Treatment of Recurrent or Progressive Malignant Glioma with a Recombinant Adenovirus Expressing...

HUMAN GENE THERAPY 12:97–113 (January 1, 2001)Mary Ann Liebert, Inc.

Clinical Protocol

Treatment of Recurrent or Progressive Malignant Glioma witha Recombinant Adenovirus Expressing Human Interferon-Beta

(H5.010CMVhIFN-b): A Phase I Trial

INVESTIGATORS:Stephen L. Eck, M.D., Ph.D. Hematology/OncologyJane B. Alavi, M.D. Hematology/OncologyKevin Judy, M.D. NeurosurgeryPeter Phillips, M.D. NeurologyAbass Alavi, M.D. Nuclear MedicineDavid Hackney, M.D. RadiologyPhilip Cross, M.S. IHGTJoseph Hughes, Ph.D. IHGTGuang-ping Gao, Ph.D. IHGTJames M. Wilson, M.D., Ph.D. IHGTKathleen Propert, Sc.D. CRCU

INSTITUTION: University of Pennsylvania Cancer Center, 3400 Spruce Street,Philadelphia, PA 19104

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1.0 PROTOCOL SUMMARY

Protocol Title: Treatment of Recurrent or ProgressiveMalignant Glioma with a RecombinantAdenovirus Expressing Human Inter-feron-Beta (H5.010CMVhIFN-b): APhase I Trial.

Study Phase: Phase IStudy Design: Single-center, open-label, inter-patient

dose escalation toxicity study.Study Objectives: Primary Objective:

To determine the toxicity of intratu-moral injection of recombinant ade-novirus expressing human interferon-beta (H5.010CMVhIFN-b) in patientswith recurrent or progressive malignantglioma and to determine the maximumtolerated dose of H5.010CMVhIFN-b.Secondary Objectives:To assess the relationship between thedose of H5.010CMVhIFN-b in patientswith recurrent or progressive malignantglioma and� The clinical/biological response as as-

sessed by physical examination, MRIscan, and survival.

� Pathologic, evidence of gene transferin resected tumor specimens.

� Serum, cerebrospinal fluid and braininterferon-beta levels.

� Immune response to adenoviral vec-tor and/or human interferon-beta.

Number of Subjects: Approximately 18 to 21.Study Population: Patients $18 years of age, with a diag-

nosis of recurrent or progressive malig-nant glioma, with tumor that isamenable to surgical resection.

Treatment Groups: A standard three-six-dose escalationscheme will be used based on toxicityparameters. Patients will undergo twointracranial administrations of H5.010-CMVhIFN-b gene therapy separatedby 1 week. The first dose will be ad-ministered by means of a stereotacticinjection into the tumor, and the sec-ond dose will be administered immedi-ately after resection of the tumor, intothe residual tumor, at the time of cran-iotomy.

Visit Schedule: Screening visit(s).Admission to the General Clinical Re-search Center (GCRC) and BaselineStudies.Follow-up visits on Days 1 to 15, in-clusive, then at Day 22, 29, and Month

2, 4, 6 and every 6 months thereafter upto two years.

Safety Parameters: Physical exams and blood testsAdverse eventsMRI scansAdenoviral cultures

Efficacy Parameters: Tumor response and time to tumor pro-gression by MRI scansTime to deathEvidence for gene transfer in tumorspecimensSerum, CSF and brain interferon-betalevelsAdenoviral and interferon-beta antibodies

2.0 INTRODUCTION

2.1 Clinical Aspects of Brain Tumors

Primary brain tumors in adults, principally the anaplastic as-trocytoma and glioblastoma multiforme, are highly malignantand nearly always fatal (Levin et al., 1997). Advances in sur-gical techniques and in radiation therapy have permitted slightlylonger survival with an improvement in quality of life. Surgi-cal advances have included intra-operative ultrasound for su-perior tumor localization, and laser that permits more preciseresection, resulting in less morbidity and more rapid recoverytime. Newer radiation therapy methods have also brought mod-est success, with improved treatment planning using computertomographic scans, multi-leaf collimators for more accuratetreatment, and high dose interstitial brachytherapy. However,brachytherapy is limited to surgically accessible tumors, andstereotactic radiosurgery is limited to small lesions (,4 cm),which probably account for only 20% of all malignant brain tu-mors. Neither radiation therapy nor chemotherapy is curativein this disease. With adjuvant chemotherapy added to radio-therapy, the median survival has been increased only from 9.4months to 12 months (Fine et al., 1993). Patients still have lit-tle chance of living longer than 2 years from diagnosis. Whenthe tumors recur, the survival is generally less than 6 months.Although primary CNS malignancies represent a small per-centage of all cancers, they account for a disproportionatelylarge amount of the annual $40 billion cost of care of cancerpatients. Clearly new therapeutic approaches are needed.

2.2 Gene Therapy in the Treatment of Brain Tumors

Relative to their prevalence, malignant gliomas have receivedsubstantial attention as targets for gene therapy (Culver, 1996).Several factors have contributed to the interest in gene therapytechniques for malignant gliomas. Most significant is that thegrowth of malignant gliomas is relatively well localized. Al-though they infiltrate the surrounding brain, making completesurgical excision impossible, they rarely metastasize outside thebrain. This tremendously simplifies the targeting of gene vec-tors to the tumor. Systemic delivery of genetic vectors to wide-spread metastases is currently not achievable with high effi-ciency and low toxicity. However, the local delivery to the bulkof the tumor can be achieved through either stereotactic injec-tion of vectors into the brain tumor, or infiltration of the tumor

with genetic vector at the time of gross total resection. In ad-dition, direct injection of genetic vectors may limit systemic ex-posure to the therapeutic agent and therefore, minimize sys-temic toxicity. Another important factor is that malignantgliomas have extremely high mortality rates that have not beensignificantly affected by newer surgical, radiation or drug ther-apies. Thus, experimental therapies become more reasonableoptions for patients with malignant glioma. Several gene ap-proaches to these tumors have been tested in animal models anda few human clinical trials. These include enzyme-prodrug ther-apy (Eck et al., 1996; Ram et al., 1997), targeting tumor sup-pressor activity (Badie et al., 1995; Hsiao et al., 1997), angio-genesis functions (Plate and Risau, 1995; Plate, 1996; Tanakaet al., 1997), immunotherapy (Dietrich et al., 1997; Glick et al.,1997; Tseng et al., 1997), and the use of oncolytic viruses (An-dreansky et al., 1996; Bischoff et al., 1996; Miyatake et al.,1997). The basic problem of vector penetration into the tumoris shared by all of these methods. This is especially true of thestrategies based on expression of tumor suppressor genes (e.g.,p53) where the effect of the therapy on the non-transduced tu-mor cells is less apparent than with a secreted protein such asinterferon-beta.

The choice of vectors capable of efficient cancer cell trans-fection in vivo has been essentially limited to retroviruses, li-posome–DNA complexes, and more recently, adenoviruses, andadeno-associated virus (Wivel and Wilson, 1998). Early genetherapy studies employed retroviral vectors almost exclusively.Although retroviruses have the potential advantage of long-termexpression via integration into the host’s chromosomal DNA(an unnecessary feature if the target cell is going to be killed)and are extremely useful for in vitro gene transfer, there are anumber of characteristics which make in vivo application ofretroviruses problematic: (1) it is difficult to produce high titerstocks of retrovirus (i.e., .107 particles/ml); (2) only activelydividing cells can integrate and effectively express the vectorDNA; and (3) retroviral DNA insertion is a random event cre-ating the possibility of insertional mutagenesis. Many tumorcells may grow slowly and may not be in active cell cycle atthe time of viral transduction. These cells are poor substratesfor retroviral gene therapy. Because of these problems, we andothers have elected to deliver the therapeutic gene using a re-combinant replication deficient adenovirus. The potential ad-vantages to this approach include: (1) recombinant adenovirusmay be produced in very large quantities and high titers (.1013

particles/ml); (2) adenovirus has no known integrative or onco-genic potential; and (3) an extremely wide diversity of tissues(both dividing and nondividing cells) have been successfullyinfected in vivo with adenoviral vectors.

2.3 Adenoviral Vector Expressing Interferon-beta

A replication defective adenovirus vector (Kozarski and Wil-son, 1993) capable of efficient gene transfer in several tumormodels has been developed. The overall approach will be to usethis adenovirus vector containing the human interferon-beta(hIFN-b) gene to transduce tumor cells. The local productionof hIFN-b protein following gene transfer has tumoricidal ef-fects in animal models (Qin et al., 1998) and extends beyondthose cells that undergo gene transfer.

The adenovirus vector described in this protocol

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(H5.010CMVhIFN-b) has been prepared at the University ofPennsylvania in collaboration with Biogen and Genovo. It is ahuman serotype 5 adenovirus in which significant portions ofthe E1 and E3 genes have been deleted, thus rendering the virusreplication incompetent. H5.010CMVhIFN-b has induced re-gression of established tumors in murine cancer models (Qin etal., 1998). Because the adenoviral vector does not integrate intothe tumor cells, the duration of gene expression is limited, al-lowing rapid eradication of tumor cells without toxicity thatmight arise from long term gene expression. The toxicity andantitumor efficacy of H5.010CMVhIFN-b have not been pre-viously studied in humans, although preliminary toxicologywork suggests that this approach is feasible. Our prior ade-novirus based deliver of the herpes virus thymidine kinase gene(H5.010RSVtk) using a similar adenoviral vector was gener-ally well tolerated in humans (unpublished studies). The phaseI study in this present application will define the toxicity ofH5.010CMVhIFN-b when administered intralesionally intobrain tumors, and will provide information about the distribu-tion of the vector and the immune response to the vector andthe transgene.

