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Optical imaging (OI) provides real-time clinical imaging capability and simultaneous molecular, morphological, and functional information of disease processes. In this study, we present a new interventional OI technique, which enables in vivo visualization of three distinct pathologic zones of ablated tumor periphery for immediate detection of residual tumors during a radiofrequency ablation (RFA) session. Rabbits with orthotopic hepatic tumors were divided into two groups (n = 8/group): incomplete RFA and complete RFA. Indocyanine green-based interventional OI was used to differentiate three pathological zones: ablated tumor, transition margin, and residual tumor or surrounding normal liver—with quantitative comparison of signal-to-background ratios among the three zones and between incompletely and completely ablated tumors. Subsequent ex vivo OI and pathologic correlation were performed to confirm the findings of interventional OI. Interventional OI could differentiate incompletely or completely ablated tumor peripheries, thus permitting identification of residual tumor. This technique may open new avenues for immediate assessment of tumor eradication during a single interventional ablation session. Significance: Interventional optical imaging can instantly visualize pathologic zones of ablated tumor peripheries to detect residual tumors, which could revolutionize current image-guided interventional oncologic ablation techniques.

Objective: To investigate the feasibility of interventional oncolytic immunotherapy with LTX-315 for residual tumors after incomplete radiofrequency ablation (iRFA) of VX2 liver tumors in a rabbit model. Methods: For in vitro experiments, VX2 tumor cells were treated with: (1) phosphate buffered saline, (2) radiofrequency hyperthermia (RFH), (3) LTX-315, and (4) RFH plus LTX-315. The residual tumors after iRFA of VX2 liver tumors were treated with: (1) phosphate buffered saline served as control, (2) 2 mg LTX-315, and (3) 4 mg LTX-315. MTS assay, fluorescence microscopy, and flow cytometry were used to compare cell viabilities and apoptosis among different groups. Ultrasound imaging was used to follow up the tumor growth, which were correlated with the optical imaging and subsequent histology. Results: For in vitro experiments, compared with the other three groups, MTS assay demonstrated the lowest cell viability, fluorescence microscopy showed the least survival cells, and apoptosis analysis revealed the highest percentage of apoptosis cells in the combination treatment groups (p < 0.001). For in vivo experiments, ultrasound imaging showed the smallest tumor volume in the group with 4 mg LTX-315 therapy compared with the other two groups (p < 0.001). The optical imaging and histopathological analysis showed complete necrosis of the tumors in the group with 4 mg LTX-315 therapy. A significant increase of CD8+ T cells and HSP70 and a significant decrease of Tregs were observed in residual tumors in the group with 2 mg LTX-315 therapy compared with the control group (p < 0.001). Conclusion: Interventional oncolytic immunotherapy with LTX-315 for residual tumors after iRFA of liver cancer is feasible, which may open up new avenues to prevent residual tumors after RFA of intermediate-to-large liver cancers.

BackgroundIt is still a challenge to prevent tumor recurrence post radiofrequency ablation (RFA) of medium-to-large hepatocellular carcinomas (HCC). Immunochemotherapy, a combination of immunotherapy with chemotherapy, has demonstrated a great potential in augmenting the treatment efficacy for some malignancies. In this study, we validated the feasibility of using radiofrequency hyperthermia (RFH)-enhanced intratumoral immunochemotherapy of LTX-315 with liposomal doxorubicin for rat orthotopic HCC.MethodsDifferent groups of luciferase-labeled rat HCC cells and rat orthotopic HCC models were treated by: (1) phosphate buffered saline; (2) RFH; (3) LTX-315; (4) RFH+LTX-315; (5) liposomal doxorubicin; (6) RFH+liposomal doxorubicin; (7) LTX-315+liposomal doxorubicin; and (8) RFH+LTX-315+liposomal doxorubicin. Cell viabilities and apoptosis of different treatment groups were compared. Changes in tumor sizes were quantified by optical and ultrasound imaging, which were confirmed by subsequent histopathology. The potential underlying biological mechanisms of the triple combination treatment (RFH+LTX-315+liposomal doxorubicin) were explored.ResultsFlow cytometry and MTS assay showed the highest percentage of apoptotic cells and lowest cell viability in the triple combination treatment group compared with other seven groups (p+T cells, CD8+/interferon (IFN)-γ+T cells, CD8+/tumor necrosis factor (TNF)-α+T cells, and natural killer cells, and a significant decrease of regulatory T cells were observed in the tumors (pConclusionsIntratumoral RFA-associated RFH could enhance the efficacy of immunochemotherapy of LTX-315 with liposomal doxorubicin for HCC, which may provide a new strategy to increase the curative efficacy of thermal ablation for medium-to-large HCC.

Background: Residual viable tumor cells after ablation at the tumor periphery serve as the source for tumor recurrence, leading to treatment failure. Purpose: To develop a novel three-dimensional (3D) multi-modal perfusion-thermal electrode system completely eradicating medium-to-large malignancies. Materials and Methods: This study included five steps: (i) design of the new system; (ii) production of the new system; (iii) ex vivo evaluation of its perfusion-thermal functions; (iv) mathematic modeling and computer simulation to confirm the optimal temperature profiles during the thermal ablation process, and; (v) in vivo technical validation using five living rabbits with orthotopic liver tumors. Results: In ex vivo experiments, gross pathology and optical imaging demonstrated the successful spherical distribution/deposition of motexafin gadolinium administered through the new electrode, with a temperature gradient from the electrode core at 80 °C to its periphery at 42 °C. An excellent repeatable correlation of temperature profiles at varying spots, from the center to periphery of the liver tumor, was found between the mathematic simulation and actual animal tumor models (Pearson coefficient ≥0.977). For in vivo validation, indocyanine green (ICG) was directly delivered into the peritumoral zones during simultaneous generation of central tumoral lethal radiofrequency (RF) heat (>60 °C) and peritumoral sublethal RF hyperthermia (

The aim of this study was to develop an interventional optical imaging (OI) technique for intraprocedural guidance of complete tumor ablation. Our study employed four strategies: 1) optimizing experimental protocol of various indocyanine green (ICG) concentrations/detection time windows for ICG-based OI of tumor cells (ICG cells); 2) using the optimized OI to evaluate ablation-heat effect on ICG cells; 3) building the interventional OI system and investigating its sensitivity for differentiating residual viable tumors from nonviable tumors; and 4) preclinically validating its technical feasibility for intraprocedural monitoring of radiofrequency ablations (RFAs) using animal models with orthotopic hepatic tumors. OI signal-to-background ratios (SBRs) among preablation tumors, residual, and ablated tumors were statistically compared and confirmed by subsequent pathology. The optimal dose and detection time window for ICG-based OI were 100 μg/mL at 24 h. Interventional OI displayed significantly higher fluorescence signals of viable ICG cells compared with nonviable ICG cells (189.3 ± 7.6 versus 63.7 ± 5.7 au, P < 0.001). The interventional OI could differentiate three definitive zones of tumor, tumor margin, and normal surrounding liver, demonstrating significantly higher average SBR of residual viable tumors compared to ablated nonviable tumors (2.54 ± 0.31 versus 0.57 ± 0.05, P < 0.001). The innovative interventional OI technique permitted operators to instantly detect residual tumors and thereby guide repeated RFAs, ensuring complete tumor eradication, which was confirmed by ex vivo OI and pathology. In conclusion, we present an interventional oncologic technique, which should revolutionize the current ablation technology, leading to a significant advancement in complete treatment of larger or irregular malignancies.

