patterns, we combined microfluidics-based gene expression analysis and genome-wide methylation profiling. We derived robust classifiers for both gene expression and methylation in cell lines and tested these classifiers in surgically resected NSCLC tumors. We validate our approach using quantitative reverse transcriptase PCR and methylation-specific PCR in formalin-fixed biopsies from patients with NSCLC who went on to fail front-line chemotherapy.Results: We show that patterns of methylation divide NSCLCs into epithelial-like and mesenchymal-like subsets as defined by gene expression and that these signatures are similarly correlated in NSCLC cell lines and tumors. We identify multiple differentially methylated regions, including one in ERBB2 and one in ZEB2, whose methylation status is strongly associated with an epithelial phenotype in NSCLC cell lines, surgically resected tumors, and formalin-fixed biopsies from patients with NSCLC who went on to fail front-line chemotherapy.Conclusions: Our data show that patterns of DNA methylation can divide NSCLCs into two phenotypically distinct subtypes of tumors and provide proof of principle that differences in DNA methylation can be used as a platform for predictive biomarker discovery and development. Clin Cancer Res; 18(8); 2360–73. ©2012 AACR.

PDF file - 1.8MB

Supplementary Tables S1-S2 from An Integrated Clinical-Genomics Approach Identifies a Candidate Multi-Analyte Blood Test for Serous Ovarian Carcinoma

PDF file - 221K, S1 Selected regions of recurrent amplification in 52 NSCLC tumor samples profiled by array CGH; S2 (A), GISTIC analysis showing combined frequency and magnitude of PIK3CA amplification in NSCLC tumor samples with squamous (upper) or adenocarcinoma (lower) histology. (B), plot of PIK3CA mRNA compared with copy number at the PIK3CA locus from a panel of NSCLC frozen tumor samples; S3 (A), array CGH data from individual squamous NSCLC tumor samples showing focal amplification of the PIK3CA locus on chromosome 3 and (B), TaqMan qPCR copy number analysis for PIK3CA for adenocarcinoma and squamous NSCLC samples,as well as matched normal lung tissue; S4 FISH assay showing additional copies of PIK3CA and, to a lesser extent, the centromeric probe CEP3, in the NSCLC cell line Calu3; S5 Western blot of selected PI3K/mTOR pathway components in NSCLC cell lines; S6 Tumor growth inhibition plot showing in vivo efficacy in H460 xenografts treated with 1mg/kg or 2.5mg/kg daily of GDC-0980

CCR Translation for This Article from DNA Methylation Profiling Defines Clinically Relevant Biological Subsets of Non–Small Cell Lung Cancer

Purpose: Tailoring cancer treatment to tumor molecular characteristics promises to make personalized medicine a reality. However, reliable genetic profiling of archived clinical specimens has been hindered by limited sensitivity and high false-positive rates. Here, we describe a novel methodology, MMP-seq, which enables sensitive and specific high-throughput, high-content genetic profiling in archived clinical samples.Experimental Design: We first validated the technical performance of MMP-seq in 66 cancer cell lines and a Latin square cross-dilution of known somatic mutations. We next characterized the performance of MMP-seq in 17 formalin-fixed paraffin-embedded (FFPE) clinical samples using matched fresh-frozen tissue from the same tumors as benchmarks. To demonstrate the potential clinical utility of our methodology, we profiled FFPE tumor samples from 73 patients with endometrial cancer.Results: We demonstrated that MMP-seq enabled rapid and simultaneous profiling of a panel of 88 cancer genes in 48 samples, and detected variants at frequencies as low as 0.4%. We identified DNA degradation and deamination as the main error sources and developed practical and robust strategies for mitigating these issues, and dramatically reduced the false-positive rate. Applying MMP-seq to a cohort of endometrial tumor samples identified extensive, potentially actionable alterations in the PI3K (phosphoinositide 3-kinase) and RAS pathways, including novel PIK3R1 hotspot mutations that may disrupt negative regulation of PIK3CA.Conclusions: MMP-seq provides a robust solution for comprehensive, reliable, and high-throughput genetic profiling of clinical tumor samples, paving the way for the incorporation of genomic-based testing into clinical investigation and practice. Clin Cancer Res; 20(8); 2080–91. ©2014 AACR.