In a prior study using H5.010RSVtk in a similar clinical trialdesign, the dose-limiting toxicity (DLT) occurred at dose of 1011

PFU (5 3 1012 particles) and resulted from increased intracra-nial pressure believed to be secondary to adenovirus-induced in-flammation. There were a total of 13 patients enrolled in thisstudy. We observed a durable complete response in one patient(disease free after more than 22 months) and prolonged freedomfrom progression (.10 months) in two other patients. These re-sults are encouraging and have led to the design of this new clin-ical trial using H5.010CMVhIFN-b. The new vector has the ad-vantage of encoding a secreted protein that will treat a largerarea of the tumor. In addition, the tumoricidal effects of hIFN-b are likely superior to that of prior gene therapy agents.

A common problem in cancer gene therapy is in obtainingefficient cell targeting in vivo. For example, using retrovirallytransferred HSV-tk, it was observed that few brain tumor cellswere transduced following vector administration (Ram et al.,1997). Similar findings were observed in a trial of adenovirusdelivered RSV-tk (our unpublished findings). The use of an ade-novirus encoding a secreted protein may overcome the limita-tions of current gene delivery in that the therapeutic protein(hIFN-b) can diffuse to areas of the tumor not transduced bythe gene vector.

2.4 Interferon-beta Therapy in Cancer

Human IFN-b, a 22.5-kDa molecular weight protein, elicitsimmunomodulatory and tumor antiproliferative effects. This bi-ologic response modifier has previously been evaluated as atherapy for malignant gliomas. Systemic administration (intra-muscular injection) of hIFN-b resulted in dose-limiting toxic-ity in patients treated at 8 million units/m2 three times per week(Fine et al., 1997). Common toxicities included asymptomaticelevations of hepatic transaminases, transient myelosuppression(anemia, thrombocytopenia, and/or leukopenia) and neurotoxi-city (seizures and encephalopathy). No complete responses andonly one partial response was observed in the 13 evaluable pa-tients (Fine et al., 1997). In a pediatric study using intravenousadministration of hIFN-b in combination with radiation ther-

apy similar results were observed (Packer et al., 1996). In thisdose escalation study up to 4 3 108 IU/m2 was administeredthree times per week. Nausea, vomiting, and grade 3 to 4 myelo-toxicity and hepatic toxicity occurred in a significant numberof patients (Packer et al., 1996). One of the 32 treated patientsdeveloped significant neurotoxicity (seizures, slurred speech,and incoordination). Skin toxicity was observed in the irradi-ated field. Two complete and 5 partial responses were observedin this study; however, there was little efficacy when judged byprogression-free survival or overall survival (Packer et al.,1996). These and earlier studies illustrate the limited antitumoractivity of hIFN-b protein and highlight the toxicity associatedwith systemic protein administration. Recently, Brem and col-leagues have demonstrated the feasibility of local intratumoraladministration of murine IFN (Wiranowska et al., 1998). Con-trolled-release polymer implants containing murine IFN-a/bwere shown to deliver most of the interferon to the hemisphereimplanted with the polymer–IFN and significantly less to thecontralateral hemisphere. Drug levels dropped off significantlyafter the first 4 days however, suggesting that the therapeuticeffect might be diminished by the limited drug exposure (Wira-nowska et al., 1998). Nonetheless, this study provides prece-dent for the use of locally delivered interferon. Delivery ofhIFN-b by gene transfer has the potential to achieve high localconcentrations of hIFN-b within the brain tumor with signifi-cantly lower levels systemically or in other areas of the brain.This may serve to widen the otherwise narrow therapeutic win-dow of this agent.

3.0 PRECLINICAL STUDIES USINGH5.010CMVhIFN-b

3.1 Preclinical Tumor Models

Recombinant adenovirus expressing hIFN-b has been exam-ined with subcutaneous human xenograft tumors in nude micein experiments performed at Biogen (Qin et al., 1998). Estab-lished human U87 glioma tumors were treated with a single intratumoral injection of a related adenovirus, H5.110CMVhIFN-b (Qin et al., 1998) and subsequently with theH5.010CMVhIFN-b vector (Qin et al., 1998). At a dose of 1 3

1011 particles, complete tumor regression was observed over aperiod of 2 weeks. All mice experienced a complete responseat this dose level. A significant response was also seen with1010 particles and limited response at the 1 3 109 dose level.Similar results were obtained in other human tumor xenograftmodels using the H5.010CMVhIFN-b adenovirus (Qin et al.,1998). These studies establish potent antiproliferative and cy-tolytic effects of interferon-b following intratumoral deliverywith an adenovirus vector.

3.2 Toxicity Studies in Primates and Mice

Two studies have been performed to examine the toxicity ofthe adenovirus expressing the IFN-b. Brief description of thesestudies and a summary of the results are outlined in Table 1.

Toxicity studies performed in mice. Mice received an in-tracerebral injection of either vehicle control or H5.010CMVhIFN-b (with the species specific murine IFN-b gene)

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over a dose range of 3 3 108, 3 3 109, or 3 3 1010 particlesper mouse delivered in 5 ml. There were 12 mice in each ofthese four groups. The vector was well tolerated during ad-ministration and throughout 61 days with no deaths; no clini-cal symptoms; limited and transient body weight decreases; andno clinical pathology changes. Histopathology of the braindemonstrated limited inflammation early; more extensive de-generation at day 18; and quiescent, dormant necrotic lesionsat day 61. There were some lesions and inflammatory responsesin the brain that were distant from the site of administration.The more diffuse lesion may have resulted from the difficultiesof scaling the vector administration to the relatively smallmurine brain. No other tissues demonstrated any significant his-tologic effects.

Toxicity in Nonhuman Primates. Four normal rhesus mon-keys each received a unilateral intracerebral injection ofH5.010CMVhIFN-b recombinant adenovirus and a contralat-eral injection of vehicle control (without adenoviral vector).Two monkeys each received 3.55 3 1011 particles ofH5.010CMVhIFN-b (“high dose”) and another two monkeysreceived 3.55 3 1010 particles (“low dose”) in 100 ml. The an-imals underwent serial clinical examinations, measurements ofvital signs, laboratory measurements including complete bloodcounts (CBC), serum chemistries, and liver function tests(LFTs), serum interferon-beta levels, and brain MRI scans. Theanimals underwent necropsy on day 45 post injection withhistopathologic analyses of multiple tissues.

The recombinant adenovirus produced localized, transientcerebral edema, as detected by MRI scan. The cerebral edemawas most evident at approximately 1 to 2 weeks following ad-ministration and rapidly diminished thereafter. The last observedMRIs (day 45) revealed normal (1 nonhuman primate [NHP]) orsignificantly improved (remaining 3 NHPs) areas of edema andenhancement. Injection of vehicle control to the contralateral sideof the brain in these four animals did not result in any signifi-cant abnormalities by MRI analysis. The animals were com-pletely asymptomatic with normal vital signs during the 45 daysfollowing injection of vector, and did not require corticosteroidsor other supportive therapy for the transient cerebral edema. Noseizures were observed at either dose. The body weights, CBCs,serum chemistries, and LFTs remained normal throughout thestudy. Serum was assayed for human interferon-b on days 1, 4,8, 11, 15, and 21 postvector injection, both by means of an ELISAand a cytopathic effect (CPE) bioassay. Serum interferon-b lev-els were below the limit of detection (three animals) and de-tectable in the 10–20 IU/ml range in one “high dose” animal us-ing the CPE assay on days 4, 8, 11, 15, and 21.

Histopathology revealed focal lesions with gitter cells (typi-cally found in recovering brain lesions) with inflammation atthe site of administration. This was accompanied by focal ar-eas of meningeal inflammation, consisting of lymphoplasma-cytic cells. Focal gliosis was also noted. Although there wassome minimal inflammation on the contralateral side, overallthe pathology seen in the rhesus monkey was more focal thanwas seen in the mouse study.

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TABLE 1. TOXICOLOGY STUDIES OF ADENOVIRUS EXPRESSING IFN-b

Study design Vector/dose Results

Study 98-54� C57BL/6 1. 3 3 108 pt General observation: No deaths. No clinical problems. Body weight� 4 mice/group/day 2. 3 3 109 pt decreases� Necropsy at days 4, 18, 61 3. 3 3 1010 pt Clinical pathology: No significant changes; no dose–response change� 5 ml into cerebrum 4. Vehicle Gross pathology: None noted� H5.010CMVmIFN-b Histopathology: � Day 4: Minimal inflammation

Histopathology: � Day 18: Degeneration noted primarily at site ofHistopathology: � administration; some spread from siteHistopathology: � Day 61: quiescent, dormant necrotic lesionsHistopathology: � Some vacuolization surrounding lesion. SomeHistopathology: � minimal cerebellum degeneration, which isHistopathology: � distant from administration siteHistopathology: � No dose effect noted

Study 98-56� Rhesus monkey 1. 3.55 3 1010 pt General observation: Vector well tolerated; no deaths; no clinical� 2/Dose 2. 3.55 3 1011 pt problems; no significant body weight change� Necropsy at day 45 Clinical pathology: No changes noted in several samples taken� 100 ml into cerebrum throughout 45 days� Vector one side MRI: Primary changes—edema and enhancement. Peaks typically� Vehicle on contralateral side at day 15–29, with significant recovery by day 45. High dose� H5.010CMVhIFN-b generally more affected at early times only

Gross pathology: No significant lesion notedHistopathology: � Focal meningeal inflammationHistopathology: � Perivascular inflammationHistopathology: � Large areas of gitter cells associated withHistopathology: � inflammationHistopathology: � Inflammation more severe on vector site;Histopathology: � limited on contralateral sideHistopathology: � No dose effect noted

Overall, the vector was well tolerated in the rhesus monkeys,and minimal focal areas could be detected by day 45 as demon-strated by MRI and histopathology. The delivered volume tothe NHPs was more easily scaled than to the mice relative tothe proposed vector administration in the human clinical trial.The results for this NHP toxicology study also closely matchedwhat was previously detected with the NHP and clinical stud-ies with the adenoviral vector expressing thymidine kinase.

