Super-resolution track density imaging of glioblastoma: histopathologic correlation.

BACKGROUND AND PURPOSE: Super-resolution track density imaging generates anatomic images with submillimeter voxel resolution by using high-angular-resolution diffusion imaging and fiber-tractography. TDI within the diseased human brain has not been previously described. The purpose of this study was to correlate TDI with histopathologic features of GBM. MATERIALS AND METHODS: A total of 43 tumor specimens (24 contrast-enhancing, 12 NE, and 7 centrally necrotic regions) were collected from 18 patients with treatment-naïve GBM by use of MR imaging-guided neurosurgical techniques. Immunohistochemical stains were used to evaluate the following histopathologic features: hypoxia, architectural disruption, microvascular hyperplasia, and cellular proliferation. We reconstructed track density maps at a 0.25-mm isotropic spatial resolution by using probabilistic streamline tractography combined with constrained spheric deconvolution (model order, 8; 0.1-mm step size; 1 million seed points). Track density values were obtained from each tissue site. A P value of .05 was considered significant and was adjusted for multiple comparisons by use of the false discovery rate method. RESULTS: Track density was not significantly different between contrast-enhancing and NE regions but was more likely to be elevated within regions demonstrating aggressive histopathologic features (P < .05). Significant correlation between relative track density and hypoxia (odds ratio, 3.52; P = .01), architectural disruption (odds ratio, 3.49; P = .03), and cellular proliferation (odds ratio, 1.70; P = .05) was observed irrespective of the presence or absence of contrast enhancement. CONCLUSIONS: Numeric values of track density correlate with GBM biologic features and may be clinically useful for identification of regions of tumor infiltration within both enhancing and NE components of GBM.

PIKE mediates EGFR proliferative signaling in squamous cell carcinoma cells.

One of the key drivers for squamous cell carcinoma (SCC) proliferation is activation of the epidermal growth factor receptor (EGFR), a known proto-oncogene. However, the mechanism of EGFR-dependent SCC proliferation remains unclear. Our previous studies indicate that epidermal growth factor (EGF)-induced SCC cell proliferation requires the SH3 domain of phospholipase C-γ1 (PLC-γ1), but not its catalytic activity. The SH3 domain of PLC-γ1 is known to activate the short form of nuclear phosphatidylinositol 3-kinase enhancer (PIKE) that enhances the activity of nuclear class Ia phosphatidylinositol 3-kinase (PI3K) required for proliferation. However, PIKE has been described for more than a decade to be present exclusively in neuronal cells. In the present study, we found that PIKE was highly expressed in malignant human keratinocytes (SCC4 and SCC12B2) but had low expression in normal human keratinocytes. Immunohistochemical analysis showed strong nuclear staining of PIKE in human epidermal and tongue SCC specimens but little staining in the adjacent non-cancerous epithelium. Treatment of SCC4 cells with EGF-induced translocation of PLC-γ1 to the nucleus and binding of PLC-γ1 to the nuclear PIKE. Knockdown of PLC-γ1 or PIKE blocked EGF-induced activation of class Ia PI3K and protein kinase C-ζ and phosphorylation of nucleolin in the nucleus as well as EGF-induced SCC cell proliferation. However, inhibition of the catalytic activity of PLC-γ1 had little effect. These data suggest that PIKE has a critical role in EGF-induced SCC cell proliferation and may function as a proto-oncogene in SCC.

Response assessment in neuro-oncology (a report of the RANO group): assessment of outcome in trials of diffuse low-grade gliomas.

Although low-grade gliomas (LGG) have a less aggressive course than do high-grade gliomas, the outcome of these tumours is ultimately fatal in most patients. Both the tumour and its treatment can cause disabling morbidity, particularly of cognitive functions. Because many patients present with seizures only, with no other signs and symptoms, maintenance of quality of life and function constitutes a particular challenge in LGG. The slow growth pattern of most LGG, and the rare radiological true responses despite a favourable clinical response to treatment, interferes with the use of progression-free survival as the primary endpoint in trials. Overall survival as an endpoint brings logistical challenges, and is sensitive to other non-investigational salvage therapies. Clinical trials for LGG need to consider other measures of patient benefit such as cognition, symptom burden, and seizure activity, to establish whether improved survival is reflected in prolonged wellbeing. This Review investigates clinical and imaging endpoints in trials of LGG, and provides response assessment in neuro-oncology (RANO) criteria for non-enhancing tumours. Additionally, other measures for patients with brain tumours that assess outcome are described. Similar considerations are relevant for trials of high-grade gliomas, although for these tumours survival is shorter and survival endpoints generally have more value than they do for LGG.

