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PubMed2000

Use of PET in neuroendocrine tumors. In vivo applications and in vitro studies.

Positron emission tomography (PET) performed with various radiolabelled compounds facilitates the study of tumor biochemistry. If the tumor uptake of an administered tracer is greater than that of surrounding normal tissue, it is also possible to localize the tumor. In initial studies, 18F-labeled deoxyglucose (FDG) was attempted to visualize the tumors, since this tracer had been successfully used in oncology, reflecting increased glucose metabolism in cancerous tissue. However, this tracer was not to any significant degree taken up by the neuroendocrine tumors. Instead, the serotonin precursor 5-hydroxytryptophan (5-HTP) labeled with 11C was used and showed an increased uptake and irreversible trapping of this tracer in carcinoid tumors. The uptake was selective and the resolution so high that we could detect more liver and lymph node metastases with PET than with CT or octreotide scintigraphy. One problem was, however, the high renal excretion of the tracer producing streaky artifacts in the area of interest. Using the decarboxylase inhibitor carbidopa, given as peroral premedication, the renal excretion decreased 6-fold and at the same time the tumor uptake increased 3-fold, hence improving the visualization of the tumors. When patients were followed during treatment with PET using 5-HTP as a tracer, a > 95% correlation between changes in urinary 5-hydroxyindoleacetic acid (U-5-HIAA) and changes in the transport rate constant for 5-HTP was observed. Thus, PET can be used to monitor treatment effects. Elevation of U-5-HIAA is considered to be uncommon in endocrine pancreatic tumors (EPTs). Initially, 11C-labeled L-DOPA was attempted as another amine important in the APUD system. With L-DOPA about half of the EPTs, mainly functioning tumors, could be detected. Recently, 5-HTP was explored as a universal tracer also for EPT and foregut carcinoids, extending the PET-examination to both thorax and abdomen (whole-body PET-examination). With this method we were able to visualize small lesions in the pancreas and thorax (e.g. ACTH-producing bronchial carcinoids) not detectable by any other method including octreotide scintigraphy, MRI and CT. Several other tracers have been investigated, e.g. the monoamineoxidase (MAO-A) inhibitor harmine with promising results in non-functioning EPTs. We are currently exploring a wide range of biochemical systems, including enzymes and receptors, both for neurotransmitters and for peptides and proteins in in vitro assays with the potential to use some of the developed tracers for in vivo visualization and tumor biological studies. In conclusion, PET is a valuable tool in the diagnosis of neuroendocrine tumors. It can detect small lesions in the thorax and abdomen not detected by other methods, which has been of great value preoperatively in several cases. It detects more lesions in the liver and lymph nodes than other methods and furthermore, it can be used to monitor treatment effects.

Gastrointestinal Neoplasms↗
PubMed2004

Association of matrilysin-2 (MMP-26) expression with tumor progression and activation of MMP-9 in esophageal squamous cell carcinoma.

Expression of matrilysin-2, matrix metalloproteinase (MMP)-26, has been implicated in the progression of several types of human cancer. Matrilysin-2 has been reported to be a physiological and pathological activator of pro-MMP-9. The aim of this study was to examine matrilysin-2 expression and determine whether it is correlated with progression of human esophageal squamous cell carcinoma (ESCC). Semi-quantitative reverse transcriptase-polymerase chain reaction, immunohistochemical analysis, zymography and an in vitro invasion assay were performed. Matrilysin-2 mRNA expression was undetectable or only faintly detected in non-tumor tissues, but its overexpression was detected in 24 of the 50 ESCC tissues. Matrilysin-2 overexpression was significantly correlated with depth of invasion, lymph node metastasis and an advance in pathological tumor node metastasis (pTNM) stage. Sections with immunostaining signals in >10% of carcinoma cells at the invasive front, which were observed in 46 of 100 cases, were judged to be positive for matrilysin-2 expression. Matrilysin-2 expression was significantly correlated with depth of invasion, lymph node and distant metastasis, advance in pTNM stage and recurrence. Expression of matrilysin-2 was significantly correlated with nuclear beta-catenin expression and MMP-9 expression. Patients with matrilysin-2-positive cancer had significantly shorter overall and disease-free survival periods than did those with matrilysin-2-negative cancer. Matrilysin-2 expression retained its significant predictive value for overall and disease-free survival in multivariate analysis. Moreover, patients with concomitant expression of matrilysin-2 and MMP-9 had the worst prognosis. Zymography revealed that matrilysin-2 expression was significantly correlated with expression of active MMP-9 in ESCC tissues. Matrilysin-2-transfected TE-1 ESCC cells showed active MMP-9 activity and were more invasive in vitro compared with mock-transfected TE-1 cells. The results of this study suggest that matrilysin-2, the expression of which is closely correlated with nuclear beta-catenin expression and active MMP-9 activity, plays a key role in the progression of ESCC.