PurposeTo validate the feasibility of using peri-tumoral radiofrequency hyperthermia (RFH)-enhanced chemotherapy to obliterate hepatic tumor margins.Method and MaterialsThis study included in vitro experiments with VX2 tumor cells and in vivo validation experiments using rabbit models of liver VX2 tumors. Both in vitro and in vivo experiments received different treatments in four groups (n=6/group): (i) RFH-enhanced chemotherapy consisting of peri-tumoral injection of doxorubicin plus RFH at 42°C; (ii) RFH alone; (iii) doxorubicin alone; and (iv) saline. Therapeutic effect on cells was evaluated using different laboratory examinations. For in vivo experiments, orthotopic hepatic VX2 tumors in 24 rabbits were treated by using a multipolar radiofrequency ablation electrode, enabling simultaneous delivery of both doxorubicin and RFH within the tumor margins. Ultrasound imaging was used to follow tumor growth overtime, correlated with subsequent histopathological analysis.ResultsIn in vitro experiments, MTS assay demonstrated the lowest cell proliferation, and apoptosis analysis showed the highest apoptotic index with RFH-enhanced chemotherapy, compared with the other three groups (pin vivo experiments, ultrasound imaging detected the smallest relative tumor volume with RFH-enhanced chemotherapy (pConclusionThis study demonstrates that peri-tumoral RFH can specifically enhance the destruction of tumor margins in combination with peri-tumoral injection of a chemotherapeutic agent. This new interventional oncology technique may address the critical clinical problem of frequent marginal tumor recurrence/persistence following thermal ablation of large (>3 cm) hepatic cancers.

Background The risk of neonatal early-onset sepsis (EOS) is traditionally assessed on maternal signs of clinical chorioamnionitis. Recently, an online EOS risk calculator was developed by Kaiser Permanente using maternal and neonatal clinical parameters. We were interested in whether an increased Kaiser sepsis risk score correlates with histologic acute chorioamnionitis or acute funisitis. Design Included in this retrospective review are 119 chorioamnionitis-exposed term neonates from January 1, 2015 and December 31, 2016. Clinical charts from mother–baby pairs were reviewed. An EOS risk score was obtained using the online Kaiser Sepsis Calculator. The presence and severity of acute chorioamnionitis and acute funisitis were recorded. A SPSS software was used for statistical analysis (IBM, New Jersey, USA). Results The Kaiser Sepsis Calculator could identify 97 of 119 (81.5%) neonates without increased risk for sepsis. Histologic acute chorioamnionitis was present in 100 of 119 cases (84%), in which 44 cases (44%) show severe acute chorioamnionitis. Acute funisitis was recognized in 87 of 119 (73.1%) cases, all of which had concurrent acute chorioamnionitis. Severe funisitis was seen in 38 of the 87 cases (43.7%). The Kaiser Sepsis risk score correlates with the presence and severity of acute funisitis ( P = .037 and P = .044, respectively) but not with the presence or the severity of acute chorioamnionitis ( P = .105 and P = .672, respectively). Conclusion Our study provides histological evidence to support that the Kaiser Sepsis Calculator may help to effectively reduce unwarranted blood culture, antibiotics exposure, and neonatal intensive care unit admission in term neonates.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) and neonatal alloimmune neutropenia (NAN) are two rare complications of newborns caused by antibodies against paternal inherited antigens. Human platelet (HPA) and neutrophil antigens (HNA) are the common targets. Human leukocyte antigen (HLA) class I proteins are also expressed on platelets and neutrophils and anti-HLA antibodies have occasionally been implicated in these complications. We report a premature twin infant who presented with severe thrombocytopenia and neutropenia clinically compatible with FNAIT and NAN, from a mother with no identifiable HPA or HNA antibodies, but with very high levels of complement-fixing antibodies against paternal inherited HLA. These antibodies were also detected in the infant. HLA antibodies are commonly present in multiparous women who deliver healthy infants. They can, however, be cytotoxic and cause clinical complications after blood products transfusion (TRALI and becoming refractory to platelets transfusion) and after organ transplantation (allogeneic organ rejection).

Molecular imaging has been advanced into the field of infectious diseases, which provides not only new insights for basic science, but also new strategies for the effective management of infectious diseases in clinical practice.

Atopic dermatitis (AD) is a chronic pruritic skin disorder affecting many people especially young children. It is a disease caused by the combination of genetic predisposition, immune dysregulation, and skin barrier defect. In recent years, emerging evidence suggests oxidative stress may play an important role in many skin diseases and skin aging, possibly including AD. In this review, we give an update on scientific progress linking oxidative stress to AD and discuss future treatment strategies for better disease control and improved quality of life for AD patients.

Claudins form a large family of tight junction proteins that have essential roles in the control of paracellular ion flux and the maintenance of cell polarity. Many studies have shown that several claudin family members are abnormally expressed in various cancers. In particular, CLDN4 (encoding claudin-4) is overexpressed in ovarian cancer. However, although CLDN4 overexpression is well established, the mechanisms responsible for this abnormal regulation remain unknown. In the present study, we delineate a small region of the CLDN4 promoter critical for its expression. This region contains two Sp1 sites, both of which are required for promoter activity. However, because of the ubiquitous expression of Sp1, these sites, although necessary, are not sufficient to explain the patterns of gene expression of CLDN4 in various ovarian tissues. We show that the CLDN4 promoter is further controlled by epigenetic modifications of the Sp1-containing critical promoter region. Cells that overexpress CLDN4 exhibit low DNA methylation and high histone H3 acetylation of the critical CLDN4 promoter region, and the reverse is observed in cells that do not express CLDN4. Moreover, the CLDN4-negative cells can be induced to express CLDN4 through treatment with demethylating and/or acetylating agents. Because CLDN4 is elevated in a large fraction of ovarian cancer, the mechanism leading to deregulation may represent a general pathway in ovarian tumorigenesis and may lead to novel strategies for therapy and an overall better understanding of the biology of this disease.

