Increased expression of neurotensin in high grade serous ovarian carcinoma with evidence of serous tubal intraepithelial carcinoma.

High grade serous ovarian carcinoma (HGSC) without identifiable serous tubal intraepithelial carcinoma (STIC) within the fallopian tube (FT) occurs in approximately 50% of patients. The objective of this study was to use a multisite tumor sampling approach to study HGSC with and without STIC. RNAseq analysis of HGSC samples collected from multiple sites e.g. ovary, FT and peritoneum, revealed moderate levels of intrapatient heterogeneity in gene expression that could influence molecular profiles. Mixed-model ANOVA analysis of gene expression in tumor samples from patients with multiple tumor sites (n = 13) and patients with a single site tumor sample (n = 11) to compare HGSC-STIC to HGSC-NOSTIC identified neurotensin (NTS) as significantly higher (> two-fold change, False Discovery Rate (FDR) < 0.10) in HGSC-STIC. This data was validated using publicly available RNA-Seq datasets. Concordance between higher NTS gene expression and NTS peptide levels in HGSC-STIC samples was demonstrated by immunohistochemistry. To determine the role of NTS in HGSC, five ovarian cancer (OvCa) cell lines were screened for expression of NTS and its receptors, NTSR1 and NTSR3. Increased expression of NTS and NSTR1 was observed in several of the OvCa cells, whereas the NTSR3 receptor was lower in all OvCa cells, compared to immortalized FT epithelial cells. Treatment with NTSR1 inhibitor (SR48692) decreased cell proliferation, but increased cell migration in OvCa cells. The effects of SR48692 were receptor mediated, since transient RNAi knockdown of NTSR1 mimicked the migratory effects and knockdown of NTSR3 mimicked the anti-proliferative effects. Further, knockdown of NTSR1 or NTSR3 was associated with acquisition of distinct morphological phenotypes, epithelial or mesenchymal, respectively. Taken together, our results reveal a difference in a biologically active pathway between HGSC with and without STIC. Furthermore, we identify neurotensin signaling as an important pathway involved in cell proliferation and epithelial-mesenchymal transition in HGSC-STIC which warrants further study as a potential therapeutic target. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Roles of the C-terminal domains of topoisomerase IIα and topoisomerase IIß in regulation of the decatenation checkpoint.

Topoisomerase (topo) IIα and IIß maintain genome stability and are targets for anti-tumor drugs. In this study, we demonstrate that the decatenation checkpoint is regulated, not only by topo IIα, as previously reported, but also by topo IIß. The decatenation checkpoint is most efficient when both isoforms are present. Regulation of this checkpoint and sensitivity to topo II-targeted drugs is influenced by the C-terminal domain (CTD) of the topo II isoforms and by a conserved non-catalytic tyrosine, Y640 in topo IIα and Y656 in topo IIß. Deletion of most of the CTD of topo IIα, while preserving the nuclear localization signal (NLS), enhances the decatenation checkpoint and sensitivity to topo II-targeted drugs. In contrast, deletion of most of the CTD of topo IIß, while preserving the NLS, and mutation of Y640 in topo IIα and Y656 in topo IIß inhibits these activities. Structural studies suggest that the differential impact of the CTD on topo IIα and topo IIß function may be due to differences in CTD charge distribution and differential alignment of the CTD with reference to transport DNA. Together these results suggest that topo IIα and topo IIß cooperate to maintain genome stability, which may be distinctly modulated by their CTDs.

Characterisation of homologous recombination deficiency in paired primary and recurrent high-grade serous ovarian cancer.

