Polarized light scanning cryomacroscopy, part I: Experimental apparatus and observations of vitrification, crystallization, and photoelasticity effects.

Cryomacroscopy is an effective means to observe physical events affecting cryopreservation success in large-size specimens. The current study aims at integrating polarized-light in the study of large-size cryopreservation, using the scanning cryomacroscope as a development platform. Results of this study demonstrate polarized light as a visualization enhancement means, including the following effects: contaminants in the CPA solution, crystallization, fracture formation, thermal contraction, and solute precipitation. In addition, photoelasticity effects are used to demonstrate the development of residual stresses and the potential for stress relaxation above the glass transition temperature. Furthermore, this study suggests that the ability to periodically switch between non-polarized light and polarized light is an essential feature of investigation. When using polarized light for example, a dark region may represent a free-of-stress and free-of-crystals material, or fully crystallized material, which may potentially experience mechanical stress; switching to a non-polarized light would help to distinguish between the different cases. The analysis of thermo-mechanical stress in cryopreservation is essentially based on four key elements: identification of physical events, knowledge of physical properties, thermal analysis of the specimen, and description of the mechanical behavior of the cryopreserved material (also known as the constitutive law). With the above knowledge, one can investigate the conditions to preserve structural integrity. While the current study aims at identification of physical events, critical knowledge on physical properties and mechanical behavior has already been developed in previous studies. The companion manuscript (Part II) aims at providing means for thermal analysis in the specimen, which will serve as the basis for a multi-scale analysis of thermo-mechanical stress in large-size specimens.

Polarized light scanning cryomacroscopy, part II: Thermal modeling and analysis of experimental observations.

This study aims at developing thermal analysis tools and explaining experimental observations made by means of polarized-light cryomacroscopy (Part I). Thermal modeling is based on finite elements analysis (FEA), where two model parameters are extracted from thermal measurements: (i) the overall heat transfer coefficient between the cuvette and the cooling chamber, and (ii) the effective thermal conductivity within the cryoprotective agent (CPA) at the upper part of the cryogenic temperature range. The effective thermal conductivity takes into account enhanced heat transfer due to convection currents within the CPA, creating the so-called Bénard cells. Comparison of experimental results with simulation data indicates that the uncertainty in simulations due to the propagation of uncertainty in measured physical properties exceeds the uncertainty in experimental measurements, which validates the modeling approach. It is shown in this study that while a cavity may form in the upper-center portion of the vitrified CPA, it has very little effect on estimating the temperature distribution within the domain. This cavity is driven by thermal contraction of the CPA, with the upper-center of the domain transitioning to glass last. Finally, it is demonstrated in this study that additional stresses may develop within the glass transition temperature range due to nonlinear behavior of the thermal expansion coefficient. This effect is reported here for the first time in the context of cryobiology, using the capabilities of polarized-light cryomacroscopy.

Thermomechanical Stress in Cryopreservation Via Vitrification With Nanoparticle Heating as a Stress-Moderating Effect.

This study focuses on thermomechanical effects in cryopreservation associated with a novel approach of volumetric heating by means on nanoparticles in an alternating electromagnetic field. This approach is studied for the application of cryopreservation by vitrification, where the crystalline phase is completely avoided-the cornerstone of cryoinjury. Vitrification can be achieved by quickly cooling the material to cryogenic storage, where ice cannot form. Vitrification can be maintained at the end of the cryogenic protocol by quickly rewarming the material back to room temperature. The magnitude of the rewarming rates necessary to maintain vitrification is much higher than the magnitude of the cooling rates that are required to achieve it in the first place. The most common approach to achieve the required cooling and rewarming rates is by exposing the specimen's surface to a temperature-controlled environment. Due to the underlying principles of heat transfer, there is a size limit in the case of surface heating beyond which crystallization cannot be prevented at the center of the specimen. Furthermore, due to the underlying principles of solid mechanics, there is a size limit beyond which thermal expansion in the specimen can lead to structural damage and fractures. Volumetric heating during the rewarming phase of the cryogenic protocol can alleviate these size limitations. This study suggests that volumetric heating can reduce thermomechanical stress, when combined with an appropriate design of the thermal protocol. Without such design, this study suggests that the level of stress may still lead to structural damage even when volumetric heating is applied. This study proposes strategies to harness nanoparticles heating in order to reduce thermomechanical stress in cryopreservation by vitrification.

