Keratin 5 overexpression is associated with serous ovarian cancer recurrence and chemotherapy resistance

This study investigated the clinical significance of keratin 5 and 6 expression in serous ovarian cancer progression and chemotherapy resistance. KRT5 and KRT6 (KRT6A, KRT6B & KRT6C) gene expression was assessed in publically available serous ovarian cancer data sets, ovarian cancer cell lines and primary serous ovarian cancer cells. Monoclonal antibodies which detect both K5/6 or only K5 were used to assess protein expression in ovarian cancer cell lines and a cohort of high grade serous ovarian carcinomas at surgery (n = 117) and after neoadjuvant chemotherapy (n = 21). Survival analyses showed that high KRT5 mRNA in stage III/IV serous ovarian cancers was significantly associated with reduced progression-free (HR 1.38, P < 0.0001) and overall survival (HR 1.28, P = 0.013) whilst high KRT6 mRNA was only associated with reduced progression-free survival (HR 1.2, P = 0.031). Both high K5/6 (≥ 10%, HR 1.78 95% CI; 1.03−2.65, P = 0.017) and high K5 (≥ 10%, HR 1.90, 95% CI; 1.12−3.19, P = 0.017) were associated with an increased risk of disease recurrence. KRT5 but not KRT6C mRNA expression was increased in chemotherapy resistant primary serous ovarian cancer cells compared to chemotherapy sensitive cells. The proportion of serous ovarian carcinomas with high K5/6 or high K5 immunostaining was significantly increased following neoadjuvant chemotherapy. K5 can be used to predict serous ovarian cancer prognosis and identify cancer cells that are resistant to chemotherapy. Developing strategies to target K5 may therefore improve serous ovarian cancer survival.Carmela Ricciardelli, Noor A Lokman, Carmen E Pyragius, Miranda P Ween, Anne M Macpherson, Andrew Ruszkiewicz, Peter Hoffmann, Martin K Oehle

IGFBP7 is associated with poor prognosis in oesophageal adenocarcinoma and is regulated by promoter DNA methylation

BACKGROUND: We examined whether silencing of IGFBP7 was associated with survival in patients with oesophageal adenocarcinoma. METHODS: Protein expression of IGFBP7 was determined using immunohistochemistry in a tissue microarray representing tumours from 65 patients with oesophageal adenocarcinoma who had not had neoadjuvant therapy. DNA methylation of the IGFBP7 promoter was determined with the melt curve analysis in cell lines and patient tissues. RESULTS: Expression of IGFBP7 was observed in the oesophageal adenocarcinoma of 34 out of 65 (52%) patients and was associated with significantly reduced median (11 vs 92 months) and 5-year survival (25% vs 52%). Multivariate analysis identified expression as an independent prognostic indicator for survival (hazard ratio=3.24, 95% confidence interval=1.58-6.67, P-value=0.0014). Hypermethylation of IGFBP7 was associated with silencing of gene expression in cell lines and patient tissues (P-value=0.0225). Methylation was observed in the squamous mucosa of 2 out of 15 (13%) patients with Barrett's oesophagus and 3 out of 17 (18%) with oesophageal adenocarcinoma. Methylation was observed in 14 out of 18 (78%) of biopsies of Barrett's mucosa and 23 out of 34 (68%) patients with oesophageal adenocarcinoma. CONCLUSION: Reduced IGFBP7 protein expression was associated with longer survival in patients with oesophageal adenocarcinoma. Methylation of the IGFBP7 promoter was associated with silencing of gene expression and was frequent in Barrett's oesophagus and oesophageal adenocarcinoma.E Smith, A R Ruszkiewicz, G G Jamieson, and P A Dre

Clinicopathological predictors of benefit from adjuvant chemotherapy for stage C colorectal cancer: microsatellite unstable cases benefit

