¹Beatson Oncology Centre, Western Infirmary, Glasgow, UK

²Department of Neurosciences, Queen Elizabeth Hospital, Birmingham, UK

³Institute of Neurological Sciences, Department of Neurosurgery, Southern General Hospital, Glasgow, UK

⁴University of Glasgow, Department of Neuropathology, Institute of Neurological Sciences, Southern General Hospital, Glasgow, UK

⁵University of Glasgow, Division of Virology, Glasgow, UK

⁶University of Glasgow, Neurovirology Research Laboratories, Institute of Neurological Sciences, Southern General Hospital, Glasgow, UK

Correspondence to: Dr R Rampling, Beatson Oncology Centre, Western Infirmary, Glasgow G11 6NT, UK

The herpes simplex virus (HSV) ICP34.5 null mutant 1716 replicates selectively in actively dividing cells and has been proposed as a potential treatment for cancer, particularly brain tumours. We present a clinical study to evaluate the safety of 1716 in patients with relapsed malignant glioma. Following intratumoural inoculation of doses up to l0⁵ p.f.u., there was no induction of encephalitis, no adverse clinical symptoms, and no reactivation of latent HSV. Of nine patients treated, four are currently alive and well 14-24 months after 1716 administration. This study demonstrates the feasibility of using replication-competent HSV in human therapy. Gene Therapy (2000) 7, 859-866.

HSV1716; clinical trial; glioma therapy

Introduction

Glioblastoma remains one of the most formidable problems in cancer medicine. Following conventional therapy with surgery, radiotherapy¹ and chemotherapy² the median survival, following diagnosis, remains approximately 1 year. Progression following primary therapy is associated with short-term survival (average 5 months).³ There is an urgent need for novel treatments and it is accepted that new regimens can be explored soon after the diagnosis of relapse.^4,5,6

We have adopted a radical approach to brain tumour therapy using the selectively replication-competent mutant 1716⁷ of herpes simplex virus type 1 (HSV1) to treat recurrent glioma. HSV 1716 is a mutant that lacks both copies of the RL1 gene⁸ which encodes the protein ICP34.5,⁹ a specific determinant of virulence. The properties of 1716 have been extensively described. It is avirulent in the normal brains of animals.⁷ Its phenotype is cell type and state dependent. It replicates in actively dividing but not in terminally differentiated cells.¹⁰ It replicates in a range of tumour models and can eliminate metastatic brain tumours while failing to replicate in normal tissue.^11,12,13 The pathological change induced in normal mouse CNS is minimal.¹⁴ In human glioblastoma cells in vitro, lytic replication of 1716 results in cell death.¹⁵

We have shown that ICP34.5 functions by complexing with proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication and repair.¹⁶ In so doing an environment is provided in which the virus can replicate. In tumour cells, where functional PCNA levels are high, ICP34.5 is not required for productive HSV replication whereas in neurons in which PCNA levels are low, ICP34.5 is an absolute requirement for the production of infectious progeny virus. In a subset of cell types, preclusion of host cell protein synthesis shut off has been shown to be a function of ICP34.5.^17,18 The host cell protein synthesis shut off induced by removal of ICP34.5 can be restored by altering the control of expression of the US 11 gene product.^19,20 However, the virulence phenotype conferred by expression of ICP34.5 is separable from the host cell protein shut off response.¹⁹ Therefore, deregulation of US 11 will not restore a virulence phenotype to 1716 and should not pose a safety issue.

We concluded that 1716 had reached the stage where a study to test its safety in patients was justified. In this report, we evaluate the toxicity of 1716 in patients with recurrent high grade glioma.