Supplementary Figure S1. Inhibition of AKT and S6 phosphorylation. Supplementary Figure S2. Cell growth inhibition by a panel of compounds. Supplementary Figure S3. Effect of GDC-0980 on AKT and S6 phosphorylation. Supplementary Figure S4. Cell growth inhibition by a panel of compounds. Supplementary Figure S5. Inhibition of AKT phosphorylation. Supplementary Figure S6. Cell growth inhibition by a panel of anti-cancer agents. Supplementary Figure S7. The alignment of C-terminus of Human p110 family. Supplementary Figure S8. Droplet digital PCR (ddPCR) detection of PIK3CB D1067Y mutation. Supplementary Figure S9. Detection of copy number variation. Supplementary Figure S10. A. Cell growth inhibition by GDC-0941. B. Droplet digital PCR (ddPCR) analysis of PTEN-null GDC-0941 resistant cell lines. Supplementary Figure S11. Droplet digital PCR detection of PIK3CB D1067V mutation. Supplementary Figure S12. PIK3CB mutant expressing EVSA-T cells are less sensitive to PI3K inhibitors. Supplementary Figure S13. Inhibition of AKT and S6 phosphorylation. Supplementary Figure S14. p110β dependency of A498. Supplementary Figure S15. ATP competition assay. Supplementary Figure S16. Relative quantification of baseline AKT phosphorylation levels in EVSA-T parental cells and resistant clones. Supplementary Figure S17. Signal profile in Rat-2 stable cell lines. Supplementary Table S1 Compound list. Supplementary Table S2 Whole exome sequencing list of genes mutated in EVSA-T GDC-0941 resistant cells.

Activation of the PI3K pathway occurs commonly in a wide variety of cancers. Experience with other successful targeted agents suggests that clinical resistance is likely to arise and may reduce the durability of clinical benefit. Here, we sought to understand mechanisms underlying resistance to PI3K inhibition in PTEN-deficient cancers. We generated cell lines resistant to the pan-PI3K inhibitor GDC-0941 from parental PTEN-null breast cancer cell lines and identified a novel PIK3CB D1067Y mutation in both cell lines that was recurrent in cancer patients. Stable expression of mutant PIK3CB variants conferred resistance to PI3K inhibition that could be overcome by downstream AKT or mTORC1/2 inhibitors. Furthermore, we show that the p110β D1067Y mutant was highly activated and induced PIP3 levels at the cell membrane, subsequently promoting the localization and activation of AKT and PDK1 at the membrane and driving PI3K signaling to a level that could withstand treatment with proximal inhibitors. Finally, we demonstrate that the PIK3CB D1067Y mutant behaved as an oncogene and transformed normal cells, an activity that was enhanced by PTEN depletion. Collectively, these novel preclinical and clinical findings implicate the acquisition of activating PIK3CB D1067 mutations as an important event underlying the resistance of cancer cells to selective PI3K inhibitors. Cancer Res; 76(5); 1193–203. ©2016 AACR.

The RAS/RAF/MEK pathway is activated in more than 30% of human cancers, most commonly via mutation in the K-ras oncogene and also via mutations in BRAF. Several allosteric mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitors, aimed at treating tumors with RAS/RAF pathway alterations, are in clinical development. However, acquired resistance to these inhibitors has been documented both in preclinical and clinical samples. To identify strategies to overcome this resistance, we have derived three independent MEK inhibitor–resistant cell lines. Resistance to allosteric MEK inhibitors in these cell lines was consistently linked to acquired mutations in the allosteric binding pocket of MEK. In one cell line, concurrent amplification of mutant K-ras was observed in conjunction with MEK allosteric pocket mutations. Clonal analysis showed that both resistance mechanisms occur in the same cell and contribute to enhanced resistance. Importantly, in all cases the MEK-resistant cell lines retained their addiction to the mitogen-activated protein kinase (MAPK) pathway, as evidenced by their sensitivity to a selective inhibitor of the ERK1/2 kinases. These data suggest that tumors with acquired MEK inhibitor resistance remain dependent on the MAPK pathway and are therefore sensitive to inhibitors that act downstream of the mutated MEK target. Importantly, we show that dual inhibition of MEK and ERK by small molecule inhibitors was synergistic and acted to both inhibit the emergence of resistance, as well as to overcome acquired resistance to MEK inhibitors. Therefore, our data provide a rationale for cotargeting multiple nodes within the MAPK signaling cascade in K-ras mutant tumors to maximize therapeutic benefit for patients. Mol Cancer Ther; 11(5); 1143–54. ©2012 AACR.