Stress-Strain Measurements in Vitrified Arteries Permeated With Synthetic Ice Modulators.

This study measures the Young's modulus in vitrified blood vessels below the glass transition temperature in conditions relevant to cryogenic storage and focuses on the cryoprotective agents (CPAs) cocktail DP6 mixed with synthetic ice modulators (SIMs). Small steplike strain changes were observed during the loading without affecting the bulk behavior, suggesting microfracture occurrences resembling previous observation on microfracture formation under compression in crystallized blood vessels. Young's modulus was measured to be 0.92-3.01 GPa, with no clear indication of SIM dependency on the Young's modulus. Instead, the range of values is attributed to variations between specimens of the same species.

Thermal expansion of vitrified blood vessels permeated with DP6 and synthetic ice modulators.

This study provides thermal expansion data for blood vessels permeated with the cryoprotective cocktail DP6, when combined with selected synthetic ice modulators (SIMs): 12% polyethylene glycol 400, 6% 1,3-cyclohexanediol, and 6% 2,3-butanediol. The general classification of SIMs includes molecules that modulate ice nucleation and growth, or possess properties of stabilizing the amorphous state, by virtue of their chemical structure and at concentrations that are not explained on a purely colligative basis. The current study is part of an ongoing effort to characterize thermo-mechanical effects on structural integrity of cryopreserved materials, where thermal expansion is the driving mechanism to thermo-mechanical stress. This study focuses on the lower part of the cryogenic temperature range, where the cryoprotective agent (CPA) behaves as a solid for all practical applications. By combining results obtained in the current study with literature data on the thermal expansion in the upper part of the cryogenic temperature range, unified thermal expansion curves are presented.

On the Effects of Thermal History on the Development and Relaxation of Thermo-Mechanical Stress in Cryopreservation.

This study investigates the effects of the thermal protocol on the development and relaxation of thermo-mechanical stress in cryopreservation by means of glass formation, also known as vitrification. The cryopreserved medium is modeled as a homogeneous viscoelastic domain, constrained within either a stiff cylindrical container or a highly compliant bag. Annealing effects during the cooling phase of the cryopreservation protocol are analyzed. Results demonstrate that an intermediate temperature-hold period can significantly reduce the maximum tensile stress, thereby decreasing the potential for structural damage. It is also demonstrated that annealing at temperatures close to glass transition significantly weakens the dependency of thermo-mechanical stress on the cooling rate. Furthermore, a slower initial rewarming rate after cryogenic storage may drastically reduce the maximum tensile stress in the material, which supports previous experimental observations on the likelihood of fracture at this stage. This study discusses the dependency of the various stress components on the storage temperature. Finally, it is demonstrated that the stiffness of the container wall can affect the location of maximum stress, with implications on the development of cryopreservation protocols.

Further experience with pancreatic stump closure using a reinforced staple line.

BACKGROUND: We previously demonstrated that pancreatic transection with a reinforced staple line results in significantly lower fistula rates than when stapling without reinforcement. (J Gastrointest Surg. 2007;11:345-349). Criticism of this initial study focused on the small size of the treated group (N = 13). We report four more years of experience with this technique with a larger sample size. METHODS: This was a before-after trial. Patients included had distal pancreatectomies with stapled stump closure. The main intervention analyzed was staple-line reinforcement with Seamguard. The experimental group consisted of a consecutive series of stapled pancreatectomies with reinforcement performed from 2005 to 2010. The control group was a consecutive series of stapled pancreatectomies without reinforcement performed between 2003 and 2005 (previously published). The main outcome measure was pancreatic fistula. RESULTS: 54 patients were included; 36 in the experimental group and 18 in the control group. Mean age was 62; 50% were males. The most common diagnoses were adenocarcinoma (31%), cystic neoplasm (24%), and neuroendocrine tumor (22%). There were no mortalities. Postoperative pancreatic leak rate was 39% in the control group, and 8% in the experimental group (P = 0.01). Seven of ten patients with leak required additional drain placement. Development of pancreatic leak resulted in prolonged hospital stays (12 vs eight days, P < 0.007). CONCLUSION: We demonstrate sustained success of reinforced stapling for pancreatic stump closure. Our technique is straightforward and results in reduced morbidity and cost. Our results suggest that surgical drains may not be needed when this technique is applied.