Most early gene therapy trials have used strategies adapted from the development of conventional cytotoxic chemical agents. These assume that a safe starting dose can be derived from animal experiments and depend upon the use of dose escalation schemes, (modified Fibonacci), to obligatory toxicity end-points. Such a design might not be appropriate for replication-competent HSV. In brain tissue ICP34.5 is an absolute requirement for HSV virulence. In model systems, deletion of the gene encoding ICP34.5 totally abrogates virulence and the mutated virus fails to cause encephalitis, no matter how high the input dose.⁷ HSV 1716 does not contain the gene and hence ICP34.5 is never expressed, irrespective of the dose. The question being asked in this study is whether the avirulent phenotype of 1716 demonstrated in model systems, pertains in the context of human therapy up to a dose at which therapeutic activity is a possibility.

We wished to show that a safe starting dose exists when 1716 is injected directly into recurrent malignant glioma. A starting dose of 10³ infectious particles (p.f.u.) was calculated to be approximately equivalent (in p.f.u. per kilogram of brain) to the dose of wild-type HSV which has been shown to cause a fatal encephalitis in mice.^7,21 With HSV Glasgow strain 17 (the wild-type parental virus of 1716), 2 p.f.u. will kill a mouse within 5 days when injected directly into the brain.⁷

As 1716 has the potential to replicate in tumours, the final titre within the brain could be several orders of magnitude higher than the inoculated dose. Extrapolating from in vitro data, 20% of infectious particles could result in a productive infection and each infected cell could give a burst size of 100 p.f.u. within a single infectious cycle. An input titre of 10⁵ p.f.u. could produce 2 ´ 10⁶ p.f.u. within 12 h.²² It was estimated that 10⁵ p.f.u., delivered under optimal conditions, could achieve a detectable level of tumour kill in some patients in a future activity study. This was the maximum dose allowed by the UK Gene Therapy Advisory Committee, (GTAC) in this study.

GTAC agreed that when safety at these dose levels had been demonstrated, permission could be sought for a separate trial to examine an activity end-point. At this stage further dose escalation could be investigated, if required. This developmental strategy is in line with the recent National Cancer Institute/European Organisation for Research and Treatment of Cancer (NCI/EORTC) workshop on phase 1 drug development, whose recommendations pointed out the need for 'new (biological) end-points for new modalities which may produce nonspecific and sporadic toxicities which are not clearly dose related'.^23,24 That severe toxicity can arise when viral treatments are extended to obligatory toxicity end-points is demonstrated by a recent report of a toxic death in a phase I study using adenovirus.²⁵ It adds further justification to GTAC's cautious approach.

Results

Patient profiles

Nine patients were treated within the protocol (Table 1). They were an unremarkable cross-section of high grade glioma patients who had relapsed following radical treatment. All had previously undergone surgery and radical radiotherapy. Two patients had received tirapazamine chemotherapy concomitant with radiotherapy and six patients had received nitrosourea chemotherapy at relapse. Patient 5 underwent surgery and repeat irradiation at his second relapse. All patients were receiving dexamethasone (4-16 mg daily) and five were taking anticonvulsants. Haematology, renal and hepatic function, chest X-ray and ECG were within study limits. Eight patients were seropositive for HSV1; only patient 4 was seronegative. He did not seroconvert. The IgG and IgM titres did not change significantly after 1716 administration in any patient (data not shown).

Eight patients had glioblastomas and one had an anaplastic astrocytoma. Tumour size is reported as the volume of enhancing tumour on gadolinium enhanced MRI (Gd MRI) (Table 2). Size varied between 8.6 and 129 ml at the time of injection. (The volume of brain involved with tumour necrosis, tumour-associated oedema and non-enhancing tumour is larger than this figure.) Also recorded are volumes measured from thallium SPECT scanning. These are larger, but are also thought to relate to the volume of actively growing tumour cells.²⁶

The patients' immune status at the time of injection is shown in Table 3. Total white cell counts (not shown) were normal in all patients. Lymphopenia was detected in all patients, most marked in patients 3 and 8. B cells were almost undetectable in four patients (Nos 3, 5, 6 and 8). T cell numbers tended to be around the lower end of the normal range with particular reductions in the CD4⁺ subset in patients 3 and 5. Cellular proliferative responses were reduced in all patients except 2 and 7 when compared with normal healthy controls. The phenotypic cytometric data together with the proliferative functional results indicate a significant degree of immuno-incompetence in this group of patients.