Doses of the H5.010CMVhIFN-b Vector for ClinicalTrial. The proposed doses for this clinical trial were selectedbased on the results from the preclinical tumor model studies,the two toxicology studies with adenovirus-IFN-b vectors, andthe previous experience with the adenovirus-TK toxicology andclinical trial studies. The dose that demonstrated minimal ef-fects in the human xenograft models in mice was at 109 parti-cles, with more complete tumor suppression or destruction at1010 to 1011 particles (pt) (Table 2). When the relevant vectorswere tested for toxicity (mIFN-b in mice, and hIFN-b in rhe-sus monkeys) with the highest possible concentrations and vol-umes for the intracerebral administrations, no significant clin-ical symptoms were noted. The affected areas of the brainmeasured by MRI and gross histology lesions in the NHPs atthe end of the study (day 45) were extremely small and local-ized to the site of injection. Based on the transient nature ofthis vector in NHP, and the similarity of these results to whatwas seen with adenoviral TK vector studies in NHP and hu-mans previously, we are proposing to administer similar dosesas were used with Ad-TK. That trial started at a very low doseof 5 3 109 pt and demonstrated some minimal effects at thenext highest dose (5 3 1010 pt); toxicity was not seen in thattrial until they reached the 5 3 1012 pt level. The present pro-tocol with H5.010CMVhIFN-b vector will use dose levels of3 3 1010 to 3 3 1012 pt/patient for a total of 5 cohort groups,with 1/2 log increments between cohorts. We believe these doseranges should provide a safe starting dose and gradual dose es-calations that will be critical to assess both the safety and po-tential benefits of hIFN-b delivered by adenoviral vector ad-ministration.

4.0 OBJECTIVES OF THE CLINICALPROTOCOL

4.1 Primary Objective

To determine the toxicity of intratumoral injection of re-combinant adenovirus expressing human interferon-beta

(H5.010CMVhIFN-b) in patients with recurrent or progressivemalignant glioma. This study will establish the maximum tol-erated dose between 3 3 1010 particles and 3 3 1012 particlesper administration.

4.2 Secondary Objectives

To assess the relationship between the dose ofH5.010CMVhIFN-b in patients with recurrent or progressivemalignant glioma and

� The clinical/biological response as assessed by physical ex-amination, MRI scan, and survival

� Pathologic evidence of gene transfer in resected tumorspecimens

� Serum, cerebrospinal fluid, and brain interferon-beta levels� Immune response to adenoviral and/or human interferon-

beta

5.0 CLINICAL TRIAL DESIGN

This will be open-label, interpatient dose escalation toxicitystudy of adenoviral interferon-beta gene therapy in patients withrecurrent or progressive malignant glioma who are undergoingsurgical tumor excision. Patients will be enrolled in cohorts ofthree and the first cohort will receive the starting dose. Each sub-sequent cohort of three patients will receive a dose that is a half-log higher than the previous cohort. This standard three-six-doseescalation scheme (Eck et al., 1996) will be used based on tox-icity parameters (Section 13.0 and Appendix 3 of protocol) to es-tablish the maximum tolerated dose. The dose levels are definedin Section 6.0. Each patient will undergo two treatments sepa-rated by 1 week. The first treatment will be a stereotactic injec-tion of H5.010CMVhIFN-b at 10 sites within the tumor. The sec-ond treatment will involve gross resection of the tumorimmediately followed by injection of H5.010CMVhIFN-b into10 sites of the residual tumor at the margins of the resection. Themaximum tolerated dose (MTD) is defined as the dose at whicheither two of three patients, or more than one of six patients, ex-periences a dose-limiting toxicity (Sections 6.0 and 13.0). Oncethe MTD has been determined, then three additional patients willbe enrolled at the next lower dose level.

Based upon our study design we anticipate treating approx-imately 18 to 21 patients over approximately 1 year on thisstudy. On the basis of our experience about 75% of eligible pa-tients offered this type of study agree to participate.


TABLE 2. COMPARISON OF VECTOR DOSES FOR EFFICACY AND SAFETY PRECLINICAL

DATA TO PROPOSED CLINICAL TRIAL COHORTS

Dose for Dose forminimal effect maximal effect

Vector Study/trial (pt) (pt)

Ad.IFN-b Efficacious dose in vivo, U-87 MG xenograft model 109 1010 to 1011

Toxicity in mice NOEL ,3 3 108 MTD .3 3 1010

Toxicity in NHP NOEL ,3.55 3 1010 MTD .3.55 3 1011

Proposed clinical trial doses with H5.010CMVhIFN-b 3 3 1010 3 3 1012

Ad.TK Doses used previously in adeno-TK clinical trial 5 3 109 5 3 1012

6.0 DOSE, DOSE SCHEDULE, AND DOSE ESCALATION

6.1 Vector Dose and Dose Schedule

The first three patients in the trial will be given 6 3 1010 par-ticles total dose, that is, 3 3 1010 particles at each of the twotreatments. Subsequent cohorts of three patients will be treatedat higher doses as shown in Table 3. For the dose escalationscheme, see Section 6.2 for guidelines.

Currently, the highest dose of adenoviral vector that can beadministered is 3 3 1012 particles in a volume of 2.0 ml, andthis quantity is based on the maximal titer of adenoviral vectorthat can currently be achieved.

6.2 Dose Escalation

6.2.1 First level of virus dose: 3 3 1010 particles/instillationor a total of 6 3 1010 particles. The first three patientswill be treated at this level. If no patients develop dose-limiting toxicity (DLT; Section 13.0), then the nexthigher dose will be used for the next three patients.The same guideline will be used for the subsequentdoses.

6.2.2 If one of three patients experiences a DLT, then threeadditional patients are enrolled at the same dose level.If only one of six patients experiences a DLT then thenext higher dose will be used for the next three pa-tients. However, if two of three or more than one ofsix patients experiences a DLT toxicity, then the MTDhas been defined, and an additional three patients areenrolled at the next lower level (Table 3). The dosethat would be used in future clinical trials (phase II) isdefined as the dose level at which zero of six or oneof six patients experiences a DLT and for which thenext higher dose level is the MTD. A minimum of sixpatients will be treated at this dose level. It is recog-nized that the MTD may not be achieved within theproposed range of doses. If this occurs, the highest dose(3 3 1012 particles/administration) will be used.

6.2.3 Patients will be entered into the study in cohorts ofthree patients, each of whom will receive the samedose. All patients at a given dose level will be followedfor at least 28 days from the time of study entry be-fore additional patients are enrolled at the next higherdose level. Treatment of subsequent patients at a higherdose level will be delayed if a current patient is expe-riencing a DLT. Thus, patients will not be enrolled at

the next dose level until the current patient improvesand it becomes apparent that the toxicity is reversible.

6.2.4 Patients who experience a DLT prior to the second vec-tor administration will receive a surgical resection (ifstill indicated) without additional vector injection.

6.2.5 There are no dose modifications for toxicity other thanas described.

7.0 ELIGIBILITY CRITERIA

7.1 Inclusion Criteria

To meet eligibility, patients must meet the following eligi-bility criteria at the time of enrollment (unless otherwise spec-ified):

7.1.1 Must be $18 years of age.7.1.2 Patients with recurrent or progressive malignant

glioma (glioblastoma multiforme and anaplastic astro-cytoma). Recurrent tumor is defined as the reappear-ance of the tumor by MRI scan following a period dur-ing which the tumor was not detectable. Progressivetumor is defined as an increase in the size of the tu-mor by MRI scan following initial treatment of the tu-mor. The diagnosis must be histologically proven ei-ther at the time of the initial presentation or at the timeof the recurrence.

7.1.3 Previous treatment with radiation therapy, interstitialradiation, radiosurgery or chemotherapy must be com-pleted more than 2 months prior to entry into the study.Prior resection is allowed but not required.

7.1.4 The tumor must be amenable to resection (tumor mustbe .70% resectable), and tumor debulking must beclinically indicated.

7.1.5 Karnofsky performance status of .70%.7.1.6 Must be on a stable dose of anticonvulsant and have

therapeutic serum levels within 2 weeks prior to entryinto the study.

7.1.7 Must be on corticosteroids (at a minimum dose equiv-alent of 8 mg twice a day of dexamethasone) for at least1 week prior to the MRI scan (Section 9.0 below).