BACKGROUND: Homologous recombination deficiency (HRD) is shown to predict response to DNA-damaging therapies in patients with high-grade serous ovarian cancer (HGSOC); however, changes in HRD during progression remains unknown. METHODS: HRD scores were evaluated in paired primary and/or recurrent HGSOC samples (N = 107) from 54 patients with adjuvant platinum-based chemotherapy. BRCA1/2 mutation, BRCA1 methylation, loss of heterozygosity (LOH), and HRD scores were characterised using tumour DNA-based next-generation sequencing assays. RESULTS: Among 50 evaluable pairs (N = 100 samples), high intra-patient correlation in HRD score was observed (r2 = 0.93). BRCA1/2 mutations, BRCA1/2 LOH, and HRD were maintained between primary and recurrent samples, except for one pair in which a BRCA1 reversion mutation was identified in the recurrent sample. Despite the reversion, both samples were classified as having high HRD scores ( ≥ 42). All samples with BRCA1/2 mutations exhibited high HRD scores; however, high HRD scores were more prevalent than BRCA1/2 mutations (55% vs. 30%, respectively). CONCLUSION: Markers of HRD were maintained between the primary and recurrent samples, regardless of other genomic changes that occurred during recurrence. HRD score/markers in primary tumours may be valuable and adequate for selection of platinum-based therapy and/or poly-ADP-ribose-polymerase (PARP) inhibitors in recurrent HGSOC.

HOXA4/HOXB3 gene expression signature as a biomarker of recurrence in patients with high-grade serous ovarian cancer following primary cytoreductive surgery and first-line adjuvant chemotherapy.

OBJECTIVES: Aberrant homeobox (HOX) gene expression is reported in high-grade serous ovarian carcinoma (HGSOC), however, its prognostic significance remains unclear. METHODS: HOX genes associated with progression-free survival (PFS) in a discovery cohort of primary HGSOC samples with RNA sequencing data, and those previously reported to be associated with clinical outcomes, were selected for qPCR testing in an independent training cohort of primary HGSOC samples (n=71). A prognostic model for PFS was developed using univariate and multivariate Cox regression. Patients were stratified into risk groups that optimized the test statistic. The model was tested in an independent HGSOC cohort from The Cancer Genome Atlas (TCGA) (n=320). The effect of selected HOX genes on drug sensitivity and reactive oxygen species (ROS) accumulation was examined in vitro. RESULTS: Of 23 HOX genes tested in the training cohort, HOXA4 (HR=1.20, 95% CI=1.07-1.34, P=0.002) and HOXB3 (HR=1.09, 95% CI=1.01-1.17, P=0.027) overexpression were significantly associated with shorter PFS in multivariate analysis. Based on the optimal cutoff of the HOXA4/HOXB3 risk score, median PFS was 16.9months (95% CI=14.6-21.2months) and not reached (>80months) for patients with high and low risk scores, respectively (HR=8.89, 95% CI=2.09-37.74, P<0.001). In TCGA, the HOXA4/HOXB3 risk score was significantly associated with disease-free survival (HR=1.44, 95% CI=1.00-2.09, P=0.048). HOXA4 or HOXB3 overexpression in ovarian cancer cells decreased sensitivity to cisplatin and attenuated the generation of cisplatin-induced ROS (P<0.05). CONCLUSIONS: HOXA4/HOXB3 gene expression-based risk score may be useful for prognostic risk stratification and warrants prospective validation in HGSOC patients.

Expression profile of COL2A1 and the pseudogene SLC6A10P predicts tumor recurrence in high-grade serous ovarian cancer.