Purpose: Elevated levels or increases in circulating tumor cells (CTC) portend poor prognosis in patients with epithelial cancers. Less is known about CTCs as surrogate endpoints or their use for predictive biomarker evaluation. This study investigated the utility of CTC enumeration and characterization using the CellSearch platform, as well as mutation detection in circulating tumor DNA (ctDNA), in patients with advanced non–small cell lung cancer (NSCLC). Experimental Design: Forty-one patients were enrolled in a single-arm phase II clinical trial of erlotinib and pertuzumab. Peripheral blood was analyzed for CTC enumeration, EGFR expression in CTCs, and detection of oncogenic mutations in CTCs and ctDNA. Changes in CTC levels were correlated with 2[18F]fluoro-2-deoxy-d-glucose–positron emission tomographic (FDG-PET) and computed tomographic (CT) imaging and survival endpoints. Results: CTCs were detected (≥1 CTC) at baseline in 78% of patients. Greater sensitivity for mutation detection was observed in ctDNA than in CTCs and detected mutations were strongly concordant with mutation status in matched tumor. Higher baseline CTC counts were associated with response to treatment by Response Evaluation Criteria in Solid Tumors (RECIST, P = 0.009) and decreased CTC counts upon treatment were associated with FDG-PET and RECIST response (P = 0.014 and P = 0.019) and longer progression-free survival (P = 0.050). Conclusion: These data provide evidence of a correlation between decreases in CTC counts and radiographic response by either FDG-PET or RECIST in patients with advanced NSCLC. These findings require prospective validation but suggest a potential role for using CTC decreases as an early indication of response to therapy and ctDNA for real-time assessment of mutation status from blood. Clin Cancer Res; 18(8); 2391–401. ©2012 AACR.

Purpose: Non–small cell lung cancers (NSCLC) comprise multiple distinct biologic groups with different prognoses. For example, patients with epithelial-like tumors have a better prognosis and exhibit greater sensitivity to inhibitors of the epidermal growth factor receptor (EGFR) pathway than patients with mesenchymal-like tumors. Here, we test the hypothesis that epithelial-like NSCLCs can be distinguished from mesenchymal-like NSCLCs on the basis of global DNA methylation patterns. Experimental Design: To determine whether phenotypic subsets of NSCLCs can be defined on the basis of their DNA methylation patterns, we combined microfluidics-based gene expression analysis and genome-wide methylation profiling. We derived robust classifiers for both gene expression and methylation in cell lines and tested these classifiers in surgically resected NSCLC tumors. We validate our approach using quantitative reverse transcriptase PCR and methylation-specific PCR in formalin-fixed biopsies from patients with NSCLC who went on to fail front-line chemotherapy. Results: We show that patterns of methylation divide NSCLCs into epithelial-like and mesenchymal-like subsets as defined by gene expression and that these signatures are similarly correlated in NSCLC cell lines and tumors. We identify multiple differentially methylated regions, including one in ERBB2 and one in ZEB2, whose methylation status is strongly associated with an epithelial phenotype in NSCLC cell lines, surgically resected tumors, and formalin-fixed biopsies from patients with NSCLC who went on to fail front-line chemotherapy. Conclusions: Our data show that patterns of DNA methylation can divide NSCLCs into two phenotypically distinct subtypes of tumors and provide proof of principle that differences in DNA methylation can be used as a platform for predictive biomarker discovery and development. Clin Cancer Res; 18(8); 2360–73. ©2012 AACR.