Effect of early enteral tube feeding on patient outcome following pancreaticoduodenectomy.

OBJECTIVE: Morbidity after pancreaticoduodenectomy (PD) is nearly 50%. In this study we analyzed if early enteral nutrition via feeding tube (FT) contributes to better patient outcomes. MATERIALS AND METHODS: Patients undergoing PD from 2003-2010. FTs were placed routinely before August 2006, and omitted thereafter. Short-term outcome measures included: time to start of oral diet, need for total parenteral nutrition (TPN), morbidity and mortality, pancreatic fistula, complications from FT, hospital length of stay, and disposition. Long-term outcome measures included time to start adjuvant therapy, and survival. RESULTS: N = 59 (25 had FT, 34 did not). Adenocarcinoma was found in 88%. Early institution of tube feeding had no positive impact on any of the outcome measures. There were three FT-related complications. CONCLUSIONS: Our results demonstrate that FT placement does not improve short-term or long-term outcomes after PD. Moreover, major complications can result from FT placement. We do not advocate the routine use of FT after PD.

Thermal expansion of the cryoprotectant cocktail DP6 combined with synthetic ice modulators in presence and absence of biological tissues.

This study explores physical effects associated with the application of cryopreservation via vitrification using a class of compounds which are defined here as synthetic ice modulators (SIMs). The general classification of SIMs includes molecules that modulate ice nucleation and growth, or possess properties of stabilizing the amorphous state, by virtue of their chemical structure and at concentrations that are not explained on a purely colligative basis. A sub-category of SIMs, referred to in the literature as synthetic ice blockers (SIBs), are compounds that interact directly with ice nuclei or crystals to modify their structure and/or rate of growth. The current study is part of an ongoing effort to characterize thermo-mechanical effects during vitrification, with emphasis on measuring the physical property of thermal expansion-the driving mechanism to thermo-mechanical stress. Materials under investigation are the cryoprotective agent (CPA) cocktail DP6 in combination with one of the following SIMs: 12% polyethylene glycol 400, 6% 1,3 cyclohexanediol, and 6% 2,3 butanediol. Results are presented for the CPA-SIM cocktail in the absence and presence of bovine muscle and goat artery specimens. This study focuses on the upper part of the cryogenic temperature range, where the CPA behaves as a fluid for all practical applications. Results of this study indicate that the addition of SIMs to DP6 allows lower cooling rates to ensure vitrification and extends the range of measurements. It is demonstrated that the combination of SIM with DP6 increases the thermal expansion of the cocktail, with implications for the likelihood of fracture formation-the most dramatic outcome of thermo-mechanical stress.

A randomized, placebo-controlled, double-blind study to evaluate the efficacy of a citrus bioflavanoid blend in the treatment of senile purpura.