Tumor recurrence, following initial response to adjuvant chemotherapy, is a major problem in women with high-grade serous ovarian cancer (HGSOC). Microarray analysis of primary tumors has identified genes that may be useful in risk stratification/overall survival, but are of limited value in predicting the >70% rate for tumor recurrence. In this study, we performed RNA-Seq analysis of primary and recurrent HGSOC to first identify unique differentially expressed genes. From this dataset, we selected 21 archetypical coding genes and one noncoding RNA, based on statistically significant differences in their expression profile between tumors, for validation by qPCR in a larger cohort of 110 ovarian tumors (71 primary and 39 recurrent) and for testing association of specific genes with time-to-recurrence (TTR). Kaplan-Meier tests revealed that high expression of collagen type II, alpha 1 (COL2A1) was associated with delayed TTR (HR = 0.47, 95% CI: 0.27-0.82, p = 0.008), whereas low expression of the pseudogene, solute carrier family 6 member 10 (SLC6A10P), was associated with longer TTR (HR = 0.53, 95% CI: 0.30-0.93, p = 0.027). Notably, TTR was significantly delayed for tumors that simultaneously highly expressed COL2A1 and lowly expressed SLC6A10P (HR = 0.21, 95% CI: 0.082-0.54, p = 0.0011), an estimated median of 95 months as compared to an estimated median of 16 months for subjects expressing other levels of COL2A1 and SLC6A10P. Thus, evaluating expression levels of COL2A1 and SLC6A10P at primary surgery could be beneficial for clinically managing recurrence of HGSOC.

Biological role and clinical implications of homeobox genes in serous epithelial ovarian cancer.

Homeobox (HOX) genes are a family of transcription factors that are essential regulators of development. HOX genes play important roles in normal reproductive physiology, as well as in the development and progression of serous carcinomas, the predominant and most aggressive subtype of epithelial ovarian cancer (EOC). This review discusses aberrant HOX gene expression in serous EOC and its impact on tumor development and progression. Further identification of HOX target genes may facilitate the development of novel diagnostic and therapeutic strategies to improve the prognosis of patients with serous EOC.

Redefining the relevance of established cancer cell lines to the study of mechanisms of clinical anti-cancer drug resistance.

Although in vitro models have been a cornerstone of anti-cancer drug development, their direct applicability to clinical cancer research has been uncertain. Using a state-of-the-art Taqman-based quantitative RT-PCR assay, we investigated the multidrug resistance (MDR) transcriptome of six cancer types, in established cancer cell lines (grown in monolayer, 3D scaffold, or in xenograft) and clinical samples, either containing >75% tumor cells or microdissected. The MDR transcriptome was determined a priori based on an extensive curation of the literature published during the last three decades, which led to the enumeration of 380 genes. No correlation was found between clinical samples and established cancer cell lines. As expected, we found up-regulation of genes that would facilitate survival across all cultured cancer cell lines evaluated. More troubling, however, were data showing that all of the cell lines, grown either in vitro or in vivo, bear more resemblance to each other, regardless of the tissue of origin, than to the clinical samples they are supposed to model. Although cultured cells can be used to study many aspects of cancer biology and response of cells to drugs, this study emphasizes the necessity for new in vitro cancer models and the use of primary tumor models in which gene expression can be manipulated and small molecules tested in a setting that more closely mimics the in vivo cancer microenvironment so as to avoid radical changes in gene expression profiles brought on by extended periods of cell culture.

Association of VEGF and VEGFR2 single nucleotide polymorphisms with hypertension and clinical outcome in metastatic clear cell renal cell carcinoma patients treated with sunitinib.

PURPOSE: Biomarkers that predict response or toxicity to antiangiogenic therapy are sought to favorably inform the risk/benefit ratio. This study evaluated the association of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) genetic polymorphisms with the development of hypertension (HTN) and clinical outcome in metastatic clear cell renal cell carcinoma (MCCRCC) patients treated with sunitinib. PATIENT AND METHODS: Sixty-three MCCRCC patients receiving sunitinib (50 mg 4/2) with available blood pressure (BP) data and germline DNA were retrospectively identified. A panel of candidate VEGF and VEGFR2 single nucleotide polymorphisms (SNPs) were evaluated for associations with the development of hypertension and clinical outcome. RESULTS: VEGF SNP -634 genotype was associated with the prevalence and duration of sunitinib-induced hypertension (as defined by systolic pressure ≥150 mmHg and/or diastolic pressure ≥90 mmHg) in both univariable analysis (P = .03 and .01, respectively) and multivariable analysis, which adjusted for baseline BP and use of antihypertension medication (P = .05 and .02, respectively). Patients with the GG genotype were estimated to have a greater likelihood of being hypertensive during treatment compared with patients with the CC genotype (odds ratio of 13.62, 95% confidence interval [CI] 3.71-50.04). No single VEGF or VEGFR SNPs were found to correlate with clinical outcome. However, the combination of VEGF SNP 936 and VEGFR2 SNP 889 were associated with overall survival after adjustment for prognostic risk group (P = .03). CONCLUSIONS: In MCCRCC patients treated with sunitinib, VEGF SNP -634 is associated with hypertension and a combination of VEGF SNP 936 and VEGFR2 SNP 889 genotypes is associated with overall survival.

