441Microfluidic real time reverse transcriptase polymerase chain reaction assay for detecting circulating cells in prostate cancer

441

442

MICROFLUIDIC REAL TIME REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION ASSAY FOR DETECTING CIRCULATING CELLS IN PROSTATE CANCER

INDIVIDUALISATION OF THE BIOPSY THE PROSTATE GLAND VOLUME DETECTION

Ross D. 1, Hart C.~, Mcintyre I.2, Raarani V.3, Brown M.I, Clarke N. 1

Eskicorapci S.. Guliyev E, Akdogan B., Dogan S., Kendi S., {)zen H.

Cancer Research UK Paterson Institute, Prompt Genito-Urinary Cancer Research Group, Manchester, United Kingdom, 2Central Manchester NHS Trust, Department of Urology, Manchester, United Kingdom, ~Christie Hospital NHS Trust, Department of Urology, Manchester, United Kingdom

Hacettepe University - Fac. of Medicine, Urology, Ankara, Turkey

INTRODUCTION & OBJECTIVES: The evolution of multi-marker quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays for the detection of circulating prostate epithelial cells (PEC) has improved the clinical potential for molecular staging of disease. However, labour intensive methodologies, inherent mxors, poor reproducibility and lack of standardisation limits the potential of this technique in large clinical trials. Commercially produced microfiuidic assays may offer solutions m these technical problems but their clinical capabilities are yet to be defined. The aim of this study was to compare the in vitro and clinical performance of a novel microfiuidic multi-marker qRT-PCR assay with an established 96 well format design. MATERIAL & METHODS: Nucleotide primers and probes for PSA, prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA) and human kallikrein 2 (HK2), were designed and optimised for a 96 well format qRT-PCR assay. A micmfluidic assay for the same markers was manufactured by Applied Biosystems TM. The integrity of both assays was confirmed using RNA derived from LNCaP cells. In vitro sensitivity was determined by serial dilution of LNCaP cells in female peripheral blood (PB). PB and bone marrow (BM) samples from patients with well characterised prostate cancer (CAP) and disease free controIs were then assayed using the two techniques. RESULTS: The expression of all markers by the LNCaP cell line was contirmed using both assays. In vitro, the micmfluidic assay was 10 fold less sensitive than the 96 well formats. Smnples from 28 men with CaP (13 hormone escaped disease, 12 hormone ngive metastases, 1 locally advanced and 2 clinically localised CaP) and 6 controls were evaluated. The qualitative results are presented below detailing the proportion of patients positive for each marker compared between the two techniques.

PROTOCOL ACCORDING TO FOR PROSTATE CANCER

INTRODUCTION & OBJECTIVES: The relative yield of 10-core mad whole range of alternative 8 and 6 core biopsy protocols over classical sextant biopsy protocol and the optimum number of cores per biopsy were detmruined according to the prostate volumes for the patients who experienced the prostate biopsy for the first time. MATERIAL & METHODS: Between Febmary 2001 and July 2004, 503 men with the indications of abnormal digital rectal examination and/or a serum PSA over 2.5 ng/ml were included in the study. All of the patients underwent a 10-core biopsy protocol with additional one core from each suspicious area detected by TRUS. Prostate volumes were divided into quartiles (14.9-35, 35.1-50, 50.1-65, 65.1-150). Optimum numbers of biopsy cores were determined for patients with different prostate volumes. RESULTS: Median age and PSA values were 63(43-83) and 7.4(1-56) for the whole group, respectively. Of 503 patients 159 (31.6 %) were positive for prostate cancer. Median age and PSA of prostate cancer patients were significantly higher than the non-cancer patients. Besides prostate and transitional zone volumes of the cancer patients were significantly lower than the non-cancer ones. Patients with larger prostates were significantly older and had lower free to total PSA ratio and higher PSA density and transitional zone PSA density values than the patients with smaller prostates. Table: Cancer detection rates of various biopsy schemes Prostate volume range(cc)

14.9-35

35.1-50

50.1-65

Cancer ratio (%)

63/127(49.6)

37/132(28)

33/119(27.7) 26/125(20.8)