BACKGROUND: Senile purpura is a common, chronic skin condition affecting more than 10 percent of individuals over the age of 50. Despite being a benign condition, the continual development of purpura lesions in afflicted patients is frequently a source of significant visual and social concern. To date, there are no known effective treatments for this condition. OBJECTIVES: To evaluate the efficacy of a novel nutraceutical citrus bioflavonoid blend in improving the skin's appearance in patients with senile purpura. METHODS: A six-week, randomized, multicenter, placebo-controlled, double-blind study was conducted to determine whether a uniquely formulated, oral citrus bioflavonoid supplement could treat active lesions of senile purpura while preventing new lesions from arising. Seventy patients with senile purpura were enrolled and 67 completed the study. Subjects were randomized into two groups receiving either a citrus bioflavonoid blend or placebo medication, which was taken orally twice daily for six weeks. Clinical evaluations were performed by blinded investigators at two locations. RESULTS: A statistically significant reduction in the number of new purpura lesions in the skin area undergoing clinical study was documented. At the end of six weeks, the citrus bioflavonoid blend treated group showed a 50 percent reduction in purpura lesions from baseline. Patient self-assessment of the effectiveness of the medication echoed the results of an investigator global assessment with a statistically significant improvement in the skin's appearance noted by the patients receiving the active medication. No adverse effects were noted by either the patients or investigators. CONCLUSION: This new treatment appears to both safely and effectively diminish skin bruising in patients with senile purpura.

Hyperthermia potentiates oncolytic herpes viral killing of pancreatic cancer through a heat shock protein pathway.

BACKGROUND: Oncolytic herpes simplex virus-1 (HSV-1) is designed to specifically infect, replicate in, and lyse cancer cells. This study investigates a novel therapeutic regimen, combining the effects of NV1066 (a recombinant HSV-1) and hyperthermia in the treatment of pancreatic cancer. METHODS: NV1066 is an attenuated HSV-1 that replicates in cells resistant to apoptosis. Heat shock protein 72 (Hsp72) is a member of a family of proteins that is upregulated after hyperthermic insult, lending cellular protection by inhibiting apoptosis. In these experiments, we test the hypothesis that increased Hsp72 expression in response to hyperthermia enhances anti-apoptotic mechanisms, thereby increasing viral replication and tumor cell kill. Hs 700T pancreatic cancer cells were treated with hyperthermia alone (42 degrees C), NV1066 alone, and combination therapy. Cell survival and viral growth were measured. The effect of siRNA-directed Hsp72 knockdown was also measured. RESULTS: Combining hyperthermia and viral treatment produced a synergistic effect on cell kill. Viral growth increased greater than 6-fold in the presence of hyperthermia (P < .05). Hyperthermia alone showed minimal cytotoxic activity against Hs 700T cells, while NV1066 infection resulted in approximately 50% cell kill. The combination of hyperthermia and viral infection significantly increased cell kill to approximately 80% (P < .01). Hsp72 knockdown attenuated this synergistic effect. CONCLUSION: Hyperthermia enhances NV1066 replication, thereby potentiating the viral oncolytic response against pancreatic cancer cells. This finding has potential clinical application in the use of heated perfusion or permissive hyperthermia for delivery of oncolytic viral therapies.

Short- and long-term costs of laparoscopic colectomy are significantly less than open colectomy.

BACKGROUND: The financial impact of laparoscopic colectomy remains poorly defined. We report the short-term costs of laparoscopic colectomy (LC) as compared with open colectomy (OC) in a high-volume tertiary care hospital, and are the first to incorporate the costs of late, colectomy-related complications in an analysis of long-term costs. METHODS: A retrospective analysis of patients undergoing elective laparoscopic (n = 76) or open (n = 162) colon resection between January 2004 and December 2006 was performed. Primary endpoints were total hospital cost of the index admission and total hospital cost for any subsequent admission for treatment of a colectomy-related complication. RESULTS: Two-hundred thirty-eight patients met inclusion criteria. Mean total hospital cost was significantly greater for patients undergoing OC (US $17,686 per patient versus US $14,518, P = 0.0003). Mean total operative costs were equivalent (US $7,451 OC versus US $7,794 LC, P = 0.274). Average length of stay was shorter for LC (5.2 versus 6.9 days, P < 0.0001). Late complication rates were 5.6% (OC) and 2.6% (LC). Integrating the cost of late complications further increased the disparity between the total cost of OC (US $18,296 per patient, 3.4% increase) as compared with LC (US $14,789, 1.9% increase). CONCLUSION: We demonstrate both short- and long-term financial benefits of LC in a high-volume tertiary care hospital.

Validation of a prediction rule to maximize curative (R0) resection of early-stage pancreatic adenocarcinoma.