Serum soluble CD44v6 levels in patients with oral and maxillofacial malignancy.

OBJECTIVE: To determine the levels of serum sCD44v6 in patients with oral cancer and evaluate the value of serum sCD44v6 in adjuvant diagnosis, staging and monitoring treatment response in these patients. MATERIALS AND METHODS: A total of 112 hospitalized patients with oral and maxillofacial malignancy and 28 healthy individuals were examined for serum sCD44v6 levels. Venous blood was collected from these patients and the healthy individuals. One week after treatment, venous blood was collected once again in 60 patients with oral and maxillofacial squamous cell carcinoma (OSCC). RESULTS: The sCD44v6 concentration was not significantly different between patients with oral and maxillofacial malignancy and control group (P > 0.05). The levels of serum sCD44v6 in patients with OSCC and salivary carcinoma showed no difference with those in control group (P > 0.05). The sCD44v6 level in patients with stage III and IV disease was higher than that of patients with stage I and II and that of the control group, but the difference was not significant (P > 0.05). Serum sCD44v6 levels in patients with OSCC after treatment became lower than that prevailed during pretreatment (P < 0.05). CONCLUSION: The possible roles of CD44v6 in the diagnosis of oral and maxillofacial malignancy deserve further elucidation and evaluation. Serum sCD44v6 may be a valuable marker in monitoring treatment response in patients with OSCC.

Analysis of metabolic indices in regions of abnormal perfusion in patients with high-grade glioma.

BACKGROUND AND PURPOSE: MR spectroscopic imaging (MRSI) and dynamic susceptibility-contrast MR imaging (DSC-MR imaging) are functional in vivo techniques for assessing tumor metabolism and vasculature characteristics. Because tumor hypoxia is influenced by tortuous, degraded, swollen, and angiogenic tumor vasculature, regions of abnormal perfusion parameters should coexist with changes in lactate and creatine metabolite levels. MATERIALS AND METHODS: DSC-MR imaging and lactate-edited MRSI were performed on 38 treatment-naive patients with high-grade gliomas (17 grade III, 21 grade IV) before surgical diagnosis. Regions of abnormal perfusion were determined from peak height and percent recovery maps for each voxel within the spectroscopic imaging volume. Choline, creatine, and lactate levels within voxels experiencing only abnormal peak height (aPH), only abnormal recovery (aRec), and both abnormal peak height and recovery (aPH+aRec) were determined and compared to the surrounding T2 hyperintensity (T2h) and normal-appearing white matter. RESULTS: There were decreasing trends in volume from aPH to aRec to aPH+aRec regions for both grade III and grade IV gliomas. Grade IV gliomas exhibited significantly elevated choline in all abnormal perfusion regions, with reduced creatine and increased lactate in the aRec region relative to the surrounding T2h. Grade III gliomas showed trends toward increased creatine within the aPH region and reduced levels within the aRec region. CONCLUSION: Depressed creatine and elevated lactate levels confirmed the lack of oxygenation within regions of compromised vascular integrity. Identification of regions with leaky or dense vasculature and metabolic markers of hypoxia and cellular proliferation could be useful in determining the more aggressive part of the tumor for targeting, monitoring, and assessing effects of treatment.

MicroPET-based pharmacokinetic analysis of the radiolabeled boron compound [18F]FBPA-F in rats with F98 glioma.

Boron neutron capture therapy (BNCT) is one of the effective methods of radiation therapy for the treatment of tumors such as malignant glioma. Boronophenylalanine ((10)B-BPA) solution has been used as a potential boron carrier for such a treatment. The aim of this study is to investigate 4-borono-2-[(18)F]-fluoro-l-phenylalanine-fructose ([(18)F]FBPA-F) in rats injected in the brain with glioma using in vivo small animal positron emission tomography (PET) imaging (microPET). Male Fischer 344 rats with F98 glioma in the left brain were used for these studies. Dynamic PET imaging of [(18)F]FBPA-F was performed on the 13th day after tumor inoculation. Arterial blood sampling was performed to obtain an input function for tracer kinetic modeling. The accumulation ratios of [(18)F]FBPA-F for the glioma-to-normal brain approached 3. The uptake characteristics of BPA-F and [(18)F]FBPA-F were similar. The results indicate that 4h after BPA-F injection would be the optimal irradiation time for BNCT. Rate constants were estimated using a three-compartment model. This study provides useful information for the clinical application of BNCT in patients with brain tumors.