7.1.8 Must give written informed consent.

7.2 Exclusion Criteria

Patients will be excluded from study entry if any of the fol-lowing exclusion criteria exist at the time of enrollment:

7.2.1 Serious uncontrolled medical illness, as judged by theinvestigator.


TABLE 3. DOSE SCHEDULE

Day 1: Day 8:particle/ particle/ Total Initial no. of

Cohort administration administration particles patients

1 3 3 1010 3 3 1010 6 3 1010 32 1 3 1011 1 3 1011 2 3 1011 33 3 3 1011 3 3 1011 6 3 1011 34 1 3 1012 1 3 1012 2 3 1012 35 3 3 1012 3 3 1012 6 3 1012 3

7.2.2 Abnormal blood tests exceeding any of the limits de-fined below:� Alanine transaminase (ALT) or aspartate transami-

nase (AST) . four times (43) the upper limit of nor-mal (ULN)

� Total white blood cell (WBC) count ,2300/mm3

� Platelet count ,80,000/mm3

� Serum creatinine . 2X ULN� Prothrombin time (PT) . 2 sec above the ULN.� Serum sodium of ,125 or .150 mEq/liter, or potas-

sium ,3.5 or .5.5 mEq/liter7.2.3 Herniation, or brainstem, optic chiasm, or subependy-

mal involvement of tumor.7.2.4 Pregnant or nursing women. In women with repro-

ductive potential, refusal to use effective contraceptivemeasures. In men, refusal to use barrier contraceptivemeasures.

7.2.5 Uncontrolled seizure disorder.7.2.6 Treatment with an investigational agent within 1 month

prior to study entry.7.2.7 History of intolerance to corticosteroids that would

preclude use during this study, or history of any med-ical condition that precludes the use of corticosteroids.

7.2.8 Unwillingness or inability to comply with proceduresrequired in this protocol.

8.0 REGISTRATION AND INFORMED CONSENT

The patient must have fulfilled the inclusion and exclusioncriteria prior to acceptance into the study. Registration will beat the time of signing the informed consent. Patients will be in-formed verbally and in writing of the nature of the research, itsgoals and potential risks. The informed consent document pre-pared for this study contains a detailed description, in lay lan-guage, of the hazards of surgery, virus injection, viral dissem-ination, and the known effects of interferon-b. A witness (otherthan an investigator) will also be present and will sign the con-sent form.

9.0 SCREENING STUDIES

The tests and evaluations described below must be performedwithin 2 weeks prior to the administration of the first dose ofgene therapy (day 1) in order to determine subject eligibility,unless otherwise indicated.

9.1 Medical history.9.2 Complete physical examination.9.3 Vital signs (pulse, blood pressure, temperature, and

weight).9.4 Complete neurological examination.9.5 Complete blood count (CBC), serum chemistry (elec-

trolytes, blood urea nitrogen [BUN], creatinine [Cr] andglucose [glu]), PT/PTT, LFTs, erythrocyte sedimenta-tion rate (ESR), and urinalysis.

9.6 Serum pregnancy test in women of reproductive poten-tial.

9.7 Serum level(s) of anticonvulsant(s).

9.8 Chest X-ray, EKG within 4 weeks prior to the first doseof gene therapy.

9.9 Review of previous brain tumor pathology by a neu-ropathologist at the study institution with confirmationof the diagnosis of malignant glioma.

9.10 Review of prior radiation therapy and chemotherapyrecords.

9.11 MRI scan of the brain with gadolinium (CT scan notacceptable).

10.0 TREATMENT

For the treatment period listed below, the patients will behospitalized in the neurosurgical ICU or GCRC, and universalprecautions will be used.

10.1 Screening StudiesAll tests listed in Section 9.0 will be completed priorto the administration of the first dose of gene therapy(day 1).

10.2 Baseline StudiesThe tests and evaluations described below must be per-formed within 2 days prior to day 1, unless otherwiseindicated.a. Medical history.b. Complete physical examination.c. Vital signs (pulse, blood pressure, temperature, and

weight).d. Complete neurological examination.e. Serum for viral immunology (anti-adenoviral anti-

bodies and lymphocyte proliferation assay).f. Serum for interferon-beta and neopterin (an inter-

feron-induced protein) measurement.g. Serum for anti-interferon-beta antibody measure-

ment.h. Cerebral spinal fluid (CSF) for interferon-beta,

anti-interferon-beta antibody and anti-adenoviralantibody measurement. If a lumbar puncture can-not be performed or is clinically contraindicatedthen this test may be omitted.

i. Adenoviral cultures (from nasal swab, rectal swab,blood, urine, and CSF). If a lumbar puncture can-not be performed or is contraindicated then the ade-noviral cultures on the CSF may be omitted.

j. MRI scan of the brain. If the MRI scan done inSection 9.0 was performed within 2 weeks prior today 1, then the MRI scan does not need to be re-peated as part of these baseline studies.

k. FDG-PET scans of the brain. To be performedwithin 2 weeks prior to day 1.

10.3 First Surgery and First Administration of Gene Ther-apy (Day 1)a. The tumor will be localized using MRI or CT scan

for the purpose of defining the stereotactic coor-dinates.

b. A tumor biopsy (minimum one site) will be ob-tained immediately prior to stereotactic injection.

c. The tumor will be infiltrated with the H5.010CMVhIFN-b vector at 10 sites, with approxi-mately 0.2 ml/site of vector, delivered over 1 min


per site, for a total vector volume of 2.0 ml, evenlyspread within the tumor volume.

d. Additional dexamethasone, anticonvulsants, andprophylactic antibiotics will be given as deemedclinically appropriate by the neurosurgeon.

e. A CT scan of the brain will be obtained within 30hr after stereotactic injection of gene therapy to as-sess for postprocedure bleeding.

10.4 Second Surgery and Second Administration of GeneTherapy (Day 8 6 2 Days)a. A follow-up MRI will be performed within 2 days

prior to the resection and vector instillation.b. Patients will undergo a craniotomy on day 8, and

the tumor will be resected as completely as possi-ble.

c. A second dose (same level) of theH5.010CMVhIFN-b vector will be injected to adepth of approximately 3 mm into the tumor bedat 10 sites approximately evenly distributedthroughout the resection cavity. The total dose willbe in 2.0 ml, evenly distributed in 0.2-ml admin-istrations at each of 10 sites.

d. Tumor biopsy and resected specimens will be sub-jected to the following tests (as described in Sec-tion 15.4):iii. Sections will be fixed in formalin and embed-

ded in paraffin for histological analyses (per-formed by the pathology lab at the institution).

iii. These same formalin fixed sections will beused for polymerase chain reactions (PCR),immunohistochemical and in situ analyses(performed by the Morphology Core of IHGT).

iii. Additional sections will be frozen, and will beassayed for levels of interferon-beta protein bymeans of an ELISA (performed at Biogen).

e. A postoperative brain MRI scan will be performedon day 9 in order to evaluate the extent of the re-section.

11.0 CLINICAL, LABORATORY, AND IMAGINGSTUDIES ON PROTOCOL

The following tests will be done following the baseline stud-ies (as outlined in Appendix 2):

11.1 The medical history and complete examination willbe performed on day 8, 15, 29, month 2, 4, 6, andevery 6 months thereafter up to 2 years.

11.2 The brief physical examination will be performed ondays 1 to 7, inclusive, then on days 9 to 14, inclu-sive, and then on day 22.

11.3 The vital signs will be performed every day from day1 to day 15, inclusive, on day 22 and day 29 and thenat month 2, 4, 6, and every 6 months thereafter up to2 years.

11.4 The complete neurological examination will be per-formed on days 1, 8, 15, 22, 29, month 2, 4, 6, andevery 6 months thereafter up to 2 years.

11.5 Laboratory tests will be performed as follows: theCBC will be done on days 3, 6, 8, 11, 15, 22, 29, then

as clinically indicated. The serum chemistry, LFTs,and PT/PTT will be done on days 3, 8, 15, 22, 29 andthen as clinically indicated. The ESR will be done onday 3, 8, and then as clinically indicated. The ESRmay be repeated on day 15 if the ESR is abnormal onday 8. The urinalysis will be done on day 8, and thenas clinically indicated. The urinalysis may be repeatedon day 15 if the urinalysis is abnormal on day 8.

11.6 The CXR will be done on day 3 and 15, and then asclinically indicated.

11.7 The lumbar puncture will be done on day 11. If thelumbar puncture cannot be performed or is clinicallycontraindicated then the lumbar puncture may beomitted.

11.8 Adenoviral cultures will be performed in samples ofblood, urine, and nasal and rectal stools taken on days2 and 9. Adenoviral cultures will be performed onCSF obtained by lumbar puncture on day 11.

11.9 MRI scans of the brain with gadolinium contrast areto be performed and evaluated by a neuroradiologist.MRI scans will be performed on day 1, 7, 9, 29, month2, 4, 6, and then every 6 months thereafter up to 2years. Additional scans will be performed as clini-cally indicated. A separate evaluation of the MRIscans for efficacy will be performed by a neuroradi-ologist blinded to the nature of the treatment (Section15.0).

11.10 PET or SPECT scans of the brain will be performed.A separate informed consent will be obtained for thePET or SPECT scans. The PET or SPECT scans willbe performed at baseline prior to vector administra-tion and as clinically indicated. A nuclear medicinephysician will evaluate all PET or SPECT studies andprovide a written report.

11.11 A CT scan of the brain will be obtained within 30 hrafter stereotactic injection of gene therapy to assessfor postprocedure bleeding.

11.12 Human-interferon-beta (hIFN-b) will be determinedat Biogen (Cambridge, MA) using an ELISA and aviral cytopathic effect (CPE) assay as previously de-scribed (Alam et al., 1997) if sufficient material isavailable, and as indicated. As a second measure ofhIFN-b, the induction of neopterin will be measured.Serum samples will be assayed at Covance Labora-tories for neopterin using a commercial assay.11.12.1 Serum will be isolated and frozen at #65°C

and then shipped to Biogen. Serum will betaken on day 4, 8, 11, 15, 22, 29, and month2 and analyzed using an ELISA and CPEassay for human interferon-beta, and usinga commercial assay for neopterin.

