Irena Klima

Abnormal p53 expression is rare in clinically localized human prostate cancer: Comparison between immunohistochemical and molecular detection of p53 mutations

We assessed the frequency and molecular basis of p53 mutations in clinically localized prostatic adenocarcinoma.

Agents Used for Chemoprevention of Prostate Cancer May Influence PSA Secretion Independently of Cell Growth in the LNCaP model of Human Prostate Cancer Progression

The aim of this study was to evaluate the inhibitory growth effects of different potential chemopreventive agents in vitro and to determine their influence on PSA mRNA and protein expression with an established screening platform.

Prostate Specific Antigen Expression Does Not Necessarily Correlate With Prostate Cancer Cell Growth

PURPOSE The antiproliferative effects of pharmacological agents used for androgen ablative therapy in prostate cancer, including goserelin, bicalutamide and cyproterone acetate (Fluka Chemie, Buchs, Switzerland), were tested in vitro. It was determined whether they affected prostate specific antigen mRNA and protein expression independent of growth inhibition. MATERIALS AND METHODS Goserelin, bicalutamide (AstraZeneca, Zug, Switzerland) and cyproterone acetate were added to prostate specific antigen expressing, androgen dependent LNCaP and androgen independent C4-2 cell line (Urocor, Oklahoma City, Oklahoma) cultures. Proliferation was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assay (Roche, Mannheim, Germany). Prostate specific antigen mRNA expression was assessed by quantitative real-time polymerase chain reaction. Secreted prostate specific antigen protein levels were quantified by microparticle enzyme-immunoassay. RESULTS Goserelin inhibited cell growth and prostate specific antigen protein secretion in LNCaP and C4-2 cells. Prostate specific antigen mRNA expression was not decreased. Bicalutamide did not affect cell growth or prostate specific antigen mRNA expression in LNCaP or C4-2 cells, although it significantly decreased prostate specific antigen protein secretion in LNCaP and to a lesser extent in C4-2 cells. Cyproterone acetate decreased the growth of C4-2 but not of LNCaP cells. It did not affect prostate specific antigen mRNA or protein expression in either cell line. CONCLUSIONS Prostate specific antigen expression does not necessarily correlate with cell growth. Without a substantial effect on cell growth bicalutamide lowers prostate specific antigen synthesis, whereas cyproterone acetate decreases cell growth with no effect on prostate specific antigen secretion. Prostate specific antigen expression may be influenced by growth inhibition but also by altered mRNA and protein levels depending on the agent, its concentration and the cell line evaluated. For interpreting clinical trials prostate specific antigen is not necessarily a surrogate end point marker for a treatment effect on prostate cancer cell growth.

CRIPTO promotes an aggressive tumour phenotype and resistance to treatment in hepatocellular carcinoma: Role of CRIPTO in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer‐related death worldwide. Despite increasing treatment options for this disease, prognosis remains poor. CRIPTO (TDGF1) protein is expressed at high levels in several human tumours and promotes oncogenic phenotype. Its expression has been correlated to poor prognosis in HCC. In this study, we aimed to elucidate the basis for the effects of CRIPTO in HCC. We investigated CRIPTO expression levels in three cohorts of clinical cirrhotic and HCC specimens. We addressed the role of CRIPTO in hepatic tumourigenesis using Cre‐loxP‐controlled lentiviral vectors expressing CRIPTO in cell line‐derived xenografts. Responses to standard treatments (sorafenib, doxorubicin) were assessed directly on xenograft‐derived ex vivo tumour slices. CRIPTO‐overexpressing patient‐derived xenografts were established and used for ex vivo drug response assays. The effects of sorafenib and doxorubicin treatment in combination with a CRIPTO pathway inhibitor were tested in ex vivo cultures of xenograft models and 3D cultures. CRIPTO protein was found highly expressed in human cirrhosis and hepatocellular carcinoma specimens but not in those of healthy participants. Stable overexpression of CRIPTO in human HepG2 cells caused epithelial‐to‐mesenchymal transition, increased expression of cancer stem cell markers, and enhanced cell proliferation and migration. HepG2‐CRIPTO cells formed tumours when injected into immune‐compromised mice, whereas HepG2 cells lacking stable CRIPTO overexpression did not. High‐level CRIPTO expression in xenograft models was associated with resistance to sorafenib, which could be modulated using a CRIPTO pathway inhibitor in ex vivo tumour slices. Our data suggest that a subgroup of CRIPTO‐expressing HCC patients may benefit from a combinatorial treatment scheme and that sorafenib resistance may be circumvented by inhibition of the CRIPTO pathway. Copyright

