Novel immunotherapeutic strategies in gynecologic oncology. Dendritic cell-based immunotherapy for ovarian cancer.

The recognition of tumor antigen loaded dendritic cells as one of the most promising approaches to induce a tumor specific immune response in vivo has recently generated widespread interest in the use of these natural adjuvants for the therapy of human malignancies refractory to standard treatment modalities. However, many cancer patients may not benefit from current strategies of cancer vaccination because an effective tumor antigen associated with their cancer has not yet been identified or because sufficient amounts of tumor tissue cannot be obtained for antigen preparation. The recent identification and cloning of a group of preferentially expressed serine proteases as novel ovarian tumor-associated antigens may offer the opportunity to test in a large group of patients the potential of DC-based immunotherapy. In this review, we describe these ovarian tumor antigens and assess the potential for therapeutic DC vaccination for the treatment of chemotherapy-resistant ovarian cancer.

Dosimetric evaluation of lung tumor immobilization using breath hold at deep inspiration.

PURPOSE: To examine the dosimetric benefit of self-gated radiotherapy at deep-inspiration breath hold (DIBH) in the treatment of patients with non-small-cell lung cancer (NSCLC). The relative contributions of tumor immobilization at breath hold (BH) and increased lung volume at deep inspiration (DI) in sparing high-dose lung irradiation (> or = 20 Gy) were examined. METHODS AND MATERIALS: Ten consecutive patients undergoing radiotherapy for Stage I-IIIB NSCLC who met the screening criteria were entered on this study. Patients were instructed to BH at DI without the use of external monitors or breath-holding devices (self-gating). Computed tomography (CT) scans of the thorax were performed during free breathing (FB) and DIBH. Fluoroscopy screened for reproducible tumor position throughout DIBH, and determined the maximum superior-inferior (SI) tumor motion during both FB and DIBH. Margins used to define the planning target volume (PTV) from the clinical target volume included 1 cm for setup error and organ motion, plus an additional SI margin for tumor motion, as determined from fluoroscopy. Three conformal treatment plans were then generated for each patient, one from the FB scan with FB PTV margins, a second from the DIBH scan with FB PTV margins, and a third from the DIBH scan with DIBH PTV margins. The percent of total lung volume receiving > or = 20 Gy (using a prescription dose of 70.9 Gy to isocenter) was determined for each plan. RESULTS: Self-gating at DIBH was possible for 8 of the 10 patients; 2 patients were excluded, because they were not able to perform a reproducible DIBH. For these 8 patients, the median BH time was 23 (range, 19-52) s. The mean percent of total lung volume receiving > or = 20 Gy under FB conditions (FB scan with FB PTV margins) was 12.8%. With increased lung volume alone (DIBH scan with FB PTV margins), this was reduced to 11.0%, tending toward a significant decrease in lung irradiation over FB (p = 0.086). With both increased lung volume and tumor immobilization (DIBH scan with DIBH PTV margins), the mean percent lung volume receiving > or = 20 Gy was further reduced to 8.8%, a significant decrease in lung irradiation compared to FB (p = 0.011). Furthermore, at DIBH, the additional benefit provided by tumor immobilization (i.e., using DIBH instead of FB PTV margins) was also significant (p = 0.006). The relative contributions of tumor immobilization and increased lung volume toward reducing the percent total lung volume receiving > or = 20 Gy were patient specific; however, all 8 of the patients analyzed showed a dosimetric benefit with this DIBH technique. CONCLUSION: Compared to FB conditions, at DIBH the mean reduction in percent lung volume receiving > or = 20 Gy was 14.3% with the increase in lung volume alone, 22.1% with tumor immobilization alone, and 32.5% with the combined effect. The dosimetric benefit seen at DIBH was patient specific, and due to both the increased lung volume seen at DI and the PTV margin reduction seen with tumor immobilization.

Increased expression of protease M in ovarian tumors.