Administration of 1716 and clinical outcome

Nine patients were treated. Three each received 10³, 10⁴ and 10⁵ p.f.u. of HSV 1716 by stereotactic injection directly into the tumour. The procedure was well tolerated with no immediate post-operative complications. All patients recovered to their preoperative state within 24 h. A summary of events in the first week is given in Table 4. Patients 3 and 6 were both prone to regular seizures and those experienced after 1716 were typical and required no additional action. Patient 4 (who was seronegative for HSV1) experienced a short-lived, self-limiting pyrexia for which no cause was found. The worsening condition in patient 8 was due to post-operative swelling and responded promptly to an increase in steroid dose. The MRIs performed 4-6 days after injection showed no changes suggestive of active infection in any patient. The tumour volumes measured on Gd MRI at this point were similar or slightly greater, as might be expected following surgery (Table 2). None satisfied Macdonald's criteria for progression.²⁷ One of the thallium SPECT scans showed an increase in volume; the remainder were smaller. The significance of this observation is doubtful.

Patients were seen weekly until week 5. No adverse clinical effects and no marked improvements which could have been attributed to the administration of 1716 were recorded. The Gd MRI volumes were measured at this point (4-9 weeks). Three satisfied the Macdonald criteria for progression.²⁷ One patient (No. 4), although clinically unchanged, requested resective surgery before his week 5 scan could be performed. The remaining five MRI were stable. The thallium SPECT volumes were smaller in one, stable in two, larger in five and not assessable in one. The HMPAO SPECT scans showed no evidence of hyperperfusion to suggest encephalitis in any patient.

Buccal swabs and serum were taken on days 2 and 6 and weekly for 4 weeks after 1716 infection and assayed for HSV. There was no evidence of HSV shedding, either 1716 or reactivated endogenous latent HSV, in any patient. No patient experienced reactivation or recrudescence of HSV in the form of skin lesions.

After completion of the formal study period, patients were followed with visits at approximately monthly intervals. The condition of all patients at the end of November 1999 is given in Table 4.

Five patients have died. Patient 2 underwent further decompression of tumour at 12 weeks after injection and remained well for more than 6 months before dying with recurrent tumour at 9 months after injection. Patients 3, 5 and 6 enjoyed short periods of clinical stability (2-5 months) before dying with clinical and image documented tumour progression. Patient 9 was well following her procedure and returned to an independent life at home. She developed a sudden onset, fulminating bacterial pneumonia and septicaemia (Listeria and E. coli) with total organ failure from which she died at 2 months. Post-mortem analysis of her tissues showed no evidence of HSV encephalitis or systemic HSV infection.

Four patients remain alive. Patient 1 has had no further antitumour treatment and is alive and well at 24 months after 1716 injection on a replacement dose of steroid. Patients 4 and 7 are both currently well and in complete remission (19 and 17 months after 1716 respectively) following further tumour decompressions at 3½ weeks and 2 months after injection, respectively. Patient 7 received, in addition, five courses of lomustine immediately following resective surgery. Patient 8 is clinically and radiologically stable at l4 months after 1716 administration. She is currently receiving chemotherapy with a single alkylating agent.

Further CT, MRI and thallium and HMPOA SPECT scans have been performed according to clinical need in all patients. On no occasion have there been changes which would suggest an encephalitis.