Human papillomavirus (HPV), particularly type 16 (HPV-16), is present in more than 99% of cervical cancers. The HPV oncoproteins E6 and E7 are constantly expressed and therefore represent ideal targets for HPV vaccine development. We previously developed DNA vaccines encoding calreticulin (CRT) linked to HPV-16 E7 and generated potent E7-specific CD8+T-cell immune responses and antitumor effects against an E7-expressing tumor. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a DNA vaccine encoding CRT linked to E6 (CRT/E6). Our results indicated that the CRT/E6 DNA vaccine, but not a wild-type E6 DNA vaccine, generated significant E6-specific CD8+T-cell immune responses in vaccinated mice. Mapping of the immunodominant epitope of E6 revealed that an E6 peptide comprising amino acids (aa) 48 to 57 (E6 aa48-57), presented by H-2Kb, is the optimal peptide and that the region of E6 comprising aa 50 to 57 represents the minimal core sequence required for activating E6-specific CD8+T lymphocytes. We also demonstrated that E6 aa48-57 contains cytotoxic T-lymphocyte epitopes naturally presented by E6-expressing TC-1 cells. Vaccination with a CRT/E6 but not a CRT/mtE6 (lacking aa 50 to 57 of E6) DNA vaccine could protect vaccinated mice from challenge with E6-expressing TC-1 tumors. Thus, our data indicate that E6 aa48-57 contains the immunodominant epitope and that a CRT/E6 DNA vaccine may be useful for control of HPV infection and HPV-associated lesions.

The distinction of metastatic mucinous carcinomas in the ovary from primary ovarian mucinous tumors (atypical proliferative/borderline and carcinoma) can be difficult because of similarities in morphology. We evaluated the immunohistochemical expression of cytokeratins 7 and 20 (CK 7, CK 20), Dpc4 (nuclear transcription factor inactivated in 55% of pancreatic carcinomas), and MUC5AC (a gastric mucin gene) in 57 primary ovarian mucinous tumors (41 atypical proliferative tumors and 16 carcinomas) and 46 metastatic mucinous carcinomas in the ovary. Primary ovarian mucinous tumors were virtually always diffusely positive for CK 7 (98%), Dpc4 (100%), and MUC5AC (98%) and often focally to diffusely positive for CK 20 (68%). Colorectal mucinous carcinomas were diffusely positive for CK 20 (100%) and Dpc4 (89%) and were distinguished from primary ovarian mucinous tumors by their frequent lack of expression of CK 7 and MUC5AC (67% were negative for each marker). Appendiceal carcinomas were diffusely positive for CK 20 (100%) and often negative for CK 7 (71%) but were often positive for MUC5AC (86%) and Dpc4 (100%). When primary ovarian and metastatic colorectal or appendiceal carcinomas shared expression of both CK 7 and CK 20, they could usually be distinguished by the pattern of positivity (diffuse CK 7 and patchy CK 20 in ovarian tumors and patchy CK 7 and diffuse CK 20 in colorectal and appendiceal tumors). Pancreatic carcinomas shared the same pattern of diffuse positivity for CK 7 (100%) and MUC5AC (92%) and focal to diffuse positivity for CK 20 (71%) as primary ovarian mucinous tumors but were negative for Dpc4 in 46%. Loss of Dpc4 expression is useful for distinguishing metastatic pancreatic carcinomas in the ovary from both primary ovarian mucinous tumors and metastatic mucinous carcinomas derived from other sites.

We encountered an extremely rare case of multiple pulmonary sclerosing hemangiomas (PSH) with extensive neuroendocrine lesions, including pulmonary neuroendocrine cell (PNC) hyperplasia, multiple carcinoid tumorlets and typical carcinoid tumors within one pulmonary lobe. To the best of our knowledge, this is the first reported case in the English medical literature of PSH combined and admixed with carcinoid tumors and extensive neuroendocrine proliferation. This case is noteworthy for several reasons. First, the lesion is multi-nodular and unusually large for a typical PSH, which may mimic malignancy on imaging studies and cause diagnostic difficulties. Second, sampling bias may lead to diagnostic errors for a lesion containing two different types of neoplasms. Third, our case displays a rare mixed and mosaic pattern of PSH with a full spectrum of pulmonary neuroendocrine lesions, which may imply a potential intrinsic association in pathogenesis between PSH and concomitant neuroendocrine neoplasms. The clinical implication of multiple PSHs is also discussed.

Background/Aims: We have previously reported a recombinant vaccinia-based vaccine (vac-Sig/E7/LAMP-1) that demonstrated a significant anti-tumor effect in a subcutaneous tumor challenge model. Since the liver is one of the most common sites for tumor metastasis and organ microenvironments may modulate tumor cell responses to therapies, the aim of the present study was to evaluate the potency of vac-Sig/E7/LAMP-1 in treating E7-expressing tumors grown in the liver. Methods: For in vivo tumor prevention experiments, mice were vaccinated intraperitoneally with vac-Sig/E7/LAMP-1 followed by intrahepatic tumor challenge. For in vivo tumor regression experiments, mice were first challenged with tumor cells and then vaccinated with vac-Sig/E7/LAMP-1 intraperitoneally. In addition, enzyme-linked immunospot assays were used to determine the frequency of E7-specific T cell precursors. Results: For in vivo tumor protection experiments, tumor growth was observed in all of the mice vaccinated with wild-type vaccinia and 60% of the mice vaccinated with wild-type E7 vaccinia. All of the mice vaccinated with vac-Sig/E7/LAMP-1 remained tumor-free 30 days after tumor challenge. For the tumor regression assays, all of the mice vaccinated with vac-Sig/E7/LAMP-1 remained tumor-free 30 days after vaccination. In contrast, all of those mice receiving culture medium, wild-type vaccinia, or wild-type E7 vaccinia developed tumors in the liver. In addition, mice vaccinated with vac-Sig/E7/LAMP-1 had the highest E7-specific CD8 + T cell precursors. Conclusions: Our data suggest that vac-Sig/E7/LAMP-1 is an effective vaccine for controlling E7-expressing tumors grown in the liver and our model suggests that antigen-specific immunotherapy may represent a powerful tool for treating liver tumors with characterized tumor-specific antigens. In addition, our data indicate that the number of E7-specific CD8 + T cell precursors directly correlated with the anti-tumor effect generated by Sig/E7/LAMP-1 vaccinia.

