John T. Lam

Enhanced therapeutic efficacy for ovarian cancer with a serotype 3 receptor-targeted oncolytic adenovirus

Oncolytic viruses that are replication competent in tumor but not in normal cells represent a novel approach for treating neoplastic diseases. However, the oncolytic potency of replicating agents is determined directly by their capability of infecting target cells. Most adenoviruses used for gene therapy or virotherapy have been based on serotype 5 (Ad5). Unfortunately, expression of the primary receptor for Ad5 (the coxsackie-adenovirus receptor, or CAR) is highly variable on ovarian and other cancer cells. By performing genetic fiber pseudotyping, we created Ad5/3-Delta24, a conditionally replicating adenovirus that does not bind CAR but facilitates entry into and killing of ovarian cancer cells. We show replication of Ad5/3-Delta24 and subsequent oncolysis of ovarian adenocarcinoma lines. Replication was also analyzed with quantitative PCR on three-dimensional primary tumor cell spheroids purified from patient samples. Moreover, in a therapeutic orthotopic model of peritoneal carcinomatosis, dramatically enhanced survival was noted. Finally, Ad5/3-Delta24 achieved a significant antitumor effect as assessed by noninvasive, in vivo bioluminescence imaging. Therefore, the preclinical therapeutic efficacy of Ad5/3-Delta24 is improved over the respective CAR- and integrin-binding controls. Taken together with promising biodistribution and toxicity data, this approach could translate into successful clinical interventions for ovarian cancer patients.

Human Immunodeficiency Virus Type 1-Mediated Syncytium Formation Is Compatible with Adenovirus Replication and Facilitates Efficient Dispersion of Viral Gene Products and De Novo-Synthesized Virus Particles

Conditionally replicative adenovirus (CRAd) vectors are designed for specific oncolytic replication in tumor tissues with concomitant sparing of normal cells. As such, CRAds offer an unprecedented level of anticancer potential for malignancies that have been refractory to previous cancer gene therapy interventions. CRAd efficacy may, however, be compromised by inefficient dispersion of the replicating vector within the tumor tissue. To address this issue, we evaluated the utility of a fusogenic membrane glycoprotein (FMG), which induces the fusion of neighboring cellular membranes to form multinucleated syncytia. We hypothesized that the FMG-mediated syncytia would facilitate dispersion of the adenovirus (Ad) gene products and viral progeny. To test this, human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, which induce syncytia in the presence of CD4+ target cells, were expressed by an Ad (Ad5HIVenv) in permissive (CD4-positive) and nonpermissive (CD4-negative) cell lines. After validating this Ad-FMG model, the efficiency of Ad replication in the presence or absence of syncytia was evaluated. The results demonstrated that syncytium formation was compatible with Ad replication and dramatically increased the dispersion of virus gene products within the cytoplasm of the syncytia as well as viral particles in the nuclei of the syncytial mass. Moreover, progeny virions were released more efficiently from syncytia compared with nonsyncytial cells. These data demonstrate the utility of FMGs as a dispersion agent and suggest that FMGs can improve the efficacy of CRAd gene therapy.

A cyclooxygenase-2 promoter-based conditionally replicating adenovirus with enhanced infectivity for treatment of ovarian adenocarcinoma.

Conditionally replicating adenoviruses (CRADs) take advantage of tumor-specific characteristics for preferential replication and subsequent oncolysis of cancer cells. The antitumor effect is determined by the capability to infect tumor cells. Here, we used RGDCRADcox-2R, which features the cyclooxygenase-2 promoter for replication control and an integrin binding RGD-4C motif for enhanced infectivity of ovarian cancer cells. RGDCRADcox-2R replicated in and killed human ovarian cancer cells effectively, while the replication in nonmalignant cells was low. Importantly, the therapeutic efficacy, as evaluated in an orthotopic model of peritoneally disseminated ovarian cancer, was significantly improved and toxicity was lower than with a wild-type virus. Thus, this CRAD could be tested for treatment of ovarian cancer in humans.

