The dl1520 virus is found preferentially in tumor tissue after direct intratumoral injection in oral carcinoma.

PURPOSE: dl1520 (also known as Onyx-015) is an E1B-deleted adenovirus designed to selectively lyse p53-deficient cancer cells. Clinical trials involving patients with recurrent squamous cell carcinoma of the head and neck have shown clinical efficacy, but no direct evidence as to the tumor or p53 selectivity of the virus was demonstrated. We wanted to determine whether dl1520 is selective for survival and replication within tumor tissue after direct injection and whether this is determined by p53 status of the tissues. We also wanted to ascertain whether the virus has any macroscopic effect on normal tissue. EXPERIMENTAL DESIGN: An open-label Phase II trial was devised in which a fixed dose of the virus was administered to 15 patients via a direct intertumoral injection before surgery for untreated oral squamous cell carcinoma. The agent was also delivered into an area of adjacent normal buccal mucosa. Specimens of the excised tumor and of biopsies of the injected normal tissue were assessed for viral presence and p53 status. RESULTS: We demonstrated that the virus replicates selectively in tumor as opposed to normal tissue after this direct injection. It was not possible to determine whether this selectivity was p53 related. It was found that dl1520 triggers an early rise in apoptosis levels in injected normal tissues. No adverse effects of viral injection were noted. CONCLUSIONS: This is the first report of injection of dl1520 into previously untreated squamous cell cancer. The data support the concept that dl1520 is replication deficient in normal, compared with cancerous, tissues and has potential as a selective anticancer agent against tumor tissues.     

Intravascular adenoviral agents in cancer patients: lessons from clinical trials

A large number of adenoviral agents are being developed for the treatment of cancer. However, the treatment-related death of a patient with ornithine transcarbamylase deficiency following adenovirus administration by hepatic artery has led to serious concerns regarding the safety of intravascular adenovirus. Both replication-incompetent (rAd.p53, e.g., SCH58500) and replication-selective (dl1520, aka Onyx-015; CG7870) oncolytic adenoviruses, by intravascular administration, are in clinical trials. We review Phases I and I/II results from these clinical trials. dl1520 and rAd.p53 were well-tolerated following hepatic artery infusion at doses of up to 2x10(12) and 2.5x10(13) particles, respectively. At a dose of 7.5x10(13) particles, rAd.p53 was associated with dose-limiting cardiac output suppression; dl1520 dose escalation did not proceed higher than 2x10(12). Intravenous (i.v.) infusions of dl1520 and CG7870 have been well tolerated by i.v. infusion at doses of 2x10(13) and 6x10(12), respectively, without identification of a maximally tolerated dose to date. Mild/moderate transaminitis was demonstrated in some patients on both the hepatic arterial and i.v. trials at doses >or=10(12) particles. Interleukin (IL)-6 and IL-10 were induced in a dose-dependent manner in most patients, but significant interpatient and intrapatient (on repeat doses) variabilities were demonstrated. Evidence of p53 gene expression (Ad.p53) or viral replication (dl1520) was demonstrated in the majority of patients receiving >or=10(12) particles. Over 100 cancer patients have been treated with intravascular adenovirus constructs to date with an acceptable toxicity profile; further clinical trial testing appears appropriate in cancer patients.     

Selective replication and oncolysis in p53 mutant tumors with ONYX-015, an E1B-55kD gene-deleted adenovirus, in patients with advanced head and neck cancer: a phase II trial