Germline and somatic DNA methylation and epigenetic regulation of KILLIN in renal cell carcinoma.

We recently identified germline methylation of KILLIN, a novel p53-regulated tumor suppressor proximal to PTEN, in >1/3 Cowden or Cowden syndrome-like (CS/CSL) individuals who are PTEN mutation negative. Individuals with germline KILLIN methylation had increased risks of renal cell carcinoma (RCC) over those with PTEN mutations. Therefore, we tested the hypothesis that KILLIN may be a RCC susceptibility gene, silenced by germline methylation. We found germline hypermethylation by combined bisulfite restriction analysis in at least one of the four CpG-rich regions in 23/41 (56%) RCC patients compared to 0/50 controls (P < 0.0001). Of the 23, 11 (48%) demonstrated methylation in the -598 to -890 bp region in respect to the KILLIN transcription start site. Furthermore, 19 of 20 advanced RCC showed somatic hypermethylation upstream of KILLIN, with the majority hypermethylated at more than one CpG island (13/19 vs. 3/23 with germline methylation, P < 0.0001). qRT-PCR revealed that methylation significantly downregulates KILLIN expression (P = 0.05), and demethylation treatment by 5-aza-2'deoxycytidine significantly increased KILLIN expression in all RCC cell lines while only increasing PTEN expression in one line. Furthermore, targeted in vitro methylation revealed a significant decrease in KILLIN promoter activity only. These data reveal differential epigenetic regulation by DNA promoter methylation of this bidirectional promoter. In summary, we have identified KILLIN as a potential novel cancer predisposition gene for nonsyndromic clear-cell RCC, and the epigenetic mechanism of KILLIN inactivation in both the germline and somatic setting suggests the potential for treatment with demethylating agents.

PLK3 amplification and tumor immune microenvironment of metastatic tumors are linked to adjuvant treatment outcomes in uterine serous cancer.

Uterine serous carcinoma (USC), an aggressive variant of endometrial cancer representing approximately 10% of endometrial cancer diagnoses, accounts for ∼39% of endometrial cancer-related deaths. We examined the role of genomic alterations in advanced-stage USC associated with outcome using paired primary-metastatic tumors (n = 29) treated with adjuvant platinum and taxane chemotherapy. Comparative genomic analysis of paired primary-metastatic patient tumors included whole exome sequencing and targeted gene expression. Both PLK3 amplification and the tumor immune microenvironment (TIME) in metastatic tumors were linked to time-to-recurrence (TTR) risk without any such association observed with primary tumors. TP53 loss was significantly more frequent in metastatic tumors of platinum-resistant versus platinum-sensitive patients and was also associated with increased recurrence and mortality risk. Increased levels of chr1 breakpoints in USC metastatic versus primary tumors co-occur with PLK3 amplification. PLK3 and the TIME are potential targets for improving outcomes in USC adjuvant therapy.

Tyrosine 656 in topoisomerase IIß is important for the catalytic activity of the enzyme: Identification based on artifactual +80-Da modification at this site.