Evaluation of biopsy and post-mortem material

Post-inoculation tissue was available from patients 2, 4 and 7 who underwent a subsequent tumour resection at 3 months, 3½ weeks and 2 months, respectively, and from patients 5 and 9 who died at 6 and 2 months, respectively, after inoculation. Histological examination of all five cases showed high grade glial tumour with no unusual features. There was no significant immunoreactivity of tumour cells or adjacent brain tissue for HSV1 using two monoclonal antibodies (Z1F11 and Novocastra HSV1). In addition, there was no evidence of 1716 or wild-type genomes by PCR. In both of the autopsied patients the location of the site of 1716 injection was identified as a cyst on the post-injection CT scan. The cysts (approximately 1 cm diameter) had persisted from the time of injection until the autopsy. There was no evidence of encephalitis involving 1716 or wild-type HSV in peritumoral brain or in limbic structures by histology, immunocytochemistry, culture or PCR analysis. Tissue cultures for virus were negative.

Discussion

Most 'gene therapy' studies for brain tumours have involved genetically modified, replication-deficient viruses expressing a transgene capable of delivering a tumour killing product. We have taken the alternative approach of using a selectively replication-competent HSV. The high proliferative activity in the tumour against a background of quiescent normal cells and the lack of systemic spread of gliomas make them good candidates for selective killing by such a virus.

Herpes simplex virus is neurotropic and introduction directly into the brain inherently carries the risk of major toxicity. A prerequisite for its development for human therapy is a demonstration of the avirulent phenotype of 1716 in its natural human host. Thus, a limited study was designed solely to look for evidence of toxicity when 1716 was injected directly into brain tumours in patients. In particular, no patient developed HSV encephalitis and no patient required the anti-herpes drug aciclovir. As a group they have lived at least as long as would be expected for a population of this type.³ Four surviving patients all received 1716 over 1 year ago.

Contrary to their previous findings²⁸ Kesari et al showed that 1716 can replicate in ependymal cells²⁹ and that intraventricular injection of high doses in nude mice is followed by death in some animals.³⁰ They concluded that these studies should serve as a warning for the use of 1716 in immunosuppressed humans. The patients in the present study were immunocompromised by previous antitumour therapy, contemporary steroids, and disease effects. However, none showed evidence of replication of 1716 in normal brain. Moreover, one was seronegative for HSV and eight seropositive and there was no difference in the response to 1716 inoculation. The seronegative patient did not seroconvert and the seropositive patients did not demonstrate any elevation of either IgG or IgM antibody titres after 1716 administration. No patient demonstrated reactivation, recrudescence or shedding of endogenous latent HSV or shedding of 1716.

Where examination of brain tissue (tumour and adjacent brain) has been possible following either tumour resection or post-mortem, no evidence of encephalitis or ongoing lytic replication was found. Immunohistochemistry using two HSV-specific antibodies failed to show immunoreactivity in either tumour or normal brain. Similarly, PCR analysis failed to detect any HSV genomes. It may be that there were no 1716 genomes in the tissue and no residual HSV antigen. As we were unable to analyse material any earlier than 3½ weeks after injection, this would not be surprising. Alternatively, it may represent a sampling problem in that the injected tumours were large and only a proportion of the cells would be actively dividing and thus able to support 1716 replication. Without the expression of a marker gene (eg -gal) it is impossible to detect virus migration and select infected tissue samples. A less likely explanation is that the assays used were not sensitive enough to detect either HSV DNA or protein at levels present in the samples.

Without doubt this study demonstrates that 1716 can be delivered intratumorally to patients with growing intracranial neoplasms to doses of l0⁵ p.f.u. without detriment to their health. Experiments in animals have shown that selective replication-competent viruses at doses of 10⁵ p.f.u. are most effective when applied to small tumours^11,12,31 but may fail to control bulky tumours. It is unlikely that obvious antitumour effects such as size reduction, will be apparent in large heterogeneous tumours using a single inoculation introduced by a simple delivery system to one site when evaluation methods such as MRI and SPECT scans are used. The present study was designed to test the hypothesis that 1716 could be introduced into the human brain without inducing encephalitis. It was not designed to test efficacy. This report provides the first evidence in support of the safety of 1716 for human use, at least in this form of cancer, and justifies further clinical research to test activity. Proposed studies will take into consideration stage of disease at which to introduce virus, mode of delivery, dose of virus and methodology required critically to assess efficacy. The animal evidence suggests that for glioma the most likely use of selectively replication-competent HSV will be as part of a combination therapy regimen in minimal residual disease.