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-transduced autologous tumor cell-based vaccines are currently one of the major forms of cancer vaccines. However, the preparation of GM-CSF-transduced autologous tumor vaccines is time-consuming and technically challenging. In addition, the host antigen presenting cells, rather than the tumor vaccine cells themselves, present tumor-specific antigens and prime the host T cells. Therefore, we tested the efficacy of antigen-specific allogeneic tumor vaccines. We used human papillomavirus 16 (HPV-16) E7 protein as a model tumor antigen, which is associated with the development of most cervical carcinoma. B16, a C57BL/6 (H-2(b)) derived melanoma cell line, was genetically engineered to produce GM-CSF alone (B16GM), HPV-16 E7 alone (B16E7), or both (B16GME7). These vaccine cells were injected into BALB/c (H-2(d)) mice (10(6) cells/mouse). Two weeks later, mice were challenged with 10(5) live HPV-16 E7(+) BL-1 (H-2(d)) tumor cells and monitored for tumor progression twice weekly. To determine the effective cell population in the antitumor immunity elicited by B16GME7, we carried out in vivo antibody depletion experiments using CD4 and CD8 specific antibodies. In addition, as a measure of the immune responses produced by B16GME7, we performed an in vitro cytotoxic T lymphocyte assay using a standard chromium release method. We found that all of the mice vaccinated with B16GME7 remained tumor free 49 days post-BL-1 challenge. In contrast, mice vaccinated with B16GM and B16E7 did not show any tumor protection against a similar dose of BL-1 cells. Furthermore, the antitumor immunity produced by B16GME7 was dependent on both CD4 and CD8 T cells. In addition, E7-specific cytotoxic T lymphocyte activity could be readily demonstrated in mice immunized with B16GME7. These results suggest that allogeneic tumor cells transduced with GM-CSF and the tumor antigen, HPV-16 E7, cannot only generate an E7-specific cytotoxic T lymphocytes response in vitro, but can also elicit a potent antitumor immune response against an E7 expressing tumor in vivo.

DNA vaccination is an attractive approach for tumor immunotherapy because of its stability and simplicity of delivery. Advances demonstrate that helper T cell responses play a critical role in initiating immune responses. The aim of the current study is to test whether targeting HPV-16 E7 to the endosomal/lysosomal compartment can enhance the potency of DNA vaccines. We linked the lysosome-associated membrane protein 1 (LAMP-1) to HPV-E7 to construct a chimeric DNA, Sig/E7/LAMP-1 DNA. For in vivo tumor prevention experiments, mice were vaccinated with E7 DNA or Sig/E7/LAMP-1 DNA via gene gun, followed by tumor challenge. For in vivo tumor regression experiments, mice were first challenged with tumor cells and then vaccinated with E7-DNA or Sig/E7/LAMP-1 DNA. Intracellular cytokine staining with flow cytometry analysis, cytotoxic T lymphocyte (CTL) assays, enzyme-linked immunoabsorbent assay (ELISA), and enzyme-linked immunospot (ELISPOT) assays were used for in vitro E7-specific immunological studies. In both tumor prevention and tumor regression assays, Sig/E7/LAMP-1 DNA generated greater antitumor immunity than did wild-type E7 DNA. In addition, mice vaccinated with Sig/E7/LAMP-1 DNA had greater numbers of E7-specific CD4+ helper T cells, higher E7-specific CTL activity, and greater numbers of CD8+ T cell precursors than did mice vaccinated with Sig/E7 or wild-type E7 DNA. Sig/E7 generated a stronger E7-specific antibody response than did Sig/E7/LAMP-1 or wild-type E7 DNA. Our results indicate that linkage of the antigen gene to an endosomal/lysosomal targeting signal may greatly enhance the potency of DNA vaccines.

An important goal of cancer immunotherapy is to prevent and treat tumor metastasis. We have previously reported a recombinant vaccinia-based vaccine (Sig/E7/LAMP-1) that demonstrated significant anti-tumor effect in a subcutaneous tumor challenge model. In this study, we investigated the potency of the Sig/E7/ LAMP-1 vaccine in preventing and treating metastatic tumors. A tumor metastasis model was generated by injecting human papillomavirus type 16 (HPV-16) E6/E7 expressing tumor cells, designated TC-1, into the tail vein of syngeneic C57BL/6 mice. All the naive mice injected with 1 x 10(6) TC-1 cells developed tumors confined exclusively to the lungs within 1 month. For in vivo tumor prevention experiments, mice were vaccinated with Sig/E7/ LAMP-1 followed by tumor challenge. While tumor growth was observed in all of the mice (10/10) in the control groups, 8 of 10 vaccinated mice (80%) remained tumor-free 2 months post-tumor challenge. For in vivo treatment experiments, mice were first inoculated with TC-1 cells and then vaccinated with Sig/E7/ LAMP-1. Treatment with Sig/E7/LAMP-1 was effective in eliminating preexisting tumor cells in 4 of 5 vaccinated mice. Most importantly, treatment with Sig/E7/LAMP-1 resulted in regression of fully established lung tumors in 10% (5/10) of vaccinated mice. Our data suggest that the Sig/E7/LAMP-1 vaccine is effective in controlling the hematogenous spread of TC-1 tumor cells. In addition, the TC-1 lung metastasis model can be used to test the efficacy of various E6/E7-specific vaccines and immunotherapeutic strategies.

A series of 202 breast cancer biopsy specimens were analysed immunohistochemically for collagen IV to demonstrate basement membrane (BM) structures and blood vessels within tumour tissue. Integrity of the BM was graded into four categories and the number of vascular channels per square millimetre of tumour tissue were counted. Defective BM structures were significantly related to high grade, lack of tubule formation, invasive disease, high S-phase fraction and variability in nuclear size and shape. High vascular channel density was related to poor tumour differentiation and a high proliferation rate of cancer cells as well as to the absence of tubule formation, inconspicuous intraductal growth and low progesterone receptor content. High vascular density and defective BM structures were signs of poor prognosis and short recurrence-free survival in the entire cohort and also in local tumours. In multivariate analysis, the vascular density had independent prognostic value, as did the diameter, axillary lymph node status and mitotic rate. The counting of vascular channels within the tumour provides additional prognostic information in breast cancer, in contrast to analysis of the BM integrity which shows hardly any prognostic information additional to that provided by the special histological features, e.g. tubule formation and intraductal growth pattern.

Formalin-fixed, paraffin-embedded biopsies of 193 women with primary breast cancer followed-up for over 10 years were analysed immunohistochemically for the expression of p53 protein. Altogether, 58% (113/193) of the tumors were positive for p53 protein. Over-expression of p53 was associated with the ductal type, high-grade tumors, dense stromal inflammatory cell infiltrate, high S-phase fraction, high mitotic frequency and high values of the nuclear factors. In univariate analysis, intense p53 over-expression predicted a poor outcome, whereas a short recurrence-free survival (RFS) was related to p53 negativity. In axillary lymph-node-negative (ANN) tumors, p53 negativity was related to short RFS, and in axillary lymph-node-positive (ANP) tumors this inverse relationship was statistically significant. In Cox's analysis, p53 protein over-expression had no independent prognostic value comparable with the well-established prognostic factors. However, p53 protein accumulation was an independent indicator of long RFS in the entire cohort, in ANP tumors and in rapidly proliferating tumors. The results indicate a dual role for p53 protein over-expression in breast cancer prognosis. The low survival probability associated with intensively p53-positive tumors is probably related to rapid cancer-cell proliferation, whereas the long RFS of p53-positive tumors might be explained by the development of circulating antibodies to p53 protein. The role of p53 protein in breast cancer is incompletely understood, and the p53 gene should be subjected to detailed analysis of specific mutations. © 1993 Wiley-Liss, Inc.