Cell migration is essential to cancer invasion and metastasis and is spatially and temporally integrated through transcriptionally dependent and independent mechanisms. Since cell migration is studied in vitro, it is important to identify genes that both drive cell migration and are biologically relevant in promoting invasion and metastasis in patients with cancer. Here, gene expression profiling and a high throughput cell migration system answers this question in human bladder cancer. In vitro migration rates of 40 microarray profiled human bladder cancer cell lines were measured by radial migration assay (RMA). Genes whose expression was either directly or inversely associated with cell migration rate were identified and subsequently evaluated for their association with cancer stage in 61 patients. This analysis identified genes known to be associated with cell invasion such as versican, and novel ones, including metallothionein E1 (MTE1) and nicotinamide N-methyltransferase (NNMT), whose expression correlated positively with cancer cell migration and tumor stage. Using loss of function analysis, we show that MTE1 and NNMT are necessary for cancer cell migration. These studies provide a general approach to identify the clinically relevant genes in cancer cell migration and mechanistically implicate two novel genes in this process in human bladder cancer.

The choice of therapy for metastatic cancer is largely empirical because of a lack of chemosensitivity prediction for available combination chemotherapeutic regimens. Here, we identify molecular models of bladder carcinoma chemosensitivity based on gene expression for three widely used chemotherapeutic agents: cisplatin, paclitaxel, and gemcitabine. We measured the growth inhibition elicited by these three agents in a series of 40 human urothelial cancer cell lines and correlated the GI50 (50% of growth inhibition) values with quantitative measures of global gene expression to derive models of chemosensitivity using a misclassification-penalized posterior approach. The misclassification-penalized posterior–derived models predicted the growth response of human bladder cancer cell lines to each of the three agents with sensitivities of between 0.93 and 0.96. We then developed an in silico approach to predict the cellular growth responses for each of these agents in the clinically relevant two-agent combinations. These predictions were prospectively evaluated on a series of 15 randomly chosen bladder carcinoma cell lines. Overall, 80% of the predicted combinations were correct (P = 0.0002). Together, our results suggest that chemosensitivity to drug combinations can be predicted based on molecular models and provide the framework for evaluation of such models in patients undergoing combination chemotherapy for cancer. If validated in vivo, such predictive models have the potential to guide therapeutic choice at the level of an individual's tumor. [Mol Cancer Ther 2007;6(2):578–86]

Expression profiling by DNA microarray analysis has provided insights into molecular alterations that underpin cancer progression and metastasis. Although differential expression of microarray-defined probes can be related to numerical or structural chromosomal alterations, it is unclear if such changes are also clustered in distinct chromosomes or genomic regions and whether chromosomal alterations always reflect changes in gene expression. Here we apply the dChip algorithm and a novel technique to test the hypothesis that expression changes occurring as a function of tumor progression and metastasis are nonrandomly distributed. Expression profiling of a human xenograff model of lung metastasis phenotype indicates that chromosomes 2, 11, and 20 contain higher percentages of differentially expressed genes (P

Cell motility is a complex biological process, involved in development, inflammation, homeostasis, and pathological processes such as the invasion and metastatic spread of cancer. Here, we describe a genomic screen designed to identify inhibitors of cell migration. A library of 10,996 small interfering RNAs (targeting 5,234 human genes) was screened for their ability to block the migration of a highly motile ovarian carcinoma cell line, SKOV-3, by using a 384-well wound-healing assay coupled with automated microscopy and wound quantification. Two or more small interfering RNAs against four genes, CDK7 , DYRK1B , MAP4K4 ( NIK / HGK ) ( MAP4K4 , mitogen-activated protein 4 kinase 4), and SCCA-1 (SerpinB3), potently blocked the migration of SKOV-3 cells, concordant with reduced transcript levels. Further studies of the promigratory role of MAP4K4 showed that the knockdown of this transcript inhibited the migration of multiple carcinoma cell lines, indicating a broad role in cell motility and potently suppressed the invasion of SKOV-3 cells in vitro . The effect of MAP4K4 on cellular migration was found to be mediated through c-Jun N-terminal kinase, independent of AP1 activation and downstream transcription. Accordingly, small molecule inhibition of c-Jun N-terminal kinase suppressed SKOV-3 cell migration, underscoring the potential therapeutic utility of mitogen-activated protein kinase pathway inhibition in cancer progression.

