¹University of California, San Francisco, Department of Surgery, USA

²University of California, San Francisco, Department of Medicine, USA

³Onyx Pharmaceuticals, Richmond, CA, USA

⁴Imperial Cancer Research Fund, Imperial College School of Medicine, Viral and Genetic Therapy Program, Hammersmith Hospital, London, UK

Correspondence to: D Kirn, Molecular Oncology Unit, Imperial Cancer Research Fund, Hammersmith Campus, DuCane Road, London W12 0HS, UK

Novel therapies are needed for locally advanced pancreatic carcinoma. ONYX-015 (dl1520) is an E1B-55 kDa region-deleted adenovirus that selectively replicates in and lyses tumor cells with abnormalities in p53 function (eg gene mutation). We carried out a phase I dose escalation study of ONYX-015 in patients with unresectable pancreatic cancer. ONYX-015 was administered via CT-guided injection (n = 22 patients) or intraoperative injection (n = 1) into pancreatic primary tumors every 4 weeks until tumor progression. Interpatient dose escalation was carried out with at least three patients per dose level from 10⁸ p.f.u. up to the 10¹¹ p.f.u. dose level (two patients treated at this dose). The majority of patients had abnormally low cellular immunity (CD4 counts and hypersensitivity skin testing). Injection of ONYX-015 into pancreatic carcinomas was well-tolerated. Mild, transient pancreatitis was noted in only one patient. Dose-escalation proceeded to the highest dose level. Neutralizing antibodies rose post-treatment in all patients. After injection, ONYX-015 was detectable in the blood 15 min later, but not between 1 and 15 days later. Viral replication was not documented, however, in contrast to trials in other tumor types. No objective responses were demonstrated. Intratumoral injection of an E1B-55 kDa region-deleted adenovirus into primary pancreatic tumors was feasible and well-tolerated at doses up to 10¹¹ p.f.u. (2 ´ 10¹²particles), but viral replication was not detectable. Gene Therapy (2001) 8, 308-315.

adenovirus; replication-selective; oncolytic virus; pancreatic cancer; phase I clinical trial

Introduction

The prognosis for patients with unresectable pancreatic carcinoma is very poor, with less than 5% still alive 3 years after diagnosis.¹ Most tumors are not amenable to surgical resection at the time of diagnosis. Unresectable pancreatic carcinomas have frequently spread through direct extension into adjacent structures (ie locally advanced) or have metastasized to the liver through the portal vein (ie regionally metastatic). Patients with locally advanced disease are treated with radiation therapy, chemotherapy or with a combination of these modalities. Unfortunately, patients almost universally experience tumor progression and death despite aggressive therapy.¹ Once these tumors have metastasized to the liver, chemotherapy with gemcitabine or 5-fluorouracil is the standard of care.² Objective tumor responses are achieved in 0-10% of these patients with either chemotherapeutic agent, and only approximately 20% remain alive after 1 year.² Therefore, novel therapies are needed for patients with locally advanced or regionally metastatic pancreatic carcinoma.

The p53 tumor suppressor gene pathway is frequently abnormal in pancreatic carcinomas.³ Approximately 40-60% will have mutations in the p53 gene.^4,5 P53 mutations have been associated with a worse prognosis and resistance to standard chemotherapeutics in numerous tumor types, including pancreatic carcinoma.⁵ In addition to mutation of the p53 gene, the p53 pathway can be disrupted through other genetic alterations including deletion of the p16/p14^arf gene locus.^6,7 Finally, overexpression of the mdm2 gene leads to increased p53 protein degradation.⁸ Therefore, the p53 pathway is disrupted in the vast majority of these tumors. Genetic alterations in the p53 pathway may contribute to the inherent resistance of these tumors to chemotherapy and radiotherapy.⁹ Novel therapies for p53-deficient pancreatic tumors are therefore needed.