BACKGROUND: The surgeon's contribution to patients with localized pancreatic adenocarcinoma (PAC) is a margin negative (R0) resection. We hypothesized that a prediction rule based on pre-operative imaging would maximize the R0 resection rate while reducing non-therapeutic intervention. METHODS: The prediction rule was developed using computed tomography (CT) and endoscopic ultrasound (EUS) data from 65 patients with biopsy-proven PAC who underwent attempted resection. The rule classified patients as low or high risk for non-R0 outcome and was validated in 78 subsequent patients. RESULTS: MODEL VARIABLES WERE: any evidence of vascular involvement on CT; EUS stage and EUS size dichotomized at 2.6 cm. In the validation cohort, 77% underwent resection and 58% achieved R0 status. If only patients in the low-risk group underwent surgery, the prediction rule would have increased the resection rate to 92% and the R0 rate to 73%. The R0 rate was 40% higher in low-risk compared with high-risk patients (P < 0.001). High risk was associated with a 67% rate of non-curative surgery (unresectable disease and metastases). CONCLUSION: The prediction rule identified patients most likely to benefit from resection for PAC using pre-operative CT and EUS findings. Model predictions would have increased the R0 rate and reduced non-therapeutic interventions.

Nectin-1 expression by squamous cell carcinoma is a predictor of herpes oncolytic sensitivity.

Oncolytic viruses based on herpes simplex virus type 1 (HSV-1) are able to infect and lyse a variety of malignant cell lines. However, there is variability in the degree of tumor susceptibility, and the cancer cell determinants of HSV sensitivity are poorly defined. Nectin-1 is a cell surface adhesion molecule that functions as a cellular receptor to HSV envelope glycoprotein D (gD). We assessed tumor nectin-1 expression as a predictor of oncolytic HSV sensitivity. A panel of human squamous carcinoma cell lines was evaluated for viral entry, replication, and cytotoxicity to an attenuated, replication-competent, oncolytic HSV (NV1023). Potential tumor determinants of HSV sensitivity were assessed, including nectin-1, herpes viral entry mediator, total gD receptor expression, S-phase fraction, and doubling time. Significant correlations between nectin-1 expression measured by quantitative fluorescence-activated cell sorting and viral sensitivity measures were identified using Pearson's coefficients. Cancer cell nectin-1 receptor blockade and nectin-1 transfection led to inhibition and enhancement of NV1023 viral entry, respectively. Cell lines with varying nectin-1 expression showed corresponding sensitivity to NV1023 therapy in vivo. Immunohistochemistry for nectin-1 was inversely related to E-cadherin staining, suggesting increased herpes sensitivity of E-cadherin-deficient tumors. These results suggest that nectin-1 may be used as a marker to predict the sensitivity of a tumor to herpes oncolytic therapy.

Intraoperative localization of lymph node metastases with a replication-competent herpes simplex virus.

OBJECTIVES: Lymph node status is the most important prognostic factor determining recurrence and survival in patients with mesothelioma and other thoracic malignancies. Accurate localization of lymph node metastases is therefore necessary to improve selection of resectable and curable patients for surgical intervention. This study investigates the potential to identify lymph node metastases intraoperatively by using herpes-guided cancer cell-specific expression of green fluorescent protein. METHODS: After infection with NV1066, a herpes simplex virus carrying green fluorescent protein transgene, human mesothelioma cancer cell lines were assessed for cancer cell-specific infection, green fluorescent protein expression, viral replication, and cytotoxicity. Murine models of lymphatic metastasis were established by means of surgical implantation of cancer cells into the preauricular (drainage to cervical lymph nodes) and pleural (mediastinal and retroperitoneal lymph nodes) spaces of athymic mice. Fluorescent thoracoscopy, laparoscopy, and stereomicroscopy were used to localize lymph node metastases that were confirmed by means of immunohistochemistry. RESULTS: In vitro NV1066 infected, replicated (5- to 17,000-fold), and expressed green fluorescent protein in all cancer cells, even when infected at a low ratio of one viral plaque-forming unit per 100 tumor cells. In vivo NV1066 injected into primary tumors was able to locate and infect lymph node metastases producing green fluorescent protein that was visualized by means of fluorescent imaging. Histology confirmed lymphatic metastases, and immunohistochemistry confirmed viral presence in regions that expressed green fluorescent protein. CONCLUSIONS: Herpes virus-guided cancer cell-specific production of green fluorescent protein can facilitate accurate localization of lymph node metastases. Fluorescent filters that detect green fluorescent protein can be incorporated into operative scopes to precisely localize and biopsy lymph node metastases.