Ral GTPases are important mediators of transformation, tumorigenesis, and cancer progression. We recently identified the metastasis-associated protein CD24, a glycosyl phosphatidyl inositol–linked surface protein, as a downstream target of Ral signaling by profiling the expression of RalA/B–depleted bladder carcinoma cells. Because CD24 is highly expressed in bladder and many other tumor types, we sought to determine if this protein plays an essential role in maintaining the malignant phenotype. Here, we show that loss of CD24 function in cell lines derived from common tumor types is associated with decreased rates of cell proliferation, clonogenicity in soft agar, changes in the actin cytoskeleton, and induction of apoptosis. Given these phenotypes, we evaluated a human bladder cancer tissue microarray by immunohistochemistry for CD24 to determine if CD24 is a prognostic cancer biomarker. Multivariate analysis showed that increased CD24 expression correlated with shorter patient disease-free survival (P = 0.07). In conclusion, we show that CD24 is a novel and functionally relevant Ral-regulated target and a potentially important prognostic marker. We suggest that these insights may lead to future therapeutic approaches that seek to eliminate CD24 function in cancer cells. (Cancer Res 2006; 66(4): 1917-22)

Purpose: Current serum testing for the detection of prostate cancer (PCa) lacks specificity. On diagnosis, the optimal therapeutic pathway is not clear and tools for adequate risk assessment of localized PCa progression are not available. This leads to a significant number of men having unnecessary diagnostic biopsies and surgery. A search for novel tumor markers identified macrophage inhibitory cytokine 1 (MIC-1) as a potentially useful marker. Follow-up studies revealed MIC-1 overexpression in local and metastatic PCa whereas peritumoral interstitial staining for MIC-1 identified lower-grade tumors destined for recurrence. Consequently, we sought to assess serum MIC-1 measurement as a diagnostic tool. Experimental Design: Using immunoassay determination of serum MIC-1 concentration in 1,000 men, 538 of whom had PCa, we defined the relationship of MIC-1 to disease variables. A diagnostic algorithm (MIC-PSA score) based on serum levels of MIC-1, total serum prostate-specific antigen, and percentage of free prostate-specific antigen was developed. Results: Serum MIC-1 was found to be an independent predictor of the presence of PCa and tumors with a Gleason sum ≥7. We validated the MIC-PSA score in a separate population and showed an improved specificity for diagnostic blood testing for PCa over percentage of free prostate-specific antigen, potentially reducing unnecessary biopsies by 27%. Conclusions: Serum MIC-1 is an independent marker of the presence of PCa and tumors with a Gleason sum of ≥7. The use of serum MIC-1 significantly increases diagnostic specificity and may be a future tool in the management of PCa.

Half of patients treated for locally advanced bladder cancer relapse with often fatal metastatic disease to the lung. We have recently shown that reduced expression of the GDP dissociation inhibitor, RhoGDI2, is associated with decreased survival of patients with advanced bladder cancer. However, the effectors by which RhoGDI2 affects metastasis are unknown. Here we use DNA microarrays to identify genes suppressed by RhoGDI2 reconstitution in lung metastatic bladder cancer cell lines. We identify such RNAs and focus only on those that also increase with tumor stage in human bladder cancer samples to discover only clinically relevant targets of RhoGDI2. Levels of endothelin-1 (ET-1), a potent vasoconstrictor, were affected by both RhoGDI2 reconstitution and tumor stage. To test the hypothesis that the endothelin axis is important in lung metastasis, lung metastatic bladder carcinoma cells were injected in mice treated with the endothelin receptor–specific antagonist, atrasentan, thereby blocking engagement of the up-regulated ET-1 ligand with its cognate receptor. Endothelin antagonism resulted in a dramatic reduction of lung metastases, similar to the effect of reexpressing RhoGDI2 in these metastatic cells. Taken together, these experiments show a novel approach of identifying therapeutic targets downstream of metastasis suppressor genes. The data also suggest that blockade of the ET-1 axis may prevent lung metastasis, a new therapeutic concept that warrants clinical evaluation.