11.12.2 CSF will be obtained from the lumbar punc-ture on day 11; it will be frozen at #65°Cand sent to Biogen for analysis using anELISA for human interferon-beta.

11.12.3 Tumor taken immediately prior to stereo-tactic injection (day 1), and at the time ofresection (day 8) will also be assayed forIFN-b levels by means of an ELISA assayat Biogen.


11.13 Measurement of human interferon-beta antibodies.Serum will be obtained on month 2, 6 and then every6 months thereafter up to 2 years, frozen at #65°Cobtained by lumbar puncture on day 11 and sent toBiogen for measurement of human interferon-beta an-tibodies. CSF will also be assayed for human inter-feron-beta antibodies.

11.14 Immunological assays for adenovirus.11.14.1 Serology and CSF samples. Serum samples

will be assayed for neutralizing adenoviralantibody, serotype analysis, and Westernblot analysis against adenoviral proteins.Serum for this assay will be obtained onday 8, 15, 22, 29, month 2, 4, 6, and every6 months thereafter up to 2 years. CSF ob-tained by lumbar puncture on day 11 willbe assayed for anti-adenoviral antibodies.

11.14.2 Lymphoproliferative assays to adenoviralvector will be determined from samplestaken on day 8, 15, 22, 29, month 2, 4, 6and every 6 months thereafter up to 2 years.

11.15 All patients will be followed until death or for a min-imum of 2 years. Follow-up visits will take place fromday 1 to day 15 inclusive, then at day 22 and day 29and month 2, 4, 6, and every 6 months thereafter upto 2 years. Clinical, laboratory and radiographic as-sessments will be performed as described above andin Appendix 2. The time-dependent outcomes to bemeasured include the tumor response, time to tumorprogression (see Section 15.0), time to death, level ofexpression of hIFN-b in the serum, CSF, and tumortissue and the immune responses to adenoviral vec-tor and hIFN-b.

12.0 CONCOMITANT THERAPY

Acetaminophen, naproxen, or ibuprofen may be adminis-tered for the prophylactic treatment of flu-like symptoms.

Patients must receive corticosteroids (at a minimum doseequivalent of 8 mg twice a day of dexamethasone) for at least1 week prior to the MRI scan (Section 9.0) and for a minimumof 1 week after the second dose of the gene therapy. After thisperiod, the steroid dose may be reduced as clinically indicated.

Concomitant treatment with another investigational drug orapproved therapy for investigational use is not allowed.

Patients may receive chemotherapy or radiotherapy nosooner than 8 weeks following the second administration ofgene therapy, unless indicated by documented progressive tu-mor growth.

13.0 DOSE-LIMITING TOXICITY

For the purpose of dose escalation, patients that develop anyof the following toxicities (as defined by the Common Toxic-ity Criteria, Appendix C) which are thought by the investiga-tor to be related to the gene therapy will be considered to havehad a dose-limiting toxicity (DLT). Unless otherwise specified,(see below) all grade 3 or 4 toxicities (as described in Appen-

dix C) will be considered dose limiting. In addition, any treat-ment-related death will be considered a DLT. The evaluable pe-riod for DLT as pertains to this dose escalation is 28 days af-ter the initial administration of gene therapy. The following willbe considered dose-limiting toxicities:

� Grade IV leukopenia, thrombocytopenia, or anemia� Grade III or grade IV hemorrhage (non-CNS) or any grade

IV CNS hemorrhage/bleeding� Grade III or grade IV infection� Grade IV fever in the absence of infection� Grade III or grade IV renal toxicity� Grade III or grade IV gastrointestinal toxicity but only grade

IV liver toxicity or precoma will be considered DLT� Grade III or grade IV pulmonary, cardiac, or blood pressure

toxicity� Only grade IV skin toxicity or grade IV wound infection will

be considered DLT� Grade III or grade IV allergic reaction� Phlebitis deep vein thrombosis, pulmonary embolism,

SIADH, and hyponatremia will be recorded but will not beconsidered a DLT because of their common occurrence in pa-tients with malignant gliomas.

� Grade III or grade IV metabolic or coagulation toxicity� Neurotoxicity will be considered dose limiting if there are:� —Clinical or MRI manifestation of generalized encephalitis

or meningitis or,� —Increased edema or mass effect by MRI scan, associated

with the development of herniation, or decrease in the levelof consciousness to Glasgow Coma Scale of 10, despiteappropriate measures or,

� —Headache lasting more than 24 hr and unrelieved by anal-gesia

� —However, if any of the above-mentioned neurotoxicitiesoccur more than 21 days following tumor resection and areassociated with radiographic findings consistent with tu-mor growth, then they will not be considered a DLT.

14.0 STATISTICAL CONSIDERATIONS

The primary objectives of this study are to evaluate the tox-icity profile, and determine the MTD of adenoviral-deliveredhIFN-b in patients with recurrent or progressive malignantglioma. This study will utilize a standard three-six phase I doseescalation clinical trial design. The primary end point for thisstudy will be dose-limiting toxicity (DLT), as defined in Sec-tion 13.0. General toxicity will be graded according to the (NCI)Common Toxicity Criteria.

The maximum tolerated dose (MTD) for this trial will be de-fined as the dose at which two of three or more than one of sixpatients (as defined in Appendix 1) demonstrate a DLT. Thedose to be used for future studies will be defined as the doselevel at which zero of six or one of six patients experiences aDLT and for which the next highest dose level is the MTD. Itis recognized that the MTD may not be achieved within the pro-posed ranges of doses. If this occurs, the highest dose will beused for future clinical studies. A minimum of six subjects willbe treated at this highest dose or one dose level below the MTD.Details of the dose escalation scheme are shown in Appendix 1.


The study will follow a standard three-six phase I escalationscheme. Three to six patients will be treated at each dose level.If the highest dose prescribed by the protocol is used withoutreaching the MTD, three additional patients will be treated atthat dose for a total of six patients. The operating characteris-tics of this design are shown in Table 4, which provides theprobability of escalation to the next highest dose for each un-derlying true DLT rate. For example, for a toxicity that occursin 10% of subjects, there is a greater than 90% probability ofescalating. Conversely, for a common toxicity that occurs witha rate of 70%, the probability of escalating is ,5%.

Table 5 shows the probability of failing to observe toxicityin a sample size of three or six patients given variuos true un-derlying toxicity rates. For example, with six patients, the prob-ability of failing to observe toxicity occurring at least 40% ofthe time is less than 5%.

Additional research questions will include measurements ofgene transfer, neutralizing antibody and other immunologicalparameters (in both CNS and serum), and viral shedding as-says. Analysis of these research questions will be primarily de-scriptive, and will involve both between-patient and within-pa-tient comparisons across doses. If applicable, comparisons willbe performed using McNemar’s test, Fisher’s exact test, andWilcoxon signed rank and rank sum tests.

15.0 MEASUREMENT OF EFFECT

15.1 Tumor response will be determined for each patientby a radiologist who is blinded to the treatment ad-ministered in this study. A complete tumor responseis defined as radiographic disappearance of all mea-surable disease on the MRI scan with stable or im-proved neurological status on stable or decreasingdoses of steroids for at least 2 months. A partial re-sponse is defined as greater than or equal to a 50%decrease in tumor size by MRI scan (on consecutiveMRI scans at least 2 months apart) with stable or im-proved neurological status on stable or decreasingdoses of steroids for at least two months. Progressivedisease is defined as a greater than or equal to 25%increase in the size of the tumor on MRI scan, newtumor on the MRI scan, or worsening neurologicalsymptoms requiring higher doses of steroids andmaintained for 2 months. Stable disease is defined aseither a less than 50% decrease in tumor size by MRIscan or a less than 25% increase in tumor size by MRIscan, with stable neurological status over a period ofat least 2 months.

15.2 Time to tumor progression (will be measured fromthe first day of treatment until the progression is doc-umented) will be determined. Tumor progression will

be determined by a radiologist that is blinded to thetreatment administered in this study. Progressive tu-mor is defined as an increase in the size of the tumorby MRI scan following the surgical resection per-formed on day 8. The PET scan of the brain will alsobe reviewed by the same radiologist and will aid indifferentiating tumor progression from inflamma-tion/edema.

15.3 Time to death will be determined.15.4 Analysis of tissue for virus effect and host response.

15.4.1 Analysis for evidence of gene transfer. Braintissue obtained at the time of palliative re-section and at autopsy (when available) willbe analyzed for the presence of DNA de-rived from the gene transfer. DNA extractsfrom paraffin sections will be tested by PCRusing primers specific for the adenoviralDNA and the hIFN-b gene to determine ifgene transfer has taken place, and how longit persists. Five to 10 sites within the tumorwill be analyzed depending on the amountand condition of the tissue obtained. Failureto detect gene transfer may indicate ineffi-cient cell transduction or may reflect tumorcell lysis by hIFN-b. A positive finding atday 8 or at autopsy (an indeterminate timelater) will provide evidence of gene transferand some indication of the duration of trans-gene persistence. PCR positive tissues willbe confirmed by immunohistochemicalstaining and/or in situ hybridization if avail-able.

In addition, hIFN-b protein levels will bemeasured in the frozen tumor sections takenat the resection, and also in the CSF andserum. The measurements of hIFN-b proteinwill be determined by means of an ELISAin the brain tissue, CSF and serum, and bymeans of a cytopathic effect (CPE) bioassayin the serum (Alam et al., 1997). These mea-surements will also help to define the levelof gene transduction and expression and po-tential for efficacy.