Topoisomerase (topo) II catalyzes topological changes in DNA. Although both human isozymes, topo IIα and ß are phosphorylated, site-specific phosphorylation of topo IIß is poorly characterized. Using LC-MS/MS analysis of topo IIß, cleaved with trypsin, Arg C or cyanogen bromide (CNBr) plus trypsin, we detected four +80-Da modified sites: tyr656, ser1395, thr1426 and ser1545. Phosphorylation at ser1395, thr1426 and ser1545 was established based on neutral loss of H(3) PO(4) (-98 Da) in the CID spectra and on differences in 2-D-phosphopeptide maps of (32) P-labeled wild-type (WT) and S1395A or T1426A/S1545A mutant topo IIß. However, phosphorylation at tyr656 could not be verified by 2-D-phosphopeptide mapping of (32) P-labeled WT and Y656F mutant protein or by Western blotting with phosphotyrosine-specific antibodies. Since the +80-Da modification on tyr656 was observed exclusively during cleavage with CNBr and trypsin, this modification likely represented bromination, which occurred during CNBr cleavage. Re-evaluation of the CID spectra identified +78/+80-Da fragment ions in CID spectra of two peptides containing tyr656 and tyr711, confirming bromination. Interestingly, mutation of only tyr656, but not ser1395, thr1326 or ser1545, decreased topo IIß activity, suggesting a functional role for tyr656. These results, while identifying an important tyrosine in topo IIß, underscore the importance of careful interpretation of modifications having the same nominal mass.

Casein kinase I delta/epsilon phosphorylates topoisomerase IIalpha at serine-1106 and modulates DNA cleavage activity.

We previously reported that phosphorylation of topoisomerase (topo) IIalpha at serine-1106 (Ser-1106) regulates enzyme activity and sensitivity to topo II-targeted drugs. In this study we demonstrate that phosphorylation of Ser-1106, which is flanked by acidic amino acids, is regulated in vivo by casein kinase (CK) Idelta and/or CKIepsilon, but not by CKII. The CKI inhibitors, CKI-7 and IC261, reduced Ser-1106 phosphorylation and decreased formation of etoposide-stabilized topo II-DNA cleavable complex. In contrast, the CKII inhibitor, 5,6-dichlorobenzimidazole riboside, did not affect etoposide-stabilized topo II-DNA cleavable complex formation. Since, IC261 specifically targets the Ca(2+)-regulated isozymes, CKIdelta and CKIepsilon, we examined the effect of down-regulating these enzymes on Ser-1106 phosphorylation. Down-regulation of these isozymes with targeted si-RNAs led to hypophosphorylation of the Ser-1106 containing peptide. However, si-RNA-mediated down-regulation of CKIIalpha and alpha' did not alter Ser-1106 phosphorylation. Furthermore, reduced phosphorylation of Ser-1106, observed in HRR25 (CKIdelta/epsilon homologous gene)-deleted Saccharomyces cerevisiae cells transformed with human topo IIalpha, was enhanced following expression of human CKIepsilon. Down-regulation of CKIdelta and CKIepsilon also led to reduced formation of etoposide stabilized topo II-DNA cleavable complex. These results provide strong support for an essential role of CKIdelta/epsilon in phosphorylating Ser-1106 in human topo IIalpha and in regulating enzyme function.

Metabolism, excretion, and pharmacokinetics of oral brivanib in patients with advanced or metastatic solid tumors.

The goal of this study was to evaluate the pharmacokinetics, mass balance, metabolism, routes and extent of elimination, and safety of a single oral dose of (14)C-labeled brivanib alaninate and the safety and tolerability of brivanib after multiple doses in patients with advanced or metastatic solid tumors. This was a two-part, single-center, open-label, single oral-dose (part A) followed by multiple-dose (part B) study in patients with advanced or metastatic solid tumors. In part A, patients received a single dose of [(14)C]brivanib alaninate and in part B patients received 800 mg of nonradiolabeled brivanib alaninate every day. Four patients (two white, two black: two with non-small-cell lung cancer, one with ovarian cancer, and one with renal cell carcinoma) were treated in both parts. The median time to reach the maximal plasma concentration of brivanib was 1 h, geometric mean maximal plasma concentration was 6146 ng/ml, mean terminal half-life was 13.8 h, and geometric mean apparent oral clearance was 14.7 l/h. After a single oral dose of [(14)C]brivanib alaninate, 12.2 and 81.5% of administered radioactivity was recovered in urine and feces, respectively. Brivanib alaninate was completely converted to the active moiety, brivanib, and the predominant route of elimination was fecal. Renal excretion of unchanged brivanib was minimal. Brivanib was well tolerated; fatigue was the most frequent adverse event occurring in all patients and the most frequent treatment-related adverse event in three (75%). The best clinical response in one patient was stable disease; the other three had progressive disease. Brivanib alaninate was rapidly absorbed and extensively metabolized after a single 800-mg oral dose; the majority of drug-related radioactivity was excreted in feces.

