MALİGNANT MESOTELIOMA GENE THERAPY

PROF DR MUSTAFA KÜRŞAT ÖZVARANİSTANBUL MEDİPOL ÜNİVERSİTESİ

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

INTRADUCTION

The tumor was once considered rare but the incidence worldwide 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.

INCIDENCE

Treatments of mesothelioma

1-radiotherapy2-chemotherapy3-surgery4-combine therapy

New therapy1-target therapy2-immunotherapy3-gene therapy

Malignant pleural mesothelioma (MPM) is an aggressive cancer with a median survival of 4-12 months.Mesothelioma is a chemoresistant malignancy. Even with combinationchemotherapy (cisplatin and/or platinum-based combination chemotherapy), only a 13 to 18% response rate has been reported. The pemetrexed/cisplatin combination achieved a response rateof 41.3%, significantly higher than the response of 16.7% incontrol group. Median survival varied from 12.1 months after pemetrexed/cisplatin therapy versus 9.3 months in the control group.

CHEMOTHERAPY

Ronan Joseph Kelly, et al. Lung Cancer. 2011 September ; 73(3): 256–263.

Price B. Am.J.Epidemiol 1997;145 (3):211.

RADİOTHERAPYRadiation is also effective in prevention of 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.

SURGERYEven 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. for a subgroup of patients with early-stage disease, epithelial histology.

Jaklitsch MT, Grondin SC, Sugarbaker DJ. World Journal of Surgery 2001;25(2):210.

TARGET therapy

Epidermal growth factor receptor inhibitors (EGFR),Angiogenesis inhibitors—VEGF inhibitionProteasome-modifying treatments inhibition of NF-κ-βHistone deacetylase inhibitors—(Vorinostat)Other targeted agents—Dasatinib,Imatinib, Everolimus

Ronan Joseph Kelly, et al. Lung Cancer. 2011 September ; 73(3): 256–263.

Gene therapyFor 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, including complementary DNA (cDNA), full-length genes, small interfering RNA (si-RNA), or oligonucleotides.

VECTORSA- Viral Vectors:Retroviruses Adeno-Associated Virus, Adenoviruses, Lentiviruses,and vaccinia/ fowl pox vektors(used as cancer vaccines) B-Nonviral VectorsLiposomas, polymers, molecular conjugatesC- Antisense therapy (for oligonucleotid)

Ronan Joseph Kelly, et al. Lung Cancer. 2011 September ; 73(3): 256–263.

Antisense therapyAntisense therapy relies on inhibition of gene expression, accomplished with a targetedoligonucleotide delivered either intravenously or intratumorallyleading to diminishedtranscription of the complementary mRNA. Results of phase studies does not well.

MPM’s location within the thoracic cavity makes the tumor uniquely accessible, facilitating directed administration of novel agents and subsequent analysis of treatment effects. MPM is local persistence or recurrence of disease rather than the development of widespread distant metastases. Gene therapy involving the pleural space offers a number of potential advantages. The pleural space has a large surface area lined by a thin layer of mesothelial cells, the ideal configuration for efficent gene transfer. Liquids or cell suspensions injected into the pleural space would disseminate rapidly and uniformly within the hemithorax.The patterns of fluid drainage from the pleural space through vascular and lymphatic channels would ensure rapid systemic uptake of any secreted proteins engendered by mesothelial transgene expression. Unlike the peritonealcavity, where adhesions and inflammation can cause severe complications.

MPM GENE THERAPY

Gene therapy approaches in malignant mesothelioma (1995):

Suicide gene therapyTransfer of immunomodulatory genesReplacement of tumor suppressor genesCombine gene therapy

Suicide gene therapy methodThe most commonly used suicide gene in human clinical trials has been the herpes simplex thymidine kinase (HSVtk) gene, whose protein product is an enzyme that can convert the nontoxic, 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 a sizable proportion of neighboring, nontransfected, cells due to intracellular transfer of phosphorylated ganciclovir and induction of antitumor immune responses .

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Sterman DH, Treat J, Litzky LA, et al. Human gene therapy 1998;9:1083-1092.

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.

confirmation of diagnosis,and placement of a chest tube. On day 2, the H5.010RSVtk viral vector( 1.6 1013 viral Particles), diluted in 50 to 100 mL normal saline, was instilled via the chest tube. Three days 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 1 (initial E1/E3-deleted Ad.HSVtk/ganciclovir trial): 21 patients

Protocol 2 (E1/E3-deleted Ad.HSVtk/ganciclovir/steroid trial): 5 patients

Intravenous corticosteroid administration (60 mg methylprednisilone) began on the night of admission and continued every 6 hours for a total of 12 doses.

Sterman DH, et al: Cancer Gene Ther 2000; 7: 1511–1518.

Protocol 3 (E1/E4-deleted Ad.HSVtk/ganciclovir trial): 5 patientsE1/E4-deleted Ad.HSVtk 2 patients ( 1.5 1013 viral Particles) 3 patients( 5 1013 viral Particles) used. Ganciclovir is same dose.

Protocol 4 (E1/E4-deleted Ad.HSVtk with escalating ganciclovir trial): 3 patientsE1/E4-deleted Ad.HSVtk ( 3 1013 viral Particles) ganciclovir dose increased from 5 mg/kg to 7.5 mg/kg.