15.4.2 Analysis for toxicity of adenovirus. Hema-toxylin and eosin stains of brain tumor willbe used to assess the extent of inflammation.This can be compared to pretreatment tumorslides to determine if there exists a large in-flammatory response, which is likely to becaused by the therapy. If a significant in-flammatory response is present, infiltratingcells will be characterized by immunohisto-


TABLE 4. TRUE UNDERLYING DLT RATE AT DOSE LEVELS

10% 20% 30% 40% 50% 60% 70% 80% 90%

Probability 0.91 0.71 0.49 0.31 0.17 0.08 0.03 0.009 0.001Pr [escalating]

chemical staining with antibodies specificfor B cells and T cells.

15.4.3 Analysis of immunologic response. Serumand cerebrospinal fluid will be collected pre-and posttherapy for detection of anti-ade-novirus antibodies by western blot and byneutralizing antibody assay. Our expecta-tions, based on prior patient specimens, isthat all patients will have nonneutralizingantibodies (western blot positive) on entry,and will likely develop high titer neutraliz-ing antibodies during therapy. The patient’shumoral immune response to adenoviruswill be studied by determining titers to ade-novirus at various times during the protocol.

Peripheral blood lymphocytes will be ob-tained to determine if the patient’s lympho-cytes will proliferate in response to aden-oviral antigen in vitro. These tests mayprovide evidence for the development of animmune response following adenovirustransduction and will play an important rolein the design of future adenovirus vectorclinical trials. The immune response to thevector may limit the efficacy of serial treat-ments, may lessen the duration of transgenicexpression, or may contribute to local, doselimiting inflammation.

Serum antibodies to human interferon-beta will also be determined (at Biogen). Itis expected that there will not be a signifi-cant response to the human protein.

16.0 RISKS AND BENEFITS OF PARTICIPATION

16.1 Expected Benefits

Since this is a phase I study it is unlikely that many (if any)patients will benefit from participating in this clinical trial. Sim-ilar viruses have, as experimental therapies, also been given topatients who have malignant gliomas. Very few patients appearto have benefited from the therapy and achieved durable com-plete responses. However, most did not appear to be benefitfrom the therapy. Patient participation in this study will advancethis field of study and may lead to benefits in the future.

16.2 Major Risks Associated with This Research

The risks imposed by this study are principally those asso-ciated with the types of surgical procedures performed in this

study and the risks associated with administration ofH5.010CMVhIFN-b. The surgical risks are well established asthese techniques are used in the delivery of the normal stan-dard of care for patients with brain tumors. These include therisk of paralysis, stroke, and difficulty with speech or visionthat may be temporary or permanent, bleeding into the brain,infection, and possible death. These risks apply equally to boththe stereotactic surgery and the surgery to remove a portion ofthe tumor. These risks are minimized by the greatest extent pos-sible through the use of neurosurgeons exclusively specializingin neuro-oncology.

The risks associated with the administration ofH5.010CMVhIFN-b include the effects of the hIFN-b proteinproduced by transduced cells and the effects of the adenoviralvector itself. Side effects directly related to the hIFN-b proteinhave been reported in related studies of patients with cancerwhen the protein was given intravenously. These toxic effectsincluded flu-like symptoms (muscle aches, fever, chills, fatigue,nausea, and or vomiting). These symptoms were brief and re-solved with standard medical care. Other side effects includedreversible injury to the liver and lowering of the number ofblood cells. A few patients developed seizures while receivingthe hIFN-b protein; however, it is not known whether theseizures were caused by the hIFN-b protein or by their braintumors.

Although the viral vector is rendered replication incompe-tent, there is a very slight possibility that viral recombinationmay occur and produce a virus that can replicate. The admin-istered virus may also contain a small amount of virus that canmultiply. The effects of these events are unknown, however itis likely that such virus will be cleared by the normal immuneresponse to adenovirus. There have been no reports of virusreplication in the prior adenovirus gene therapy studies con-ducted to date.

17.0 ADVERSE EVENTS AND PARTICIPANT WITHDRAWALS

17.1 Adverse Event Definitions

The terms “relationship,” “serious,” and “severity” usedthroughout this section are defined in Table 6.

For the purposes of this study, adverse events are defined asany signs (including the clinical manifestations of abnormallaboratory results) or medical diagnoses noted by medical per-sonnel, or symptoms reported by the patient, regardless of re-lationship to study drug, that:

1. Have onset anytime after the start of study drug treatment(signs, symptoms, and diagnoses occurring prior to the firstdose of study drug are not considered to be adverse eventsif they do not increase in severity).


TABLE 5. TRUE UNDERLYING DLT RATE AT DOSE LEVELS

Sample size 10% 20% 30% 40% 50% 60% 70% 80% 90%

n 5 3 0.73 0.51 0.34 0.22 0.13 0.064 0.027 0.080 0.001n 5 6 0.53 0.26 0.12 0.047 0.016 0.0041 ,0.001 ,0.001 ,0.001

OR

2. Have worsened since the event was previously reported (thisincludes worsening of signs, symptoms, or diagnoses thatwere present prior to dosing).

Treatment-Emergent Adverse Event: A treatment-emergentadverse event is defined as any adverse event not present priorto gene instillation or any event already present that worsens ineither intensity or frequency following gene instillation.

Baseline-Emergent Adverse Event: A baseline-emergent ad-verse event is defined as any adverse event which occurs orworsens during the screening process (after informed consent)before gene instillation on Day 1.

17.2 Eliciting and Recording Adverse Events

Patients must be given the name and phone number of per-sonnel at the study site who can be called in the event of anemergency or to report an adverse event that is of concern tothe subject.

Reporting of adverse events will be accomplished by col-lecting information on adverse experiences during the screen-ing process and at scheduled follow-up visits. In order to avoid

bias in eliciting adverse events, patients will be asked “Haveyou had any changes in your health since last visit.” All ad-verse events (serious and nonserious; treatment-emergent andbaseline-emergency) must be recorded on the Adverse EventReport Form and also the Serious Adverse Event Report Formif applicable. Adverse events are to be recorded regardless ofrelationship to study drug. The information to be recorded willinclude, but not be limited to, relationship of the event to studydrug, and severity of the event.

The investigators will provide a written report of all seriousand unexpected adverse events to the FDA within 15 calendardays of the event. If the event is a death or life-threatening andunexpected, the FDA will be notified by phone or fax withinseven calendar days, followed by the written reports within 15days of the event.

In order to facilitate timely reporting of serious adverseevents to the FDA by the investigators, clinical research staffwill contact the Quality Assurance Unit (QAU) of the institu-tion immediately and fax the completed Adverse Event ReportForm and initial Serious Adverse Event Report Form prior tothe close of the following business day. All serious and unex-pected adverse events will be reported to the QAU, regardlessof the intervention-related assessment. In addition, the Princi-


TABLE 6. ADVERSE EVENT DEFINITIONS

Serious Adverse EventSerious adverse events are defined as the following (in accordance with 21 CFR §312.32 and the recommendations of theInternational Conference on Harmonization [Federal Register, October 7, 1997, Vol. 62, No. 194, pp. 52239-45]):� Any death.� Any life-threatening event, i.e., an event that places the patient, in the view of the investigator, at immediate risk of death

from the event as it occurred (does not include an event that, had it occurred in a more severe form, might have causeddeath).

� Any event that requires or prolongs in-patient hospitalization.� Any event that results in persistent or significant disability/incapacity.� Any congenital anomaly/birth defect diagnosed in a child of a subject who participated in this study and received study

drug� Any event that necessitates medical or surgical intervention to prevent one of the outcomes listed above.

Relationship of Adverse Event to Study Drug

Unrelated Any reaction that does not follow a reasonable temporal sequence from administration of study drug AND thatis likely to have been produced by the patient’s clinical state or other modes of therapy administered to thesubject.

Unlikely Any reaction that does not follow a reasonable temporal sequence from administration of study drug OR thatdoes not follow a known response pattern to the suspected drug OR that is likely to have been produced by thepatient’s clinical state or other modes of therapy administered to the patient.

Likely A reaction that follows a reasonable temporal sequence from administration of study drug AND that follows aknown response pattern to the suspected drug AND that could not be reasonably explained by the knowncharacteristics of the patient’s clinical state or other modes of therapy administered to the patient.

Definite A reaction that follows a reasonable temporal sequence from administration of study drug AND that follows aknown response pattern to the suspected drug AND that recurs with rechallenge, and/or is improved by stoppingthe drug or reducing the dose.

Severity of Adverse Event

Mild Symptom(s) barely noticeable to patient or does not make patient uncomfortable; does not influenceperformance or functioning; prescription drug not ordinarily needed for relief of symptom(s) but may be givenbecause of personality of patient.

Moderate Symptom(s) of a sufficient severity to make patient uncomfortable; performance of daily activity is influenced;patient is able to continue in study; treatment for symptom(s) may be needed.

Severe Symptom(s) cause severe discomfort; severity may cause cessation of treatment with study drug; treatment forsymptom(s) may be given and/or patient hospitalized.


pal Investigator will report all adverse events to the InstitutionalIRB following their regulations regarding SAE reporting.