65.1-150

Marke

Sample

96 Well Format

Microfluidic

% Apex/Midgland/Base (Classical sextant)

84

78

70

62

PSA

PB

68%

4%

% Apex/Midgland/Lat.B ase

87

86

79

69

BM

54%

29%

% Apex/B ase/Lat.Midgland

92

86

79

73

94

95

88

81

PSMA PSCA HK2

PB

50%

7%

% Apex/Lat.Midgland/Lat.Base

BM

96%

29%

% Apex/Midgland/Base/Lat.Midgland

95

89

82

81

PB

61%

0%

% Apex/Midgland/B ase/Lat.Base

89

89

85

81

BM

88%

8%

% Apex/Midgland/Lat.Midgland/Lat.Base

97

97

9i

88

PB

32%

0%

% Apex/Base/Lat.Midgland/Lat.Base

95

97

94

92

BM

33%

29%

% 10-core

98

100

97

100

CONCLUSIONS: Despite the methodological advantages of microfiuidic qRT-PCR over established techniques, we have demonstrated that its utility may be limited by poor sensitivity. In vitro sensitivity was 10 fold lower than standard qRT-PCR. Clinically it failed to detect the presence of target RNA in a significant proportion of patients who were marker positive using a standard assay. Levels of sensitivity beyond the capability of this current assay will be required for the identification of men at risk of micrometastatic CaR

CONCLUSIONS: Patients with larger prostates had lower cancer detection rates. 10-core biopsy protocol increased the cancer detection rate over sextant and alI probable 6 and 8-core biopsy protocoIs and must be used for all group of patients except for the patients with prostate volumes of 14.9-35 ce. Patients with I4.9-35 cc, 8-core biopsy protocol consisting of apex, midglandular, lateral midglandular and lateral base can be used since it revealed similar results with the 10-core biopsy protocol.

443 RACIAL CANCER

ORIGIN IS IN UK MEN

LINKED

TO

KNOWLEDGE

OF

PROSTATE

Chandrasekera S. 1, Z h u G. I, D e z y l v a S. 1, Grunfeld B. 2, M u i r G ? 1King's College Hospital, Urology, London, United K i n g d o m , 2Guys K i n g ' s and St T h o m a s Medical School, Psychology, London, United K i n g d o m I N T R O D U C T I O N & O B J E C T I V E S : Prostate cancer is the second m o s t c o m m o n cause o f cancer death in U K men. A l t h o u g h there has been a h i g h e r incidence and later stage disease in black British men, there is no evidence r e g a r d i n g their awareness o f prostate cancer and national advice on screening. We assessed the level o f prostate cancer awareness and attitudes in black and white m e n in the U K , and to see i f written information would m o d i f y awareness. M A T E R I A L & M E T H O D S : Information w a s collected f r o m two groups o f 650 m e n without prostate cancer u s i n g a new, validated, prostate cancer awareness questionnaire. The first group w e r e a s k e d to fill in the questionnaire at the first interview, while in the second phase o f the study, a second group (which w a s a m a t c h e d sample to those in phase 1) were initially g i v e n printed information on prostate cancer (provided b y the U K Prostate Cancer Charity) and requested to fill out the questionnaire within 4-6 w e e k s o f r e c e i v i n g such information, D a t a were c o m p a r e d b e t w e e n the two ethnic groups u s i n g SPSS statistical package. D a t a f r o m the second phase w a s then c o m p a r e d to those o f phase 1 to analyse changes in k n o w l e d g e and attitudes after p r o v i d i n g prostate health education. R E S U L T S : The overall response rate w a s 65%, with a significantly lower response f r o m black men. A m o n g significant differences in the first group, whites w e r e almost four times m o r e likely than blacks to h a v e been tested for prostate cancer. (p<0.001). Black m e n were u n a w a r e o f their increased risk o f developing the disease, although they w e r e m o r e likely to k n o w a victim. Blacks demonstrated poorer k n o w l e d g e about the s y m p t o m s o f prostate cancer (p< 0.001) and also m o r e n e g a t i v e attitudes about its control and treatment (]9 < 0.01). In the second group, there were no significant differences in demographics from group one. H o w e v e r (by A N O V A ) there were significant improvements in knowledge, risk awareness and attitudes following targeted education: this was true for black and white men. C O N C L U S I O N S : While black British m e n have a high risk o f prostate cancer their "knowledge o f the disease is poor. Simple education methods can significantly i m p r o v e awareness and k n o w l e d g e in both black and white men.

