- Email: [email protected]
Combined suicide gene therapy and oncolytic viral therapy for prostate cancer
26
25 SELECTIVE $;;zVIRUS,
CYTOTOXICITY OF A REPLICATION-SELECTIVE dl 118, FOR PROSTATIC CANCER CELL LINES
Moneiat-Artus
IN
P.l, Villette J.M.‘, Ramon Y Cajal S.2, Teillac P.‘. Cussenot 0.’
‘CeRePP and Urology Department, Saint Louis Hospital, University of Paris, Paris, 2Pathology Department, Clinica Puerta De Hierro, Madrid, Spain INTRODUCTION & OBJECTIVES: Replication selective microbial agents hold promise as a novel cancer treatment platform. EIB gene mutated adenovirus (adv) were the first such genetically engineered agent to be tested in humans. These EIB mutant adv have been engineered to replicate in and kill cancer cells where ~53 protein is not functional, while having no cytopathic effect on cells with functional ~53 pathway. Over 200 cancer patients have been treated to date and the virus was generally well tolerated at doses of up to 2”12 particles by diverse routes of administration and replication was tumor-selective and transient. In a phase II clinical trial, dll520, an EIB-55kD gene deleted adv, was combined with a standard chemotherapy in patients with recurrent radio and chemo-resistant squamous cell cancer of the head and neck. It resulted in a 63% objective response and a 27% complete response with no progression during 6 months follow up. According to our knowledge, no evaluation of replication selective adv in urological cancer have been reported.
WITHDRAWN
MATERIAL & METHODS: We used dl I 18, a complete El B gene deleted adv, which has shown, in vitro and in viva, specific cytotoxicity for a range of human cancer cells. Primary cultures of normal prostate epithelial cells, prostate cell lines (CaHPVIO, PNTlA, PNT2) and prostate cancinoma cell lines (LnCaP, DUl45, PC3) were exposed 3 days to 0,2 to 200 pfu of dlIl8. Number of surviving cells was determined by counting nuclei after cell lysis in a Coulter counter. Results are expressed in percentage of surviving cells. RESULTS: No significant cytolysis (0,9%) was observed in primary cultures. But cytolysis was marked (from 27,6% to 93,5%) in all tested cell lines. Althought dll 18 was supposed to be specific to ~53 deficient cells, ~53 status was not a predictive factor for adv induced cytotoxicity in functional ~53 (LnCaP, PNTlA, PNTZ) or deficient ~53 (PC3, DUl45, CaHPVlO) cell lines. CONCLUSIONS: We show for the first time that an ElB deleted adv, dllI8, have no toxicity for primary culture of epithelial prostate cells but have a specific and significant cytotoxicity for prostate, normal and carcinoma, cell lines. This toxicity is independent upon ~53 cell status. This preliminary result needs in vivo confirmation, but holds promise for the use of replication-selective adv in the treatment of prostate cancer.
27 LOH
ANALYSES IN THE REGION OF THE PUTATIVE GENE Cl3 ON CHROMOSOME 13Q13
TUMOR
SUPPRESSOR
Fiissel S., Fiedler U., Stade .I., Meye A., Ehlers W.. Failer
G..
Schmidt
U.,
Wirth M.P.
Department of Urology, Technical University Dresden, Dreaden, Germany INTRODUCTION & OBJECTIVES: Genetic alterations including allelic losses and increase in gene copy number are involved in carcinogenesis. Recently we could
isolate the putative tumour suppresser gene (TSG) Cl3 local&d between the wellknown TSG BRCA-2 and RB-I on chromosome l3q. Loss of heterozygosity (LOH) studies of this chromosomal region should elucidate the relevance of Cl3 within prostate carcinoma
(PCs) development.
MATERIAL & METHODS: Genomic DNA was Isolated from paraffin embedded prostate tissues (tumour and non-tumorous) as well as from peripheral blood of 21 primary PCs patients. LOH studies were carried out using 14 different microsatellite markers including the loci of BRCA-2, Cl 3 and RB- I on chromosome 13ql3. PCR
fragments obtained by amplification with Texasred-labeled marker-specific primers were resolved on a 6% sequencing gel and analysed by the automated Vistra DNA sequencer (Amersham Pharmacia). Allelic imbalance (Al) was defined as a difference of peak height of IO-306 between tumour and non tumorous tissue and LOH as differences
higher than 30%
RESULTS: Altogether,
II
For 18 of 21 patients (86%) chromosomal alterations were found. of 21 patients (52%) showed LOH and I7 patients (81%) had AI for one or more markers. Three to nine alterations (LOH orA1) per marker were detected in affected tumours. of the seven markers
Our data indicate
a high genomic
instability
rate for at least six
tested in and around the Cl3 locus. The overall allelic alteration of 47% for these seven C 13.associated markers was higher than for the four markers of the RB-I locus (39%) and the three BRCA-2 markers (25%). For Cl3 and RB-I we identified a LOH cluster region (LCR) in ten of the 21 cases (48%), eight of these cases showed a combined loss of both regions. In a second step. from nine of
frequency
the 21 investigated patients we analysed 24 pairs of manually microdissected tumorous and tumourfree tissue samples. In comparison to the LOH results of the original tumour specimens analysed in 88% of the cases a confirmation or an increase of the microsatellite instability rate was obtained.