Vascular endothelial growth factor polymorphisms: role in response and toxicity of tyrosine kinase inhibitors.

Angiogenesis is central to the growth of normal tissues and tumors. Inhibiting this pathway has been a strategy for drug development for tumors not responsive to most agents used in chemotherapy. Notably, signaling mediated by vascular endothelial growth factor (VEGF) is a key target because aberrant signaling via this pathway is frequently associated with neoangiogenesis in tumors. The drug-discovery effort to blunt VEGF signaling has led to the approval of bevacizumab and several receptor tyrosine kinase inhibitors (TKIs) that have shown efficacy in the clinical management of breast, colorectal, lung, and kidney cancer. Understanding the genetic variability in VEGF and VEGF receptor has led to identifying genotypic variations (single nucleotide polymorphisms [SNPs]) associated with treatment outcome and toxicity. Notably, identification of SNPs in VEGF associated with angiogenesis inhibitor treatment-induced hypertension and outcome provides exciting opportunities for personalized medicine to improve outcome and reduced toxicity with these novel TKIs.

RNA Immune Signatures from Pan-Cancer Analysis Are Prognostic for High-Grade Serous Ovarian Cancer and Other Female Cancers.

Immune cell infiltrates within the tumor microenvironment can influence treatment response and outcome in several cancers. In this study, we developed RNA-based immune signatures from pan-cancer analysis that could serve as potential markers across tumor types and tested them for association with outcome in high-grade serous ovarian cancer (HGSOC) and other female cancers. Pan-cancer RNA-Seq cluster analysis of immune-related gene expression profiles in The Cancer Genome Atlas (TCGA) from 29 different solid tumors (4446 specimens) identified distinct but concordant gene signatures. Among these immune signatures, Cytotoxic Lymphocyte Immune Signature (CLIS), T-cell trafficking (TCT), and the TCT to M2 tumor-associated macrophage (M2TAM) ratio (TCT:M2TAM) were significantly (p < 0.05) associated with overall survival (OS), using multivariable Cox proportional hazards regression models, in a discovery cohort and two independent validation cohorts of HGSOC patients. Notably, the TCT:M2TAM ratio was highly significant (p ≤ 0.000001) in two HGSOC cohorts. Immune signatures were also significant (p < 0.05) in the presence of tumor cytoreduction, BRCA1/2 mutation, and COL2A1 expression. Importantly, the CLIS and TCT signatures were also validated for prognostic significance (p < 0.05) in TCGA cohorts for endometrial and high tumor mutational burden (Hi-TMB) breast cancer. These immune signatures also have the potential for being predictive in other cancers and for patients following different treatment strategies.

Targeting Aurora A Kinase (AAK) in Platinum-Resistant High Grade Serous Ovarian Cancer.