444 LAPAROSCOPIC RADICAL PROSTATECTOMY A MULTI-INSTITUTIONAL STUDY WITH MORE THAN 5500 PATIENTS -

RassweiIer J.~, Stolzenburg j.2, Sulser T?, Deger S, 4, Zumb6 J.S, Hofnrockel G. 6, John H. 7, Janetschek G. 8, Fehr J.L. 9, Hatzinger M. 1°, Probst M? 1, Rothenberger K.H. 12, Poulakis VJ 3, Truss M. 14, Popken G. ~5, Westphal j.16, Anes U. 17, Fnrnara E 18, Laparoscopic Working Group of DGU, Dtisseldnrf, Germany ~SLK Kliniken Heilbronn, University of Heidelberg, Urology, Heilbronn, Germany, ZUniversity of Leipzig, Urology, Leipzig, Germany, 3University of Basel, Urology, Basel, Switzerland, 4University of Berlin-Chafit6, Urology, Berlin, Germany, 5Klinikum Leverkusen, Urology, Leverkusen, Germany, 6Klinikum Worms, Urology, Leverkusen, Germany, 7University of Zfirich, Urology, Schaffhausen, Switzerland, 8EIisabethenkrankenhans, Urology, Linz, Austria, 9Kantonsspital Schaffuausen, Urology, Leverkusen, Switzerland, mUniversity of Mannheim, Urology, Mannheim, Germany, ltUniversity of Frankfurt, Urology, Frankfurt, Germany, ~2Kreiskrankenhans Landshut, Urology, Landshut, Germany, ZJNord-West Krankenhaus, Urology, Frankfurt, Germany, ~4University of Hannover, Urology, Hannover, Germany, ~fHelioskliniken Buch, Urology, Berlin, Germany, t6Josephshospital, UrNogy, Krefeld, Germany, lVKlinikum Kaiserslautem, Urology, Kaiserslantem, Germany, t SUniversity of Halle, Urology, Halle, Germany INTRODUCTION & OBJECTIVES: Laparnscopic radical prnstatectnmy has become one option in the management of localized prostate cancer. In order to evaluate the transferability of this difficult technique, we present the results of a survey of the laparoeopic working group of the German Urological Association (DGU). MATERIAL & METHODS: We analyzed the data of 5585 patients who underwent LRP in 18 different centres performed by 48 urologists from March 1999 to August 2004. Three centres started in the year 1999 and 2000 respectively, and four centres in 2001, 2002, and 2003 respectively. Three centres performed more than 700 and five more than 350 cases. A transperitnneal descending technique with primary dissection of the seminal vesicles was used in 2000, a transperitoneal ascending technique in 1211, an extraperitoneal descending technique in 2000, and an extraperitoneal ascending technique in 98 cases. The data of all eentres were pooled focusing on operative data, complications, reintervention rates as well as functional and nncological results. RESULTS: The average of mean operating time 211 (131-292) minutes, with shorter duration of the extraperitoneal descending technique. Conversion to open surgery ranged from 0 to 10.3 % with an average of 1.9 %. Reinterventiun rate amounted to 2.95 (0.3-7-7) %, and overall complication rate ranged from 1.8 to 10.8 % including bleeding (0.3-2.5%) and rectal lesion (1.5-2.5%) as most frequent complication. The rate of positive margins averaged 8.5 (3.2-18) % for pT2- and 31 (20-38) % for pT3-tumours Continence after i2 months (no pad) was 85 (72-94) %. Data about potency were available only from 8 centres: 42.9 (36-67) % following bilateral nerve-sparing. Long-term data from one centre showed 5 years PSA-recurrence rate of I0.5 %. CONCLUSIONS: Our results clearly dcnrunstrate the safe transferability of this technically demanding operation to the interested urological community with at least equal outcomes compared to open surgery.

European Urology Supplements 4 (2005) No. 3, pp. 113