ONYX-015 is an E1B-55kDa gene-deleted adenovirus engineered to selectively replicate in and lyse p53-deficient cancer cells. To evaluate the selectivity of ONYX-015 replication and cytopathic effects for the first time in humans, we carried out a Phase II clinical testing of intratumoral and peritumoral ONYX-015 injection in 37 patients with recurrent head and neck carcinoma. Patients received ONYX-015 at a daily dose of 1 x 10(10) plaque-forming units (pfu) via intratumoral injection for 5 days during week 1 of each 3-week cycle (n = 30; cohort A), or 1 x 10(10) pfu twice a day for 10 days during weeks 1 and 2 of each 3-week cycle. Posttreatment biopsies documented selective ONYX-015 presence and/or replication in the tumor tissue of 7 of 11 patients biopsied on days 5-14, but not in immediately adjacent normal tissue (0 of 11 patients; P = 0.01). Tissue destruction was also highly selective; significant tumor regression (>50%) occurred in 21% of evaluable patients, whereas no toxicity to injected normal peritumoral tissues was demonstrated. p53 mutant tumors were significantly more likely to undergo ONYX-015-induced necrosis (7 of 12) than were p53 wild-type tumors (0 of 7; P = 0.017). High neutralizing antibody titers did not prevent infection and/or replication within tumors. ONYX-015 is the first genetically engineered replication-competent virus to demonstrate selective intratumoral replication and necrosis in patients. This agent demonstrates the promise of replication-selective viruses as a novel therapeutic platform against cancer.     

Interactions between adenovirus proteins and the p53 pathway: the development of ONYX-015

dl1520 (ONYX-015) is an adenovirus mutant that lacks the E1b 55K gene. As a result it cannot neutralize p53. Therefore dl1520 should only grow in cells that lack p53, and should replicate selectively in cancer cells. However, there is no correlation between replication and p53 status, in cancer cells. This is for two reasons: (1) E1B 55K has additional functions, that are necessary in some tumor cells and not in others. (2) p53 function can be lost by alternative mechanisms such as loss of p14ARF. In normal cells, dl1520 induces p53, and is generally strongly attenuated for replication. ONYX-015 is currently being tested in clinical trials, and is a promising new therapeutic agent in cancer.     

Activity of the adenoviral E1A deletion mutant dl922-947 in ovarian cancer: comparison with E1A wild-type viruses, bioluminescence monitoring, and intraperitoneal delivery in icodextrin

The adenoviral mutant dl922-947 has potent activity in a variety of tumors. We investigated the efficacy of dl922-947 in ovarian carcinoma; compared its activity to wild-type adenovirus, dl309, and dl1520; and investigated the use of icodextrin to enhance activity in vivo. We also assessed the utility of luciferase bioluminescence imaging to quantify the response of human ovarian carcinoma xenografts to dl922-947. Ovarian carcinoma cell lines were transfected in vitro with dl922-947, adenovirus 5 wild-type (Ad5 WT), dl309, and dl1520 and monitored for S-phase induction, viral protein expression, replication, and overall survival. In vivo, the efficacy of dl922-947 when delivered in PBS or icodextrin to female nude mice bearing IGROV1 xenografts was determined. In vitro, dl922-947 induced lysis with greater efficacy than Ad5 WT, dl309, or dl1520 in all ovarian carcinoma cell lines tested, which was associated with earlier expression of viral proteins and S-phase induction. The lytic effect in immortalized ovarian surface epithelial cells confirmed that cellular retinoblastoma pathway status is a strong determinant of dl922-947 activity. In vivo, i.p. delivery of dl922-947 (5 x 10(9) particles daily x 5) increased median survival from 20 to 96 days (P < 0.0001) and delivery in icodextrin-enhanced survival further. However, delayed hepatic toxicity was evident in some dl922-947-treated mice, which was not dependent upon viral replication within tumor cells or the liver. dl922-947 has potency in ovarian carcinoma and i.p. delivery in icodextrin may enhance this activity. Immunocompetent models of ovarian carcinoma are required for further evaluation of hepatotoxicity.     

p53 selective and nonselective replication of an E1B-deleted adenovirus in hepatocellular carcinoma.