Aim: In colorectal cancer (CRC), adjuvant therapy is offered on the basis of stage and attempts to identify factors to better target treatment have not been successful. Recent work suggested that mismatch repair deficient CRCs may not benefit from 5FU adjuvant chemotherapy but studies remain conflicting. We aimed to determine if gender, tumor site, tumor pathological characteristics and microsatellite instability (MSI) predict survival benefit from adjuvant chemotherapy in stage C CRC. Methods: Data were collated on ACPS (Australian Clinico-pathological Staging System) stage C CRC cases that underwent curative resection over a 23-year period. Pathology was reevaluated, DNA was extracted from the formalin-fixed paraffin specimen, and MSI status was established by BAT26 instability. Multivariate analysis was performed using Cox proportional hazard model and effects modification interaction testing. Results: In total 814 unselected cases were included, of whom 37% received chemotherapy. Seventy-seven cases exhibited MSI. Overall, adjuvant chemotherapy produced a cancer-specific survival benefit (HR 0.52, 95% CI 0.39-0.70; P <0.0001). On interaction testing, none of the examined parameters significantly influenced the magnitude of that survival benefit. Chemotherapy was beneficial in both the MSI (HR 0.08, 95% CI 0.02-0.27; P = <0.0001) and the microsatellite stable cohort (HR 0.62, 95% CI 0.47-0.81; P = 0.001). Conclusion: These results suggest that survival benefit from 5FU adjuvant chemotherapy for stage C CRC does not vary according to gender, site of tumor, pathological characteristics or MSI status. This study suggests that it would be unwise to exclude patients from being offered adjuvant chemotherapy on the basis of MSI.Michelle L. Thomas, Peter J. Hewett, Andrew R. Ruszkiewicz, and James W.E. Moor

Measuring the combinatorial expression of solute transporters and metalloproteinases transcripts in colorectal cancer

Published: 19 August 2009Background: It was hypothesised that colorectal cancer (CRC) could be diagnosed in biopsies by measuring the combined expression of a small set of well known genes. Genes were chosen based on their role in either the breakdown of the extracellular matrix or with changes in cellular metabolism both of which are associated with CRC progression. Findings: Gene expression data derived from quantitative real-time PCR for the solute transporter carriers (SLCs) and the invasion-mediating matrix metalloproteinases (MMPs) were examined using a Linear Descriminant Analysis (LDA). The combination of MMP-7 and SLC5A8 was found to be the most predictive of CRC. Conclusion: A combinatorial analysis technique is an effective method for both furthering our understanding on the molecular basis of some aspects of CRC, as well as for leveraging well defined cancer-related gene sets to identify cancer. In this instance, the combination of MMP-7 and SLC5A8 were optimal for identifying CRC.Caroline A. Kerr, Robert Dunne, Barney M. Hines, Michelle Zucker, Leah Cosgrove, Andrew Ruszkiewicz, Trevor Lockett and Richard Hea

Metallothionien 3 expression is frequently down-regulated in oesophageal squamous cell carcinoma by DNA methylation

BACKGROUND: Metallothionein 3 (MT3) inhibits growth in a variety of cell types. We measured MT3 gene expression by RT-PCR, and DNA methylation in the MT3 promoter by combined bisulphite restriction analysis, in four oesophageal cancer cell lines and the resected oesophagus from 64 patients with oesophageal squamous cell carcinoma (SCC). RESULTS: MT3 expression was not detected in one of the four oesophageal cell lines. The MT3 promoter was methylated in all of the oesophageal cell lines, but the degree of methylation was greater in the non-expressing cell line. After treatment with 5-aza-2'-deoxycytidine there was a reduction in the degree of methylation, and an increase in MT3 expression, in each of the cell lines (p < 0.01). Methylation was detected in 52% (33 of 64) of primary SCC and 3% (2 of 62) of histologically normal resection margins. MT3 expression was measured in 29 tumours, 17 of which had methylation of MT3. The expression of MT3 was significantly less in the methylated tumours compared to either the unmethylated tumours (p = 0.03), or the matched margin (p = 0.0005). There was not a significant difference in MT3 expression between the tumour and the margin from patients with unmethylated tumour. No correlations were observed between methylation of MT3 and survival time, patient age, gender, smoking or drinking history, tumour stage, volume, or lymph node involvement. CONCLUSION: We conclude that MT3 expression is frequently down-regulated in oesophageal SCC, by DNA methylation, but that this is not a prognostic indicator

Methylation levels of LINE-1 repeats and CpG island loci are inversely related in normal colonic mucosa

Division of Translational and Clinical Oncolog

Desmoglein 2 promotes vasculogenic mimicry in melanoma and is associated with poor clinical outcome