CONCLUSIONS: Chromosome I3 is one of the most frequently altered chromosomes in cancer including carcinoma of the prostate gland. Since deletions of the region around the well-known TSG BRCA-2 and RB-I often do not correlate with the expression of these genes, further TSG are supposed. With C I3 we could identify a new putative TSG candidate between microsatellite instability and a frequent
support the involvement
these
two
genes
that
shows
a high
rate of
loss along with the RB-I locus. These data
of Cl3 in prostate carcinogenesis.
26 COMBINED SUICIDE GENE THERAPY THERAPY FOR PROSTATE CANCER Young James’. Young Lawren&. Nicholas’
AND ONCOLYTIC
VIRAL
Searle Pete?, Doherty Alan’, Wallace Michael’, James
‘Urology, University Hospital Birmingham, Birmingham. United Kingdom, 2Gene & Immunotherapy Group, CRC Institute for Cancer Studies, Birmingham, United Kingdom, ‘Cancer Studies, CRC Institute for Cancer Studies, Birmingham, United Kingdom INTRODUCTION & OBJECTIVES: A conditionally replicating adenovirus, named dll520, which selectively replicates in cancer cells only, has been produced by deletion of the adenoviwl E I B sequence that normally encodes a ~53 binding protein. Clinical trials of dl I520 in head & neck cancer metastases in conjunction with conventional chemotherapy have produced responses even in previously chemoresistant cancers. Adenoviral oncolytic therapy by itself is unlikely however to produce sufficient cell killing to eradicate significant tamour bulk. In addnion, traditional adenoviral gene therapy vectors cannot be administered in a single sufficiently high dose to hope to eradicate turnours and the effects of subsequent administration are diminished by secondary immune responses. To overcome these hurdles to successful cancer therapy. we have generated variants of dll520 containing the prodrugconverting enzyme E. Coli nitroreductase (NR). which converts the weak monofunctional alkylating agent 5.(aziridin-I-yl)-2.4 dinitrobenzamide, also called CBl954, into a highly cytotoxic bifunctional alkylating agent. A similar vector expressing the marker transgene enhanced green fluorescent protein (eGFP) has also been produced to assay transgene expressmn. Both transgenes are under the control of a prostate specific transcriptional regulatory region (TRR). MATERIAL & METHODS: The genes for eGFP and NR were cloned downstream from a 632bp PSA promoter sequence and a single 1469bp PSA enhancer sequence into the El region of the adenoviral genome. Conditionally replicating adenovirus containing the tranagene was then rescued by standard methods of eukaryotic cell transfection. LNCaP cells were mfected with virus encoding eCFP and transgene expression analysed by ultraviolet (UV) microscopy and fluorescence activated cell-sorting (FACS) analysis. NR expression was analysed by Western blotting of protein lysates of LNCaPcells infected with adenovirua encodmg NR. Sensitisation of LNCaP cells to the prodrug CB 1954 following infection with the virus encoding NR was examined using the Celltiter assay. RESULTS: Rephcation of the adenoviral vector within LNCaP cells was demonstrated on dot blot analysis of viral DNA preparadons. Infection of LNCaP cells with virus encoding eGFP demonstrated high levels of transgene expression by both UV microscopy and FACS analysis. Western blot analysis confirmed rising NR expression levels over a j-day time course. Infection of LNCaP cells with NR encoding adenovirus and addition of CBl954 produced additive cell killing, greater than that produced by virus or prodrug alone. CONCLUSIONS: Novel adenovlruses that are capable of replicating and expressing marker and therapeutic transgenes in the prostate cancer cell line LNCaP have been produced. Transgene expressmn levels from the PSA TRR can be significantly increased by viral replication. Additive cell killing effects have been achieved using this vector in its capacity as gene therapy vector and viral oncolytic agent.
