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MALİGNANT MESOTELIOMA GENE THERAPY PROF DR MUSTAFA KÜRŞAT ÖZVARAN İSTANBUL MEDİPOL ÜNİVERSİTESİ

MALİGNANT MESOTELIOMA GENE THERAPY PROF DR MUSTAFA KÜRŞAT ÖZVARAN İSTANBUL MEDİPOL ÜNİVERSİTESİ. INTRADUCTION.

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MALİGNANT MESOTELIOMA GENE THERAPY PROF DR MUSTAFA KÜRŞAT ÖZVARAN İSTANBUL MEDİPOL ÜNİVERSİTESİ

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  1. MALİGNANT MESOTELIOMA GENE THERAPY PROF DR MUSTAFA KÜRŞAT ÖZVARAN İSTANBUL MEDİPOL ÜNİVERSİTESİ

  2. INTRADUCTION Malignant mesothelioma is an aggressive neoplasm arising from the mesothelial surfaces ofthe pleural and peritoneal cavities, pericardium, or the tunica vaginalis. Up to 80% of all cases are pleural in origin and aredefined as malignant pleuralmesotheliomas (MPM).

  3. INCIDENCE The tumor was once considered rare but the incidenceworldwide of MPM is anticipated to increase by more than 50% over the next 10 years. Because of patterns of occupational and environmental asbestos exposure. Hodgson JT, et al. Br J Cancer. 2005; 92(3):587–93.

  4. Treatments of mesothelioma 1-radiotherapy 2-chemotherapy 3-surgery 4-combine therapy New therapy 1-target therapy 2-immunotherapy 3-gene therapy

  5. CHEMOTHERAPY Malignant pleural mesothelioma (MPM) is an aggressive cancer with a median survival of 4-12 months. Mesothelioma is a chemoresistant malignancy. Even with combination chemotherapy (cisplatin and/or platinum-based combinationchemotherapy), only a 13 to 18%response rate has been reported. Thepemetrexed/cisplatin combination achieved a response rate of 41.3%, significantly higher than the response of 16.7% in control group. Median survival varied from 12.1 months after pemetrexed/cisplatintherapy versus 9.3 months in the control group. Ronan Joseph Kelly, et al. Lung Cancer. 2011 September ; 73(3): 256–263. Price B. Am.J.Epidemiol 1997;145 (3):211.

  6. RADİOTHERAPY Radiation is also effective in preventionof local recurrence after thoracentesis or thoracoscopic biopsy. Postoperative radiation was found to benefit patients with positive resection margins, after extrapleural pneumonectomy demonstrated excellent local control. SURGERY Even with combined surgery, chemotherapy and radiation, only a minority of patients are rendered prolonged disease-free survivors. An encouraging 45% 5-year survival rate has been reported by Sugarbaker et al. fora subgroup of patients with early-stage disease, epithelial histology. Jaklitsch MT, Grondin SC, Sugarbaker DJ. World Journal of Surgery 2001;25(2):210.

  7. TARGET therapy Epidermal growth factor receptor inhibitors (EGFR), Angiogenesis inhibitors—VEGF inhibition Proteasome-modifying treatments inhibition of NF-κ-β Histone deacetylase inhibitors—(Vorinostat) Other targeted agents—Dasatinib,Imatinib, Everolimus Ronan Joseph Kelly, et al. Lung Cancer. 2011 September ; 73(3): 256–263.

  8. Gene therapy For therapeutic purposes base upon the transfer of genetic material in to tumor cells. The requirement for successful gene therapy is efficient gene delivery, gene transfer efficiency, duration of expression, and induction of inflammation. Gene therapy should not cause any different disease in host. Based upon the transfer of genetic material, includingcomplementary DNA (cDNA), full-length genes, small interfering RNA (si-RNA), or oligonucleotides.

  9. VECTORS A- Viral Vectors: Retroviruses Adeno-Associated Virus, Adenoviruses, Lentiviruses, and vaccinia/ fowl pox vektors(used as cancer vaccines) B-Nonviral Vectors Liposomas, polymers, molecular conjugates C- Antisense therapy (for oligonucleotid) Ronan Joseph Kelly, et al. Lung Cancer. 2011 September ; 73(3): 256–263.

  10. Antisense therapy Antisense therapy relies on inhibition of gene expression, accomplished with a targeted oligonucleotide delivered either intravenously or intratumorallyleading to diminished transcription of the complementary mRNA. Results of phase studies does not well.

  11. MPM GENE THERAPY MPM’s location within the thoracic cavitymakes the tumor uniquely accessible, facilitating directedadministration of novel agents and subsequent analysis oftreatment effects. MPM is local persistence or recurrence ofdisease rather than the development of widespread distantmetastases. Gene therapy involving the pleural space offers a numberof potentialadvantages. The pleural space has a largesurface area lined by a thin layer of mesothelial cells, theideal configuration for efficent gene transfer. Liquids orcell suspensions injected into the pleural space would disseminaterapidly and uniformly within the hemithorax. The patterns of fluiddrainage from the pleural space through vascular andlymphatic channels would ensure rapid systemic uptakeof any secreted proteins engendered by mesothelial transgeneexpression. Unlike the peritonealcavity, where adhesions and inflammation can cause severecomplications.