Proteases are known to play important roles in tumor invasion and metastasis. Protease M, which was originally identified by Anisowicz and colleagues in 1996, is a new member of the serine protease family. We also identified the protease M transcript in a differential PCR screen of ovarian tumors and have investigated its expression in 44 ovarian tumors (12 low malignant potential tumors, 32 carcinomas) and 10 normal ovaries using quantitative PCR. The PCR product was labeled with (32)P and a phosphoimager was used to determine the relative expression of the protease M gene compared to internal control beta-tubulin. mRNA expression levels of protease M were significantly elevated in 9 of 12 low malignant potential tumors and 30 of 32 carcinomas. Northern blot hybridization showed that the 1.7-kb protease M transcript was abundant in carcinoma but not detected in normal ovary. Immunohistochemical staining of normal ovary and ovarian tumor tissue sections with antibodies generated to protease M derived peptides corroborated the semi-quantitative PCR and Northern analysis data. Our results suggest that protease M is frequently overexpressed in ovarian tumors and may therefore contribute to the invasive nature or growth capacity of ovarian carcinomas.

Ovarian tumor cells express a transmembrane serine protease: a potential candidate for early diagnosis and therapeutic intervention.

Proteases have been implicated in the extracellular modulation required for tumor growth and invasion. In an effort to categorize those proteases contributing to ovarian carcinoma progression, we have utilized redundant primers to conserved amino acid (AA) domains surrounding the catalytic triad of His, Asp and Ser to amplify serine proteases that are differentially expressed in carcinomas. Using this method, we have identified and cloned a serine protease named TADG-15 (tumor-associated differentially expressed gene 15) that is overexpressed in ovarian tumors. TADG-15 is a transmembrane multidomain serine protease which includes ligand binding domains and a serine protease in the extracellular space.

The CA 125 gene: an extracellular superstructure dominated by repeat sequences.

CA 125 has long presented problems to both clinicians and investigators because there was no definitive information on its structure and function. Here, we describe our work on cloning the CA 125 gene with the anticipation that such information will provide the basis for understanding its structure and its physiologic role in both normal and malignant tissues. The CA 125 protein core is composed of a short cytoplasmic tail, a transmembrane domain and an extraordinarily large glycosylated extracellular structure. This structure is dominated by a repeat domain composed of 156 amino acid repeat units which encompass the epitope binding sites. The molecule also includes an amino terminal domain of serine/threonine-rich sequences which would account for most of the O-glycosylation known to be present in CA 125. CA 125 is an unusually large transmembrane glycoprotein. Its release from the surface of the cell is most probably dependent on cytoplasmic phosphorylation followed by proteolytic cleavage. The extracellular domain is characterized by a large number of repeat units (probably 60+) which encompass an interactive disulfide bridged cysteine-loop and the site of OC125 and M11 binding. Sequencing the gene provides us with the ability to initiate the quest to understand the biological function of CA 125.

Expression of the protease inhibitor antileukoprotease and the serine protease stratum corneum chymotryptic enzyme (SCCE) is coordinated in ovarian tumors.

We have previously reported that the stratum corneum chymotryptic enzyme (SCCE) is overexpressed in ovarian cancers and that SCCE has potential as a useful marker and/or a therapeutic target for ovarian carcinoma. Antileukoprotease (ALP) has been shown to be a specific inhibitor of SCCE. The objective of this study was to investigate the potential cotranscription and overexpression of ALP in carcinoma of the ovary. The expression of ALP transcript was evaluated by Northern blot hybridization and by semiquantitative polymerase chain reaction (PCR) technique. The presence of the ALP protein in ovarian tumor cells was evaluated by immunohistochemistry. Northern blot hybridization showed that the ALP transcript was abundant in ovarian carcinomas but was not detected in the normal ovary. Semi-quantitative PCR examination revealed that the mRNA level of ALP was significantly elevated in low-malignant-potential tumors and in ovarian carcinomas compared with that in normal ovaries (P < 0.01). There was significant positive correlation between SCCE and ALP mRNA overexpression status in ovarian tumor cases (P < 0.01). Immunohistochemical expression of ALP protein was observed in ovarian tumor cells, whereas little or no staining was observed in normal ovarian surface epithelium. Like SCCE, ALP is highly overexpressed in ovarian tumor cells, which begs the question of whether it remains an effective inhibitor of SCCE or whether it is discordant in time or space and is ineffective as an inhibitor of the SCCE enzyme.

Overexpression of testisin, a serine protease expressed by testicular germ cells, in epithelial ovarian tumor cells.