When reporting fatal, life-threatening, or serious and unex-pected adverse events, the research center personnel will pro-vide as much of the following information as is available at thetime:

a. Patient name, numberb. Investigator and primary physician’s name (if different)c. Protocol title and numberd. Patient’s date of birth, sex, and ethnicitye. Current day in treatment protocolf. Concurrent medications, including dose, route of ad-

ministration, and duration of therapyg. Information regarding the event, including the following

details:� Description� Date of onset and end date (if appropriate)� Whether hospitalization or prolonged hospitalization

was required� Treatment required for the AE� Treatment outcome� Investigator’s determination of relationship to the gene

instillation� Whether the AE is life-threatening

17.2.1 Expedited Reports

Adverse events are recorded on the Adverse Event case re-port form. The form is continually updated as events happen.The AE form is faxed to the Associate Director of the QAUwithin 48 hr.

17.2.2 Patient Withdrawal

A patient will be withdrawn from the study if the patientwishes to withdraw from the trial or if the subject develops se-rious concurrent illness unrelated to their malignant glioma.

18.0 PATIENT RECRUITMENT ANDFINANCIAL CONSIDERATIONS

18.1 Patient Recruitment

The patients will be recruited from the population of gliomapatients followed at the University of Pennsylvania and at a sec-ond institution or referred by other physicians. There will beno advertisements for subjects. Patients will be recruited fromboth genders and all races, without bias. The disease under studyaffects all races and both genders equally. The University ofPennsylvania Hospital is located in a large urban area and servesas a general hospital for West Philadelphia (which has a largeAfrican-American population) as well as a referral center for awide three-state region. Therefore, we expect a mixture of racesin the patients eligible for study, as seen in similar brain tumorstudies at HUP.

Patients will be screened to meet the inclusion and exclusioncriteria detailed in this protocol. Due to the novelty of genetherapy, and due to the invasiveness and the time commitmentrequired for the study, it is expected that the investigators willneed to spend a significant amount of time explaining the na-ture of this study.

18.2 Financial Considerations

The cost of the H5.010CMVhIFN-b, the stereotactic surgeryand the first week of hospital stay will be covered by resourcesavailable to conduct the study. The costs of the tumor resectionand subsequent recovery will be billed to the patient’s insur-ance company in accordance with their policy. The patient’s in-surance company will only be billed for those portions of thestudy that is part of the standard of care for the treatment ofpatients with malignant glioma. Tests and procedures that arepart of the experimental therapy will not be billed to the pa-tient’s insurance company. Depending on the patient’s insur-ance coverage, the patient may incur costs related to the partof this study that is part of the standard of care for patients withmalignant gliomas.

19.0 REFERENCES

Alam J. et al. (1997). Pharmacokinetics and pharmacodynamics of in-terferon beta-1a (IFNb-1a) in healthy volunteers after intravenous,subcutaneous and intramuscular injection. Clinical Drug Investiga-tion 14(1): 35–43.

Andreansky S. S. et al. (1996). The application of genetically engi-neered herpes simplex viruses to the treatment of experimental braintumors. Proceedings of the National Academy of Sciences of theUnited States of America 93(21): 11313–11318.

Badie B. et al. (1995). Adenovirus-mediated p53 gene delivery inhibits9L glioma growth in rats. Neurological Research 17(3): 209–216.

Bischoff J. R. et al. (1996). An adenovirus mutant that replicates se-lectively in p53-deficient human tumor cells. Science 274: 373–376.

Culver, K. W. (1996). Gene therapy for malignant neoplasms of theCNS. Bone Marrow Transplantation 18(3).

Dietrich P. Y. et al. (1997). Immunobiology of gliomas: new perspec-tives for therapy. Annals of the New York Academy of Sciences 824:124–140.

Eck S. L. et al. (1996). Clinical protocol: treatment of advanced CNSmalignancies with the recombinant adenovirus H5.010RSVTK: aphase I trial. Human Gene Therapy 7: 1465–1482.

Fine H. et al. (1993). Meta-analysis of radiation therapy with and with-out adjuvant chemotherapy for malignant gliomas in adults. Cancer71: 2585–2597.

Fine H. A. et al. (1997). A phase I study of a new recombinant humanb-interferon (BC9015) for the treatment of patients with recurrentgliomas. Clinical Cancer Research 3: 381–387.

Glick R.P. et al. (1997). Intracerebral versus subcutaneous immuniza-tion with allogeneic fibroblasts genetically engineered to secrete in-terleukin-2 in the treatment of central nervous system glioma andmelanoma. Neurosurgery 41(4): 898–906.

Hsiao M. et al. (1997). Intracavitary liposome-mediated p53 gene trans-fer into glioblastoma with endogenous wild-type p53 in vivo resultsin tumor suppression and long-term survival. Biochemical and Bio-physical Research Communications 233(2): 359–364.

Kozarski, K. F. and J. M. Wilson (1993). Gene therapy: adenovirusvectors. Current Opinion in Genetics and Development 3: 499–503.

Levin, V.A., Leibel, S.A. and Gutin, P.H. (1997). Neoplasms of theCentral Nervous System. in Cancer, Principles and Practice of On-cology, 5th Edition, V.T. DeVita, Jr., S. Hellman and S.A. Rosen-berg, Lippincott-Raven, New York.

Miyatake S. et al. (1997). Defective herpes simplex virus vectors ex-pressing thymidine kinase for the treatment of malignant glioma.Cancer Gene Therapy 4(4): 222–228.

Packer R.J. et al. (1996). Treatment of children with newly diagnosedbrain stem gliomas with intravenous recombinant b-interferon andhyperfractionated radiation therapy. A Children’s Cancer Groupphase I/II study. Cancer 77(10): 2150–2156.

CONSENT TO ACT AS A SUBJECT IN AN INVESTIGATIONAL STUDY

Title:

Treatment of Recurrent or Progressive Malignant Glioma with a Recombinant Adenovirus Expressing Human Interferon-Beta (H5.010CMVhIFN-b ): A Phase I Trial (UPCC#: 2399)

Purpose of Study.

The purpose of this research study is to determine the effectiveness and safety of a recombinant (laboratory-produced) adenovirusinjected into brain tumors. The virus has been especially designed to carry a gene called human interferon beta (hIFNb). Thestudy will investigate whether this gene (a gene is part of the nucleus of a cell) will enter brain tumor cells and kill them. Thisstudy may not benefit you in any way. Its principal goal is to help researchers gather information about the use of gene therapyand the use of human interferon beta in the treatment of brain tumors and to evaluate the safety and toxicity of this therapy.

You have been given the opportunity to participate in this study because you have a brain tumor that was not cured by yourinitial treatments. Patients with brain tumors who are not cured by their initial therapy usually are not cured by subsequent ther-apies. In this study, the insertion of a new gene (the hIFNb gene) into a portion of the brain tumor cells may result in tumor celldeath.

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Detailed description of participation.

Pre-gene therapy evaluation:

You will be evaluated to determine if you are a suitable subject to participate in the gene therapy study. This will involve a fullmedical history and physical examination, blood tests, MRI scan of the brain and chest x-ray. Approximately 30 ml (2 table-spoons) of blood will be taken from an arm vein. A pregnancy test will be performed if you are a woman with childbearing po-tential.

Administering the gene into the brain tumor:

You will be admitted to the Clinical Research Center or the Neurosurgery Service of the Hospital of the University of Pennsyl-vania. A positron emission tomography scan (PET scan) of the brain will be performed. For this scan, you will lie on a com-fortable reclining chair in the Nuclear Medicine Section of the hospital, and scans (pictures) will be taken of your brain. Yourhead will not be enclosed during this procedure. Prior to the scans, a needle will be in an arm vein (in the hand or elbow area)and used for injection of a radioactive of form of sugar, F-18 deoxyglucose. The purpose of the PET scan is to determine if yourbrain tumor uses sugar, as many malignant tumors do. The purpose of the PET scan is to determine if your brain tumor usessugar, as many malignant tumors do. The PET scan will be repeated after gene therapy, to see if the treatment has changed thecharacteristics of the tumor.

On the first hospital day you will undergo a lumbar puncture (spinal tap). You will lie on your side or will be sitting up dur-ing this procedure. Your lower back will be cleaned and you will receive a local anesthetic by needle injection into your lowerback. A small amount of spinal fluid will be removed with a needle from your lower back and this fluid will be analyzed in thelaboratory. Need to lie flat for about 6 hours after this procedure.

On the second hospital day you will be taken to the operating room. Under general anesthesia, a stereotactic frame will beplaced on the outside of your head. This is a large ring attached to the scalp temporarily, to permit precise measurements of thetumor location. You will be transported to the Radiology Department for a MRI or CT scan of the brain, then back to the oper-ating room. While still under general anesthesia, the neurosurgeon will perform surgery by stereotactic technique. In other words,the brain tumor will be entered through small holes rather than through a large incision in the brain. The surgeon will inject intothe tumor the recombinant adenovirus, containing the hIFNb gene. The stereotactic frame will be removed after surgery before


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you leave the operating room (while you are still under anesthesia). After the surgery, you will be taken to the Intensive CareUnit for routine postoperative care. You will be transferred back to the Clinical Research Center (CRC) in 1 or 2 days. You willhave a MRI scan of the brain on the first and sixth postoperative days.

Seven days after the stereotactic surgery, you will have a second operation. This will be a standard neurosurgical operation(does not use a stereotactic frame) under general anesthesia, to remove as much of the brain tumor as is safe and possible. Af-ter the tumor is removed, the surgeon will inject another dose of the same adenovirus into the area of remaining tumor. Afterthis operation you will again be transferred to the Intensive Care Unit and after 1 or 2 days to the CRC for further care. MRIscans will be performed 1 and 14 days after surgery. You will be discharged from the hospital after 14 days, or whenever yourcondition is considered medically stable.