ONYX-015 (dl1520) is an E1B-55 kDa gene-deleted adenovirus that replicates selectively in and destroys cancer cells with abnormalities in the p53 pathway, including pancreatic carcinoma cell lines.^10,11 Injection of this mutant adenovirus into p53-deficient human tumors grown in nude mice results in viral replication and necrosis within the tumor masses, including pancreatic tumors.¹⁰ We have also shown that ONYX-015 can spread hematogenously to distant tumor sites in this model, and that replication and necrosis occurs at these distant sites, as well.¹² Efficacy has also been documented following intravenous administration in these nude mouse-human tumor xenograft models.¹² Clinical trials in patients with recurrent squamous cell carcinoma of the head and neck have documented ONYX-015 replication and necrosis induction within some injected tumor masses; replication within, or ONYX-015-mediated damage to, normal tissues adjacent to the injected tumors has not been reported.^13,14 However, durable objective responses occurred in less than 15% of patients. In contrast, combinations of ONYX-015 and chemotherapy have resulted in durable responses in over half of all head and neck tumors treated.¹⁴ Given the antitumoral activity of ONYX-015 against p53-deficient tumors following direct injection or hematogenous spread, and given its favorable toxicity profile alone and in combination with chemotherapy, ONYX-015 appears to be well-suited for clinical trials in patients with locally or regionally advanced pancreatic carcinoma.

We therefore carried out a phase I dose escalation study of intratumoral injection of ONYX-015 into unresectable carcinomas within the pancreas. Safety, feasibility, and maximally tolerated dose were the primary study end-points. In addition, biological data on intravascular viral dissemination, the humoral immune response and antitumoral activity were also obtained.

Results

Baseline patient characteristics

The schedule of treatment and evaluation activities is listed in Table 1. Baseline patient characteristics are described in Table 2. Approximately 50% of screened patients were enrolled on to the study. The median age of patients on study was 66.5 years old (range 33-79), and 27% were female. Eight patients had locally advanced disease (35%) and 15 had distant metastases present (65%). Previous treatment for pancreatic carcinoma included chemotherapy in 10 patients (43%), radiotherapy in four patients (17%), and surgery for their primary tumor in 12 patients (52%). Most patients had a KPS of 80-90%. Seventeen of 23 (74%) had neutralizing antibodies against ONYX-015 at baseline. p53 gene status could be obtained in 16 tumors (70%); seven tumors had mutant sequences and nine had wild-type gene sequences (exons 5-9).

MTV, MTD and toxicity determination

At a dose of 10 ⁸ p.f.u., the relative injection volume was escalated from 10 to 20% (determined as a percentage of the estimated tumor volume). While a relative volume of 10% was easily injected, the 20% relative volume was difficult to administer completely into the tumor mass. Because of the difficulty in administering the entire injection volume into the tumor mass at the 20% level, the 30% injection volume was not attempted; therefore, the 20% relative injection volume was the maximally tolerated volume (MTV) for subsequent injections (Table 3).

The MTV was then used to administer escalating doses of ONYX-015 as described in Table 3. Only a single dose-limiting toxicity was noted on study. A patient at the 10¹⁰ dose level developed rapidly progressive liver function test abnormalities, including grade 3 hyperbilirubinemia and transaminitis. Although pre-treatment liver abnormalities existed due to metastatic disease, and progression of intrahepatic metastatic tumor foci was believed to have caused these liver abnormalities, the patient died prior to confirmation of progressive tumor in the liver. Therefore, since involvement of ONYX-015 could not be ruled out, the adverse event was categorized as a DLT, and a total of six patients was accrued at this dose level. None of the other five patients at this same dose level, or any of the four patients treated at higher dose levels, had any significant liver toxicity. Therefore, it appears highly unlikely that the DLT was actually associated with ONYX-015. Doses of 3 ´ 10¹⁰ p.f.u. were therefore well-tolerated in five patients (6 ´ 10¹¹ particles), and the maximum dose that could be administered based on manufacturing capabilities was the MTD for the study (10¹¹ p.f.u.; 2 ´ 10¹² particles) (Tables 4 and 5).

No clinically significant pancreatitis was noted on this study (Table 4). A single patient developed mild pancreatitis requiring increased oral pain medication usage for 4 days (3 ´ 10⁹ dose level). Pancreatic enzymes peaked on day 5 after the first cycle (peak amylase, 188; peak lipase, 48) and returned to baseline within approximately 2 weeks. This patient did not require hospitalization. The pancreatitis did not recur despite repeat treatment. Most patients (91%) reported some flu-like symptoms, including fever, myalgias, asthenia and/or chills. Chills, myalgias and flu-like symptoms were mild (grade 1) in almost all cases, and the duration of these symptoms was typically short (<48 h). Fevers were variable in severity: grade 3 (35%), grade 2 (30%) and grade 1 (26%). No patients discontinued therapy on the basis of flu-like symptoms. Of note, despite the potential for drainage of virus directly into the portal circulation, no significant liver toxicity attributed to ONYX-015 was noted.