Real-time intraoperative detection of breast cancer axillary lymph node metastases using a green fluorescent protein-expressing herpes virus.

OBJECTIVE: To investigate the use of a green fluorescent protein (GFP)-expressing oncolytic herpes virus to enable real-time intraoperative detection of breast cancer lymph node metastases. SUMMARY BACKGROUND DATA: Axillary lymph node status is the most important factor determining treatment, recurrence, and overall survival for women with breast cancer. The current methods of determining nodal status, however, have limitations. NV1066 is a novel oncolytic herpes viral strain that specifically infects cancer cells and expresses GFP. METHODS: Seven human breast cancer cell lines were infected in vitro with NV1066 and assessed for GFP expression, viral replication, and cytotoxicity. An in vivo model of breast cancer lymphatic metastasis was established in mice. Tumor-bearing mice were treated with NV1066 via injection into the primary tumor. Axillary lymph nodes were analyzed using an in vivo fluorescent imaging system. Histologic and molecular assessment of lymph nodes were performed using immunohistochemistry and reverse transcriptase PCR and operating characteristics were determined. RESULTS: NV1066 infected, expressed GFP, replicated within, and killed all human breast cancer cell lines in vitro. Injection of NV1066 into primary breast tumors resulted in viral transit to axillary lymph nodes, infection of lymphatic metastases, and GFP expression that was visualized with in vivo fluorescent imaging. Histologic and molecular confirmation demonstrated favorable operating characteristics of this method (sensitivity 80%; specificity 96%). CONCLUSIONS: We introduce a novel, sensitive, and specific method of lymphatic mapping that utilizes NV1066-guided cancer cell-specific viral production of GFP to enable real-time intraoperative detection of lymphatic metastases.

Estrogen enhances the efficacy of an oncolytic HSV-1 mutant in the treatment of estrogen receptor-positive breast cancer.

Oncolytic herpes simplex virus-1 (HSV-1) mutants selectively replicate in and lyse tumor cells. Viral replication is dependent on the cellular proliferative mechanism. Estrogen increases cellular proliferation and decreases apoptosis in estrogen receptor-positive (ER+) human breast cancer cells. We hypothesize that the cellular changes produced by estrogen may enhance oncolytic viral replication and improve the treatment of ER+ breast cancer cells. Estrogen increased proliferation and replication of the HSV-1 mutant, NV1066, in ER+ breast cancer cells. Additionally, cells grown with estrogen had lower rates of apoptosis and higher bcl-2 levels at baseline and after infection. Estrogen enhanced the oncolytic effect of NV1066, with cell kills of 95% and 97% at MOIs of 0.1 and 0.5, compared to 53 and 87% respectively without estrogen (p<0.001). Therapy of ER+ human breast cancer cells with a replication-competent HSV-1 mutant is improved in the presence of estrogen, in contrast to more standard therapies, such as chemotherapy and radiation, which demonstrate decreased efficacy in similar conditions. These data provide the mechanistic basis for the use of oncolytic HSV-1 in patients with hormone receptor-positive breast cancer, particularly if the disease progresses with conventional therapies.

Oncolytic herpes viral therapy is effective in the treatment of hepatocellular carcinoma cell lines.