EGFR overexpression and radiation response in glioblastoma multiforme.

PURPOSE: Recent studies have suggested relative radioresistance in glioblastoma multiforme (GM) tumors in older patients, consistent with their shorter survival. Two common molecular genetic abnormalities in GM are age related: epidermal growth factor receptor (EGFR) overexpression in older patients and p53 mutations in younger patients. We tested whether these abnormalities correlated with clinical heterogeneity in GM response to radiation treatment. METHODS AND MATERIALS: Radiographically assessed radiation response (5-level scale) was correlated with EGFR immunoreactivity, p53 immunoreactivity, and p53 exon 5-8 mutation status in 170 GM patients treated using 2 prospective clinical protocols. Spearman rank correlation and proportional-odds ordinal regression were used for univariate and multivariate analysis. RESULTS: Positive EGFR immunoreactivity predicted poor radiographically assessed radiation response (p = 0.046). Thirty-three percent of tumors with no EGFR immunoreactivity had good radiation responses (>50% reduction in tumor size by CT or MRI), compared to 18% of tumors with intermediate EGFR staining and 9% of tumors with strong staining. There was no significant relationship between p53 immunoreactivity or mutation status and radiation response. Significant relationships were noted between EGFR score and older age and between p53 score or mutation status and younger age. CONCLUSION: The observed relative radioresistance of some GMs is associated with overexpression of EGFR.

The influence of pH and cadmium sulfide on the photocatalytic degradation of 2-chlorophenol in titanium dioxide suspensions.

The influence of pH and cadmium sulfide on the photocatalytic degradation of 2-chlorophenol (2-CP) in titanium dioxide suspensions was investigated to evaluate the feasibility of mixed semiconductors on the photodegradation of chlorinated organics in aqueous solution. Apparent first-order rate constants (k(obs)) and initial rate constants were used to evaluate the degradation efficiency of 2-CP. Higher degradation efficiency of 2-CP was observed at higher pH values. The apparent pseudo-first-order rate constant was 0.036 min(-1) at pH 12.5 in TiO2/UV system, while a 2- to 9-fold decrease in k(obs) was observed over the pH range of 2.5-9.5. The addition of phosphate buffer solutions at different pH values have different effects on the degradation of 2-CP. H2PO4- has little effect on the photodegradation of 2-CP, while HPO4(2-) could inhibit the photodegradation efficiency of 2-CP. Chlorocatechol, hydroquinone, benzoquinone and phenol were identified as the predominant aromatic intermediates for the photocatalytic degradation of 2-CP. Moreover, less aromatic intermediates at higher pH were observed. Direct oxidation contributed significantly to the photodegradation of 2-CP. An addition of a semiconductor decreased the initial and apparent first-order rate constants of 2-CP. The cutoff of wavelength of 320nm could diminish the contribution of direct photolysis of 2-CP. The combination of cadmium sulfide and titanium dioxide can lead to an enhanced rate of disappearance of 2-CP compared to those in single semiconductor system. A 1.2 to 2.5-fold increase in rate constant in coupled semiconductor system relative to the single semiconductor system was obtained.

Preoperative proton MR spectroscopic imaging of brain tumors: correlation with histopathologic analysis of resection specimens.

