P53 Gene Therapy Research Articles

Gene therapy for esophageal squamous cell carcinoma

Despite improvement of surgical treatment and application of multi-modality therapies to advanced esophageal cancer, the prognosis is extremely poor in patients with T4 tumors. Based on the genetic background of esophageal cancer, we have developed various gene therapy strategies against human esophageal cancer cells. In this article, we reviewed molecular events of esophageal cancer and gene therapy approaches for its treatment. First, we analyzed p53 genetic alterations and angiogenesis in esophageal cancer. Second, we evaluated an impact of p53 recombinant adenoviral vector (Ad5CMV-p53) on esophageal cancer cells. Significant growth suppression was observed following infection with Ad5CMV-p53 in human esophageal squamous cell carcinoma cell lines. This observation suggests that Ad5CMV-p53 may be a potentially effective therapeutic agent for locally advanced esophageal cancer. Promising avenues for investigation include double gene therapy and adjuvant use of gene therapy with radiation therapy. Third, we have performed a clinical study for p53 gene therapy for un-resectable advanced esophageal cancer. This clinical trial was planned to evaluate vector tolerability and efficacy. A total of 10 patients were enrolled into this phase I/II trial.

Molecular diagnosis and treatment for esophageal carcinoma with p53

We reviewed molecular diagnosis and molecular treatment using p53 as a target for esophageal squamous cell carcinoma (SCC). First, we analyzed serum p53 antibodies (s-p53 Abs) in patients with esophageal SCC. Positive rate was 31% in all patients analyzed (n = 292) and 23% in stage I patients (cTNM/UICC stages) (n = 48). Presence of s-p53 Abs was significantly associated with p53 protein overexpression in resected tumor specimens. Seropositive patients were more likely to be resistant to chemoradiation and had a worse prognosis than seronegative patients. Second, we performed a clinical study of p53 gene therapy in 10 patients with unresectable chemoradiation-resistant esophageal SCC. In 9 patients, stability of the local tumor was achieved. No serious adverse events related to Ad5CMV-p53 have occurred in these patients, and the trial was safely conducted. Thus, intratumoral injection of Ad5CMV-p53 is safe, feasible, and biologically active when administered in multiple doses to patients with esophageal cancer. Observations from these clinical studies indicate that p53 is a useful molecular target in the diagnosis and treatment of esophageal SCC.

Tumor response criteria and biomarkers associated with increased survival following adenoviral p53 gene therapy (ADVEXIN) in patients with recurrent squamous cell carcinoma of the head and neck (SCCHN)

6057 Background: There is a growing body of data indicating that conventional WHO and RECIST criteria do not optimally identify tumor responses associated with increased survival. Methods: We compared several response criteria to assess the efficacy of intratumoral adenoviral p53 gene therapy (Advexin) in a Phase 2 trial of 106 patients with recurrent SCCHN. Results: The percentage of patients with tumor responses defined by reductions in bi-dimensional tumor area by CT scans of 50%, 30%, 25%, 10% or stable disease of > 3 months were 8%, 10%, 11%, 16% and 20% respectively. The median survival for the entire population was 5.9 months while the responder populations defined by tumor decreases of 50%, 30%, 25%, 10% or stable disease > 3 months had median survivals of 40.8, 17.0, 13.7, 12.3 and 11.4 months respectively. There was a statistically significant increase in median survival for each of the responder populations compared to non-responders (p < 0.0009 for each comparison by logrank test). Statistical significance was maintained for the response definitions in landmark analyses excluding patients with survivals less than 3 and 6 months respectively. Locoregional disease control (CR + PR + SD > 3 months) was a stronger predictor of survival by Cox Proportional Hazard analysis (p = 0.0002) than conventional > 50% tumor reduction CR + PR response criteria (p = 0.005). With respect to abnormal p53 detected by immunohistochemistry (≥20% positive cells), 75% of patients (12/16) with p53+ tumors demonstrated locoregional disease control compared to only 18% (2/11) with p53− tumors p=0.0063 Fisher’s Exact Test. In addition, the median survival of patients with abnormal p53 was 11.6 months compared to only 3.5 months in patients with normal p53 (p=0.0007 Logrank Test). Conclusions: Our findings indicate that abnormal p53 is a predictive biomarker that identifies a subset of patients most likely to benefit from p53 gene therapy and that recurrent SCCHN tumor response definitions based upon smaller reductions in tumor size or the absence of progression (stable disease > 3 months) more accurately identified Advexin treated patients with increased survival than conventional response criteria. [Table: see text]