  12. Gene therapy approaches in malignant mesothelioma (1995): Suicide gene therapy Transfer of immunomodulatory genes Replacement of tumor suppressor genes Combine gene therapy

  13. Suicide gene therapy method The most commonly used suicide gene in human clinical trialshas been the herpes simplex thymidine kinase (HSVtk) gene,whose protein product is an enzyme that can convert thenontoxic, clinically used, antiviral drug ganciclovir into a highly cytotoxic phosphorylated form. One attractive feature of the HSVtk/ganciclovir system is the presence of a‘‘bystander effect,’’ where tumor cell killing is elicited in asizable proportion of neighboring, nontransfected, cells dueto intracellular transfer of phosphorylated ganciclovir and induction of antitumor immune responses .

  14. Sterman and colleagues(1998 and 2000) initiated a series of Phase I clinical trials of adenovirus (Ad.HSVtk/GCV) gene therapy in advanced MPM patients. They used 4 protocol in 2 studies. Protocol 1 (initial E1/E3-deleted Ad.HSVtk/ganciclovir trial): 21 patients confirmation of diagnosis,and placement of a chest tube. On day 2, the H5.010RSVtk viral vector( 1.6 1013viral Particles), dilutedin 50 to 100 mL normal saline, was instilled via the chest tube. Threedays after vector instillation (on day 5), a videothoracoscopy was done for tumor specimen acquisition.The following morning (day 6), i.v.ganciclovir was initiated at 5 mg/kg twice daily (bid) for 14 days. 3-month intervals control. . Protocol 2 (E1/E3-deleted Ad.HSVtk/ganciclovir/steroid trial): 5 patients Intravenous corticosteroid administration (60 mg methylprednisilone) began on the night of admissionand continued every 6 hours for a total of 12 doses. Sterman DH, Treat J, Litzky LA, et al. Human gene therapy 1998;9:1083-1092.

  15. Protocol 3 (E1/E4-deleted Ad.HSVtk/ganciclovir trial): 5 patients E1/E4-deleted Ad.HSVtk 2 patients ( 1.5 1013viral Particles) 3 patients( 5 1013viral Particles) used. Ganciclovir is same dose. Protocol 4 (E1/E4-deleted Ad.HSVtk with escalating ganciclovir trial): 3 patients E1/E4-deleted Ad.HSVtk ( 3 1013viral Particles) ganciclovir dose increased from 5 mg/kg to 7.5 mg/kg. Sterman DH, et al:Cancer Gene Ther 2000; 7: 1511–1518.

  16. RESULTS Dose related intratumoralHSVtk gene transfer was demonstrated in 23 of 30 patients, Having evidence of HSVtk protein expression at tumor surfaces and up to 30–50 cell layers deep. No gene transfer informationis available from protocol 4, because post-vectorinstillation biopsies were not obtained.Anti-tumor antibodies and anti-adenoviral immune responses, includinghigh titers of anti-adenoviral neutralizing antibody and proliferative T-cell responses weregenerated in both serum and pleural fluid.In comparisonto patients treated with the same dose of IP Ad. tk,corticosteroid-treated patients demonstrated decreased localized and systemic inflammatory responses andshowed a trend towards increased intratumoral genetransfer. Intravenous methylprednisilone failed to inhibitthe generation ofanti-Ad antibodies or Ad-induced peripheral blood mononuclear cell activation. E1/E4-deleted Ad.tk vector, two patients treated at the higherdose level of 5 1013 particles of H5.001RSVtk (and standarddose ganciclovir) are still alive, >6.5 years after completion of the protocol. Each of the patients had stage I epithelioid mesothelioma at diagnosis. Sterman DH, et al. Clin Cancer Res. 2005; 11(20):7444–7453.

  17. RESULT Using the current strategy, therapeutic efficacycould primarily be expected in patients with a relatively small tumor burden. An alternative treatment schema might involve administration of IP Ad. tk as neoadjuvanttherapy followed by surgical ‘debulking’ to minimize the tumor burden. Another method of improving intratumoral gene transfer would be repeated administration ofvector and GCV, the use of adenoviral vectors capable ofselective replication in mesothelioma cells, or combination therapy of IP Ad.tk gene transfer with standard chemotherapy Sterman DH, et al. Clin Cancer Res. 2005; 11(20):7444–7453.