Identification of biomarkers to recognize individuals with Barrett's esophagus (BE) predisposed to develop malignancy is currently a pressing issue. We utilized gene expression profiling to compare molecular signatures of normal esophagus and stomach, BE, and adenocarcinoma (AC) to identify such potential biomarkers. Over 22,000 genes were analyzed by oligonucleotide microarrays on 38 unique RNA Unsupervised and supervised clusterings were performed on a subset of 2849 genes that varied most significantly across the specimens. Immunohistochemistry (IHC) for two of the significantly differentially expressed gene products was performed on tissue microarrays. Unsupervised clustering identified two discernable molecular BE profiles, one of which was similar to normal gastric tissue ("BE1"), and another that was shared by several of the AC specimens ("BE2"). The BE1 profile included expression of several genes that have been described as tumor-suppressor genes, most notably trefoil factor 1 (TFF-1). The BE2 profile included expression of genes previously found overexpressed in cancers, such as carboxylesterase-2 (CES-2). IHC demonstrated the loss of TFF-1 late in the progression of BE to AC. It also revealed CES-2 as being upregulated in AC documented to have arisen in the presence of BE. These potential biomarkers, as well as the relative expression of genes from BE1 versus those from BE2, may be validated in the future to aid in risk stratification and guide treatment protocols in patients with BE and associated AC.

Pulmonary metastases frequently develop in patients with aggressive bladder cancer, yet investigation of this process at the molecular level suffers from the poor availability of human metastatic tumor tissue and the absence of suitable animal models. To address this, we developed progressively more metastatic human bladder cancer cell lines and an in vivo bladder-cancer lung-metastasis model, and we successfully used these to identify genes of which the expression levels change according to the degree of pulmonary metastatic potential. By initially intravenously injecting the poorly metastatic T24T human urothelial cancer cells into nude mice, and then serially reintroducing and reisolating the human tumor cells from the resultant mouse lung tumors, three derivative human lines with increasingly metastatic phenotypes, designated FL1, FL2, and FL3, were sequentially isolated. To identify the genes associated with the most lung-metastatic phenotype, the RNA complement from the parental and derivative cells was evaluated with oligonucleotide microarrays. In doing so, we found 121 genes to be progressively up-regulated during the transition from T24T to FL3, whereas 43 genes were progressively down-regulated. As expected, many of the genes identified in these groups could, according to the ascribed functions of their protein product, theoretically participate in tissue invasion and metastasis. In addition, the magnitude of gene expression changes observed during the metastatic transition correlated with the in vivo propensity for earlier lung colonization and decreased host survival. To additionally define which genes found in the experimental system were of relevance to human bladder cancer lung metastasis, we evaluated gene expression profiles of 23 primary human bladder tumors of various stages and grades, and then we compared these gene expression profiles to the altered profiles in our model cell lines. Here we found that the expression of epiregulin, urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP)14, and tissue inhibitor of metalloproteinase (TIMP-2) were consistently and progressively up-regulated when viewed as a function of tumor stage in tissues of patients versus the metastatic potential seen in the mouse lung model. The strong correlation of these four markers between the experimental and clinical situations helps validate this system as a useful tool for the study of lung metastasis and defines targets of therapy that may reduce the incidence of this process in patients.