European
Urology
Supplements
1 (2002) No. 1, pp. 9
25 SELECTIVE $;;zVIRUS,
CYTOTOXICITY OF A REPLICATION-SELECTIVE dl 118, FOR PROSTATIC CANCER CELL LINES
Moneiat-Artus
IN
P.l, Villette J.M.‘, Ramon Y Cajal S.2, Teillac P.‘. Cussenot 0.’
‘CeRePP and Urology Department, Saint Louis Hospital, University of Paris, Paris, 2Pathology Department, Clinica Puerta De Hierro, Madrid, Spain INTRODUCTION & OBJECTIVES: Replication selective microbial agents hold promise as a novel cancer treatment platform. EIB gene mutated adenovirus (adv) were the first such genetically engineered agent to be tested in humans. These EIB mutant adv have been engineered to replicate in and kill cancer cells where ~53 protein is not functional, while having no cytopathic effect on cells with functional ~53 pathway. Over 200 cancer patients have been treated to date and the virus was generally well tolerated at doses of up to 2”12 particles by diverse routes of administration and replication was tumor-selective and transient. In a phase II clinical trial, dll520, an EIB-55kD gene deleted adv, was combined with a standard chemotherapy in patients with recurrent radio and chemo-resistant squamous cell cancer of the head and neck. It resulted in a 63% objective response and a 27% complete response with no progression during 6 months follow up. According to our knowledge, no evaluation of replication selective adv in urological cancer have been reported.
WITHDRAWN
MATERIAL & METHODS: We used dl I 18, a complete El B gene deleted adv, which has shown, in vitro and in viva, specific cytotoxicity for a range of human cancer cells. Primary cultures of normal prostate epithelial cells, prostate cell lines (CaHPVIO, PNTlA, PNT2) and prostate cancinoma cell lines (LnCaP, DUl45, PC3) were exposed 3 days to 0,2 to 200 pfu of dlIl8. Number of surviving cells was determined by counting nuclei after cell lysis in a Coulter counter. Results are expressed in percentage of surviving cells. RESULTS: No significant cytolysis (0,9%) was observed in primary cultures. But cytolysis was marked (from 27,6% to 93,5%) in all tested cell lines. Althought dll 18 was supposed to be specific to ~53 deficient cells, ~53 status was not a predictive factor for adv induced cytotoxicity in functional ~53 (LnCaP, PNTlA, PNTZ) or deficient ~53 (PC3, DUl45, CaHPVlO) cell lines. CONCLUSIONS: We show for the first time that an ElB deleted adv, dllI8, have no toxicity for primary culture of epithelial prostate cells but have a specific and significant cytotoxicity for prostate, normal and carcinoma, cell lines. This toxicity is independent upon ~53 cell status. This preliminary result needs in vivo confirmation, but holds promise for the use of replication-selective adv in the treatment of prostate cancer.
27 LOH
ANALYSES IN THE REGION OF THE PUTATIVE GENE Cl3 ON CHROMOSOME 13Q13
TUMOR
SUPPRESSOR
Fiissel S., Fiedler U., Stade .I., Meye A., Ehlers W.. Failer
G..
Schmidt
U.,
Wirth M.P.
Department of Urology, Technical University Dresden, Dreaden, Germany INTRODUCTION & OBJECTIVES: Genetic alterations including allelic losses and increase in gene copy number are involved in carcinogenesis. Recently we could
isolate the putative tumour suppresser gene (TSG) Cl3 local&d between the wellknown TSG BRCA-2 and RB-I on chromosome l3q. Loss of heterozygosity (LOH) studies of this chromosomal region should elucidate the relevance of Cl3 within prostate carcinoma
(PCs) development.
MATERIAL & METHODS: Genomic DNA was Isolated from paraffin embedded prostate tissues (tumour and non-tumorous) as well as from peripheral blood of 21 primary PCs patients. LOH studies were carried out using 14 different microsatellite markers including the loci of BRCA-2, Cl 3 and RB- I on chromosome 13ql3. PCR
fragments obtained by amplification with Texasred-labeled marker-specific primers were resolved on a 6% sequencing gel and analysed by the automated Vistra DNA sequencer (Amersham Pharmacia). Allelic imbalance (Al) was defined as a difference of peak height of IO-306 between tumour and non tumorous tissue and LOH as differences
higher than 30%
RESULTS: Altogether,
II
For 18 of 21 patients (86%) chromosomal alterations were found. of 21 patients (52%) showed LOH and I7 patients (81%) had AI for one or more markers. Three to nine alterations (LOH orA1) per marker were detected in affected tumours. of the seven markers
Our data indicate
a high genomic
instability
rate for at least six
tested in and around the Cl3 locus. The overall allelic alteration of 47% for these seven C 13.associated markers was higher than for the four markers of the RB-I locus (39%) and the three BRCA-2 markers (25%). For Cl3 and RB-I we identified a LOH cluster region (LCR) in ten of the 21 cases (48%), eight of these cases showed a combined loss of both regions. In a second step. from nine of
frequency
the 21 investigated patients we analysed 24 pairs of manually microdissected tumorous and tumourfree tissue samples. In comparison to the LOH results of the original tumour specimens analysed in 88% of the cases a confirmation or an increase of the microsatellite instability rate was obtained.