Need for long term follow up:

Gene therapy is a very new form of treatment and the long-term effects are not known. To gain the most information and to pro-vide the greatest safety, periodic evaluations will be needed. Your agreement to participate in this study means that you recog-nize the need for prolonged relationship with the study and appreciate the need for continued elevations. This will entail monthlyevaluations that include history, physical examination, MRI scans and routine blood tests.

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Consent for autopsy:

To fully evaluate the effects and safety of gene therapy, it will be necessary to obtain as much information as possible. In theeventual occurrence of your death, evaluation of your brain will be a very valuable method to see the full effects of gene ther-apy. Therefore your participation in this study means that you agree to an autopsy. This agreement must also be known by yournext of kin so that person can follow your wishes.

Expected benefits:

You may not benefit in any way from this research study. Similar viruses have, as experimental therapies, also been given to pa-tients who have the same type of brain tumor. A few of these patients appear to have benefited from the therapy; however, mostdid not appear to benefit from the therapy. This experimental therapy is designed to kill the brain tumor cells. The tumor maybecome smaller for a period of time, but it is not known if this will occur or for how long any benefit will last.

Risks, discomforts and inconveniences of the research

Blood tests:

Blood tests will be performed frequently, daily while in the hospital, less often thereafter. You may develop a bruise or tender-ness at the site of needle punctures. Over the entire course of the study, less than one unit (less than one pint) of blood will betaken for all tests. Patients sometime become light-headed or even faint when blood is drawn.

Lumbar Puncture:

You will need to sign a separate consent form for the lumbar puncture (spinal tap). The potential risks include infection, bleed-ing, headache or an allergic reaction to the local anesthetic. There is a very rare possibility of paralysis or stroke.

Surgery:

You will be asked to sign a separate consent form for the neurosurgery. The potential risks include paralysis, stroke, and diffi-culty with speech or vision that may be temporary or permanent, bleeding into the brain, infection, and possible death. Theserisks apply equally to both the stereotactic surgery and the surgery to remove a portion of the tumor.

PET scans:

The arterial and venous catheters (tubes) could cause some bleeding at the site of the injection. This tube could become discon-nected causing blood loss or the artery or vein could become clotted. This could cause circulation problems in the hand makingit cold and painful. Pieces of the tube or clot could break off and lodge elsewhere requiring treatment. These complications areextremely rare. The PET scan uses radioactive material and therefore you will receive a radiation dose. The radiation dose youreceive is within the limits permitted for human research volunteers. While the radioactive material is in your body it is not haz-ardous to persons near you. Also, it will have largely disappeared within 6 hours and will have entirely disappeared within abouta day. These studies will not be performed on pregnant women. A blood pregnancy test will be done on any woman of child-bearing potential.

Complications caused by the virus:

a) Production of the hIFNb protein. The gene that will be inserted into the brain tumor cells, and potentially into normal braincells, is not normally made by these cells. Although a very similar protein is made by some human cells, it is not known if thisprotein can cause toxic effects when produced in brain tumors. Tests in animals indicate that it is not likely to be toxic. Symp-toms directly related to the hIFNb protein have been reported in related studies of patients with cancer when the protein was


given intravenously. These side effects included flu-like symptoms (muscle aches, fever, chills, fatigue, nausea and or vomiting).These symptoms were brief and resolved with standard medical care. Other side effects included reversible injury to the liverand lower of the number of blood cells. A few patients have developed seizures while receiving the hIFNb protein, however itis not known whether the seizures were caused by the hIFNb protein or by their brain tumors. The possibility of toxicity existsthat may include new toxicities not previously reported.

b) Multiplication of the virus. The virus that is being used has been made so that it cannot reproduce by itself. There is a veryslight possibility that something may happen during the study that will allow the virus to multiply or that the virus may containa small amount of virus that can multiply. The effects of this event are unknown. It is possible that your immune system will killthe virus after it has been there for several days to weeks. Other brain tumor patients have received a very similar adenovirusand no ill effects were observed related to virus replication. Virus replication has not been detected in other cancer patients re-ceiving this type of adenoviral gene therapy.

c) Spread of the virus. Your immune system may not be functionally normally because of your disease and the medicationsyou are taking. Therefore, it is possible that the virus could spread from your body to your surroundings. The virus has been al-tered so that it can not reproduce by itself. If however, something should happen that the virus does spread and reproduce, itwould likely behave no differently from similar types of viruses that are already in the environment around us. Spread of thistype of virus has not been observed in prior patients treated with this type of virus. However, if there are signs that virus is be-ing spread, you may be asked to remain in the hospital longer than 2 weeks for further monitoring.

d) Changes in the body’s eggs or sperm. There is a remote possibility that the virus could change genes in eggs or sperm.Therefore, patients who are pregnant are not permitted to enter this study, and all patients in this study are advised to take pre-cautions not to bear children in the next few months following treatment.

e) Damage to brain cells. It is possible that the virus or the proteins it produces could injure normal brain cells. This could re-sult in temporary or permanent damage to normal brain cells.

f) Participation in multiple studies may be hazardous to you. If you are already participating in another research study, pleaseinform us fully. You should not participate in multiple studies, unless you and the investigators agree that your health and theoutcome of the study will not be jeopardized.

Withdrawing from the study

Your participation in this project is voluntary. You may refuse to participate in or withdraw form the study at any time withoutpenalty or loss of benefits to which you may otherwise be entitled. In case you decide to withdraw from the study you may suf-fer the following consequences. 1) Loss of the ability to monitor for side effects of gene therapy. 2) Failure to find out if thereare any unforeseen risks from gene therapy that may not be obvious without repeated evaluation. If you withdraw from the studyafter the gene is placed into your brain, it will not be possible to evaluate fully the safety of gene therapy. Should complicationsoccur, it will not be possible to detect them promptly and institute therapy. Should you decide to withdraw from this study, yourphysicians will still be available to provide you with the best possible care.

Loss of privacy:

You will not be identified in any reports on this study. The records will be kept confidential to the extent provided by federal,state and local law. The Federal Food and Drug Administration may inspect the records of this investigation. Gene therapy isvery interesting to the general public. Every effort will be made to maintain your privacy.

Alternatives to participation

Alternative treatments for your brain tumor may exist and may include standard surgery, chemotherapy, or implantations of ra-dioactive sources into the brain. Other experimental therapies may also be available. Your participation in this study is entirelyvoluntary and you can withdraw from the study at any time. A decision not to participate in the study will not jeopardize yourhealth care, and you will continue to be able to receive other therapies if they are available. The physicians involved in this studywill explain the other treatments available to you and their potential risks and benefits.

Costs to subject or insurance carrier resulting from participation in the study

There will be no cost to you for your participation in the study. Any costs resulting from care you require as part of the studywill be provided through the Clinical Research Center at the Hospital of the University of Pennsylvania (HUP) Medical Centeror special research funds made available to conduct this study. Medical care (including diagnostic test) that are part of the rou-tine care for patients with brain tumors (e.g., MRI scans, surgical resection of the tumor) will be billed to your insurance com-pany.

Payments to patients who participate in the study

Patients will not be paid for participating in this study.

Injury/Complications.

Complications may arise during this study either due to your illness or to the treatment of your illness. If there is a physical in-jury that results from research procedures, the University will provide medical treatment. Costs associated with such care will be


borne by either third party payors (your insurance company) or the Clinical Research Center at the Hospital of the University ofPennsylvania. Medical treatment will be provided in accordance with the determination by the University of its responsibility toprovide such treatment. However, the University does not provide compensation to a person who is injured while participatingas a subject in research.

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Significant new findings

Any significant new findings resulting from this study will be made known to you and your family. To find out more about anyaspect of this study you may contact the persons whose names addresses and telephone numbers appear below. If significant newknowledge is obtained during this study that may relate to your willingness to continue participation, you will be informed ofthis knowledge.

Stephen L. Eck, M.D., Ph.D. Jane Alavi, M.D.Assistant Professor Associate Professor215-898-4178 215-662-6319

Department of Medicine, Division of Hematology and Oncology16 Penn TowerThe Hospital of the University of Pennsylvania3400 Spruce StreetPhiladelphia, PA 19104

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Availability of further information.

If you wish further information regarding your rights as a research subject, you may contact the Office of Regulatory Affairs atthe University of Pennsylvania by telephoning (215) 898-2614. If significant new knowledge is obtained during this researchstudy that may relate to your willingness to continue participation, you will be informed of this knowledge. To find out moreabout any aspect of the study including your rights, you may contact the investigators.

Documentation of consent

One copy of this document will be kept together with our research records on this study. A second copy will be placed in yourhospital record. A third copy will be given to you to keep.

Voluntary consent of the subject. I have read the information given above or it has been read to me. I understand the meaningof the information. The investigator has satisfactorily answered my questions concerning the study. I hereby consent to partici-pate in the study. I will also have my next of kin sign a provisional consent to an autopsy to be used when I eventually die. Theconsent for an autopsy may be withdrawn at any time.

Names and signatures of consenting persons and witnesses.

My signature below means that I am freely giving my permission to participate in this study and that I have received a copy ofthis informed consent document.

___________________________________________________ _______________Subject’s signature Date

I have explained this consent form and the nature of the study to the patient. I have explained all possible risks and potentialbenefits although there may be additional unforeseen risks that I am unaware of, and have answered any questions asked by thesubject.

___________________________________________________ _______________Investigator’s signature Date

___________________________________________________ _______________Witness’ signature Date


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