Elevated partial thromboplastin times developed after treatment in three patients. No bleeding episodes occurred in these patients. A test for anticardiolipin antibody was performed in one of these patients and was positive. No evidence of hypercoagulability was noted clinically, and these patients had no abnormalities in PT or platelet levels.

Immunological endpoints

By laboratory parameters, patients were relatively immunosuppressed at the time of entry on to this study. CD4 counts were abnormally low in most patients (Table 2). DTH skin testing to commonly recognized antigens (eg candida, mumps) was low or absent in 83% of patients with evaluable test results.

Neutralizing antibody titers to ONYX-015 (Ad5 protein coat) were positive in 17 of 23 cases (74%) at baseline (Table 2). Titers increased and/or became positive in all patients following a single injection with ONYX-015 (Table 6). These titers also continued to rise after a second cycle of treatment. Patients with pre-existing antibody levels had the most rapid increases in antibody levels, consistent with a memory antibody response. No clear correlation was demonstrated between neutralizing antibody titers and viral dose, antitumoral activity or PCR positivity in the blood.

Viral genome dissemination

Quantitative PCR testing of the blood for ONYX-015 was performed at the following time-points after injection (cohorts 2-6): 15 min, 24 h, 5 days (±1 day) and 15 days (±2 days). Ten of 11 patients with sample(s) at the 15 min time-point had virus detectable in the blood at this time. The level of ONYX-015 in the blood correlated with the dosage administered. The mean concentration of viral genomes in the blood at the 3 ´ 10¹⁰-10¹¹ p.f.u. dose level was higher than at the 10⁹-3 ´ 10⁹ p.f.u. dose level (P = 0.09). Therefore, in almost all evaluable cases virus dissemination into the systemic circulation was acutely detectable.

Detection of viral replication

Viral replication was assessed by two methods. Since virus becomes undetectable in the blood within 6 h after intratumoral injection, positive genome detection within the blood at 24 h would be highly suggestive of viral replication and shedding into the bloodstream.¹⁵ A second more invasive approach is to obtain cellular material from injected tumors, in this case fine needle aspirations (FNA). Clinical trials of intratumoral injection in head and neck cancer patients documented equivalent detection of replication with both methods.^13,16,17

At the 15-min time-point after injection, 80% of patients had positive blood samples at the three top dose levels. None of the blood samples drawn at the 1-, 5- or 15-day time-points were positive for ONYX-015. Post-treatment fine needle aspirates were evaluable for viral replication in approximately 50% of cases (day 22). The vast majority of inevaluable samples taken post-treatment consisted of necrotic debris associated with very few viable cells. Evaluable samples were uniformly negative for viral replication.

Antitumoral activity

No objective responses were documented (Table 3). Six patients had shrinkage of 35-45% (minor responses) of the injected tumor. In addition, 10 patients maintained stable disease within the injected target tumor for at least 3 months (median time to target tumor progression 5.5 months). Six patients had liver metastases present at baseline. No objective responses were demonstrated in liver metastases; a minor response was noted in one patient. Anecdotal decreases in pain intensity and pain medication usage were noted in three patients. Reductions in pain medication usage lasted approximately 3-4 weeks. The percentage of patients alive over time following treatment initiation was as follows: 40% at 6 months and 9% at 12 months (range 2-18+ months). The cause of death was tumor progression in all cases.

Discussion

Novel therapies are needed for locally and regionally advanced pancreatic cancer. We have shown that injection of an E1B-55 kDa gene-deleted adenovirus (ONYX-015) into carcinomas within the pancreas is feasible and well-tolerated. A maximally tolerated dose could not be reached; the highest dose level that could be manufactured was well-tolerated and did not cause significant toxicity. Five patients received doses of 3 ´ 10¹⁰ p.f.u. (6 ´ 10¹¹ particles) without dose-limiting toxicity. In addition to the notable lack of systemic toxicity, no significant local complications occurred either. Of note, pancreatitis was not seen at the highest dose levels, and the single case that did occur was mild and resolved within 4 days. Pancreatitis did not occur again in this patient despite repeat treatment. No significant local complications occurred. Of note, ONYX-015 was well-tolerated despite systemic dissemination immediately after intratumoral injection.