Aurora A kinase (AAK) involved in G2-M transition is functionally involved in centrosome maturation and maintaining an active spindle assembly checkpoint. We tested the hypothesis that in platinum-taxane resistant high grade serous ovarian cancer (HGSOC) inhibition of AAK involved in G2-M transition would enhance the anti-tumor activity of cisplatin (CP) or paclitaxel (PT). Using HGSOC cell lines from platinum-taxane refractory patients that do not harbor BRCA1/2 mutations, we tested the anti-tumor activity of CP, or PT alone or in combination with the AAK inhibitor alisertib (AL). Treatment with CP for 3 h or PT for 6 h followed sequentially by AL for 48 h led to a significant decrease in cell survival (p < 0.001) compared to treatment with either drug alone in HGSOC cells but not in immortalized normal human ovarian surface epithelium or normal human fallopian tube secretory epithelium cells. The treatment with CP or PT followed by AL also led to a significant increase in reactive oxygen species (p < 0.05), apoptosis (p < 0.001) and accumulation of cells in G2/M that was accompanied by a modest increase in expression of AAK. Downregulation of AAK, but not aurora B kinase, with targeted siRNAs also significantly enhanced apoptosis by CP or PT, suggesting that AL specifically targeted AAK. In summary, in HGSOC without BRCA1/2 mutations, CP, or PT resistance can potentially be circumvented by sequential treatment with AL that inhibits AAK involved in G2-M transition.

Association of immunophenotype with expression of topoisomerase II α and ß in adult acute myeloid leukemia.

Anthracyclines used in the treatment of acute myelogenous leukemia (AML) inhibit the activity of the mammalian topoisomerase II (topo II) isoforms, topo II α and topo IIß. In 230 patients with non-M3 AML who received frontline ara-C/daunorubicin we determined expression of topo IIα and topo IIß by RT-PCR and its relationship to immunophenotype (IP) and outcomes. Treatment outcomes were analyzed by logistic or Cox regression. In 211 patients, available for analysis, topo IIα expression was significantly lower than topo IIß (P < 0.0001). In contrast to topo IIα, topo IIß was significantly associated with blast percentage in marrow or blood (P = 0.0001), CD7 (P = 0.01), CD14 (P < 0.0001) and CD54 (P < 0.0001). Event free survival was worse for CD56-negative compared to CD56-high (HR = 1.9, 95% CI [1.0-3.5], p = 0.04), and overall survival was worse for CD-15 low as compared to CD15-high (HR = 2.2, 95% CI [1.1-4.2], p = 0.02). Ingenuity pathway analysis indicated topo IIß and immunophenotype markers in a network associated with cell-to-cell signaling, hematological system development/function and inflammatory response. Topo IIß expression reflects disease biology of highly proliferative disease and distinct IP but does not appear to be an independent variable influencing outcome in adult AML patients treated with anthracycline-based therapy.

von Hippel-Lindau gene status and response to vascular endothelial growth factor targeted therapy for metastatic clear cell renal cell carcinoma.

PURPOSE: The von Hippel-Lindau (VHL) gene is often inactivated (by mutation or promoter hypermethylation) in renal cell carcinoma but the relation to therapeutic outcome is unclear. MATERIALS AND METHODS: Patients with metastatic clear cell renal cell carcinoma with available baseline tumor samples who received vascular endothelial growth factor targeted therapy were included in analysis. Patient characteristics, VHL gene status and clinical outcome were documented. Our primary end point was to test for response rate in relation to VHL inactivation. Progression-free survival and overall survival in relation to VHL status were investigated as secondary end points. RESULTS: A total of 123 patients were evaluable. Response rate, median progression-free survival and median overall survival were 37% (95% CI 28-46), 10.8 (95% CI 7.7-14.8) and 29.8 (CI not estimable) months, respectively. Patients with VHL inactivation had a response rate of 41% vs 31% for those with wild-type VHL (p = 0.34). Patients with loss of function mutations (frameshift, nonsense, splice and in-frame deletions/insertions) had a 52% response rate vs 31% with wild-type VHL (p = 0.04). On multivariate analysis the presence of a loss of function mutation remained an independent prognostic factor associated with improved response. Progression-free survival and overall survival were not significantly different based on VHL status. CONCLUSIONS: To our knowledge this is the largest analysis investigating the impact of VHL inactivation on the outcome of vascular endothelial growth factor targeted agents in metastatic renal cell carcinoma. We did not find a statistically significant increase in response to vascular endothelial growth factor targeted agents in patients with VHL inactivation. Loss of function mutations identified a population of patients with a greater response. Investigation of downstream markers is under way.