Adenocarcinoma↗
PubMed1988

Structural requirements for hydantoins and 2-thiohydantoins to induce lymphoproliferative popliteal lymph node reactions in the mouse.

The ability of a large number of hydantoins and 2-thiohydantoins to induce primary local lymphoproliferative popliteal lymph node (PLN) reactions has been investigated, as judged by PLN weight enlargement, in an attempt to evaluate the discriminating potential of the PLN reaction to low mol. wt chemicals and to establish structure-activity relationships. Among a series of nineteen hydantoins and related compounds only 5,5-diphenylhydantoin (phenytoin), its major metabolite 5-(p-hydroxyphenyl)-5-phenylhydantoin, 5,5-diphenyl-2-thiohydantoin and N-(5-nitro-2-furfurylidene)-1-aminohydantoin (nitro-furantoin) elicited marked PLN reactions in C57BL/6J mice. In DBA/2 mice, PLN responses to the aforementioned compounds were considerably less or virtually absent. A number of hydantoin derivatives and related compounds with one phenyl group and/or other substituents at the 1,3 or 5 position induced only slightly elevated or suppressed PLN responses in C57BL/6J mice. The influence of polar, and lipophilic aliphatic and aromatic substituents at the 5 position were compared among a series of 22 3-methyl-2-thiohydantoin as well as 21 3-phenyl-2-thiohydantoin amino acid derivatives for their ability to elicit primary PLN reactions in C57BL/6J mice. Substitution with only one aromatic group at the 5 position seemed to be necessary to induce PLN enlargements to 2-thiohydantoins already substituted at the 3 position with a methyl group or even more pronounced when substituted with a phenyl group. p-Hydroxylation of 5-benzyl-3-phenyl-2-thiohydantoin significantly diminished the PLN response. In contrast, p-hydroxylation of one of two phenyl groups as in 5-(p-hydroxyphenyl)-5-phenylhydantoin had little effect on lymphoproliferative PLN reactions. The presence of a hydroxyl group in a non-aromatic cyclic substituent as in hexahydro-6-hydroxy-2-methyl-3-thioxo-1H-pyrrolo[1,2-c]imidazol-1- one had no effect on the PLN reaction. A series of aliphatic substituents in the 5 position of 2-thiohydantoins showed that the number of carbon atoms of the substituents as well as the position of side chains in the isomer, rather than the methyl or phenyl group in the 3 position of the 2-thiohydantoin molecule, determined the strength of the PLN enlargement. It is concluded that the PLN weight increase assay appears to be able to discriminate between subtle chemical differences as studied with a large series of hydantoin and 2-thiohydantoin derivatives. The PLN assay may therefore be useful as a preliminary short-term screening method for identification of (classes of) compounds able to induce lymphoproliferative reactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

4-1 BB stimulation inhibits allergen-specific immunoglobulin E production and airway hyper-reactivity but partially suppresses bronchial eosinophilic inflammation in a mouse asthma model.