Concurrently with our study, another clinical study has been started in glioma patients using the HSV1 strain F mutant G207 which has mutations in both ribonucleotide reductase (RR) and ICP34.5.³² As the mutation in RR also impairs virulence of HSV although not abolishing it, use of this double mutant provides a further safety dimension. However, the replication competence of G207 in tumour cells is markedly reduced compared with that of 1716 and hence to achieve tumour cell killing, substantially higher doses will be required. Consequently doses of G207 are being given which are several orders of magnitude higher (eg 10⁹ p.f.u.) than those of 1716.

Patients and methods

Virus production

A low passage laboratory grade stock of 1716⁷ was the starting material for production of GMP grade virus for human use. Production of 1716 in BHK-21/C13 cells from a Master Seed Cell Bank under cGMP conditions was carried out by Q-One Biotech Ltd, Glasgow, UK. The final clinical grade virus preparation was titrated for infectivity, tested for contamination and stored at -70°C in identical pairs of vials. Each vial contained 1716 at the appropriate concentration (10³-10⁵ p.f.u.) in 1 ml total volume of human serum albumen, HSA 0.5-1.0% in isotonic PBS. One week before the intended injection date, the titre of virus in one of the paired vials was confirmed on BHK21/C13 cells.

Titration of 1716 was carried out by standard methods on BHK21/C13 cells.³³ Viral titres were expressed as concentration of p.f.u. in comparison to the TCID₅₀ values obtained from Q-One Biotech.

Trial procedure

The study protocol was approved by the UK Gene Therapy Advisory Committee (GTAC) and the Medicines Control Agency (MCA), the Glasgow Southern General Hospital Research Ethics Committee and the Health and Safety Executive Committee.

Patient population

Patients were considered for study if they had biopsy proven high grade glioma, previously treated with surgery and radiotherapy and had exhausted all other conventional treatments at relapse. They must have had no chemotherapy or radiotherapy within 6 weeks. Proof of recurrence was on the basis of deteriorating clinical symptoms and unequivocal worsening in two successive scans of the same type (CT or MRI) with further confirmation using thallium SPECT. Subjects were aged between 18 and 70 with Karnofsky status >60 and a life expectancy >8 weeks. Their haematological, renal, hepatic and coagulation functions were within normal limits for our laboratory and their general condition satisfactory.

Trial subjects were required to be neurologically stable on an appropriate dose of steroid and able to give informed written consent. Consent was given by both the patient and a chosen relative or carer to an independent physician who was not otherwise concerned with the design or immediate conduct of the study.

Patients considered for the study underwent a comprehensive work-up including general clinical, and neurological evaluation, microbiological, immunological, haematological and biochemical assessments. Neuroradiology included CT scanning, gadolinium-enhanced MRI and thallium-201 and Tc^99mHMPOA SPECT scans.

Administration of 1716

Three patients were treated at each dose level (10³, 10⁴, 10⁵ p.f.u.). The study was to be terminated if any patient experienced symptoms or signs of HSV encephalitis or if any grade 3 or 4 toxicity was encountered. Stereotactic injection of 1716 was carried out using a specially designed inner needle with a curved distal end that was passed down a standard sedan outer biopsy needle. The curve allowed the needle to penetrate laterally into the tissues. The extent of penetration into the tissue was controlled by a stop set on the needle. The procedure was carried out in a designated CT imaging suite. The patients underwent premedication and routine anaesthesia with alfentanyl and propofol. The recurrent tumour was localised using a 'Lecksell' stereotactic frame and an initial biopsy was taken. An immediate cytological smear was prepared and reported within 10 min to confirm positioning of the needle within active tumour. Following confirmation, the biopsy trocar was replaced by the delivery needle and multiple radial injections (6-10) at different levels within the tumour were performed as the probe was withdrawn in a stepwise fashion after a single pass. Between 5 and 10 aliquots of virus to a total of 1 ml were delivered into the tumour. An immediate post-operative CT scan without contrast was taken to ensure no internal haemorrhage and to confirm delivery of the virus within the tumour.