Purpose: RhoGDI2 was recently shown to be a metastasis suppressor gene in models of bladder cancer. We sought to further understand its importance in human cancer by determining the level of its expression and the distribution of its encoded protein in normal human tissues and cell lines and to evaluate whether its protein expression is a determinant of human bladder cancer progression. Experimental Design: RhoGDI2 mRNA and protein expression was evaluated in cell lines and human tissues using Affymetrix and tissue microarrays, respectively. Tissue microarrays represented most human normal adult tissues and material from 51 patients that had undergone radical cystectomy for bladder cancer. In these 51 patients, the χ2 test was used to test for associations between RhoGDI2 and stage, grade of urothelial carcinoma, histological type, and disease-specific survival status. Cox proportional hazards regression analyses were used to estimate the effect of RhoGDI2 expression level on time to development of metastatic disease and disease-specific survival time, adjusting for grade, stage, and histological type. Results: In normal tissues, there was strong RhoGDI2 protein expression in WBCs, endothelial cells, and transitional epithelium. RhoGDI2 mRNA expression was inversely related to the invasive and metastatic phenotype in human bladder cancer cell lines. In patients with bladder cancer, univariate analysis indicated that reduced tumor RhoGDI2 protein expression was associated with a lower actuarial 5-year disease-free and disease-specific survival (P = 0.01). In addition, patients with tumors that had low or absent RhoGDI2 had a shorter time to disease-specific death (P ≤ 0.01). When tumor grade, stage, histological type, and RhoGDI2 staining level were examined using multivariate analysis, RhoGDI2 expression was found to be an independent predictive factor for disease-specific death (P = 0.03). Conclusions: These results suggest that RhoGDI2 is an independent predictor of prognosis for patients with bladder cancer and provide clinical evidence in support of its involvement in cancer metastasis.

The dynamic balance between histone acetylation and deacetylation plays a significant role in the regulation of gene transcription. Much of our current understanding of this transcriptional control comes from the use of HDAC inhibitors such as trapoxin A (TPX), which leads to hyperacetylated histone, alters local chromatin architecture and transcription and results in tumor cell death. In this study, we treated tumor cells with TPX and HDAC1 antisense oligonucleotides, and analysed the transcriptional consequences of HDAC inhibition. Among other genes, the small GTPase RhoB was found to be significantly upregulated by TPX and repressed by HDAC1. The induction of RhoB by HDAC inhibition was mediated by an inverted CCAAT box in the RhoB promoter. Interestingly, measurement of RhoB transcription in approximately 130 tumor-derived cell lines revealed low expression in almost all of these samples, in contrast to RhoA and RhoC. Accumulating evidence indicates that the small GTPase Rho proteins are involved in a variety of important processes in cancer, including cell transformation, survival, invasion, metastasis and angiogenesis. This study for the first time demonstrates a link between HDAC inhibition and RhoB expression and provides an important insight into the mechanisms of HDAC-mediated transcriptional control and the potential therapeutic benefit of HDAC inhibition.

Cdx2 has been identified as a marker of colon cancer in RNA-profiling experiments. We show here that the detection of Cdx2 protein by immunohistochemistry correlates well with RNA transcript levels as detected by oligonucleotide microarrays. Using tissue microarrays containing most normal tissue types and an antibody to the Cdx2 protein, strong diffuse Cdx2 staining was only seen in the nuclei of small and large intestinal epithelium and portions of the pancreatic duct system. In tissue microarrays containing 745 cancers from many anatomic sites, colonic adenocarcinomas showed strong extensive staining in 90% of cases, with adenocarcinomas of the stomach, esophagus, and ovary (endometrioid and mucinous types) showing extensive staining in only 20-30% of cases. Other types of carcinomas showed extensive staining in only

Oncogenesis occurs through the acquisition and selection of multiple somatic mutations--each contributing to the growth, survival and spread of the cancer. Key attributes of the malignant phenotype, such as unchecked proliferation and cell survival, can often be "reversed" by the selective diminution of dominant oncogenes by chemical or genetic means (e.g. beta-catenin in colorectal carcinomas; bcr-abl in chronic myelogenous leukemias (CMLs)). These observations suggest that the products of oncogenes, or of secondary genes that mediate and maintain tumor phenotypes, might be revealed through the systematic disruption of each and every gene in tumor-derived cells. Some of these genes may encode proteins amenable to therapeutic intervention, thus fueling the cancer drug discovery process. However, a functional assessment of each known or predicted gene in mammalian cells is a daunting task and represents the rate-limiting step in drug target identification and validation. In this regard, RNA interference (RNAi) by small interfering RNAs (siRNA) holds great promise as the "tool of choice" to mediate the selective attenuation of mammalian gene expression and protein function. Here, we review strategies by which RNAi might be used to determine the genetic alterations that contribute to malignant transformation via large-scale cell-based screens, and propose how this information can be used in conjunction with small molecule screens to identify pathways critical to cancer cell survival.