Summary As a part of a larger programme to search for the prognostic factors in cervical cancer, quantitative xzorphonxetry, demonstration of AgNORs and expression of different cytokeratin polyprptide (SK2–27, SK1, A 53–B/A2) were used to study a series o/85 cervical squamous cell carcinomas, previously analysed for the presence of human papillomavirus (HPV) DNA by in situ hybridization and polymonarase chain reaction (PCR). The following nuclear profile parameters were calculated: nuclear area, perimeter, maximum diameter, ellipsoidity (form Pe), regularity (form Ar) and roundness (form Pe). In each case, the number of small ( 3 µm), the total number and the ratio large/small AgNORs were registered. The cancer cell density and the lymphoid cell density were assessed. In the survival analysis, neither the expression of different cytokeratin polypeptides or the pattern of cytokeratin staining proved to be an independent variable. Similarly, none of the nuclear profile parameters analysed possessed an independent prognostic value in the survival analysis. The ratio of large/small AgNORs proved to be a significant independent prognostic predictor (p = 0. 0104), second only to the lymphoid cell density. Also the total number of AgNORs was a prognostic indicator. This suggests that AgNOR size and ratio reflect tumor proliferation also in cervical squamous cell carcinoma, as shown in other human malignancies. Similarly, the density of cancer cell nuclei proved to bean independent prognostic predictor (p=0.0601) in that the tumours in patients with longer survival showed lower density of the nuclei. On the contrary, higher density of peritumoral lymphoid cell nuclei mascorrelated with longer survival up to the point that the intensity of such infiltrate was the most powerful single prognostic factor in the present series (p=0.0026). The mechanism responsible for this prognostic significance of the lymphoid cell infiltration needs further assessment, e.g. subpopulation analysis by monoclonal antibodies, and functional tests of these subpopulations. The results clearly indicate that histoquantitative, morphometry and cytokeratin expression seem to be of little or no value as prognostic predictors in cervical cancer, and further work is still needed to elucidate the factors determining the disease outcome more accurately than the currently available means.

To evaluate flow rate dependence of CO2 gas and saline perfusion for sapphire probe ablation, all together 204 human arterial specimens of atheroma and normal vessel were irradiated with Nd-YAG laser, in an experimental circulation-occlusion model within 37°C flowing whole blood. During lasing procedures, various flow rates of CO2 gas (0.2–2.01 min−1) and saline (2.0–20.0 ml min−1), and various lasing powers (7, 12 and 17 W) and lasing time (1–20 s) were used. Histological changes of all specimens irradiated were microscopically examined. The results showed that the laser ablation area enlarged with increasing CO2 flow rates and decreasing saline flow rates. Relative ablation efficiency on atheromatous plaque, in comparison to those on normal vessel wall and surrounding tissue site, increased slightly with increasing lasing power and lasing time. In this experimental setting, the mode of action of the sapphire probe ablation on human arterial atheroma seems to be more satisfactory with CO2 gas perfusion than with saline perfusion.

Uterine serous carcinomas typically have a characteristic morphology (papillary architecture, high-grade nuclei) and immunoprofile (diffuse/strong p53 expression, loss of hormone receptor expression) that distinguish them from most endometrial endometrioid carcinomas. However, glandular variants of serous carcinoma can simulate Federation Internationale de Gynecologie et d'Obstetrique (FIGO) grade 2 endometrioid carcinomas, and some serous carcinomas lack p53 expression and retain hormone receptor expression, making classification difficult. P16 expression patterns distinguish endometrioid carcinomas (patchy) from human papillomavirus (HPV)-related endocervical adenocarcinomas (diffuse/strong) but utility for distinction of serous carcinomas from endometrioid carcinomas and endocervical adenocarcinomas has not been evaluated in a large series. Immunohistochemical analysis of p16 expression was performed on 201 uterine and endocervical adenocarcinomas in hysterectomy specimens, including 49 serous carcinomas, 101 endometrial endometrioid carcinomas (44 FIGO grade 1, 40 FIGO grade 2, and 17 FIGO grade 3), and 51 HPV-related endocervical adenocarcinomas. All serous carcinomas demonstrated diffuse/moderate-strong p16 expression, with percentage of positive tumor cells ranging from 90% to 100% (mean/median: 95%/100%). In contrast, endometrial endometrioid carcinomas exhibited less diffuse and less intense expression, with percent of positive tumor cells ranging from 10% to 90% (mean/median: 38%/30%; staining intensity: variable). Similar to serous carcinomas, all endocervical adenocarcinomas exhibited diffuse/moderate-strong p16 expression, with percentage of positive tumor cells ranging from 90% to 100% (mean/median: 94%/90%). P16 can serve as an additional diagnostic marker, used as part of an immunohistochemical panel, including p53 and hormone receptors, for distinction of uterine serous carcinomas from endometrioid carcinomas. Distinction of serous carcinomas from endocervical adenocarcinomas (HPV-related type), both of which share diffuse p16 expression and frequently lack hormone receptor expression, relies on morphology and diffuse/strong p53 expression in the former and detection of HPV in the latter.

A long-term prospective follow-up program has been conducted for genital human papillomavirus (HPV) infections in 532 women in Kuopio since 1981. By regular examinations (at 6-month intervals) using colposcopy, cytology and/or punch biopsy, 426 of these women have been followed up for the minimum of 48 months (mean 71 +/- 19 months). These 426 women were included in the present report. The colposcopic data of all women at the first and the latest visits were recorded from the colpophotographs, analysed and correlated with cytology, histology, HPV DNA detection and clinical behavior (i.e. prognosis) of the disease. Colposcopic abnormalities were found in 313 (73.5%) women at the first visit and in 138 (32.4%) patients at the latest visit. HPV infections were diagnosed in 368 (86.4%) women at the first visit and in 75 (17.9%) at the latest visit on light microscopy (i.e. biopsy and/or PAP smear). CIN was found in 54.7% of the women at the first visit. HPV DNA was detected by in situ hybridization in 38.2% of the biopsies assayed. Colposcopical multicentric lesions were found in one third of the women upon examination of the entire lower genital tract. The majority of the lesions were situated within the transformation zone. HPV infection and CIN were most frequently encountered in women with multiple lesions. Progression to CIN III was most frequent in large lesions (28.6%). No colposcopic pattern was able to distinguish HPV infections from CIN lesions, or predict the progression of the former. The results implicate that: (1) colposcopy is an essential method to disclose abnormalities due to CIN and HPV infections, but histological changes cannot be predicted on a single colposcopy; (2) HPV infection is a multicentric disease, making the careful examination of the entire lower genital tract mandatory on every colposcopy; and (3) the HPV progressors cannot be predicted on the basis of the colposcopic patterns.