RESULTSDose relatedintratumoralHSVtk 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-vector instillation biopsies were not obtained. Anti-tumor antibodies and anti-adenoviral immune responses, including high titers of anti-adenoviral neutralizing antibody and proliferative T-cell responses were generated in both serum and pleural fluid.In comparison to patients treated with the same dose of IP Ad. tk, corticosteroid-treated patients demonstrated decreased localized and systemic inflammatory responses and showed a trend towards increased intratumoral gene transfer. Intravenous methylprednisilone failed to inhibitthe generation of anti-Ad antibodies or Ad-induced peripheral blood mononuclear cell activation. E1/E4-deleted Ad.tk vector, two patients treated at the higher dose level of 5 1013 particles of H5.001RSVtk (and standard dose ganciclovir) are still alive, >6.5 years after completion ofthe protocol. Each of the patients had stage I epithelioid mesothelioma at diagnosis.

Sterman DH, et al. Clin Cancer Res. 2005; 11(20):7444–7453.

RESULTUsing the current strategy, therapeutic efficacy could primarily be expected in patients with a relatively small tumor burden. An alternative treatment schema might involve administration of IP Ad. tk as neoadjuvant therapy followed by surgical ‘debulking’ to minimize thetumor burden. Another method of improving intratumoral gene transfer would be repeated administration of vector and GCV, the use of adenoviral vectors capable of selective replication in mesothelioma cells, or combinationtherapy of IP Ad.tk gene transfer with standard chemotherapy

Sterman DH, et al. Clin Cancer Res. 2005; 11(20):7444–7453.

Schwarzenberger et al. at the LouisianaState University (LSU). The study was a phase I clinical trial involving direct IP infusion of escalating doses of HSVtk suicide gene-modified PA1STK cells in 15 patients with MM followed by 7 days of intravenous GCV. The first two cohorts received a single IP dose via an indwelling pleural catheter; cohorts 3–5 received three weekly doses. The treatment was well tolerated without any grade 3 or 4 toxicity. Significant inductions of both Th1 and Th2 cytokines up to 20-fold over baseline were observed. No objective radiographic responses were observed in any of the treated patients .

Schwarzenberger P, et al. Cancer Gene Ther 2011; 18:906–912.

SUİCİD GENE THERAPY WİTH SECOND VECTOR

Cytokine Gene TherapyThe rationale for this approach is that expression of cytokine genes by tumor cells generates a high level of intratumoral cytokines in an autocrineand paracrine fashion, inducing powerful local cytokine effects while minimizing systemic toxicity. Prolonged local cytokine expression can induce direct tumor cell apoptosis and inhibit tumor angiogenesis but, perhaps most importantly, induces anti-tumor immune responses.The rationale for cytokine gene therapy is that high level expression of immunostimulatory cytokines (such as interleukin-2 [IL-2], IL-12, tumor necrosis factor [TNF], GM-CSF, or interferons [α, β, or γ]) from tumor cells will activate the immune system in situ resulting in a more effective anti-tumor immune response without having to target specific antigens.

Mukherjee S., et al. Cancer Gene Therapy 2000;7(5):663-670.

human IL-2 gene Study The first human clinical trial of direct intratumoral delivery of cytokine genes in malignant pleural mesothelioma (MPM) was conducted by investigators, using a recombinant vaccinia virus (VV) expressing the human IL-2 gene. The VV-IL-2 vector was serially injected into palpable chest wall lesions of 6 patients with advanced MM. Toxicities were minimal. VV-IL-2 mRNA was detected by RT-PCR in serial tumor biopsies for up to 6 days after injection. Unfortunately, no significant tumor regression was seen in any of the patients, and only modest intratumoral T-cell infiltration was detected on tumor biopsies.

Sterman DH, et al:Clin Cancer Res 2007; 13: 4456–4466

IFN-β gene transfer The study evaluated the safety and feasibility of a single-dose IP IFN-β genetransfer using an adenoviral vector (Ad.IFN- β) in patients with MPM and metastatic pleural effusions. Ad.IFN- β was administered via an indwelling pleural catheter in escalating doses in two cohorts of patients – MPM (7 patients) and MPE (3 patients) . Subjects were evaluated for toxicity, gene transfer, immune responses, and anti-tumor responses. IP Ad.IFN- β was generally well tolerated, with transient lymphopenia as the most common side effect. Gene transfer was documented in 7 of the 10 patients by demonstration of IFN- β mRNA or protein expression inpleural fluid. Four of 10 patients showed meaningful clinical responses defined as disease stability and/or regression .

two doses of Ad.IFN-β vector studyUniversity of Pennsylvania Medical Center to determine whether two doses of Ad.IFN-β vector would be superior to a single dose [75] . Ten patients with MPM and 7 with MPE (underlying primary malignancies: 3 lung cancer, 2 ovarian cancer, and 2 breast cancer), received two doses of Ad.IFN- β (3 ×10 12 viral particles) through an indwelling pleural catheter. Because of potential safety concerns, the trial used a dose interval of 14 days initially for 13 patients. After demonstrating no additional toxicity, the remaining 4 patients were treated with a 7-day dosing interval. Repeated doseswere generally well tolerated. In this repeat dose gene transfer study, high levels of IFN- β were detected in pleural fluid after the first dose; however, absent IP IFN- β expression after the second dose correlated with the rapid induction of neutralizing Ad antibodies(Nabs). There were 7 patients with survival times longer than 18 months.Overall, repeated IP instillation of Ad.IFN- β vector was safe and induced immune responses and some evidence of clinical responses. However, rapid development of Nabs prevented 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.

Ad.IFN- α 2b gene therapyAd.IFN- α 2b was instilled IP on study days 1 and 4 via a tunneled pleural catheter. The starting vector dose was 1 x10 12 viral particles of Ad.hIFN- α 2b, but this dose was reduced to 3x10 11 after the first 3 patients developedsignificant CRS symptoms after the first dose of IP vector. Subjects were assessed for anti-tumor responses approximately 60 days after the initial treatment using CT and PET scans. Pleural fluid and serum IFN- α 2b levels, 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 of the study was the demonstration of lower levels of Nabs at 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.