BACKGROUND AND PURPOSE: Tumor progression is often difficult to distinguish from nonneoplastic treatment response on the basis of MR images alone. This study correlates metabolite levels measured by preoperative MR spectroscopic (MRS) imaging with histologic findings of biopsies, obtained during image-guided resections of brain mass lesions, to clarify the potential role of MRS in making this distinction. METHODS: Twenty-nine patients with brain tumors underwent high-resolution (0.2-1 cc) 3D proton MRS imaging and MR imaging before undergoing surgery; 11 had a newly diagnosed neoplasm, and 18 had recurrent disease. Surgical biopsies were obtained from locations referenced on MR images by guidance with a surgical navigation system. MR spectral voxels were retrospectively centered on each of 79 biopsy locations, and metabolite levels were correlated with histologic examination of each specimen. RESULTS: All mass lesions studied, whether attributable to tumor or noncancerous effects of previous therapy, showed abnormal MR spectra compared with normal parenchyma. When the pattern of MRS metabolites consisted of abnormally increased choline and decreased N-acetyl aspartate (NAA) resonances, histologic findings of the biopsy specimen invariably was positive for tumor. When choline and NAA resonances were below the normal range, histologic findings were variable, ranging from radiation necrosis, astrogliosis, and macrophage infiltration to mixed tissues that contained some low-, intermediate-, and high-grade tumor. CONCLUSION: This study demonstrated that 3D MRS imaging can identify regions of viable cancer, which may be valuable for guiding surgical biopsies and focal therapy. Regions manifesting abnormal MR spectra had a mixture of histologic findings, including astrogliosis, necrosis, and neoplasm.

A Phase II study of paclitaxel in patients with recurrent malignant glioma using different doses depending upon the concomitant use of anticonvulsants: a North American Brain Tumor Consortium report.

BACKGROUND: The primary objective of the current study was to determine the response rate of paclitaxel in patients with recurrent malignant glioma by using different doses dependent on the concomitant use of anticonvulsants. Secondary objectives were to determine the time period to treatment failure, to evaluate toxicities, and to obtain pharmacokinetic data. METHODS: Adult patients who had recurrent malignant glioma were treated with paclitaxel. Patients were treated at different doses depending on the concomitant use of anticonvulsants known to induce the p450 hepatic enzyme system. Patients on such agents were treated at a dose of 330 mg/m2, whereas those not on these anticonvulsants were treated at a dose of 210 mg/m2. Tumor response was assessed at 6-week intervals. Treatment was continued until documented tumor progression or unacceptable toxicity occurred, or a total of 12 paclitaxel infusions was completed. RESULTS: From January 1997 to June 1997, 23 patients were treated with paclitaxel. Four patients were ineligible for the current study. Of the 19 eligible patients, there were no responses seen. Four (21%) had stabilization of disease. Median time to treatment failure was 1 month (95% confidence interval [CI], 1-2 mos) and median survival was 7 months (95% CI, 6-10 mos). Three patients were removed from the current study because they had toxicity. Pharmacokinetic studies demonstrated that drug levels and clearance values were consistent with previously reported findings. CONCLUSION: Even though higher doses were administered to patients who had recurrent malignant glioma and who were on concomitant anticonvulsants, there were no objective responses to paclitaxel. Time to tumor progression was 1 month. Further testing of paclitaxel at this dose schedule does not appear to be warranted in this patient population.

Phase III trial of accelerated hyperfractionation with or without difluromethylornithine (DFMO) versus standard fractionated radiotherapy with or without DFMO for newly diagnosed patients with glioblastoma multiforme.

PURPOSE: To report the results of a prospective Phase III trial for patients with newly diagnosed glioblastoma multiforme (GBM), treated with either accelerated hyperfractionated irradiation with or without difluromethylornithine (DFMO) or standard fractionated irradiation with or without DFMO. METHODS AND MATERIALS: Adult patients with newly diagnosed GBM were registered and randomized following surgery to one of 4 treatment arms: Arm A, accelerated hyperfractionation alone using 2 fractions a day of 1.6 Gy to a total dose of 70.4 Gy in 44 fractions; Arm B, accelerated hyperfractionation as above plus DFMO 1.8 gm/m2 by mouth every 8 h beginning one week before radiation until the last fraction was given; Arm C, single-fraction irradiation of 1.8 Gy/day to 59.4 Gy; Arm D, single-fraction irradiation as in Arm C plus DFMO given as in Arm B. Patients were followed for progression-free survival (PFS) and overall survival (OS), as well as for toxicity. Eligibility required histologically proven GBM, age > or =18, Karnofsky performance status (KPS) > or =60, and no prior chemotherapy or radiotherapy. Adjuvant chemotherapy was not used in this protocol. RESULTS: A total of 231 eligible patients were enrolled. There were 95 men and 136 women with a median age of 57 years, and median KPS of 90. Extent of resection was total in 23, subtotal in 152, and biopsy only in 56 patients. The 4 arms were balanced with respect to age, KPS, and extent of resection. Times to event measurements are from date of diagnosis. Median OS and PFS were 40 and 19 weeks for Arm A; 42 and 22 weeks for Arm B; 37 and 16 weeks for Arm C; and 44 and 19 weeks for Arm D (p = 0.48 for survival; p = 0.32 for PFS). Comparison of the 2 arms treated with DFMO to the 2 arms without DFMO revealed no difference in OS (37 weeks vs. 42 weeks, p = 0.12) or PFS and thus no benefit to the use of DFMO. Comparison of the 2 standard fractionation arms to the 2 accelerated hyperfractionation arms also resulted in no difference in OS (42 weeks vs. 41 weeks, p = 0.75) or PFS, showing no benefit to accelerated hyperfractionated irradiation. CONCLUSION: In this prospective Phase III study, no survival or PFS benefit was seen with accelerated hyperfractionated irradiation to 70.4 Gy, nor was any benefit seen with DFMO as a radiosensitizer. Standard fractionated irradiation to 59.4 Gy remains the treatment of choice for newly diagnosed patients with glioblastoma multiforme.