An E1B gene-attenuated adenovirus (dl1520) has been proposed to have a selective cytolytic activity in cancer cells with a mutation or deletion in the p53 tumor suppressor gene (p53-null), a defect present in almost half of human hepatocellular carcinomas (HCCs). In this study, the in vitro and in vivo antitumor activity of dl1520 was investigated focusing on two human HCC cell lines, a p53-wild type (p53-wt) cell line and a p53-null cell line. dl1520 was tested for in vitro cytopathic effects and viral replication in the human HCC cell lines Hep3B (p53-null) and HepG2 (p53-wt). The in vivo antitumor effects of dl1520 were investigated in tumors grown s.c. in a severe combined immunodeficient mouse model. In addition, the combination of dl1520 infection with systemic chemotherapy was assessed in these tumor xenografts. At low multiplicities of infection, dl1520 had an apparent p53-dependent in vitro viral growth in HCC cell lines. At higher multiplicities of infection, dl1520 viral replication was independent of the p53 status of the target cells. In vivo, dl1520 significantly retarded the growth of the p53-null Hep3B xenografts, an effect augmented by the addition of cisplatin. However, complete tumor regressions were rare, and most tumors eventually grew progressively. dl1520 had no effect on the in vivo growth of the p53-wt HepG2 cells, with or without cisplatin treatment. The E1B-deleted adenoviral vector dl1520 has an apparent p53-dependent effect in HCC cell lines. However, this effect is lost at higher viral doses and only induces partial tumor regressions without tumor cures in a human HCC xenograft model.     

E1B55kDa缺陷型腺病毒对人肝癌细胞的杀伤研究

目的 探讨E1B55kDa缺陷型腺病毒dl1520对肝癌细胞的体内外杀伤作用。方法 用dl1520分别感染p53基因型不同的人肝癌细胞,感染后第4天用染色的方法检测存活细胞。用RT－PCR检测细胞内p53和p21^Waf-1基因表达的改变,通过检测腺病毒衣壳蛋白hexon基因的表达证实腺病毒的感染。在SCID裸鼠瘤体内注射dl1520,观察dl1520对肝癌细胞的体内杀伤作用。结果 p53基因缺失的肝癌细胞Hep3B对dl1520诱导的细胞毒性作用最敏感,超过60％的细菌被杀伤,而不足20％的PLC／PRF／5（p53基因突变型）和HepG2(p53基因野生型）肝癌细胞被杀伤。腺病毒感染后,HepG2细胞内p53和p21^Waf-1基因表达水平均明显升高。瘤体内注射dl1520,可显著抑制Hep3B裸鼠移植瘤的生长,而对PLC／PRF／5和HepG2的裸鼠移植瘤则无明显的生长抑制作用。结论 E1B55kDa缺失的腺病毒可以选择性地杀伤p53基因缺失的肝癌细胞,是一种潜在的肿瘤治疗手段。     

In vivo antitumor activity of ONYX-015 is influenced by p53 status and is augmented by radiotherapy

The E1B-deleted, replication-competent ONYX-015 (dl1520) adenovirus was originally described as being able to selectively kill p53-deficient cells due to a requirement of p53 inactivation for efficient viral replication. This hypothesis has become controversial because subsequent in vitro studies have demonstrated that the host range specificity of ONYX-015 is independent of p53 gene status. Using a pair of isogenic cell lines that differ only in their p53 status, we demonstrate here that although ONYX-015 can replicate in both p53 wild-type and mutant cells in vitro, the virus demonstrates significantly greater antitumor activity against mutant p53 tumors in vivo. Moreover, ONYX-015 viral therapy can be combined with radiation to improve tumor control beyond that of either monotherapy. The results demonstrate that ONYX-015 can discern in vivo between tumors having a different p53 status and that it may be an effective neoadjuvant to radiation therapy.     

E1B缺陷腺病毒对鼻咽癌CNE-2细胞杀伤作用及其机理研究

为总结我国肿瘤内科临床与基础研究成果 ,促进肿瘤内科学发展 ,由中华医学会、中华医学杂志编辑委员会主办的“首届中国肿瘤内科论坛” ,定于 2 0 0 2年第三季度召开 (具体时间、地点另行通知 )。本次会议将由出版社出版正式专著《中国肿瘤内科学研究进展》和光盘版。一、征文内容 :(1)肿瘤化疗与化疗药物的合理应用 ;各种恶性肿瘤化疗方案的制定与临床应用 ;化疗并发症的防治等。(2 )肿瘤耐药与耐药逆转剂的研究与临床应用。 (3)各类抗肿瘤药物的研究及进展。 (4 )肿瘤的生物治疗、免疫治疗与基因治疗等。 (5 )实体瘤和白血病高剂量化疗及…     