Human adenoviruses are pro-inflammatory, inducing both innate cytokine-mediated responses and cell-mediated cytotoxicity.^18,19,20 Adenoviral replication within a tissue leads to an acute neutrophilic infitration followed by an influx of monocytes, macrophages and eventually lymphocytes.^21,22,23 A critical objective of this study was therefore to assess the risk of pancreatitis and other local complications following intratumoral injection of ONYX-015. The distinct lack of significant pancreatitis following treatment may be due to several factors. First, ONYX-015 replication was not detectable by the methods employed, in contrast to data from patients with other tumor types. The risk of local complications might theoretically increase with the level of viral replication. Secondly, anatomical barriers to viral spread and generalized inflammation in the pancreas may also have served to limit local toxicity. By the time these patients are diagnosed, the pancreas itself is frequently fibrotic, scarred and shrunken due to chronic inflammation and tumor progression. Frequently, only small amounts of relatively normal pancreatic tissue remain. In addition, these tumors are generally fibrotic at the interface between the tumor periphery and normal pancreatic tissue. Viral spread beyond the tumor mass itself may be inhibited by these fibrotic bands. Therefore, other novel biological agents may also be tolerated following intratumoral injection within the pancreas. Future clinical trials with such agents may be indicated.

Viral replication was not detectable in patients on this study. The sensitivity of the available tests for detecting viral replication is unknown. However, viral replication was detected by both methods during similar clinical trials of intratumoral ONYX-015 injection in patients with either colorectal or head and neck carcinomas.^13,15,16,17 Interestingly, the prevalence of viral replication in head and neck cancer patients (days 5-8 after treatment initiation) based on either test was equivalent at approximately 70%.^13,16,17 Similarly, intrahepatic colorectal carcinoma metastatses supported detectable viral replication by blood testing in over 50% of high-dose patients. Therefore, the lack of any evidence for replication on this study is notable, although low level viral replication below the limit of detection cannot be ruled out. One potential explanation for the lack of viral genome detection in venous blood samples, if replication did occur, would be hepatic clearance of the virus after virus entry into the portal vein. Fine needle aspirates could miss focal low level replication, as well. Nevertheless, these data clearly indicate a lack of robust intratumoral viral replication.

The apparent pancreatic carcinoma resistance to replication must be overcome on future studies in order to maximize biological activity. Interestingly, pancreatic tumor lines were extremely sensitive to adenoviral therapy in vitro,^10,11 thus highlighting a major difference between results with cell lines versus tumors in patients. Whether pancreatic tumor cells in patients are relatively resistant to ONYX-015 uptake, subsequent replication and/or release into the systemic circulation is unclear at this time and will require further study. CAR levels in various tumor types should be measured, for example, as these results might predict sensitivity to adenoviral agents such as ONYX-015. Potential clearance mechanisms include cytokine-mediated effects (eg tumor necrosis factor), cytotoxic T cell or natural killer cell-mediated immune responses. The relatively crude immunologic parameters assessed in these studies cannot explain the differences seen in replication; CD4 counts and DTH responsiveness were reduced to a greater degree in head and neck or pancreatic cancer patients on ONYX-015 clinical trials than in colorectal carcinoma patients (D Kirn, unpublished data). However, these tests cannot rule out the possibility that intratumoral cytokine or immunologic parameters may differ between tumor types. Since objective regressions were demonstrated in head and neck cancer patients with high-titer neutralizing antibody levels in previous studies, neutralizing antibodies per se cannot explain these findings. Finally, dense fibrosis and a high ratio of normal cells to tumor cells within a tumor would be predicted to reduce viral uptake, replication and spreading efficiency. Pancreatic tumors are highly fibrotic and a significant fraction of the cells within the mass are not malignant in many cases. Therefore, methods to enhance spread and/or extracellular matrix breakdown within the tumor mass should be considered.