  18. SUİCİD GENE THERAPY WİTH SECOND VECTOR Schwarzenberger et al. at the LouisianaState University (LSU). The study was a phase I clinical trial involving direct IPinfusion of escalating doses of HSVtk suicide gene-modifiedPA1STK cells in 15 patients with MM followed by 7days of intravenous GCV. The first two cohorts receiveda single IP dose via an indwelling pleural catheter; cohorts3–5 received three weekly doses. The treatment waswell tolerated without any grade 3 or 4 toxicity. Significantinductions of both Th1 and Th2 cytokines up to 20-fold over baseline were observed. No objective radiographicresponses were observed in any of the treated patients . Schwarzenberger P, et al. Cancer Gene Ther 2011; 18:906–912.

  19. Cytokine Gene Therapy The rationale for this approachis that expression of cytokine genes by tumor cellsgenerates a high level of intratumoral cytokines in anautocrineand paracrine fashion, inducing powerful localcytokine effects while minimizing systemic toxicity. Prolongedlocal cytokine expression can induce direct tumorcell apoptosis and inhibit tumor angiogenesis but, perhapsmost importantly, induces anti-tumor immune responses. The rationale for cytokine gene therapy is that high level expression ofimmunostimulatorycytokines (such as interleukin-2 [IL-2], IL-12, tumor necrosis factor [TNF], GM-CSF, orinterferons [α, β, or γ]) from tumor cells will activate the immune system in situ resulting ina more effective anti-tumor immune response without having to target specific antigens.

  20. human IL-2 gene Study The first human clinical trial of direct intratumoraldelivery of cytokine genes in malignant pleural mesothelioma(MPM) was conducted by investigators, using a recombinantvaccinia virus (VV) expressing the human IL-2 gene. The VV-IL-2 vector was seriallyinjected into palpable chest wall lesions of 6 patients withadvanced MM. Toxicities were minimal. VV-IL-2 mRNA was detected by RT-PCRin serial tumor biopsies for up to 6 days after injection. Unfortunately, no significanttumor regression was seen in any of the patients,and only modest intratumoral T-cell infiltration was detected on tumor biopsies. Mukherjee S., et al. Cancer Gene Therapy 2000;7(5):663-670.

  21. IFN-β gene transfer The study evaluatedthe safety and feasibility of a single-dose IP IFN-β gene transfer using an adenoviral vector (Ad.IFN- β) in patients with MPM and metastatic pleural effusions. Ad.IFN- β was administered via an indwelling pleural catheterin escalating doses in two cohorts of patients – MPM(7 patients) and MPE (3 patients) . Subjects wereevaluated for toxicity, gene transfer, immuneresponses, and anti-tumor responses. IP Ad.IFN- βwas generally well tolerated, with transient lymphopeniaas the most common side effect. Genetransfer was documented in 7 of the 10 patients by demonstrationof IFN- β mRNA or protein expression inpleural fluid. Fourof 10 patients showed meaningful clinicalresponses definedas disease stability and/or regression . Sterman DH, et al:Clin Cancer Res 2007; 13: 4456–4466

  22. two doses of Ad.IFN-β vector study University of Pennsylvania Medical Center to determinewhether two doses of Ad.IFN-β vector would be superiorto a single dose [75] . Ten patients with MPM and 7 withMPE (underlying primary malignancies: 3 lung cancer, 2ovarian cancer, and 2 breast cancer), received two dosesof Ad.IFN- β (3 ×10 12 viral particles) through an indwelling pleuralcatheter. Becauseof potential safety concerns, the trial used a doseinterval of 14 days initially for 13 patients. After demonstratingno additional toxicity, the remaining 4 patientswere treated with a 7-day dosing interval. Repeated doses were generally well tolerated. In this repeat dose gene transfer study, high levels ofIFN- β were detected in pleural fluid after the first dose;however, absent IP IFN- β expression after the second dosecorrelated with the rapid induction of neutralizing Ad antibodies(Nabs). There were7 patients with survival times longer than 18 months. Overall, repeated IP instillation of Ad.IFN- β vector wassafe and induced immune responses and some evidence ofclinical responses. However, rapid development of Nabsprevented effective gene transfer after the second dose,even with a dose interval as short as 7 days . Sterman DH, et al. Mol Ther 2010; 18: 852–860.

  23. Ad.IFN- α 2b gene therapy Ad.IFN- α 2b was instilled IP on study days 1 and 4 viaa tunneled pleural catheter. The starting vector dose was1 x10 12 viral particles of Ad.hIFN- α 2b, but this dosewas reduced to 3x10 11 after the first 3 patients developed significant CRS symptoms after the first dose of IPvector. Subjects wereassessed for anti-tumor responsesapproximately 60 days after the initial treatment usingCT and PET scans. Pleural fluid and serum IFN- α 2blevels, In general,Ad.IFN- α 2b vector instillation was well tolerated, At the time of first radiographic assessment (60 days),3 subjects had progressive disease, 4 had stable disease,and 2 had PR. The most important outcome ofthe study was the demonstration of lower levels of Nabsat the shortened dosing interval with prolonged IP expression of the interferon transgene. Ad.IFNα induced much higher levels of gene transfer than Ad.INFβ. Sterman DH, et al. Am J Respir Crit Care Med 2011; 184: 1395–1399.

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