E1B缺陷腺病毒对鼻咽癌CNE-2细胞杀伤作用及其机理研究

目的：研究E1B缺陷腺病毒d11520对鼻咽癌CNE-2细胞的杀伤作用及其作用机理。方法：用MTT法和细胞病变试验（CPE）测量d11520在体外对CNE-2细胞的抑制和杀伤作用。并通过测定病毒在CNE-2细胞中的复制产量和DAPI染色试验探讨其杀伤机理；瘤内注射d11520，观察其对CNE-2裸鼠移植瘤的治疗效果。用免疫织化方法检测肿瘤组织中病毒的复制。结果：体外实验结果显示：d11520能在CNE-2细胞中复制，导致明显的细胞病变，显著抑制CNE-2细胞的生长，在病变细胞中可见明显的核膨胀，瘤内注射d11520能明显抑制裸鼠移植瘤的生长，在肿瘤组织中可检测到病毒复制。结论：d11520能在CNE-2细胞中复制并使之裂解，从而在体内外有效地抑制和杀伤CNE-2细胞。     

A phase I/II trial of rAd/p53 (SCH 58500) gene replacement in recurrent ovarian cancer

PURPOSE: To determine the safety, gene transfer, host immune response, and pharmacokinetics of a replication-deficient adenovirus encoding human, recombinant, wild-type p53 (SCH 58500) delivered into the peritoneal cavity (i.p.) alone and sequentially in combination with platinum-based chemotherapy, of patients with recurrent ovarian, primary peritoneal, or fallopian tube cancer containing aberrant or mutant p53. METHODS: SCH 58500 was administered i.p. to three groups of patients with heavily pretreated recurrent disease. Group 1 (n=17) received a single dose of SCH 58500 escalated from 7.5 x 10(10) to 7.5 x 10(12) particles. Group 2 (n=9) received two or three doses of SCH 58500 given alone for one cycle, and then with chemotherapy for two cycles. The SCH 58500 dose was further escalated to 2.5 x 10(13) particles/dose in group 2. A third group (n=15) received a 5-day regimen of SCH 58500 given at 7.5 x 10(13) particles/dose per day i.p. alone for cycle 1 and then with intravenous carboplatin/paclitaxel chemotherapy for cycles 2 and 3. RESULTS: No dose-limiting toxicity resulted from the delivery of 236/287 (82.2%) planned doses of SCH 58500. Fever, hypotension abdominal complaints, nausea, and vomiting were the most common adverse events. Vector-specific transgene expression in tumor was documented by RT-PCR in cells from both ascitic fluid and tissue biopsies. Despite marked increases in serum adenoviral antibody titers, transgene expression was measurable in 17 of 20 samples obtained after two or three cycles of SCH 58500. Vector was detectable in peritoneal fluid by 24 hours and persisted for as long as 7 days whereas none was detected in urine or stool. There was poor correlation between CT scans and CA125 responses. CA125 responses, defined as a greater than 50% decrement in serum CA125 from baseline, were documented in 8 of 16 women who completed three cycles of the multidose regimen. CONCLUSION: CT scans are not a valid measure of response to i.p. SCH 58500 due to extensive adenoviral-induced inflammatory changes. Intraperitoneal SCH 58500 is safe, well tolerated, and combined with platinum-based chemotherapy can be associated with a significant reduction of serum CA125 in heavily pretreated patients with recurrent ovarian, primary peritoneal, or fallopian tube cancer.     

Thymidine kinase gene therapy with concomitant topotecan chemotherapy for recurrent ovarian cancer