PD33-06 MCAM SUPPORTS THE AGGRESSIVE PHENOTYPE IN HUMAN PROSTATE CANCER

METHODS: Formalin-fixed paraffin embedded (FFPE) primary prostate cancer samples were obtained from the Welsh Cancer Bank. Targeted-NGS was performed using the Life Technologies Ion Torrent: Ion AmpliSeq Cancer Hotspot Panel v2 and the Ion Personal Genome Machine sequencer. The hotspot panel covers ~2800 COSMIC mutations of 50 oncogenes and tumour suppressor genes. Standard IHC techniques were also used concentrating on markers of the Wnt, PI3Kinase (PI3K) and MAP-Kinase (MAPK) oncogenic signalling pathways. RESULTS: 61 primary prostate cancer samples were sequenced, 58 from radical retropubic prostatectomy (RRP) specimens and 3 from transurethral resection of prostate (TURP) sections, with a range of Gleason Scores (GS). 21/61 (34.4%) samples harboured a mutation in a cell cycle pathway gene such as TP53 or RB1 and 3/61 (4.9%) in a DNA repair gene such as ATM. 10/61 (16.5%) of samples harboured a mutation in a gene associated with the Wnt pathway such as APC or CTNNB1. 14/61 (23.0%) of samples analysed had a mutation in a gene commonly associated with the PI3K pathway such as PTEN or AKT1. 5/61 (8.2%) had a mutation in a gene associated with the MAPK pathway such as KRAS. IHC profiles were analysed on 317 prostate samples: 73 normal and 244 cancers. There was greater expression of markers associated with Wnt, PI3K and MAPK signalling pathways in prostate cancer samples when compared to normal samples. There was greater expression in high-risk GSs with some markers associated with biochemical recurrence following RRP. Furthermore, we were able to separate lowand high-risk GS samples based on molecular profiles using markers of the Wnt, PI3K and MAPK and principle components analysis. CONCLUSIONS: Targeted NGS and IHC can identify recurrent mutations and signalling pathway aberrations within primary prostate cancer samples, which have potential to be targeted and used in routine clinical practice. In addition, the molecular signatures of lowand highrisk are different and can be separated using a combination of markers and IHC. This finding could explain the marked difference in the behaviours of these tumours types.

MP44-17 PATIENT DERIVED XENOGRAFTS AS PRECLINICAL MODELS OF UROLOGICAL MALIGNANCIES

INTRODUCTION AND OBJECTIVES: The cross-talk of mammalian target of rapamycin (mTOR) pathway is clinical limitation of mTOR inhibitor for the treatment of urothelial carcinoma (URCa). This study is to search mTOR pathway downstream genes to overcome cross-talk at non muscle invasive low grade (LG)-URCa of the bladder. METHODS: Gene expression patterns, gene ontology, and gene clustering by dual (p70S6K and S6K) siRNAs or rapamycin in 253J and TR4 cell lines were investigated by microarray analysis and we selected mTOR/S6K pathway downstream genes which were suppressed to siRNAs, and rapamycin up-regulated or rapamycin down-regulated. We validated mTOR downstream genes with immunohistochemistry using tissue microarray of 90 non-muscle invasive LG-URCa patients whether genes can predict clinical outcomes, and knockout study to evaluate the synergistic effect with rapamycin. RESULTS: In the microarray analysis, we selected mTOR pathway downstream genes which consisted of 4 rapamycin downregulated (FOXM1, KIF14, MYBL2, and UHRF1), and 4 rapamycin upregulated (GPR87, NBR1, VASH1 and PRIMA1). In tissue microarray, FOXM1, KIF14, and NBR1 were more expressed at T1, and MYBL2 and PRIMA1 were more expressed at tumor size more than 3 cm. In multivariate Cox regression model, KIF14 and NBR1 were significant predictors of recurrence in non-muscle invasive LG-URCa of the bladder. In NBR1 knock out model, rapamycin treatment showed synergistic effect to inhibit cell viability and colony forming ability compared to rapamycin only. CONCLUSIONS: KIF14 and NBR1 were mTOR/S6K pathway downstream genes to predict recurrence in non-muscle invasive LGURCa of the bladder and NBR1 knockout overcome the rapamycin coss-talk.