Expression of functional luteinizing hormone (LH) receptor and its messenger ribonucleic acid in bovine endometrium: LH augmentation of cAMP and inositol phosphate in vitro and human chorionic gonadotropin (hCG) augmentation of peripheral prostaglandin in vivo.

Bovine endometrium contains LH/hCG binding sites and LH increases endometrial production of prostaglandin H synthase-2 (PGHS-2) and prostaglandin synthesis. This study showed that uterine endometrium contained both LH receptor mRNA transcript and a 93-kDa immunoreactive protein that bound to anti-rat LH receptor antibody. LH receptor and its mRNA were expressed maximally in the endometrium of cows from the luteal phase compared to the follicular phase of the estrous cycle. Furthermore, there was a response shown when incubation of endometrial minces from both pre-estrus/estrus and luteal phase (but not post-ovulatory phase) with LH or oxytocin (20 ng/ml) that resulted in a significant (p<0.02) increase in cAMP and total inositol phosphates. When Day 15 cows were injected i.v. with 3000 units hCG, the increase in peripheral 13,14-dihydro-15-keto PGF(2alpha) was 2.5-fold higher than saline controls or oxytocin. We conclude that LH stimulates endometrial cAMP and total inositol phosphates which results in increased formation of uterine PGHS-2 similar to LH effect on ovarian PGHS-2. The increased 13,14-dihydro-15-keto PGF(2alpha) production induced in vivo by injections of hCG indicates that LH may have a reinforcing role in luteolysis.

Age and radiation response in glioblastoma multiforme.

OBJECTIVE: Advanced age is a strong predictor of shorter survival in patients with glioblastoma multiforme (GM), especially for those who receive multimodality treatment. Radiographically assessed tumor response to external beam radiation therapy is an important prognostic factor in GM. We hypothesized that older GM patients might have more radioresistant tumors. METHODS: We studied radiographically assessed response to external beam radiation treatment (five-level scale) in relation to age and other prognostic factors in a cohort of 301 GM patients treated on two prospective clinical protocols. A total of 223 patients (74%) were assessable for radiographically assessed radiation response. A proportional odds ordinal regression model was used for univariate and multivariate analysis. RESULTS: Younger age (P = 0.006), higher Karnofsky Performance Scale score before radiotherapy (P = 0.027), and more extensive surgical resection (P = 0.028) predicted better radiation response in univariate analyses. Results were similar when clinical criteria were used to classify an additional 61 patients without radiographically assessed radiation response (stable versus progressive disease). In multivariate analyses, age and extent of resection were significant independent predictors of radiation response (P < 0.05); Karnofsky Performance Scale score was of borderline significance (P = 0.07). CONCLUSION: Older GM patients are less likely to have good responses to postoperative external beam radiation therapy. Karnofsky Performance Scale score before radiation treatment and extent of surgical resection are additional predictors of radiographically assessed radiation response in GM.

Clinical infection control in gene therapy: a multidisciplinary conference.

Gene therapy is being studied for the treatment of a variety of acquired and inherited disorders. Retroviruses, adenoviruses, poxviruses, adeno-associated viruses, herpesviruses, and others are being engineered to transfer genes into humans. Treatment protocols using recombinant viruses are being introduced into clinical settings. Infection control professionals will be involved in reviewing the safety of these agents in their clinics and hospitals. To date, only a limited number of articles have been written on infection control in gene therapy, and no widely available recommendations exist from federal or private organizations to guide infection control professionals. The goals of the conference were to provide a forum where gene therapy experts could share their perspectives and experience with infection control in gene therapy and to provide an opportunity for newcomers to the field to learn about issues specific to infection control in gene therapy. Recommendations for infection control in gene therapy were proposed.