Patient evaluation

Patients were monitored in a high dependency unit for 2-3 h and then in an isolated room in a routine ward. During the 6 day inpatient stay, patients were assessed every 4 h for blood pressure, heart rate, peripheral O₂ saturation, Glasgow Coma Scale and focal neurological features. Neurological and general physical examinations were performed twice daily. Haematological, biochemical and immunological status were monitored routinely. Full protocol neuroradiology was repeated on day 6. Additional scanning was performed only if there was a deterioration in neurological status. Evidence of HSV encephalitis was sought through detailed assessment of neurological condition and vital signs. Other toxicities were routinely assessed and judged according to the NCI Common Toxicity Criteria (CTC) checklist. An anti-HSV protocol based on the delivery of aciclovir was available in event of concern over encephalitis. Patients were discharged on day 7 provided they remained well. They were followed weekly for 4 weeks at which times clinical, haematological, biochemical, and virological assessments were performed. At the end of week 5, a full radiological assessment was performed with MRI and SPECT. Tumour response was monitored using the patient's clinical condition and radiological response by volumetric analysis and reported according to conventional criteria.²⁷

Investigations on patient samples

Immunological investigations: A routine nephalometric (Behring BNA II, Milton Keynes, UK) assay was used to measure the immunoglobulins IgA, G and M. Lymphocyte populations and subsets were measured in whole blood incubated with dual-labelled (fluorescein and phycoerythrin) monoclonal antibodies. Lysed specimens were analysed on a Becton Dickinson FACScan.

Lymphocyte function was measured in a whole blood stimulation assay using serial doses of phytohaemagglutinin, concanavalin A and pokeweed mitogen. Stimulated cells were pulse labelled with tritiated thymidine for 4 h and counted by liquid scintillation counting. Results are expressed as stimulation index (SI) - the ratio of the stimulated counts to the nonstimulated count at any dose of mitogen. Each assay was controlled using whole blood from volunteers. Normal ranges are not available. Stimulation is unequivocally reduced if the SI is less than 10. Values above 10 are difficult to interpret on individual samples and are best compared directly with counts obtained from a healthy control run in parallel. It is also important to note that where lymphocyte numbers are low the accuracy of this assay declines.

ELISA for HSV-1 IgG and IgM was carried out using a Virotech HSV-1-specific kit. Values are calculated for IgG and IgM levels in Virotech (Russelsheim, Germany) enzyme (VE) units where >11 VE units is classified as positive.

Immunohistochemistry for HSV was performed on paraffin sections using monoclonal antibodies recognising the HSV1 65K DNA binding protein (Z1F11, a gift from Howard Marsden) and an unspecified epitope of HSV1 strain Stoker (Novocastra, Newcastle upon Tyne, UK). Sections were pre-treated by microwaving for antigen retrieval. The primary antibodies were titrated using sections from wild-type HSV1 encephalitis and were both subsequently used at a concentration of 1:1000. Bound antibody was visualised using a multilink kit (Menarini, Wokingham, UK) and diaminobenzidine. Sections from wild-type HSV1 encephalitis were used as positive controls. Omission of primary antibody constituted the negative control.

PCR assay: DNA was extracted from biopsy and post mortem samples using a Nucleon Biosciences ST (Coatbridge, UK) (soft tissue) Genomic DNA Extraction Kit. The DNA pellet was resuspended in TE buffer and the concentration measured by agarose gel electrophoresis and spectrophotometry before being used for the PCR assay. DNA extracted from BHK cells infected with strain 17 or 1716 was used as controls. On the assumption of a burst size of 100 p.f.u. per cell and a particle:p.f.u. ratio of 100:1, the level of sensitivity was 4 ´ 10⁵ HSV genomes/ml of total DNA as estimated from reconstruction experiments using DNA from infected BHK cells. (Using diagnostic primers which amplify gB of HSV1 and 2,³⁴ the level of sensitivity was the same.)