Tumors can develop a blood supply not only by promoting angiogenesis but also by forming vessel-like structures directly from tumor cells, known as vasculogenic mimicry (VM). Understanding mechanisms that regulate VM is important, as these might be exploitable to inhibit tumor progression. Here, we reveal the adhesion molecule desmoglein 2 (DSG2) as a novel mediator of VM in melanoma. Analysis of patient-derived melanoma cell lines and tumor tissues, and interrogation of The Cancer Genome Atlas (TCGA) data, revealed that DSG2 is frequently overexpressed in primary and metastatic melanomas compared to normal melanocytes. Notably, this overexpression was associated with poor clinical outcome. DSG2+ melanoma cells self-organized into tube-like structures on Matrigel, indicative of VM activity, which was inhibited by DSG2 knockdown or treatment with a DSG2-blocking peptide. Mechanistic studies revealed that DSG2 regulates adhesion and cell-cell interactions during tube formation, but does not control melanoma cell viability, proliferation or motility. Finally, analysis of patient tumors revealed a correlation between DSG2 expression, VM network density and expression of VM-associated genes. These studies identify DSG2 as a key regulator of VM activity in human melanoma and suggest this molecule might be therapeutically targeted to reduce tumor blood supply and metastatic spread.Lih Yin Tan, Chris Mintoff, M. Zahied Johan, Brenton W. Ebert, Clare Fedele, You Fang Zhang, Pacman Szeto, Karen E. Sheppard, Grant A. McArthur, Erwin Foster-Smith, Andrew Ruszkiewicz, Michael P. Brown, Claudine S. Bonder, Mark Shackleton, Lisa M. Eber

The La antigen is over-expressed in lung cancer and is a selective dead cancer cell target for radioimmunotherapy using the la-specific antibody APOMAB(R)

Background: The lupus-associated (La)-specific murine monoclonal antibody DAB4 (APOMAB®) specifically binds dead cancer cells. Using DAB4, we examined La expression in human lung cancer samples to assess its suitability as a cancer-selective therapeutic target. We evaluated the safety and effectiveness of radioimmunotherapy (RIT) using DAB4 radiolabeled with Lutetium-177 (177Lu) in the murine Lewis Lung (LL2) carcinoma model, and determined whether combining RIT with DNA-damaging cisplatin-based chemotherapy, a PARP inhibitor (PARPi), or both alters treatment responses. Methods: The expression of La mRNA in human lung cancer samples was analysed using the online database Oncomine, and the protein expression of La was examined using a TissueFocus Cancer Survey Tissue Microarray. The binding of DAB4 to cisplatin-treated LL2 cells was assessed in vitro. LL2 tumour-bearing mice were administered escalating doses of 177Lu-DAB4 alone or in combination with chemotherapy, and tumour growth and survival measured. Biodistribution analysis was used to determine tissue uptake of 177Lu-DAB4 or its isotype control (177Lu-Sal5), when delivered alone or after chemotherapy. PARPi (rucaparib; AG-014699) was combined with chemotherapy and the effects of combined treatment on tumour growth, tumour cell DNA damage and death, and intratumoural DAB4 binding were also analysed. The effect of the triple combination of PARPi, chemotherapy and 177Lu-DAB4 on tumour growth and survival of LL2 tumour-bearing mice was tested. Results: La was over-expressed at both mRNA and protein levels in surgical specimens of human lung cancer and the over-expression of La mRNA conferred a poorer prognosis. DAB4 bound specifically to cisplatin-induced dead LL2 cells in vitro. An anti-tumour dose response was observed when escalating doses of 177Lu-DAB4 were delivered in vivo, with supra-additive responses observed when chemotherapy was combined with 177Lu-DAB4. Combining PARPi with chemotherapy was more effective than chemotherapy alone with increased tumour cell DNA damage and death, and intratumoural DAB4 binding. The combination of PARPi, chemotherapy and 177Lu-DAB4 was well-tolerated and maximised tumour growth delay. Conclusions: The La antigen represents a dead cancer cell-specific target in lung cancer, and DAB4 specifically targeted tumour tissue in vivo, particularly after chemotherapy. Tumour uptake of DAB4 increased further after the combination of PARPi and chemotherapy, which generated new dead tumour cell-binding targets. Consequently, combining 177Lu-DAB4 with PARPi and chemotherapy produced the greatest anti-tumour response. Therefore, the triple combination of PARPi, chemotherapy and RIT may have broad clinical utility.Alexander H Staudacher, Fares Al-Ejeh, Cara K Fraser, Jocelyn M Darby, David M Roder, Andrew Ruszkiewicz, Jim Manavis and Michael P Brow

Low expression of gamma-glutamyl hydrolase mRNA in primary colorectal cancer with the CpG island methylator phenotype

Division of Translational and Clinical Oncolog