Enhanced tumor cell kill by combined treatment with a small-molecule antagonist of mouse double minute 2 and adenoviruses encoding p53

Strategies to treat cancer by restoring p53 tumor suppressor functions are being actively investigated. These approaches range from expressing an exogenous p53 gene in p53 mutant cancers to antagonizing a p53 inhibitor in p53 wild-type (WT) cancer cells. In addition, exogenous p53 is used to strengthen the anticancer efficacy of oncolytic adenoviruses. Many cancers express high levels of the major negative regulator of p53, mouse double minute 2 (MDM2) protein. Recently, a novel class of highly potent and specific MDM2 antagonists, the Nutlins, was identified. We envisioned that Nutlins could protect both endogenous and exogenous p53 from MDM2-mediated inactivation. We therefore investigated treating human cancer cells with a combination of adenovirus-mediated p53 gene therapy and Nutlin. Combination treatment resulted in broadly effective cell kill of p53 WT and p53-negative cancer cells. Cytotoxicity was associated with profound cell cycle checkpoint activation and apoptosis induction. We also tested Nutlin in combination with oncolytic adenoviruses. Nutlin treatment accelerated viral progeny burst from oncolytic adenovirus-infected cancer cells and caused an estimated 10- to 1,000-fold augmented eradication of p53 WT cancer cells. These findings suggest that Nutlins are promising compounds to be combined with p53 gene therapy and oncolytic virotherapy for cancer.

45. Prognostic Determinants Associated with Efficacy of Adenoviral p53 Gene Therapy (Advexin) in Patients with Recurrent Squamous Cell Carcinoma of the Head and Neck (SCCHN)

45. Prognostic Determinants Associated with Efficacy of Adenoviral p53 Gene Therapy (Advexin) in Patients with Recurrent Squamous Cell Carcinoma of the Head and Neck (SCCHN)

Study of p53 gene therapy combined with radiotherapy in tumor

野生型p53基因的导入不仅可诱导肿瘤细胞凋亡,而且还可以提高肿瘤细胞对放射线的敏感性,从而增强放射治疗对肿瘤细胞的杀伤作用,但其作用机制尚未完全明了.野生型p53基因和放射线的协同作用有助于提高肿瘤疗效,从而使针对p53基因的基因治疗和放射线联合治疗方案具有良好的应用前景。

INGN 201

INGN 201

Trans-arterial gene therapy for hepatocellular carcinoma in a rabbit model

To study the effect of adenovirus (Ad)-p53 gene therapy on hepatocellular carcinoma (HCC) in a rabbit model. VX2 tumor was grown in the liver of 24 rabbits. Animals were divided into four groups: group A receiving trans-arterial gene therapy (Ad-p53) only, group B receiving combined Ad-p53 therapy and trans-arterial embolization (lipiodol), group C receiving trans-arterial chemoembolization (lipiodol + mitomycin C), control group (D) receiving sodium chloride. Tumor volume (V1) was measured by using MRI (d 13). Interventional procedure was applied (d 14). Tumor volume (V2) was assessed by MRI (d 21) and the mean ratio (V2/V1) was calculated. After the second MRI, specimens of the liver were abstained and examined immunohistochemically using mutant-type p53 antibody. The positive expression was scored. Compared with control group (chi= 3.14 +/- 0.64), therapeutic groups all showed a significant decrease in the tumor growth ratio (P<0.05). A slight difference was found between group A (chi = 2.35 +/- 0.59) and group B (chi = 1.75 +/- 0.28) (P=0.048). No statistically significant difference was observed between group B and group C (chi = 2.00 +/- 0.44). The positive expression rate of mutant-type p53 was the lowest in group B and significantly different between group A and group C (P<0.05). Compared to the control subjects, groups A and C both showed a decrease in the expression of mutant-type p53, but there was no significant difference between them. Trans-arterial Ad-p53 gene therapy can reduce tumor growth of HCC in rabbit model.