Based on these encouraging safety and feasibility results, further testing of viral agents is indicated in patients with locally or regionally advanced pancreatic cancer. Phase II studies might eventually be designed to determine whether such agents have activity against both the injected (primary) tumor and micro- or macroscopic metastases within the liver. Portal venous shedding of the virus must have occurred during this trial since virus was documented in the peripheral blood. The efficacy of replication-selective adenoviruses can be optimized through several approaches. First, the intratumoral distribution of ONYX-015 has a major impact on its efficacy. Physical approaches to distributing the virus within the tumor mass have led to enhanced efficacy.^17,24 Physical distribution can be improved by increasing the frequency of dosing and/or the number of needle passes at the time of each treatment.²⁴ We are exploring novel approaches to intratumoral injection of pancreatic tumors. Endoscopic ultrasound, for example, is routinely performed for the diagnosis and staging of pancreatic carcinomas. Injection of adenoviruses such as ONYX-015 through endoscopes into pancreatic tumors is feasible.²⁵ In addition, synergistic or additive efficacy of ONYX-015 and chemotherapy has been documented both in animal tumor models and in clinical trials.^10,15,16 Of note, ONYX-015 can be effectively combined with either 5-fluorouracil or gemcitabine. Current clinical trials are exploring combinations of these agents with ONYX-015. Finally, other replication-selective agents are being developed for cancer treatment including herpesviruses, vaccinia, Newcastle disease virus, VSV, poliovirus and reovirus.²⁶

Methods

Objectives

The primary objectives of this study were as follows: to determine the safety of a single and repeated injections of ONYX-015 administered directly into carcinomas of the pancreas; to determine the maximally tolerated dose and/or dose-limiting toxicity of ONYX-015 administered as described above. Secondary objectives of the study included the following: to determine the humoral immune response to ONYX-015 injection; to determine the magnitude and duration of viral replication and shedding into the bloodstream as a function of viral dose and tumor characteristics; to determine the antitumoral effects of ONYX-015 injection (as above) on solid tumor growth at the injection site and at metastatic sites, especially within the liver.

Eligibility criteria

Inclusion criteria included the following: histologically or cytologically confirmed carcinoma of the exocrine pancreas; cancer that was not considered resectable for potential cure (ie locally advanced or metastatic); the primary pancreatic tumor mass was amenable to biopsy and direct injection at laparotomy or via CT guidance; Karnofsky performance status of 60%; life expectancy of 3 months; 18 years of age; the patient was to (a) undergo laparotomy for clinical indications (including attempted resection and/or palliative choledochojejunostomy, gastrojejunostomy) or (b) the patient was able to undergo CT-guided intratumoral injection; consent form for study participation signed; must have been using a reliable method of contraception if sexually active; creatinine <2.0 mg/dL; AST and ALT <2.5-fold upper limit of normal; PT/INR <2.0 and PTT within normal limits; neutrophils >1500/ml, hemoglobin >9 g/dl, platelets >100000/ml.

Patients with any of the following were excluded from the study: known chronic liver dysfunction before the development of pancreatic cancer (eg cirrhosis, chronic hepatitis) which in the estimation of the principal investigator put the patient at high risk for liver complications; >50% liver replacement by tumor (estimated radiographically); active infection, including human immunodeficiency virus; any viral syndrome diagnosed within the previous 2 weeks; chemotherapy within the previous 3 weeks (6 weeks for nitrosoureas or mitomycin-C); radiotherapy to the target tumor site within the last 4 weeks; concomitant hematological malignancy; chronic immunosuppressive medication; pregnant or lactating females; prior participation in any research protocol which involved administration of adenovirus vectors; treatment with any other investigational therapy within the last 4 weeks. Since no single gold-standard test for p53 function within a tumor existed, p53 tumor status did not affect enrollment. However, the p53 gene status of the tumor was assessed by gene sequencing (exons 5-9).

Protocol review and approvals

This protocol was approved by the Institutional Review Board at the University of California, San Francisco and was filed to an existing IND for test article with the US Food and Drug Administration.

Test article

ONYX-015 (dl1520) is a chimeric human group C adenovirus (Ad2 and Ad5) that does not express the 55 kDa product of the E1B gene; the virus was constructed in the laboratory of Arnold Berk.²⁷ The virus contains a deletion between nucleotides 2496 and 3323 in the E1B region encoding the 55 kDa protein. In addition, a C to T transition at position 2022 in E1B generates a stop codon at the third codon position of the protein. These alterations eliminate expression of the E1B 55 kDa gene in ONYX-015 infected cells. ONYX-015 was grown and titered on the human embryonic kidney cell line HEK293 as previously described.¹⁰

Treatment regimen

For administration of ONYX-015 by radiographic guidance, patients were treated in CT radiology suites. Vialed virus solution was thawed and diluted with electrolyte-48 solution to the appropriate titer for each patient's dose level. Vials of ONYX-015 were opened and diluted in biological safety cabinets. Thawed virus was maintained at 2° to 8°C during dilution and handling, except for warming to room temperature immediately before administration. The virus solution was then further diluted to a final volume of 10-20% of the volume of the tumor to be injected. The tumor volume was estimated by taking the product of the maximal tumor diameter, its perpendicular and the estimated depth, and dividing by two. Dilutions were performed immediately before tumor injection using plastic syringes.