Eighty-one women treated for an invasive squamous cell carcinoma of the uterine cervix during the period from 1964 to 1987 were studied to assess the prognostic value of human papillomavirus (HPV) types 6,11, 16 and 18, as well as the nuclear DNA ploidy pattern, analyzed using HPV amplification by polymerase chain reaction (PCR) and flow cytometry, respectively. The mean age of the women was 57.7 ± 13.4 years, and the mean follow-up until the patients death or January 1990 was 99 ± 87 months. Altogether, 46 women died; 38 (82.6%) of these deaths were due to cervical cancer. The 5-year survival was significantly correlated with age (P= 0.01), and the FIGO stage of the tumors (P= 0.015), but not with tumor differentiation. Diploid tumor was found in 40 (63.5%) cases, and aneuploid in 23 (36.5%) cases. A DNA index 3= 1.5 was found in 47.8% (11/23) of the cases of aneuploid tumors. The 5-year survival rate in diploid tumors was 60.0% (21/35), as compared to 54.5% (12/22) in aneuploid ones, and in patients with a DNA index of < 1.5, the 5-year survival rate was 58.7% (27/46), as compared to 54.5% (6/11) in those with a DNA index ≥ 1.5. Amplified HPV DNA was found in 30 cases (37.0%) with the pairs of HPV DNA primers for HPV types of 6, 11, 16 and 18. When repeated with the anticontamination primers, only 19 (23.5%) cases remained HPV DNA positive. HPV 16 was the most frequent type present in 57.9% (11/19) of the cases, followed by HPV 18 in 36.8% (7/19). Neither the HPV DNA-positivity nor HPV type proved to be of prognostic significance. The results suggest that despite an intimate association of HPV 16 and 18 in cervical carcinogenesis, the presence of their DNA in cancer biopsies does not seem to have any prognostic value. The most significant prognostic factors are still the age and the FIGO stage at diagnosis. Aneuploid tumors or those with DNA index ≥ 1.5 seem to have a slightly (not statistically significantly) impaired prognosis as compared with the diploid tumors and those with DNA index < 1.5.

CO2 gas has been proposed as a new perfusion medium for laser angioplasty. To compare CO2 gas with conventional saline perfusion, 146 fresh specimens of normal and atheromatous human artery were irradiated with a neodymium: yttrium-aluminum-garnet laser with a sapphire probe in flowing whole blood in an experimental circulation-occlusion model. The dimensions of the ablation crater and the extent of the surrounding tissue damage were measured microscopically. Significantly better ablation of atheromatous plaque was achieved with CO2 perfusion than with saline perfusion: mean ablation areas were 5.0 mm2 versus 2.8 mm2, respectively (P = .001, Student t test). In contrast, the ablation areas on normal vessel wall were identical (mean, 3.4 mm2) with the two perfusion media. Moreover, CO2 gas functioned as a negative contrast agent and facilitated direct monitoring of the laser recanalization procedure. On an experimental basis, CO2 gas perfusion seems to improve the efficiency and safety of laser ablation in human arteries.

To assess the prevalence of HPV infection in the genital tracts of women with normal PAP smears, a random series of 109 women was reexamined using colposcopy, a further PAP smear, and punch biopsies taken from the cervix (in 33 cases), vagina (212 cases), and anus (20 cases). The biopsy material was examined using routine histological investigations, in situ hybridization (ISH) with a 35S-labelled DNA probe cocktail (HPV 6, 11, 16, 18), and the polymerase chain reaction (PCR) to detect HPV DNA. Changes consistent with HPV infection were seen in 6.9% (18/262) of the biopsy specimens. Seven biopsy specimens (2.7%) from seven different women were found to contain HPV DNA using ISH. All of these ISH-positive lesions were diagnosed as morphologically characteristic HPV lesions: six flat condylomas and one papillary condyloma. Using PCR, the HPV DNA detection rate was highest in the cervical biopsy specimens (50%) and lowest (28.6%) in the anal biopsy specimens. A total of 35.5% of the 93 biopsy specimens studied using PCR contained HPV DNA. The commonest type was HPV 11 (54.5%), followed by HPV 18 (33.3%). Four of the nine biopsy specimens (44.4%) from colposcopically normal areas proved HPV DNA-positive using PCR. Of 17 biopsy specimens in which the histology was normal, seven were examined using PCR and three were DNA-positive. The discovery of HPV DNA using PCR in 32/92 of the biopsy specimens (34.8%) which had been found to be HPV DNA-negative when routine ISH was used is noteworthy. The results suggest that the light microscopy criteria currently used in diagnosing HPV infections are of no value in predicting latent HPV infections and that acetowhite staining is unable to distinguish between subclinical and latent infections on the one hand and changes unrelated to HPV on the other.

The effectiveness of CO2 gas as a perfusion medium was compared to that of saline in laser ablation of human atheromatous plaque. In an experimental circulation-occlusion model using flowing whole blood, human cadaveric arterial samples were irradiated by a sapphire probe with the Nd-YAG laser. The following experiments were performed: 1) lasing without perfusion, 2) lasing with saline perfusion of the probe, and 3) lasing with CO2 perfusion. Different perfusion flow rates of saline and CO2 were used. Results showed that the mean ablation area was 1.6-fold larger with CO2 than with saline perfusion (P less than 0.05, Student's t test). The mean lateral injury at the site adjacent to the ablation crater and at the area directly facing the probe was not significantly different with either perfusion medium. The larger ablation area with CO2 was probably due to the fact that CO2 is a good insulator for maintaining a higher probe temperature and keeps the probe free of blood debris. In conclusion, our results show that CO2 perfusion facilitates more effective laser ablation of atheromatous plaque than saline perfusion by the sapphire probe with the continuous wave Nd-YAG laser.