MP41-10 MODELLING PROSTATE CANCER USING PRIMARY AND METASTATIC CANCEROIDS

INTRODUCTION AND OBJECTIVES: A recent study showed that adipose-derived stem cells are able to counteract urethral stricture formation in rats. The aim of this study was to evaluate the feasibility of autologous adipose derived stromal vascular fraction (SVF) transplantation into male urethra stricture walls after direct vision incision of urethra (DVIU). METHODS: A prospective clinical study was undertaken after ethics approval and appropriate patient consent. The inclusion criteria were: male patients older than 18 years, with single short recurrent not-obliterating urethral stricture (<2 cm). The exclusion criteria were: patients not willing to consent, multiple strictures and those not deemed suitable for endoscopic management. Failure was defined as need for further interventions. Preoperative workup included history, examination, retrograde urethrogram (RGU), voiding cystourethrogram (VCUG), urine culture, renal function tests, AUA score, IIEF, PROM. Plastic surgery team performs liposuction to extract 50 mls of fat from the patient’s abdominal wall. 50ml fat-SVF was obtained using a Goog Manufacturing Practice collagenase (Celase , Cytori Therapeutics, San Diego, USA) according to a standard protocol. SVF was diluted in 5 ml saline solution for the injection. A cystoscopy was performed and the stricture evaluated, a glide wire was placed and an urethrotomy performed at 6 o’clock position in bulbar urethra. Gide wire was left in situ. The SVF solution was injected at the site of the stricture and on either side of the stricture. A 12 Fr urinary catheter was placed. The urinary catheter was removed after 24 hrs. Follow up was of 3.5 months. RESULTS: Two patients were included in the study. The main characteristics are reported in table 1. No local or systemic side effects or complications were recorded. No recurrence of urethral stricture was detected in both patients after 3.5 months. Table 1 CONCLUSIONS: This is the first study to demonstrate a successful autologous SVF transplantation in male urethral stricture after DVIU. Further studies are necessary to confirm the efficacy of SVF in preventing urethral stricture recurrence.

ALK1Fc Suppresses the Human Prostate Cancer Growth in in Vitro and in Vivo Preclinical Models

Prostate cancer is the second most common cancer in men and lethality is normally associated with the consequences of metastasis rather than the primary tumor. Therefore, targeting the molecular pathways that underlie dissemination of primary tumor cells and the formation of metastases has a great clinical value. Bone morphogenetic proteins (BMPs) play a critical role in tumor progression and this study focuses on the role of BMP9- Activin receptor-Like Kinase 1 and 2 (ALK1 and ALK2) axis in prostate cancer. In order to study the effect of BMP9 in vitro and in vivo on cancer cells and tumor growth, we used a soluble chimeric protein consisting of the ALK1 extracellular domain (ECD) fused to human Fc (ALK1Fc) that prevents binding of BMP9 to its cell surface receptors and thereby blocks its ability to activate downstream signaling. ALK1Fc sequesters BMP9 and the closely related BMP10 while preserving the activation of ALK1 and ALK2 through other ligands. We show that ALK1Fc acts in vitro to decrease BMP9-mediated signaling and proliferation of prostate cancer cells with tumor initiating and metastatic potential. In line with these observations, we demonstrate that ALK1Fc also reduces tumor cell proliferation and tumor growth in vivo in an orthotopic transplantation model, as well as in the human patient derived xenograft BM18. Furthermore, we also provide evidence for crosstalk between BMP9 and NOTCH and find that ALK1Fc inhibits NOTCH signaling in human prostate cancer cells and blocks the induction of the NOTCH target Aldehyde dehydrogenase member ALDH1A1, which is a clinically relevant marker associated with poor survival and advanced-stage prostate cancer. Our study provides the first demonstration that ALK1Fc inhibits prostate cancer progression, identifying BMP9 as a putative therapeutic target and ALK1Fc as a potential therapy. Altogether, these findings support the validity of ongoing clinical development of drugs blocking ALK1 and ALK2 receptor activity.

OP1-06 IN MOLECULAR DETECTION OF OCCULT LYMPH NODE METASTASES EPITHELIAL MARKERS ARE HERE TO STAY

abnormal promoter DNA methylation for selected genes using mRNA expression by quantitative RT real time PCR. Immunohistochemistry was used to confirm testicular expression and potential importance in spermatogenesis RESULTS: Differentially methylated CpG sites (w20K) were identified using an F Test (p 30% difference in methylation within promoter region of men with NOA and fertile controls. Of the aberrantly methylated CpGs, 13 were hypomethylated and 11 were hypermethylated groups. From the top 11 hypermethylated genes, six genes (MRI1, DCAF12L1, TMEM95, CECR2, DDR1, NPHS2) were selected for validation since they were shown to be expressed in testis. Of the 6 genes validated with qPCR, DDR1 showed aberrant gene expression pattern. Four (21%) patients out of the 19 NOA men had lower expression levels (1.8x) of DDR1, whereas two (10.5%) men had higher expression levels (2.5x) of DDR1compared to fertile men (p<0.05). Immunohistochemical analysis suggests presence of DDR1 within cytoplasm of germ cells in fertile men and men with maturation arrest histology. DDR1 protein is absent in men with Sertoli cell only or germ cell aplasia. CONCLUSIONS: Aberrant expression of DDR1 is associated with NOA. The functional relevance of abnormal methylation of DDR1 to NOA warrants further investigation