CONCLUSIONS: Chromosome I3 is one of the most frequently altered chromosomes in cancer including carcinoma of the prostate gland. Since deletions of the region around the well-known TSG BRCA-2 and RB-I often do not correlate with the expression of these genes, further TSG are supposed. With C I3 we could identify a new putative TSG candidate between microsatellite instability and a frequent
support the involvement
these
two
genes
that
shows
a high
rate of
loss along with the RB-I locus. These data
of Cl3 in prostate carcinogenesis.
26 COMBINED SUICIDE GENE THERAPY THERAPY FOR PROSTATE CANCER Young James’. Young Lawren&. Nicholas’
AND ONCOLYTIC
VIRAL
Searle Pete?, Doherty Alan’, Wallace Michael’, James
‘Urology, University Hospital Birmingham, Birmingham. United Kingdom, 2Gene & Immunotherapy Group, CRC Institute for Cancer Studies, Birmingham, United Kingdom, ‘Cancer Studies, CRC Institute for Cancer Studies, Birmingham, United Kingdom INTRODUCTION & OBJECTIVES: A conditionally replicating adenovirus, named dll520, which selectively replicates in cancer cells only, has been produced by deletion of the adenoviwl E I B sequence that normally encodes a ~53 binding protein. Clinical trials of dl I520 in head & neck cancer metastases in conjunction with conventional chemotherapy have produced responses even in previously chemoresistant cancers. Adenoviral oncolytic therapy by itself is unlikely however to produce sufficient cell killing to eradicate significant tamour bulk. In addnion, traditional adenoviral gene therapy vectors cannot be administered in a single sufficiently high dose to hope to eradicate turnours and the effects of subsequent administration are diminished by secondary immune responses. To overcome these hurdles to successful cancer therapy. we have generated variants of dll520 containing the prodrugconverting enzyme E. Coli nitroreductase (NR). which converts the weak monofunctional alkylating agent 5.(aziridin-I-yl)-2.4 dinitrobenzamide, also called CBl954, into a highly cytotoxic bifunctional alkylating agent. A similar vector expressing the marker transgene enhanced green fluorescent protein (eGFP) has also been produced to assay transgene expressmn. Both transgenes are under the control of a prostate specific transcriptional regulatory region (TRR). MATERIAL & METHODS: The genes for eGFP and NR were cloned downstream from a 632bp PSA promoter sequence and a single 1469bp PSA enhancer sequence into the El region of the adenoviral genome. Conditionally replicating adenovirus containing the tranagene was then rescued by standard methods of eukaryotic cell transfection. LNCaP cells were mfected with virus encoding eCFP and transgene expression analysed by ultraviolet (UV) microscopy and fluorescence activated cell-sorting (FACS) analysis. NR expression was analysed by Western blotting of protein lysates of LNCaPcells infected with adenovirua encodmg NR. Sensitisation of LNCaP cells to the prodrug CB 1954 following infection with the virus encoding NR was examined using the Celltiter assay. RESULTS: Rephcation of the adenoviral vector within LNCaP cells was demonstrated on dot blot analysis of viral DNA preparadons. Infection of LNCaP cells with virus encoding eGFP demonstrated high levels of transgene expression by both UV microscopy and FACS analysis. Western blot analysis confirmed rising NR expression levels over a j-day time course. Infection of LNCaP cells with NR encoding adenovirus and addition of CBl954 produced additive cell killing, greater than that produced by virus or prodrug alone. CONCLUSIONS: Novel adenovlruses that are capable of replicating and expressing marker and therapeutic transgenes in the prostate cancer cell line LNCaP have been produced. Transgene expressmn levels from the PSA TRR can be significantly increased by viral replication. Additive cell killing effects have been achieved using this vector in its capacity as gene therapy vector and viral oncolytic agent.
European
Urology
Supplements
1 (2002) No. 1, pp. 9