Preclinical evaluation of a class of infectivity-enhanced adenoviral vectors in ovarian cancer gene therapy

Ovarian carcinoma cells are often infected inefficiently by adenoviruses (Ad) due to low expression of coxsackie–adenovirus receptors (CAR), hindering the application of adenovirus-mediated gene therapy in ovarian cancer. In this study, we explored a class of infectivity-enhanced Ad vectors, which contain CAR-independent targeting motifs RGD (Ad5.RGD), polylysine (Ad5.pK7), or both (Ad5.RGD.pK7), for their utility in ovarian cancer gene therapy using in vitro and in vivo model systems. We found that these vectors infected established ovarian carcinoma cell lines and primary ovarian cancer cells with significantly enhanced infectivity. Among them, Ad5.RGD.pK7 appeared to be most efficient. Further, we evaluated their gene delivery efficiency using two different ovarian cancer mouse models – subcutaneous and intraperitoneal human ovarian cancer xenografts. All of the modified vectors appeared to be more efficient than the unmodified Ad5 vector in both models, although some of the differences are not statistically significant. Of these, Ad5.RGD.pK7 exhibited the highest efficacy in the subcutaneous tumor model, while Ad5.pK7 worked most efficiently in the intraperitoneal tumor model. These preclinical results suggest that Ad5.RGD.pK7 and Ad5.pK7 may be very useful in ovarian cancer gene therapy.

Replication of an integrin targeted conditionally replicating adenovirus on primary ovarian cancer spheroids

Replication competent viruses hold promise for treatment of advanced cancers resistant to available therapeutic modalities. Although preliminary clinical results have substantiated their efficacy, preclinical development of these novel approaches is limited by assay substrates. The evaluation of candidate agents could be confounded by differences between primary tumor cells and tumor cell lines, as discordance in the levels of surface receptors relevant for viral entry has been reported. Since primary tumor cells are difficult to analyze ex vivo for longitudinal observation of virus replication, we developed three-dimensional aggregates or spheroids of unpassaged and purified ovarian cancer cells as a means for prolonging primary tumor cell viability and as a three-dimensional in vitro model for replicative viral infection. Ovarian cancer cells purified from ascites samples were sustained for 30 days while retaining the infection profile with tropism modified and unmodified adenoviruses (Ads). Cell line and primary cell spheroids were used to quantitate the replication and oncolytic potency of replicative Ads in preclinical testing for human ovarian cancer trials. Therefore, spheroids provide a method to sustain purified unpassaged primary ovarian cancer cells for extended periods and to allow evaluation of replicative viruses in a three-dimensional model.

Utilization, Reliability, and Clinical Impact of Point-of-Care Testing during Critical Care Transport: Six Years of Experience

The use of point-of-care testing (POCT) has been reported in the setting of critical care transport (1)(2)(3)(4)(5)(6), although the overall benefits have not been evaluated in depth. In addition, problems with testing reliability may be uncovered only after an extended period of field use. This report describes the use of POCT by our critical care transport program over 6 years. All critical care transports made from January 1996 to December 2001 were reviewed. Transport vehicles were ambulances or twin-engine jets. The transport teams consisted of a physician on transport or with radio contact, a respiratory therapist, and a registered nurse. All transports were equipped with i-STAT® portable analyzers (i-STAT® Corporation) and disposable cartridges for testing. The analyzer and cartridges were stored in an insulated bag for temperature control during the trip. The analytical performance verification protocol (electronic controls) recommended in the i-STAT System Manual was followed before each patient test. Liquid controls were run monthly. Proficiency testing was completed in accordance with the requirements of the College of American Pathologists. The manufacturer’s test cartridges were the G3, 6+, EG7+, and glucose. Tests included pH, P co2, P o2, calculated bicarbonate, total CO2, base excess, oxygen saturation, sodium, potassium, chloride, urea, glucose, hematocrit, and calculated hemoglobin and glucose. Each cartridge requires 65 μL of whole blood for testing. The blood was drawn and analyzed by physician order. From 1997 through 2001, the team filled out an evaluation form for quality review after cases where POCT was performed. Patient test results and charts for each POCT episode were reviewed retrospectively to identify changes in treatment linked to test results. Other data were extracted from transport department and quality-control records. This research was approved by …

Modulation of renal glomerular disease using remote delivery of adenoviral‐encoded solubletype II TGF‐β receptor fusion molecule

Systemic adenoviral (Ad) gene therapy for renal disorders is largely hampered by the unique architecture of the kidney. Consequently, currently available Ad vectors are of only limited therapeutic utility in the context of glomerular and fibroproliferative renal diseases.