Identification and Functional Characterization of the Hepatic Stellate Cell CD38 Cell Surface Molecule

The activation of hepatic stellate cells (HSCs) is a critical event in hepatic fibrosis, because these cells are the main producers of extracellular matrix proteins in the liver and contribute to the modulation of inflammatory responses via the secretion of several cytokines and the expression of adhesion molecules. The goal of the present study was to characterize cell surface proteins that regulate HSC activation. To this end, a panel of monoclonal antibodies (mAbs) was generated. mAb 14.27 recognized a protein of 45 kd that was highly expressed on HSCs. Affinity purification of this protein followed by sequencing revealed that protein to be CD38. We subsequently demonstrated that CD38 was constitutively expressed by HSCs and that its expression increased after in vitro and in vivo activation. mAb 14.27 induced an increase in cytosolic Ca2+ levels in HSCs, showing that it functions as an agonistic antibody. Moreover, the effects mediated by the CD38 mAb included induction of the proinflammatory cytokine interleukin-6 and up-regulation of the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and neural cell adhesion molecule. Collectively, our data suggest that CD38 can act as a regulator of HSC activation and effector functions.

Importance of p53 for cancer onset and therapy

Cancer predisposition, onset and therapeutic response can be critically determined by the integrity of the tumor suppressor p53. The majority of human cancers appear to exhibit either abnormal p53 or disrupted p53 activation pathways. Intervention to restore wild-type p53 activities is an attractive approach for cancer therapy. The manipulation of p53 and its targets is a challenging field that is still in its infancy, but witnessing some notable developments in the areas of p53 gene therapy, mutant reactivation and suppression of the negative p53 regulator Mdm2 using small molecules. In addition, wild-type p53 manipulation in healthy tissues of cancer patients in the context of chemotherapy and radiation therapies is offering the potential of enhanced patient recovery.

Eradication of p53-Mutated Head and Neck Squamous Cell Carcinoma Xenografts Using Nonviral p53 Gene Therapy and Photochemical Internalization

Photochemical internalization (PCI) technology has been used for PEI-mediated p53 gene transfer in mice bearing head and neck squamous cell carcinoma (HNSCC) xenografts. Using luciferase as a reporter gene, PCI led to a 20-fold increase in transgene expression 48 h after transfection and sustained transgene expression for 7 days. Therefore, iterative p53 gene transfer was performed by means of a weekly single injection of PEIGlu4/p53 complexes alone or with PCI for 5 (group A) or 7 (group B) weeks. The efficiency of p53 gene therapy was evaluated by following tumor growth and expression of P53-related downstream proteins (P21, MDM2, Bcl2, Bax). Apoptosis induction was evidenced through caspase-3 activation and PARP cleavage. Using PCI, tumor growth inhibition was observed in all transfected animals. Further, successful tumor cure was achieved in 17% (group A) and 83% (group B) of animals. PCI-mediated p53 gene transfer led to higher P53 protein expression that was correlated with induction of Bax and P21 proapoptotic proteins, repression of Bcl2 as well as activation of caspase-3, and cleavage of PARP. The present study demonstrates that PCI enhances the in vivo efficiency of PEI-mediated p53 gene transfer and can be proposed for p53 gene therapy in HNSCC.

298. Combination Effect of Telomerase-Specific Replication-Competent Adenovirus (Telomelysin, OBP-301) and p53 Gene Therapy in Human Non- Small Cell Lung and Colon Cancer Cells

Gene therapy was investigated as a promising treatment for cancer. However, low transduction efficiency limits the efficacy of gene therapy in clinics. We previously demonstrated that telomerase- specific replication-competent adenovirus (Telomelysin, OBP-301), in which the human telomerase reverse transcriptase (hTERT) promotor element drives expression of E1A and E1B genes linked with an IRES, induced selective E1 expression and efficient antitumor effect in human cancer cells, but not normal human fibroblasts. In this study, we evaluated the anticancer effect of the combination therapy of E1-deleted replication-deficient adenovirus expressing the wild-type p53 tumor suppressor gene (ADVEXIN, Ad-p53) and OBP-301 in human H1299 non-small cell lung cancer cells and SW620 colon cancer cells by using cell proliferation assay, immunoblotting, and cell-cycle analysis. We found that E1-deficient Ad-p53 could replicate in human cancer cells in the presence of OBP-301 and kill infected cells more efficiently than Ad-p53 alone or OBP-301 alone. Western Blotting Analysis demonstrated that OBP-301 plus Ad-p53 induced high levels of p53 protein expression, leading to cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). In addition, flowcytometry showed that the sub-G1 population was dramatically increased with OBP-301 and Ad-p53 infection. The in vivo combination effect of intratumoral injection of Ad-p53 and OBP-301 in a human tumor xenograft model will be also reported. Our data demonstrate that OBP-301 in combination with Ad-p53 efficiently enhances antitumor activity, and provide the potential to affect the development of oncolytic virotherapy and gene therapy.