INTRODUCTION: Patients with recurrent ovarian cancer were treated with a replication-deficient recombinant adenovirus containing the herpes simplex virus thymidine kinase gene administered intraperitoneally (i.p.) followed by administration of an anti-herpetic prodrug and topotecan. MATERIALS AND METHODS: A total of 10 patients with stage IIIc epithelial ovarian cancer underwent secondary debulking to < or =0.5 cm residual tumor. Patients with normal i.p. flow received i.p. delivery of adenovirus. Two patients each were treated on dose level 1 (2 x 10(10) vector particles (VP)), dose level 2 (2 x 10(11) VP), and dose level 3 (2 x 10(12) VP); four patients were treated on dose level 4 (2 x 10(13) VP). Acyclovir and topotecan were started 24 hours after vector delivery. RESULTS: No patient treated at any dose level incurred unanticipated toxic effects, and all side effects resolved. The most common adverse event was myelosuppression: grade 3 or 4 thrombocytopenia with grade 2-4 anemia in three patients and grade 3 or 4 neutropenia in eight patients. Three patients developed thrombocytosis and three patients had a mild elevation of serum glutamic pyruvic transaminase/alanine aminotransferase. Temperature elevations that were not associated with detectable infection occurred in two patients. DISCUSSION: I.p. delivery of adenoviral vector with concomitant topotecan chemotherapy was well tolerated without significant lasting toxicities. Side effects were independent of the dose of adenoviral vector.     

In vivo studies of adenovirus-mediated p53 gene therapy for cis-platinum-resistant human ovarian tumor xenografts.

We have recently reported that mutations of the tumor suppressor p53 gene are associated with the development of resistance to cis-platinum in human ovarian cancer cells, and that adenovirus-mediated reintroduction of the wild-type p53 (wtp53) gene in ovarian tumor cells resulted in the sensitization of tumor cells to cis-diamminedichloroplatinum (II) (CDDP). The purpose of this study was to evaluate whether i.p. treatment of CDDP-resistant tumor cells expressing mutant p53 (mutp53) with a recombinant adenovirus expressing wtp53 (Adwtp53) would result in the sensitization of resistant cells to CDDP. In order to determine whether i.p. injection of a recombinant adenovirus would result in expression of the transgene in tumor cells growing intraperitoneally, we first injected A2780/CP cells in nude mice and 10 days later the mice were injected i.p. with a recombinant adenovirus expressing beta-galactosidase (Ad beta-gal). Twenty-four hours following i.p. injection of Ad beta-gal, tumors were removed and stained for beta-gal. While tumors showed extensive staining for beta-gal, indicating internalization of adenovirus and the expression of the transgene in tumors, no expression of beta-gal protein was detected in liver. I.p. treatment of A2780/CP tumor xenografts with Adwtp53 caused extensive tumor cell death, which was further enhanced by CDDP. Treatment with Adwtp53 (5 x 10(7) pfu/day, 3-5 treatments) resulted in a significant decrease in tumor volume and increase in animal survival compared to either no treatment or treatment with vector alone without p53 gene. Additional therapy with CDDP (1 mg/kg/day x 3-4) further reduced tumor volume and increased survival (30-40%), suggesting that combination therapy of Adwtp53 and CDDP was better than single agents alone. Our results indicate that i.p. dosing with adenovirus-mediated wtp53 gene therapy could be beneficial in combination with CDDP for the treatment of ovarian tumors expressing mutp53.     

Liver-directed viral therapy for cancer p53-targeted adenoviruses and beyond

Loss of p53 function is one of the most frequent genetic alterations in human cancers. Both replication-incompetent (rAd.p53, or SCH58500) and replication-selective (dl1520, or Onyx-015) adenoviruses are being developed for the treatment of p53-deficient cancers. Hepatic arterial infusion (HAI) has historically been used to selectively target colorectal tumors within the liver; consequently, regional therapy with adenovirus in this setting is an attractive approach. This article reviews Phase I and I/II HAI trial results with these adenovirus constructs.     

Potentiation of radiation therapy by the oncolytic adenovirus dl1520 (ONYX-015) in human malignant glioma xenografts

In spite of aggressive surgery, irradiation and/or chemotherapy, treatment of malignant gliomas remains a major challenge in adults and children due to high treatment failure. We have demonstrated significant cell lysis and antitumour activity of the E1B-55 kDa-gene-deleted adenovirus ONYX-015 (dl1520, CI-1042; ONYX Pharmaceuticals) in subcutaneous human malignant glioma xenografts deriving from primary tumours. Here, we show the combined efficacy of this oncolytic therapy with radiation therapy. Total body irradiation (5 Gy) of athymic nude mice prior to intratumoral injections of ONYX-015 1 x 10(8) PFU daily for 5 consecutive days yielded additive tumour growth delays in the p53 mutant xenograft IGRG88. Radiation therapy was potentiated in the p53 functional tumour IGRG121 with a 'subtherapeutic' dose of 1 x 10(7) PFU daily for 5 consecutive days, inducing significant tumour growth delay, 90% tumour regression and 50% tumour-free survivors 4 months after treatment. These potentiating effects were not due to increased adenoviral infectivity or replication. Furthermore, cell lysis and induction of apoptosis, the major mechanisms for adenoviral antitumour activity, did not play a major role in the combined treatment strategy. Interestingly, the oncolytic adenovirus seemed to accelerate radiation-induced tumour fibrosis. Potentiating antitumour activity suggests the development of this combined treatment for these highly malignant tumours.     