OBJECTIVE: In a continued effort to identify and characterize secreted proteases that are overexpressed in ovarian carcinomas, we discovered the testisin protease as such a candidate. When this discovery was originally made, no data existed in the literature or in the GenBank database that identified such a gene. Our main objective was to determine whether this gene was overexpressed exclusively in ovarian tumor tissues compared with normal ovary and whether it was expressed in any other normal tissues. METHODS: mRNA was isolated and cDNA was prepared from 34 ovarian tumors (four adenomas, three low malignant potential tumors, and 27 carcinomas) and seven normal ovaries. The testisin mRNA expression level relative to internal control, beta-tubulin, was determined by Northern blot analysis and semiquantitative polymerase chain reaction (PCR). RESULTS: Northern blot hybridization showed that the testisin transcript was abundant in ovarian carcinoma but was not detected in normal ovary. On examination of Northern blots from normal fetal and adult tissues, only adult testis showed abundant transcripts of testisin. Semiquantitative PCR examination showed that the testisin mRNA levels in ovarian tumors of low malignant potential and in ovarian carcinomas were significantly higher than in normal ovaries (P <.01). Testisin mRNA level in ovarian carcinomas was also significantly higher than in ovarian adenomas (P <.05). Testisin overexpression rates in advanced stage (stage 2 or 3) diseases were significantly higher than that in early stage diseases (stage 1) in ovarian carcinoma samples (P <.05). CONCLUSIONS: The induction of the testisin transcript might contribute to the development, progression, and invasive or metastatic capacity of ovarian carcinomas.

Ovarian tumor cells express a novel multi-domain cell surface serine protease.

Serine proteases serve many functions in normal biological processes. These functions are often usurped by cancer cells to allow progression of tumors by increasing the growth and metastatic potential of the neoplasia. Here, we have used a polymerase chain reaction (PCR)-based strategy to clone Tumor Associated Differentially-expressed Gene-12 (TADG-12), a new serine protease from ovarian carcinoma. This technique also revealed a variant splicing form of TADG-12 that could lead to a truncated protein product. Semi-quantitative PCR showed that TADG-12 is overexpressed in 41 of 55 ovarian cancer specimens relative to normal expression, and the variant form, TADG-12V is found at increased levels in 8 of 22 carcinomas examined. Northern blot revealed three transcripts, the largest of which is approximately 2.4 kb. An ovarian tumor cDNA library was screened, and the entire cDNA of TADG-12 has been identified. This sequence encodes a putative protein of 454 amino acids which includes a potential transmembrane domain, an LDL receptor-like domain, a scavenger receptor cysteine-rich domain, and a serine protease domain. These features imply that TADG-12 will be at the cell surface, and it may be useful as a molecular target for therapy or a diagnostic marker.

The stratum corneum chymotryptic enzyme that mediates shedding and desquamation of skin cells is highly overexpressed in ovarian tumor cells.

BACKGROUND: Proteases play essential roles in the process of tumor invasion and metastasis. The serine protease stratum corneum chymotryptic enzyme (SCCE) has been purified from human stratum corneum and is known to contribute to the cell shedding process by catalyzing the degradation of intercellular cohesive structures at the skin surface. The presence of SCCE on the surface of tumor cells suggests it also may contribute to the process of tumor cell shedding, resulting in early metastasis of carcinoma. METHODS: Gene expression of SCCE was investigated in 44 ovarian tumors (12 low malignant potential tumors and 32 carcinomas) and 10 normal ovaries by quantitative polymerase chain reaction (PCR). The PCR product was labeled with (32)P and a phosphoimager was used to determine the relative expression of SCCE compared with an internal control Beta-tubulin. mRNA transcripts were studied by Northern blot hybridization and protein expression and localization was examined by Western blot analysis and immunohistochemistry. RESULTS: mRNA expression levels of SCCE were elevated significantly in 66.7% of 12 low malignant potential tumors and 78.1% of 32 carcinomas. Furthermore, SCCE protein was abundant in tumor cells and tumor cell lines that overexpressed the mRNA transcript. CONCLUSIONS: The results of the current study suggest that SCCE frequently is overexpressed in ovarian tumors and therefore may contribute to tumor cell growth, tumor spread, and the metastatic potential of ovarian tumor cells.