Under CT guidance, a 22-gauge thin wall needle was passed into the target tumor mass. Investigators confirmed that the needle was in the mass by documenting tumor cells on a cytology specimen obtained by fine needle aspiration. The number of intratumoral injection sites was dependent on the maximal diameter of the target mass: 3 cm (one central injection), >3 cm and 6 cm (two injection sites, each roughly in the center of the respective tumor halves), >6 cm (three injection sites). In divided dose injections the dose/volume of each injection was equally distributed between injection sites. The needle was gradually withdrawn during injection of the virus-containing solution into the tumor mass (in order to optimize virus distribution). At the discretion of the principal investigator, patients were observed overnight in the hospital post-injection.

Definitions of maximum tolerated dose (MTD), maximum tolerated volume (MTV) and dose-limiting toxicity (DLT)

The MTD was defined as the dosage immediately preceding (lower than) the dose at which two patients experienced a DLT after the first treatment with ONYX-015, or the maximally administered dose based on virus production (the 10¹¹ p.f.u. dose). The MTV was defined as the highest safe volume for ONYX-015 injection (as defined in cohorts 1a-c) that could be completely injected into the tumor mass.

DLT was defined as any one of the following: grade 4 toxicity for flu-like symptom(s) (eg fever, fatigue, or myalgia) or pancreatitis attributed to ONYX-015 treatment; grade 4 liver toxicity (of any duration) attributed to ONYX-015 treatment, or grade 3 liver toxicity lasting 7 days attributed to ONYX-015 treatment; any other toxicity of grade 3 severity attributed to ONYX-015 treatment.

Dose-escalation scheme

Before dose escalation, the MTV was defined by treating patients in cohort 1 with a constant amount of ONYX-015 (10⁸ p.f.u.) while increasing the volume injected from 10% of the tumor volume to 20% of the tumor volume; the 30% volume was never attempted. Once the MTV was defined (20%), patients in cohorts 2-6 received ONYX-015 injections in the MTV.

Eligible patients were enrolled into one of the following ONYX-015 treatment cohorts, according to the injection volume-escalation and dose-escalation scheme described below. The maximum possible dose of 10¹¹ p.f.u. was limited by total virus production capacity:

Cohort 1a: 10⁸ p.f.u. injected in a volume equal to 10% of the tumor volume

Cohort 1b: 10⁸ p.f.u. injected in a volume equal to 20% of the tumor volume

Cohort 1c: 10⁸ p.f.u. injected in a volume equal to 30% of the tumor volume (never reached)

Cohort 2: 10⁹ p.f.u. injected in the maximum tolerated volume (MTV = 20% of the estimated tumor volume)

Cohort 3: 3 ´ 10⁹ p.f.u. injected in the MTV

Cohort 4: 10¹⁰ p.f.u. injected in the MTV

Cohort 5: 3 ´ 10¹⁰ p.f.u. injected in the MTV

Cohort 6: 10¹¹ p.f.u. injected in the MTV

Treatment cohorts were enrolled sequentially with at least a 3-week interval between treatment of the last patient of one cohort and treatment of the first patient in the next cohort. At least three patients were entered per dose level (except at the 10¹¹ p.f.u. dose level, due to limited production capacity for treatment of more than two patients). If no DLT occurred in all three patients, then patients were enrolled at the next dose level. If one of the three patients entered into a cohort had a DLT, up to three additional patients were enrolled at the same dose level; at least a 2-week interval between treatment of each of these patients was required. If none of these three additional patients had a DLT during the first 3 weeks following treatment, patients were then enrolled at the next dose level. If one or more of these additional patients were to have had a DLT (ie two patients enrolled at the dose level have a DLT), this would define the MTD; no other patient(s) would have been enrolled at this dose level, and no further dose escalation would have occurred.