The primers chosen to distinguish between HSV1 strain 17 and the mutant 1716 were F1 (20-mer) and R2 (18-mer). Sequences are F1 (CAG GCA CGG CCC GAT GAC CG) and R2 (CTT TAA AGC GGT GGC GGC). Primer F1 is homologous to the region 125170-125189 of the HSV-1 genome. Primer R2 is complementary to the region 125993-125976 of the same genome. These primers amplified a 64 bp fragment which was diagnostic for 1716 and absent from wild-type 17. Primers were supplied by Genosys (Cambridge, UK) and made-up as 1 nM solutions.

PCR reagents used per reaction were 1.5 l 10 mM dNTPs, 5 l Pfx Amplification buffer; 0.5 l Platinum Pfx DNA polymerase (Gibco BRL, Paisley, UK); l l 50 mM Mg₂SO₄; 1 l each of a 0.1 nM stock of primers F1 and R2 and 2 l DNA template, made up with H₂O to 50 l total volume.

PCR conditions were 94°C for 2 min; 30 cycles ´ {94°C for 15 s; 72°C for 2 min; 72°C for 2 min}; 72°C for 10 min; store at 4°C.

Assay for infectious HSV in patient samples: This was carried out by adding the growth medium in which swabs were placed to monolayers of BHK21/C13 cells which were then examined over 7 days for evidence of cytopathic effect and/or HSV plaques. Serum samples were assayed similarly.

Tumour volume measurement using MRI

MRI data sets were acquired on a 1.5 Tesla Siemans Magnetom. Tumour enhancement volumes were measured using a threshold method applied to co-registered, pre- and post-contrast, 3D, T1-weighted MR data sets. Subtraction of these sets resulted in images showing only enhancement. The background noise in the subtraction images was estimated using regions of interest (ROI) from the opposite hemisphere to the neoplasm. The lower value of the threshold was set to 2.5 standard deviations above this average noise level. A large ROI was drawn around the general region of the tumour to exclude other enhancing structures, such as veins, and all voxels within this ROI that were above the previously set threshold were counted. As the voxel size is known the volume of enhancement was calculated by multiplying the number of enhancing voxels by the number of partitions which showed them, to give an overall enhancing tumour volume.

Tumour volume measurement using thallium SPECT

Since thallium SPECT is less affected than MRI by non-neoplastic processes it may be considered a more specific indicator of tumour growth. A semi-quantitative measurement of tumour volume was obtained using a slice by slice ROI area measurement technique (volume = area ´ slice spacing). Tumour edges were defined using a thresholding technique. Normal brain uptake was assessed in the contralateral hemisphere in at least three slices at the same anatomical level as the tumour. A threshold level was empirically determined as being 1.7 ´ normal brain uptake. All areas in the brain with an uptake exceeding the threshold level were outlined. The total area above threshold was calculated for each slice, excluding areas considered to represent 'normal' high uptake regions, eg choroid plexus.

Acknowledgements

This work was supported by the UK Medical Research Council project grant No. G9539438N to RR, GC and MB. GMP grade 1716 was generously supplied without charge from Q1 Biotech, Glasgow and we are indebted to David Onions and Gillian Lees for their support. We are grateful to Howard Marsden, MRC Virology Unit, Glasgow who kindly supplied the ZlF11 antibody. Dr A Farrell and Mr E Galloway gave invaluable help and advise with respect to the immunology measurements. We also thank the GTAC committee and the MCA for allowing this study to proceed.

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Table 1 Patient characteristics prior to their recruitment into the study

Table 2 Tumour volumes at three time-points during the study period

Table 3 Routine immunology profiles for all study patients at the time of injection with HIV 1716

Table 4 The condition of the patients during the study and at November 1999