Cloning of tumor-associated differentially expressed gene-14, a novel serine protease overexpressed by ovarian carcinoma.

The family of enzymes known as serine proteases supports many biological functions for cancer cells, including activation of growth and angiogenic factors and activation of other proteases for invasion and metastasis. In addition, many of these serine proteases are secreted by cells into the extracellular space to serve these functions. Therefore, serine proteases are excellent candidate tumor markers. To examine serine proteases expressed by ovarian carcinoma, we designed degenerate PCR primers corresponding to the conserved regions of these genes and used them in reverse transcriptase-PCR experiments with normal and tumor cDNA as a template. The PCR products were subcloned and sequenced, and one of these clones was found to encode a novel serine protease, named tumor-associated differentially expressed gene-14 (TADG14). Northern blot analysis indicated that the mRNA for TADG14 is 1.4 kb long and that it is highly overexpressed in ovarian carcinoma compared with normal ovary. The entire cDNA has been obtained, and based on sequence homology, it encodes a 260-amino acid serine protease. Semiquantitative PCR indicates that TADG14 is overexpressed in 24 of 40 tumors studied. Northern blot data confirm this overexpression, and immunohistochemical staining suggests that this protein is secreted. As such, the TADG14 protease may be useful as a diagnostic tool or as a molecular target for therapy.

The matrix metalloprotease pump-1 (MMP-7, Matrilysin): A candidate marker/target for ovarian cancer detection and treatment.

Matrix metalloproteases are known to play an important role in tumor invasion by mediating degradation of extracellular matrix. In this study, we have investigated the expression of the matrix metalloprotease pump-1 gene (also referred to as MMP-7, Matrilysin) in 44 ovarian tumors (12 low malignant potential tumors, 32 carcinomas) and 10 normal ovaries using quantitative PCR. The PCR product was labelled with 32P and a phosphoimager was used to determine the relative expression of pump-1 compared to an internal control beta-tubulin. mRNA expression levels of pump-1 were significantly elevated in 9 of 12 low malignant potential tumors and 26 of 32 carcinomas. Northern blot hybridization showed that the 1. 1-kb pump-1 transcript was abundant in carcinoma but seldom expressed in normal adult tissues including normal ovary. Immunohistochemical localization of the pump-1 protein confirms its expression by ovarian tumor cells. Our results suggest that pump-1 is frequently overexpressed in ovarian tumors and may contribute to its invasive nature or growth capacity, therefore pump-1 may serve as a useful marker for early detection of disease and/or a target for therapeutic intervention in downregulation of tumor progression.

Expression of HOXD10 gene in normal endometrium and endometrial adenocarcinoma.

OBJECTIVE: Hox genes encode DNA transcription regulatory proteins that contain a conserved 61 amino acid protein called the homeodomain. Although best known for their role in cellular differentiation during embryonic development, aberrant expression of these genes has been associated with hematologic and solid neoplasms. The purpose of this study was to determine the relative expression of HOXD10 in human endometrial adenocarcinomas. METHODS: mRNA was isolated from 7 normal endometrial specimens and 28 endometrial adenocarcinoma specimens. cDNA was synthesized using random hexamer primers. The expression of HOXD10 relative to beta-tubulin (internal control) was assessed by densitometric comparison of co-amplified Phosphorus-32 (32P) labeled gene products separated by agarose gel electrophoresis. Direct sequencing of purified HOXD10 polymerase chain reaction product was also performed. RESULTS: The sequence of the purified HOXD10 product corresponds to the known DNA sequence reported in the National Institute of Health Gene Bank. mRNA expression of HOXD10 relative to beta-tubulin is significantly lower in endometrial carcinomas than in normal endometrium. Furthermore, the ratio of HOXD10 to beta-tubulin expression varies inversely with the histologic grade of the tumor (P = .0009). CONCLUSION: Cancer is a multistep process involving the aberrant expression of genes that regulate cell growth and differentiation. Human HOXD10 gene expression is altered in endometrial carcinoma and varies with the histologic grade of differentiation. This observation supports the theory that homeobox genes play a role in oncogenesis.