Ovarian cancer immuno-reactive antigen domain containing 1 (OCIAD1), a key player in ovarian cancer cell adhesion.

OBJECTIVES: To identify proteins unique to metastatic ovarian cancer and test their potential involvement in cell adhesion. METHODS: We purified plasma membrane from paired metastatic and primary tumor tissues from patients with stage IIIC ovarian cancer. Membrane proteins unique to metastases were identified by liquid chromatographic mass spectrometry (LC-MS). The role of one of the identified proteins, ovarian cancer immuno-reactive antigen domain containing 1 (OCIAD1) in cell adhesion was determined in the presence of LPA using both over-expression and down regulation approaches. RESULTS: We identified a differentially expressed 29 kDa protein as OCIAD1 over-expressed in metastatic tissues, when compared to primary tumor tissues. OCIAD1 over-expression in HEY ovarian cancer cells increased LPA-induced, but not basal level cell adhesion to extracellular matrix proteins collagen I and laminin 10/11. This enhancement was not blocked by LY294002 and GF109203X, suggesting that OCIAD1 does not use PKC and PI3K signaling pathways to exert its effect on adhesion. In addition, LPA induced cell adhesion to collagen I was unaffected by paclitaxel (5 microM) in OCIAD1 overexpressing cells. CONCLUSIONS: This is the first report that OCIAD1 is over-expressed in metastatic ovarian cancer tissues. The effect of OCIAD1 on cell adhesion may be related to its function in ovarian cancer. Failure of paclitaxel to affect ovarian cancer cell adhesion in presence of OCIAD1 raises the possibility of OCIAD1's role in tumor metastasis. Ongoing studies using a mouse orthotopic LPA-dependent ovarian cancer metastasis model are focused on strategies to inhibit the potential role of OCIAD1 in tumor metastasis.

Phase I/II trial of 5-fluorouracil and a noncytotoxic dose level of suramin in patients with metastatic renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is recognized as a neoplasm resistant to chemotherapy. In vitro experiments demonstrated that suramin, at noncytotoxic doses, enhanced the activity of chemotherapy including 5-fluorouracil (5-FU) in xenograft models. PATIENTS AND METHODS: A phase I/II trial of noncytotoxic suramin in combination with weekly 5-FU in patients with metastatic RCC was conducted. The treatment consisted of intravenous (i.v.) suramin followed by a 500 mg/m2 i.v. bolus of 5-FU given 4.5 hours after starting suramin. In the phase I portion, a cohort of 6 patients received a suramin dose calculated to achieve a plasma level of 10-50 micromol/L. Therapy was administered once weekly for 6 doses, followed by 2 weeks off. This was followed by a phase II portion in which the primary goal was to determine the objective response rate. RESULTS: Twenty-three patients were enrolled in the study: 6 in the phase I portion and 17 in phase II. Seventy-eight percent of patients were men, the mean age was 58.8 years, 96% had previous nephrectomy, and 70% had received previous systemic therapy. Histologic subtype was clear cell in 91%. Dose-limiting toxicity was observed in 1 of 6 patients (grade 3 hypersensitivity related to suramin infusion). The suramin dosing nomogram used in phase I and II portions of the trial yielded the desired plasma level of 10-50 micromol/L from 4.5 hours to 48 hours after infusion in 94 of 115 treatments. No objective responses were noted, and the median time to treatment failure was 2.5 months. The major toxicities (all grades) were fatigue (83%), nausea/vomiting (78%), diarrhea (61%), and chills (61%). CONCLUSION: Suramin levels expected to reverse fibroblast growth factor-induced resistance can be achieved with the dosing regimen used in this study. The toxicity observed with suramin and 5-FU was acceptable. The combination does not have clinical activity in patients with metastatic RCC.