A three-dimensional assay for measurement of viral-induced oncolysis

Oncolytic viruses represent a novel cancer treatment strategy. Despite their promising preclinical data, however, corresponding clinical trials have disappointed. To aid preclinical analyses, we hypothesized that three-dimensional tumor cell clusters or spheroids might provide an assay system superior to conventional monolayer cell cultures. Spheroids show viral infection, replication and oncolytic patterns distinct from conventional monolayer assays. Therefore, viral tumor penetration and oncolysis measurements may be improved with such three-dimensional models. Also, preclinical analyses of oncolytic viruses frequently measure mitochondrial activity, but more accurate measures of oncolysis might involve quantitation of intracellular protein release. Therefore, we measured luciferase released from luciferase-expressing spheroids and found unique patterns that maintained consistency with various viruses and doses. The relative variations between viruses and doses may represent temporal differences in oncolysis dynamics. Analysis of five recombinant replicative adenoviruses with promise for clinical application showed that Ad5/3-Δ24 produced the most luciferase release 1 week after infection and achieved the earliest and highest peak luciferase release level. Ad5/3-Δ24 also effected the earliest subtotal spheroid cell death. These findings closely parallel monolayer oncolysis assays with these agents. Therefore, the luciferase-expressing tumor spheroid assay represents a promising three-dimensional model for preclinical analysis of replicative oncolytic agents.

Transcriptional Blocks Limit Adenoviral Replication in Primary Ovarian Tumor

Purpose: Despite the success of conditionally replicating adenoviruses in tumor models, clinical success has been limited when they are used as a single modality agent. Overcoming the disparity in efficacy between in vivo animal models and human use is a key hurdle for better conditionally replicating adenovirus therapy in humans. We endeavored to identify biological blocks to adenoviral infection and replication in tumor cells. Experimental Design: We hypothesized that the differences in adenoviral replication between ovarian cancer cell lines and patient tumor samples are the result of a block in viral RNA transcription. To test this hypothesis, established ovarian cancer cell lines and purified patient ovarian cancer cells were infected with wild-type adenovirus. RNA for early adenoviral genes E1A and E1B as well as the late transcripts for fiber and hexon were measured using real-time PCR. Results: Established ovarian cancer cell lines treated with wild-type virus had a lower E1A:E1B ratio than the patient samples. Additionally, the levels of fiber and hexon relative to E1A were also decreased in the patient samples compared with the established cell lines. These findings were consistent with an early- to late-phase block in the adenovirus replication cycle. Conclusions: These data suggest that the biology of abortive infection in the patient samples may be linked to a defect in the production of early and late viral transcripts. Identification of factors leading to abortive infection will be crucial to understanding the low viral replication in patient samples.

A Leukemia/Lymphoma With Lymphoblastic Morphology And L3 Morphologic Relapse

High-grade lymphoid leukemias/lymphomas occasionally show inconsistent or discordant morphologic and immunophenotypic features that challenge diagnostic efforts. We present a case with fluctuating findings that illustrates such difficulties. A 20-year-old human immunodeficiency virus (HIV)-negative male presented with hepatosplenomegaly, leukemia (white blood cell [WBC] count 5.1 × 103/μL; 12% blasts; hemoglobin 7.5 g/dL; hematocrit 22.8%; platelets 20 × 103/μL), and no lymphadenopathy. The marrow had 96% FAB L1 leukemic blasts (Figure 1). Flow cytometry showed expression of CD10, CD19, and CD79a with surface and cytoplasmic immunoglobulin (cIg and sIg) light chain restriction and absence of CD34 and TdT. Immunohistochemical stains showed expression of CD43, BCL2, and Ki67 (52% positivity) and confirmed CD34 and TdT negativity. Marrow culture failure limited cytogenetic studies. Cyclophosphamide, doxorubicin, and etoposide (CAE) and intrathecal methotrexate were administered for treatment of a high-grade B-cell leukemia/lymphoma (BL/L) and led to a complete remission [1]. Disease recurred 23 weeks after presentation with 84% marrow blasts of L3 morphology (Figure 1). Oil red O stains were negative. The Ki-67 immunostain marker showed 73% positivity. An Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1) immunostain was uninterpretable. The relapsed disease showed persistent CD43, BCL2, and cIg expression with absence of CD34 and sIg and gain of TdT (Table 1). The marrow yielded 5 metaphase cells including 2 with trisomy 21. A second relapse sample revealed a third MYC signal in 15 of 300 (5%) cells by fluorescence in situ hybridization (FISH). Ifosfamide, carboplatin, and etoposide (modified ICE) and highdose methotrexate was administered. This patient’s WBC count reached 63.2 × 103/μL with 91% blasts before he expired about 1 year after presentation. The presenting findings were most consistent with acute lymphoblastic leukemia (ALL). Most ALL cases express CD34 or TdT, but absence of both is possible. Also, many ALL cases express CD43. Rare ALL cases express sIg, but most of these are TdT positive [2–4]. Leukemic BL/L is rare but characteristically associated with advanced disease or HIV infection [5]. Also, the proliferation rate (52% Ki-67 positivity) was inadequate for diagnosis of BL/L. No MYC abnormalities were identified, but up to 10% of BL/L