The claudin (CLDN) genes encode a family of proteins involved in the formation and function of tight junctions. CLDN gene expression is frequently altered in several human cancers, and in particular, CLDN3 and CLDN4 are commonly overexpressed in ovarian cancer. However, the mechanisms leading to the deregulation of these genes in cancer remain unclear. In the present study, we have examined the CLDN3 promoter and have identified a minimal region containing an Sp1 site crucial for its activity. In addition, we find that the CLDN3 promoter is regulated through epigenetic processes. Cells that express high levels of CLDN3 exhibit low DNA methylation and high histone H3 acetylation of the critical CLDN3 promoter region, and the reverse is observed in cells that do not express this gene. CLDN3-negative cells can be induced to express CLDN3 through treatment with DNA methyltransferase or histone deacetylase inhibitors. Interestingly, in vitro binding experiments, as well as chip assays show that Sp1 binds the unmethylated promoter much more efficiently, providing a mechanism for CLDN3 silencing in non-expressing cells. Finally, siRNA-mediated knockdown of Sp1 led to a significant decrease of CLDN3 expression at both the mRNA and protein levels, demonstrating a crucial role for this transcription factor in the regulation of CLDN3. Our data provide a basis for CLDN3 expression in ovarian cancer cells, as well as a mechanism for the silencing of this promoter in tumors lacking expression of claudin-3.

Human papillomavirus (HPV) E6 and E7 are consistently expressed and are responsible for the malignant transformation of HPV-associated lesions. Thus, E6 and E7 represent ideal targets for therapeutic HPV vaccine development. We have previously used the gene gun approach to test several intracellular targeting and intercellular spreading strategies targeting HPV-16 E7. These strategies include the use of the sorting signal of lysosome-associated membrane protein (LAMP-1), Mycobacterium tuberculosis heat shock protein 70 (HSP70), calreticulin (CRT) and herpes simplex virus type 1 (HSV-1) VP22 proteins. All of these strategies have been shown to be capable of enhancing E7-DNA vaccine potency. In the current study, we have characterized DNA vaccines employing these intracellular targeting or intercellular spreading strategies targeting HPV-16 E6 for their ability to generate E6-specific CD8+ T cell immune responses and antitumor effects against an E6-expressing tumor cell line, TC-1, in C57BL/6 mice. We found that all the intracellular targeting strategies (CRT, LAMP-1, HSP70) as well as the intercellular spreading strategy (VP22) were able to enhance E6 DNA vaccine potency, although the orientation of HSP70 linked to E6 antigen in the E6 DNA vaccine appears to be important for the HSP70 strategy to work. The enhanced E6-specific CD8+ T cell immune response in vaccinated mice also translated into potent antitumor effects against TC-1 tumor cells. Our data indicate that all of the intracellular targeting and intercellular spreading strategies that have been shown to enhance E7 DNA vaccine potency were also able to enhance E6 DNA vaccine potency.

We report the pathologic finding of a multinucleated foreign body giant cell reaction to squames and fetal hair in the placental membranes in a 37-week 1-day intrauterine gestation. This reaction appeared to have developed in association with repeated intrauterine procedures performed in the third trimester, including cordocentesis for fetal blood sampling, intrauterine blood transfusion, and amnioreduction for polyhydramnios. This type of reaction most likely was directed to prolonged amniotic fluid leakage that occurred spontaneously or after intrauterine procedures in the second half of the second trimester and the third trimester. Careful examination of the placental membranes and recognition of the foreign body giant cell reaction may provide etiologic insight in cases of unexplained oligohydramnios.

Objective. To evaluate the clinico-pathologic characteristics and survival outcome associated with overexpression of Her-2/neu in patients with uterine serous carcinoma (USC). Methods. Twenty-five patients with a confirmed pathologic diagnosis of USC and available paraffin embedded tissue samples treated at the Johns Hopkins Medical Institutions from 1/1/1992 through 12/31/2000 were identified retrospectively. Her-2/neu expression was evaluated by immunohistochemistry using HercepTest (DAKO). Clinical data were abstracted from medical records. Clinico-pathologic characteristics associated with Her-2/neu overexpression like staging, histology, lymph-vascular space involvement, and myometrial invasion were evaluated using logistic regression analysis and Fisher's exact test. Analyses of overall survival time were performed using the Kaplan–Meier method and Cox proportional hazards regression models. Results. Twelve (48%) of the 25 USC cases demonstrated Her-2/neu overexpression. There was a significant difference in Her-2/neu overexpression and surgical staging (81.8% vs. 28.6%, P = 0.01). Survival analysis according to primary tumor characteristics revealed that overexpression of Her-2/neu was significantly associated with a worse survival outcome (HR = 6.58, 95%CI: 1.36–31.89, P = 0.02). Conclusions. Her-2/neu overexpression is associated with advanced surgical stage USC and poor survival outcome. These data may be useful in guiding the clinical management of patients with USC and have potential implications for the development of novel treatment strategies.

Metastatic pancreatic mucinous adenocarcinomas in the ovaries can be difficult to distinguish from primary ovarian mucinous neoplasms because the former can simulate the latter grossly and histologically and both tumor types share the same cytokeratin 7/cytokeratin 20 immunoprofile. We previously reported the utility of loss of Dpc4 expression in distinguishing metastatic pancreatic carcinomas from primary ovarian mucinous tumors. Recently several new pancreatic carcinoma markers have been identified, including mesothelin, fascin, and prostate stem cell antigen (PSCA). In this study we investigate the expression patterns of these markers in 35 primary ovarian mucinous tumors (28 atypical proliferative [borderline] tumors and 7 invasive carcinomas) and 11 metastatic pancreatic mucinous carcinomas in the ovary. Primary ovarian mucinous tumors expressed mesothelin (17%), fascin (26%), and PSCA (43%) less frequently than metastatic pancreatic adenocarcinomas (73%, 73%, and 82%, respectively). Expression of all three markers was seen only in metastatic pancreatic adenocarcinomas (45%), and coexpression of at least two markers was observed significantly more frequently in metastatic (82%) than primary ovarian mucinous tumors (17%). Our results indicate that an immunohistochemical panel including Dpc4, mesothelin, fascin, and PSCA is useful for evaluating difficult mucinous tumors in the ovary when the differential diagnosis includes metastatic pancreatic adenocarcinoma.