The 40-kilodalton processed glycoprotein, mesothelin, is highly expressed in epithelial mesotheliomas and adenocarcinomas of the ovary (serous papillary) and pancreas, but its expression in a large series of other common carcinomas has not been completely explored. In the present study, we used oligonucleotide and tissue microarrays to profile the expression of the mesothelin gene (MSLN) and encoded protein, respectively. Among 150 carcinomas of multiple anatomic sites, we found the highest average expression of MSLN in serous carcinomas of the ovary and adenocarcinomas of the pancreas, consistent with previous reports, as well as measurable but less-striking expression in pulmonary, gastric/esophageal, and colorectal adenocarcinomas. On tissue microarrays containing 621 carcinomas derived from the same and additional sites as those profiled by gene expression, mesothelin immunoreactivity was highest in cancers of the ovary (serous papillary, endometrioid, and undifferentiated) and pancreas, with less frequent staining seen in adenocarcinomas of the endometrium, lung, and stomach/esophagus. Some immunopositivity was observed in 42% of pulmonary adenocarcinomas, including 18% that had >50% of tumor cells that were immunoreactive. Some 14% of breast and 30% of colorectal adenocarcinomas showed immunopositivity, but no case contained >50% tumor cells that were immunoreactive. Mesothelin was either entirely absent or present in

Aberrant expression of the apoptosis inhibitor bcl-2 provides a survival advantage throughout oncogenesis and can facilitate chemotherapeutic resistance in a variety of human cancers. Follicular lymphoma (FL) for example, is characterized by the chromosomal translocation t(14;18), which results in bcl-2 overexpression and initiates lymphomagenesis. Although FL cells possess ample amounts of bcl-2, they respond remarkably well to standard first-round chemotherapy. However, the vast majority of patients relapses and becomes progressively resistant to therapy. We obtained cell lines derived from chemosensitive and chemoresistant FL patients, that are characterized by the chromosomal translocation t(14;18) and expression of bcl-2, to investigate how chemotherapeutic drugs can circumvent bcl-2 anti-apoptotic function and to identify alterations in those pathways that may facilitate resistance to DNA damaging drugs. In chemosensitive FL cells, we found that DNA damaging drugs promote apoptosis through p53-dependent upregulation of the TRAIL-DR5 receptor, resulting in activation of caspase-8 and downstream executioner caspases, thereby evading bcl-2 mediated suppression of apoptosis. Examination of drug resistant FL cell lines revealed that at least two defects in this pathway can contribute to chemotherapeutic resistance: 1. p53 gene mutations that disable the transcriptional response to DNA damaging drugs, including expression of the TRAIL-DR5 receptor, and 2. transcriptional repression of the cell-death executioner enzyme caspase-3.

The advent of DNA microarray technology has ushered in an era of systems biology whereby researchers can study the transcriptional behavior of thousands of genes in parallel. Advances in manufacturing techniques and informatics, and the availability of several genome sequences have furthered these capabilities to the point where whole-transcriptome studies can be accomplished in yeast, flies and plants, and soon will be possible in mammals. Concomitant with the expanding ability of the technology has been the development of novel techniques and their application towards the study of cellular biology.

With the development and application of DNA microarrays, the expression of almost all human genes can now be systematically examined in human malignancies. This can lead to the identification of candidate molecular targets for therapeutic intervention and biomarkers for early detection of these diseases. However, perhaps the most exciting result to come from this research has been the demonstration that patterns of gene expression can distinguish between tumors of different anatomical origin, and define new subgroups of cancer with similar histological appearance, but distinct molecular profiles. Some of these new molecular subclasses of tumor appear to correlate with clinical behavior. If substantiated in larger studies, this might form a basis for stratifying patients so that they receive optimal therapeutic treatment and follow-up.