Recent advances on p53 gene therapy

人类p53基因位于17p13.1,全长约16～20 kb,由11个外显子和10个内含子组成,编码393个氨基酸,是相对分子质量为53 000的核磷酸化蛋白[1].它与肿瘤的发生、发展以及肿瘤患者的预后有着十分密切的关系.大约50%的人类肿瘤中存在p53基因的变化, 因此,针对p53进行肿瘤基因治疗逐渐成为研究的热点。

943. Prognostic Determinants Associated with Efficacy of Adenoviral p53 Gene Therapy in Patients with Recurrent Squamous Cell Carcinoma of the Head and Neck

Patients with recurrent SCCHN need effective therapies that do not compound tumor morbidity. We investigated the long term safety and prognostic factors influencing efficacy of adenoviral p53 gene therapy in a large series of SCCHN patients.

Adenoviral Gene Therapy in Head and Neck Cancer

Despite advances in surgical techniques, improvement in radiation therapy and the addition of new biological agents such as cetuximab to traditional chemotherapy, the median survival of patients with head and neck cancer has changed little over the past few decades. However, recent advances in the fundamental understanding of head and neck cancer biology suggest that targeting molecular pathways underlying carcinogenesis may provide alternative or additional approaches to the treatment of head and neck cancer. Viruses, particularly adenoviruses, have been critical in the application and development of these molecular approaches. Adenoviruses have been engineered to function as vectors for delivering therapeutic genes for gene therapy. The purpose of this review is to provide a prospective on the use of adenoviruses in head and neck cancer therapy by examining clinical trials of adenovirus-mediated p53 gene therapy and by reviewing the application of a promising oncolytic adenovirus, ONYX-015, in head and neck cancer.

289. Adenoviral-Mediated p53 Gene Therapy (Advexin) in a Patient with Li-Fraumeni Syndrome with a Mixed Germ Cell Tumor: Tolerability and Response

Li-Fraumeni syndrome is an autosomal dominant disorder characterized by familial clustering of malignancies, predominantly sarcomas, breast cancer, brain tumors and adrenocortical carcinoma; an inordinate pattern of multiple primaries; and occurrence at a young age. Approximately 70% of the cases can be attributed to TP53 germ-line mutation 73% of which are located in the coding regions of exons 5 to 8. The index patient is a 25 year old woman presenting in 2002 with abdominal pain, anorexia and vomiting leading to the diagnosis of granulosa cell tumor confirmed by IHC-the second such case reported with the syndrome. TP53 sequencing showed 151delG, a frameshift mutation in exon 4. The mutation results in a stop codon downstream. Results of IHC for gene expression are pending. She was initially treated with bleomycin, cisplatin and velban. Following recurrence in 2004, the patient was received Ifosfamide, cisplatin and etoposide followed by the substitution of paclitaxel for Ifosfamide after development of encephalopathy. Disease progression eventuated in pelvic radiation and, with the development of CNS metastases, whole brain radiation.

726. Clinical Trial of Recombinant Adenovirus-p53 (Gendicine) Combined with Radiotherapy in Nasopharyngeal Carcinoma Patients

The study was to evaluate the safety and the efficacy of recombinant adenovirus-p53 (Adp53, trademarked as Gendicine) combined with radiotherapy in nasopharyngeal carcinoma (NPC) patients. A controlled clinical trial on Gendicine combined with radiotherapy, that is, gene therapy + radiotherapy (GTRT), was conducted in 24 patients, and 25 patients treated with radiotherapy alone (RT) formed the control group. In the GTRT group, Gendicine 1|[times]|1012 virus particle (vp) was intratumorally injected once a week over to eight weeks, and concurrent irradiation was given. For both groups, the conventional fractionation 2Gy/day, five fractions a week, with a total dose of 70Gy in 35 fractions were given to either primary tumor or neck lymph node. Patients were monitored for adverse event and serum anti-adenoviral antibody, and tumors were monitored for response. A comparative study was performed between both groups on the immediate response rate by CT at the end of 4th week, 7th week and 2 months after the treatment. The median follow-up interval of surviving patients was 34.5 months (24|[sim]|45 months). Wild-type p53 gene therapy enhanced significantly radiotherapy efficacy by 1.59 times in patients with NPC at 4- week time point. Two months after the treatment complete response rate of GTRT group was 2.1 times that of RT group (62.5% v.s. 29.6%). The 3-year overall survival rate of GTRT group was 14.4% higher than that of GT group. No dose-limiting toxicity and adverse events were noted, except for transient fever after Gendicine administration. Intratumoral injection with Gendicine was safe and effective for patients with NPC.