Tumor formation in immunocompetent hosts is believed to be dependent on the ability of tumor cells to evade the immune system, as suggested by the alterations of expression of the major histocompatibility complex (MHC) and related molecules in a number of cancers. Our previous serial analysis of gene expression (SAGE) study revealed that HLA-DRA (encoding the alpha chain of HLA-DR) is one of the most highly overexpressed genes in ovarian cancer. This finding was unanticipated, as overexpression of MHC molecules would be expected to increase tumor immunogenicity, therefore compromising tumor growth. We have now examined the expression of HLA-DR alpha chain in ovarian and a variety of other cancers using tissue arrays and found it overexpressed in a majority of the cancer tissues investigated. In contrast, the HLA-DR beta chain, which together with the alpha chain forms the functional HLA-DR complex, was not frequently found expressed in cancer, resulting to a lack of mature HLA-DR in these tissues. Interestingly, HLADRA and HLADRB transcripts were both found expressed in many other cancer types, including ovarian cancer, suggesting that the downregulation of HLADR beta chain is a post-transcriptional or post-translational mechanism. In addition, we observed high levels of the invariant chain (Ii/CD74) expression in both the cytoplasm and plasma membrane of ovarian tumor cells, possibly contributing to the lack of mature HLA-DR protein expression. Interestingly, we found that IFN-gamma could induce mature HLA-DR at the surface of normal ovarian cells, while this ability was reduced in tumor cells. Together, these data suggest that, while ovarian tumors overexpress HLA-DR alpha, perhaps as a result of inflammatory events in the tumor microenvironment, the tumor cells may have compensatory mechanisms to reduce the production of functional MHC class II molecules, thus reducing immunogenicity and favoring tumor growth. In addition, because of its ubiquitous expression in ovarian and other cancers, HLA-DR alpha may represent a novel biomarker for malignancy.

Intravascular magnetic resonance (MR) imaging to guide interventional procedures is a rapidly growing field. A primary concern with these new techniques is their thermal safety. The purpose of this study was to evaluate, in vivo, the thermal effect of an MR imaging-guidewire (MRIG) for intravascular MR imaging (IVMRI).Two indications of potentially adverse local heating were investigated: blood coagulation disorders and pathologic changes in target vessels. Experiments were performed on ten rabbits with a 1.5 T MR scanner. Using a 0.64-mm MRIG as the RF receiver, we imaged the target aorta using a fast spin-echo pulse sequence with an average specific absorption rate (SAR) of 0.6 W/kg. The total MR imaging time was approximately 70 minutes.There were no abnormal value changes of the coagulation factors between pre- and post-IVMRI, no clinical manifestations of blood coagulation disorders, and, histopathologically, no thermal damage in target vessels.This study demonstrates, from a pathophysiological point of view, the potential safe use of the MR imaging-guidewire for intravascular MR imaging. Further study is required to precisely define the boundaries of these safe operating parameters.

Antigen-specific cancer immunotherapy involves the delivery of tumor-associated antigen to the host for the generation of tumor-specific immune responses and antitumor effects. We hypothesized that different delivery systems may influence the pattern of antigen-specific immune response and the outcome of antitumor effect. We therefore evaluated recombinant vaccinia virus and naked DNA for the generation of antigen-specific immune responses and antitumor effects. We previously found that recombinant vaccinia and naked DNA vaccines containing the chimeric Sig/E7/LAMP-1 gene were capable of controlling the growth of HPV-16 E7-expressing tumor cells (TC-1). In this study, we performed a head-to-head comparison of optimized delivery of Sig/E7/LAMP-1 vaccinia and DNA vaccines using dose-escalating tumor challenge. At a dose of 1 x 10(6) TC-1 cells per mouse, Sig/E7/LAMP-1 DNA provided 100% protection against subcutaneous growth of tumors, while Vac-Sig/E7/LAMP-1 protected only 40% of the mice. Furthermore, Sig/E7/LAMP-1 DNA vaccines are capable of protecting against challenge with a more stringent subclone of TC-1 (TC-1 P2) established from TC-1 tumors that survived initial Sig/E7/LAMP-1 vaccinia vaccination. Immunological assays revealed that both vaccines induced comparable levels of CD8(+) T cell precursors and anti-E7 antibody titers. Interestingly, Sig/E7/LAMP-1 vaccinia induced both E7-specific IFN-gamma- and IL4-secreting CD4(+) T cell precursors while Sig/E7/LAMP-1 DNA induced only E7-specific IFN-gamma-secreting CD4(+) T cell precursors. We also found that IL-4 knockout C57BL/6 mice vaccinated with Sig/E7/LAMP-1 vaccinia exhibited a more potent antitumor effect than vaccinated wild-type C57BL/6 mice in our tumor protection experiments. These results suggest that IL-4 may play a detrimental role in the antitumor effect mediated by vaccinia vaccines. Our findings suggested that DNA vaccines may provide better tumor protection than vaccinia vaccines employing the same gene, which may have implications in the future design of antigen-specific cancer immunotherapy.

DNA vaccination has emerged as an attractive approach for tumor immunotherapy. The aim of this study was to evaluate the potency of DNA vaccines in preventing and treating the liver and lung metastases of a human papillomavirus-16 (HPV-16) E7-expressing murine tumor (TC-1). We used the gene gun method to vaccinate C57BL/6 mice intradermally with DNA vaccines containing the HPV-16 E7 gene, the E7 gene linked to the sorting signals of the lysosome-associated membrane protein-1 (Sig/E7/ LAMP-1), or the ‘empty’ plasmid vector. The in vivo antitumor immunity was analyzed in both tumor prevention and tumor regression experiments. In addition, cytotoxic T lymphocyte (CTL) assays, enzyme-linked immunospot assay and enzyme-linked immunoabsorbent assay were used to assess the E7-specific T cell-mediated and humoral immunity. Mice vaccinated with Sig/E7/LAMP-1 DNA generated the strongest E7-specific CTL activities, the highest numbers of E7-specific CD8+ cell precursors and the highest titers of E7-specific antibodies. While both E7 DNA and Sig/E7/LAMP-1 DNA generated potent antitumor immunity in the liver and lung metastases models, the Sig/E7/LAMP-1 DNA was more potent under stringent conditions. DNA vaccination with E7-expressing plasmids was effective in controlling liver and lung metastases of an E7-expressing murine tumor. Our data suggest that antigen-specific DNA vaccination can potentially be applied to control liver and lung metastases of tumors with defined tumor-specific antigens.

One of the recent controversies with substantial clinical interest is the role of HPV in pathogenesis of ovarian cancer. The available highly conflicting data are based on analysis of 175 ovarian carcinomas so far. As an attempt to further elucidate this issue, the first systematic study of HPV detection in ovarian cancer was carried out using a highly sensitive general primer PCR (confirmed by hybridization for low- and high-risk HPV types separately) in a series of 98 histologically and clinically well-characterized epithelial ovarian malignancies. Despite the high (fg) sensitivity and a wide HPV type coverage of the technique used, all 98 ovarian carcinomas failed to demonstrate any signs of HPV DNA whatsoever. The preexisting 12 reports comprising a total of 175 ovarian tumors analyzed for HPV were summarized, giving highly discrepant results (i.e., detection rates from 0 to 100%) with the overall HPV DNA detection rate of 25.7%. The reasons for these discrepant findings are most probably technical. Our data are consistent with those of the majority of the most recent reports failing to disclose HPV DNA in ovarian neoplasia. The present completely negative results make the authors inclined to conclude that HPV is highly unlikely to play any causal role in the pathogenesis of epithelial ovarian neoplasia.