Patient evaluation on study

The schedule of activities for patients on study is outlined in Table 1. Blood was drawn on days 8, 15 and 22 of each cycle for serum chemistry, complete blood count, PT/PTT, amylase, lipase and liver function tests (AST, ALT, total and direct bilirubin, alkaline phosphatase). In addition, blood was tested for the presence of ONYX-015 by polymerase chain reaction (PCR) 15 min, 24 h, 5 days, 8 days, 15 days and 22 days after intratumoral injection. Neutralizing antibodies to ONYX-015 were assessed on days 8, 15 and 22. Tumor response assessment included measurement of CA19-9 levels in blood and abdominal CT scanning on day 22. Tumor size was the product of the maximal cross-sectional tumor diameter and its perpendicular. At the time of each tumor injection, a fine needle aspirate sample was obtained from the tumor mass for assessment.

Fine needle aspirates were assessed for the degree of tumor necrosis and for evidence of viral replication. Tumor samples were considered evaluable if if at least 50 viable cells were present on the cytology slide. Viral replication was assessed by in situ hybridization for viral DNA as previously described.¹⁶ Briefly, in situ hybridization was performed on formalin-fixed cytology slides hydrated through ethanols, digested with proteinase K and post-fixed in 4% paraformaldehyde. Hybridization was carried out overnight at 37°C with 0.5 g/ml biotinylated adenovirus DNA probe (Enzo Diagnostics, Farmingdale, NY, USA). After three successive washes in 1 ´ SSC at 55°C, an alkaline phosphatase conjugated-anti-biotin antibody (Vector Laboratories, Burlingame, CA, USA) was applied. NBT/BCIP was used as the chromagen and slides were counterstained with nuclear fast red.

Quantitative PCR testing of the blood for ONYX-015 was performed at the following time-points after injection (cohorts 2-6): 15 min, 24 h, 5 days (±1 day) and 15 days (±2 days). PCR for ONYX-015 was performed using the TaqMan assay which quantitates the number of ONYX-015 genomes in human plasma (the amplicon overlaps the E1B region deletion and does not detect wild-type adenovirus sequences). Viral DNA is extracted from patient samples, standards and controls using QIAamp DNA minikit. The lower limit of detection is 1.05 ´ 10⁴ particles of ONYX-015 per ml of plasma. The presence of PCR inhibitors in the sample is monitored using an independent PCR reaction.

Neutralizing antibody titers against ONYX-015 were determined on blood samples as follows. Patient and control samples were incubated at 55°C for 30 min to inactivate complement. Clinical plasma samples previously determined to produce high, midrange and negative titers were designated as plasma controls. Each dilution was mixed with adenovirus stock at a titer prequalified to produce 15-20 plaques per well of a 12-well dish in DMEM growth medium. The patient's samples and controls were inoculated for 1 h at room temperature, and applied to 70-80% confluent JH393 cells in 12-well dishes. After 2 h of incubation at 37°C, 5% CO₂ plasma-virus mix was removed and 2 ml of 1.5% agarose in DMEM was added to each well. Plates were read on day 7 post-inoculation by counting the number of plaque forming units (p.f.u.) per well. The titer of neutralizing antibody for each sample was reported as the dilution of plasma that reduced the number of plaques to 60% of the number of plaques in the virus control without antibody.

Pretreatment tumor biopsies were taken for p53 sequencing from the recurrent tumor mass that was to be injected. Exons 5-9 were sequenced completely (Oncormed, Gaithersburg, MD, USA).

Acknowledgements

We would like to acknowledge the assistance of the following individuals: Margaret Uprichard, Sherry Toney, Olga Diri, Britta Randlev, Brian Breitbard, Dianne Davies, Chris Maack, Larry Romel, Amy Waterhouse, Fran Kahane, Patrick Trown. This study was sponsored by Onyx Pharmaceuticals, Richmond, California.

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Table 1 Schedule of activities and evaluations

Table 2 Baseline patient characteristics

Table 3 Patient treatment, dose-limiting toxicity and tumor response data

Table 4 Percentage of patients experiencing adverse events, regardless of relationship to ONYX-015 (reported in >20%)

Table 5 Percentage of patients experiencing adverse events - possibly or probably related to ONYX-015 as judged by the principal investigator (toxicities with >10% of patients reporting)

Table 6 Humoral immune response to intratumoral injection of ONYX-015