Detection of p53 gene change and serum antibody level in phase II clinical trial of ad p53 gene therapy

To determine the alteration of p53 gene in tumor tissues and to monitor the immunoresponsiveness to anti adenovirus in patients following treatment of recombinant adenovirus-p53 (Adp53, SBN-1) combined with radiation. Tumor tissues were collected from 22 patients with malignant tumors at advanced stage before and after treatment with recombinant adenovirus-p53 (Adp53, SBN-1) combined with radiation. Immunohistochemistry was used to detect the p53 expression. Microdissection, PCR, and denaturation high performance liquid chromatography (DHPLC), and DNA sequencing were used to detect the mutation of p53 gene. The levels of the anti-adenovirus antibody SBN-1, IgG and IgM in the serum were detected by using ELISA. p53 positive staining was shown in the nuclei of tumor cells in 6 of 15 tumor samples showing the increase of stability of protein caused by gene mutation. DHPLC and DNA sequencing showed point mutation in 7 of 22 tumor samples. Six of the 7 cases with p53 gene mutation showed complete and partial remission at the degree > 50%, and the 8 cases without p53 gene mutation only showed partial remission or stability of condition. The IgG positive rate was 50% (8/ 16), showing that 50% of the patients had been infected by adenovirus (mainly the types 3, 7, and 11) before. The IgM positive rate was 6% (1/16), showing that most of the patients had not been infected by adenovirus (mainly the types 3, 7, and 11) at that time. The anti-SBN-1 antibody was negative in all patients before the rAd-53 therapy and became positive since the 4th week after the beginning of treatment and the intensity of positivity increased along with the time. An effective gene diagnosis system for detecting the p53 gene alteration has been developed.

Mutations in p53 cDNA sequence introduced by retroviral vector

The high mutation rates of retroviruses are a potential problem with retroviral vectors. We studied the mutation rates and spectra of p53 sequences transduced with a retroviral vector in a cancer gene therapy model. When p53-deficient H358 non-small cell lung cancer cells were treated with a retroviral vector carrying normal p53 cDNA, most of transduced cells were killed by apoptosis. However, a small number of clones escaped p53-mediated apoptosis. We examined the p53 cDNA structure in these resistant clones. PCR-based analysis showed that 88/102 clones had detectable mutations in p53, including gross rearrangements, deletions/insertions, and base substitutions. To study the mutation rate of the p53 sequence in all transduced clones, the retroviral vector containing the non-functional p53 gene and the Neo-resistant marker gene was introduced into H358 cells. Only one of 95 isolated clones showed a base substitution. These results indicate that the mutation rate of p53 is not particularly high, but there is a significant risk that cancer cells will resist p53 gene therapy as a result of retroviral replication errors.

Mild hyperthermia plus adenoviral p53 over-expression additively inhibits the viability of human malignant glioma cells

Adenoviral replacement of the p53 gene has already been proved effective for the treatment of various tumours, including malignant gliomas. However, it is difficult to treat malignant glioma with p53 gene therapy alone because of problems with resistance or a less-than-satisfactory response to the treatment. This study investigated whether heat shock at 43°C (mild hyperthermia) augments the cytotoxic effect of p53 gene transfer on malignant glioma cells expressing wild-type p53 (D54) or mutant p53 (U373-MG and U251-MG). The combination of mild hyperthermia and adenoviral p53 over-expression had an additive inhibitory effect on cellular proliferation in all three cell lines studied. Further, both cell cycle analysis and a DNA fragmentation assay showed that apoptosis was induced by p53 over-expression alone but not by heat shock at 43°C alone. However, p53 over-expression followed by mild hyperthermia additively increased the proportion of cells in which apoptosis was induced, regardless of the endogenous p53 status of the tumour cells. Interestingly, a caspase-independent mechanism was observed to be involved in the p53-induced apoptosis in U251-MG and D54 cells. Taken together, the findings showed that combining adenoviral p53 transfer with mild hyperthermia inhibits the proliferation of malignant glioma cells in an additive manner, irrespective of their endogenous p53 status, suggesting a novel treatment strategy for this malignancy.