Hepatic arterial infusion of a replication-selective oncolytic adenovirus (dl1520): phase II viral,immunologic, and clinical endpoints

Replication-selective oncolytic adenoviruses are being developed for the treatment of cancer, but the safety and feasibility of repeated adenovirus delivery to tumors via the bloodstream was unknown, particularly in light of a patient death after hepatic artery infusion of a replication-defective adenovirus vector. We performed a Phase II trial of an oncolytic replication-selective adenovirus (dl1520, also known as Onyx-015) administered by hepatic artery infusion in patients with gastrointestinal carcinoma metastatic to the liver (n = 27). dl1520 was infused into the hepatic artery (2 x 10(12) particles) on days 1 and 8 as a single agent, and thereafter starting on day 22 in combination with i.v. 5-fluorouracil and leucovorin every 28 days. Repeated viral infusions were feasible, and no deaths occurred on study; reversible grade 3/4 hyperbilirubinemia occurred in 2 patients. Systemic inflammatory cytokine responses varied greatly between patients and even between cycles within a given patient. Proinflammatory cytokines [e.g., tumor necrosis factor, IFN-gamma, and interleukin (IL) 6] typically rose within 3 h and were followed at 18 h by a rise in IL-10. However, in the single patient who suffered a severe but reversible systemic inflammatory response, a unique cytokine profile was detected: marked acute increases of IL-6 (20-fold higher than average for all of the patients) and inhibition of IL-10 production. Delayed secondary peaks of viremia were reproducibly detected 3-6 days after treatment, even in the presence of high level neutralizing antibody titers and antiviral cytokines. Mathematical modeling was used to calculate the number of virus particles produced and shed into the blood with each replication cycle. The combination of virotherapy and chemotherapy had antitumoral activity in some chemotherapy-resistant colorectal tumors. The intra-arterial infusion of oncolytic adenoviruses warrants additional study.     

Assessment of growth inhibition and morphological changes in in vitro and in vivo hepatocellular carcinoma models post treatment with dl1520 adenovirus

BACKGROUND AND AIMS: E1B-deleted virus dl1520 (ONYX-015) has been previously used in clinical trials mainly for treatment of head and neck tumors, and has been shown to have beneficial effects independent of p53 status. The main aim of this investigation was to carry out a preclinical study for assessment of the use of dl1520 in in vitro and in vivo hepatocellular carcinoma (HCC) models with various p53 status (deleted, mutant, and wild type), and study the ultrastructural changes in the carcinoma cells during and following treatment with dl1520. METHODS: dl1520 (ONYX-015) virus was used for treatment of three HCC cell lines in culture, then for treatment of developed xenografts in SCID mice. The effects of dl1520 on HCC cell growth and accompanied morphological changes were assessed by various techniques including transmission electron microscopy. dl1520 infection was confirmed using polymerase chain reaction and immunolabeling at transmission electron microscopy level. RESULTS: dl1520 was effective in killing cells and inhibiting HCC cell growth both in vitro and in vivo. The cell killing was at higher levels in cells possessing abnormal p53. Survival rates in SCID mice treated with dl1520 were statistically significantly higher in HCC tumors with deleted and mutant p53, than in tumors with wild-type p53. CONCLUSIONS: The findings in this study suggest that dl1520 could be safely and effectively used for treatment of HCC dependent on the p53 status of the cells in vivo. Characteristic morphological changes that took place in the dl1520-treated HCC cells/tumors were distinct at transmission electron microscopy level and are the first of their kind to be reported.