The rising incidence of hepatocellular carcinoma (HCC) in western countries, along with the poor prognosis offered by present-day treatment modalities, makes novel therapies for this disease necessary. Oncolytic herpes simplex viruses (HSV) are replication-competent viruses that are highly effective in the treatment of a wide variety of experimental models of human malignancies. This study seeks to investigate the effectiveness of oncolytic herpes viruses in the treatment of primary HCC cell lines. Sixteen commercially available human HCC cell lines were studied. G207 is an attenuated, replication-competent, oncolytic HSV engineered to selectively replicate within cancer cells. Cell lines were tested for viral sensitivity to G207 and their ability to support viral replication using standard cytotoxicity and viral replication assays. Eleven of 16 cell lines were moderately to highly sensitive to G207 viral oncolysis. HCC cell lines additionally demonstrated the ability to support viral replication in vitro with as high as 800-fold amplification of the administered viral dose observed. G207 is cytotoxic to, and efficiently replicates within, HCC cell lines in vitro. From these data, we suggest that oncolytic HSV therapy may have a role in the treatment of HCC, and in vivo studies are warranted.

Real-time diagnostic imaging of tumors and metastases by use of a replication-competent herpes vector to facilitate minimally invasive oncological surgery.

Current efforts on expanding minimally invasive techniques into the realm of oncological surgery are hindered by lack of accurate visualization of tumor margins and failure to detect micro metastases in real time. We used a systemic delivery of a herpes viral vector with cancer-selective infection and replication to precisely differentiate between normal and malignant tissue. NV1066 is a genetically modified, replication-competent herpes simplex virus carrying a transgene for enhanced green fluorescent protein (GFP). We tested the potential of NV1066 in delineating tumor tissue in vitro and in vivo in a wide range of cancers and whether NV1066-induced GFP expression can detect small foci of tumors and metastases in in vivo models using an operating endoscope with fluorescent filters. Our findings indicate that NV1066 can be used for real-time intraoperative imaging and enhanced detection of early cancers and metastases. We demonstrate that a single dose of NV1066, administered either locally (intratumoral or intracavitary) or systemically, will detect loco-regional and distant disease throughout the body. Such cancer selectivity is confirmed in 110 types of cancer cells from 16 different primary organs. Fluorescence-aided minimally invasive endoscopy revealed microscopic tumor deposits unrecognized by conventional laparoscopy/thoracoscopy. Furthermore, NV1066 ability to transit and infect tumor and metastases is proven in syngenic and transplanted tumors in different animal models, both immunocompetent and immunodeficient. Cancer-selective GFP expression is confirmed by histology, immunohistochemistry, and qRT-PCR. These studies form the basis for real-time, intraoperative diagnostic imaging of tumor and metastases by minimally invasive endoscopic technology.

Imaging and therapy of malignant pleural mesothelioma using replication-competent herpes simplex viruses.

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive cancer that is refractory to current treatment modalities. Oncolytic herpes simplex viruses (HSV) used for gene therapy are genetically engineered, replication-competent viruses that selectively target tumor cells while sparing normal host tissue. The localized nature, the potential accessibility and the relative lack of distant metastasis make MPM a particularly suitable disease for oncolytic viral therapy. METHODS: The infectivity, selective replication, vector spread and cytotoxic ability of three oncolytic HSV: G207, NV1020 and NV1066, were tested against eleven pathological types of MPM cell lines including those that are resistant to radiation therapy, gemcitabine or cisplatin. The therapeutic efficacy and the effect on survival of NV1066 were confirmed in a murine MPM model. RESULTS: All three oncolytic HSV were highly effective against all the MPM cell lines tested. Even at very low concentrations of MOI 0.01 (MOI: multiplicity of viral infection, ratio of viral particles per cancer cell), HSV were highly effective against MPM cells that are resistant to radiation, gemcitabine and cisplatin. NV1066, an oncolytic HSV that expresses green fluorescent protein (GFP), was able to delineate the extent of the disease in a murine model of MPM due to selective infection and expression of GFP in tumor cells. Furthermore, NV1066 was able to reduce the tumor burden and prolong survival even when treatment was at an advanced stage of the disease. CONCLUSION: These findings support the continued investigation of oncolytic HSV as potential therapy for patients with therapy-resistant MPM.