Phase III randomized study of postradiotherapy chemotherapy with alpha-difluoromethylornithine-procarbazine, N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosurea, vincristine (DFMO-PCV) versus PCV for glioblastoma multiforme.

Although the efficacy of the nitrosourea-based combination chemotherapy procarbazine, N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosurea, and vincristine (PCV) has been previously demonstrated in the setting of anaplastic/intermediate-grade gliomas, the benefit for glioblastoma patients remains unproven. In the current study, we sought to determine whether the addition of alpha-difluoromethylornithine (eflornithine), an inhibitor of ornithine decarboxylase, which has shown encouraging results in the setting of recurrent glioma patients, to a nitrosourea-based therapy (PCV) would constitute a more effective adjuvant therapy in the treatment of glioblastoma multiforme patients in the postradiation therapy setting. Following conventional radiation therapy, 272 glioblastoma (GBM) patients were randomized to receive either alpha-difluoromethylornithine-PCV (DFMO-PCV; 134 patients) or PCV alone (138 patients), with survival and time to tumor progression being the primary endpoints. The starting dosage of DFMO was 3.0 g/m2 p.o. q8h for 14 days before and after treatment with N-(2-chloroethyl)-N-cyclohexyl-N-nitrosurea; PCV was administered as previously described1. Clinical and radiological (Gadolinium-enhanced MRI) follow-ups were nominally at the end of each 6 or 8 week cycle (PCV at 6 weeks; DFMO-PCV at 8 weeks). Laboratory evaluations for hematologic and other adverse effects were at 2 week intervals. There was no difference in median survival or median time-to-tumor progression between the two treatment groups, as measured from day of commencement of postradiotherapy chemotherapy [MS (months): DFMO-PCV, 10.5; Overall survival, as measured from time of tumor diagnosis at first surgery, was 13.3 and 14.2 months at the median and 6.2 and 8.7% at 5 years, respectively, for the DFMO-PCV and PCV arms. The treatment effect was unchanged after adjustment for age, performance status (KPS), extent of surgery, and other factors using the multivariate Cox proportional hazard model. Adverse effects associated with DFMO consisted of gastrointestinal (diarrhea nausea/vomiting), cytopenias, and minimal ototoxicity (limited to tinnitus) at the dose range tested. The addition of DFMO to the nitrosourea-based PCV regimen in this phase III study demonstrated no additional benefit in glioblastoma patients, underscoring the resistance of glioblastoma multiforme tumors to alkylating agents. For patients with anaplastic (intermediate grade) gliomas, in which the previously demonstrated benefit of post-radiation chemotherapy is more substantial, the evaluation of DFMO-PCV vs. PCV is still ongoing and hopefully will yield more encouraging results.

Generation of retroviral packaging and producer cell lines for large-scale vector production and clinical application: improved safety and high titer.

For many applications, human clinical therapies using retroviral vectors still require many technological improvements in key areas of vector design and production. These improvements include higher unprocessed manufacturing titers, complement-resistant vectors, and minimized potential to generate replication-competent retrovirus (RCR). To address these issues, we have developed a panel of human packaging cell lines (PCLs) with reduced homology between retroviral vector and packaging components. These reduced-homology PCLs allowed for the use of a novel high multiplicity of transduction ("high m.o. t.") method to introduce multiple copies of provector within vector-producing cell lines (VPCLs), resulting in high-titer vector without the generation of RCR. In a distinct approach to increase vector yields, we integrated manufacturing parameters into screening strategies and clone selection for large-scale vector production. Collectively, these improvements have resulted in the development of diverse VPCLs with unprocessed titers exceeding 2 x 10(7) CFU/ml. Using this technology, human Factor VIII VPCLs yielding titers as high as 2 x 10(8) CFU/ml unprocessed supernatant were generated. These cell lines produce complement-resistant vector particles (N. J. DePolo et al., J. Virol. 73: 6708-6714, 1999) and provide the basis for an ongoing Factor VIII gene therapy clinical trial.