BACKGROUND: 4-1 BB, a member of the tumour necrosis factor receptor superfamily, functions as a co-stimulatory molecule. Recently, stimulation of the 4-1 BB pathway was shown to suppress antigen-specific CD4(+) T cell and subsequent T cell-dependent humoral immune responses. OBJECTIVE: We examined the effect of agonistic anti-4-1 BB monoclonal antibody (mAb) treatment on allergic asthma, in which allergen-specific type 2 helper T cells (Th2) have been shown to play an important role. METHODS: BALB/c mice were systemically sensitized with intraperitoneal injections of ovalbumin (OVA) and alum on days 0 and 14, and then challenged with inhaled OVA on days 28, 29 and 30. In test groups, the agonistic anti-4-1 BB mAb was administered at the time of initial systemic sensitization with OVA. On day 31, mice were challenged with inhaled methacholine, and enhanced pause was measured as an index of airway hyper-responsiveness (AHR). Levels of OVA-specific IgE in serum, and levels of various cytokines in bronchoalveolar lavage (BAL) fluids were measured. The severity of airway inflammation was determined by differential cell counts in BAL fluids and histopathologic lung analysis. To evaluate local immunity, we cultured lymphocytes from draining perihilar lymph nodes and evaluated the proliferative response to OVA and the levels of IL-5 in the culture supernatant. In addition, the functional mechanism of 4-1 BB stimulation was evaluated in splenocytes obtained at day 7 after systemic OVA sensitization. RESULTS: We found that treatment with the anti-4-1 BB mAb significantly decreased AHR and the production of allergen-specific IgE. Bronchial inflammation, however, had only partially improved and the levels of IL-4 and IL-5 in BAL fluids showed only a small degree of reduction compared with the control Ig-treated mice. Thoracic lymphocytes from anti-4-1 BB-treated mice showed significant suppression of OVA-induced proliferation and IL-5 production. In anti-4-1 BB-treated mice, splenocytes exhibited poor proliferation and marked apoptosis 7 days after systemic OVA challenge. CONCLUSION: These results suggest that stimulation of the 4-1 BB pathway effectively suppresses some features of allergic asthma, including allergen-specific IgE production and AHR, through deletion of allergen-specific Th2 cells. However, we found that bronchial allergic inflammation was not strictly mediated by suppression of the Th2 immune response in this murine model of asthma. Despite these somewhat contradictory effects, intervention in the 4-1 BB pathway might provide a potential novel immunotherapeutic approach for treatment of allergic asthma.

Allergens↗
PubMed2005

Mast cells protect mice from Mycoplasma pneumonia.

RATIONALE: As the smallest free-living bacteria and a frequent cause of respiratory infections, mycoplasmas are unique pathogens. Mice infected with Mycoplasma pulmonis can develop localized, life-long airway infection accompanied by persistent inflammation and remodeling. OBJECTIVE: Because mast cells protect mice from acute septic peritonitis and gram-negative pneumonia, we hypothesized that they defend against mycoplasma infection. This study tests this hypothesis using mast cell-deficient mice. METHODS: Responses to airway infection with M. pulmonis were compared in wild-type and mast cell-deficient Kit(W-sh)/Kit(W-sh) mice and sham-infected control mice. MEASUREMENTS AND MAIN RESULTS: Endpoints include mortality, body and lymph node weight, mycoplasma antibody titer, and lung mycoplasma burden and histopathology at intervals after infection. The results reveal that infected Kit(W-sh)/Kit(W-sh) mice, compared with other groups, lose more weight and are more likely to die. Live mycoplasma burden is greater in Kit(W-sh)/Kit(W-sh) than in wild-type mice at early time points. Four days after infection, the difference is 162-fold. Titers of mycoplasma-specific IgM and IgA appear earlier and rise higher in Kit(W-sh)/Kit(W-sh) mice, but antibody responses to heat-killed mycoplasma are not different compared with wild-type mice. Infected Kit(W-sh)/Kit(W-sh) mice develop larger bronchial lymph nodes and progressive pneumonia and airway occlusion with neutrophil-rich exudates, accompanied by angiogenesis and lymphangiogenesis. In wild-type mice, pneumonia and exudates are less severe, quicker to resolve, and are not associated with increased angiogenesis. CONCLUSIONS: These findings suggest that mast cells are important for innate immune containment of and recovery from respiratory mycoplasma infection.

Animals↗
PubMed2004

Laparoscopic radical prostatectomy: is intact organ removal attainable? Study of margin status.

PURPOSE: To determine the initial oncologic results (pathology) of specimens removed by laparoscopic radical prostatectomy (LRP) by examining the surgical margins. PATIENTS AND METHODS: The 70 consecutive LRP procedures performed for clinically localized prostate cancer at Eastern Virginia Medical School from April 2001 to November 2002 were reviewed for preoperative and important intraoperative variables. The initial histopathology report and a prospective review by a single genitourinary pathologist for margin status as well parenchymal exposure of benign glands were assessed. Postoperative prostate specific antigen (PSA) levels were noted. RESULTS: The clinical stage distribution was as follows: T1c = 59, T2a = 10, and T2b = 1. The preoperative median PSA value was 6.96 ng/mL. The mean operative time was 307 minutes. The mean estimated blood loss was 298 mL. The prospective pathologic review results were as follows: 1 pT0 (1.4%), 60 pT2 (85.7%), and 9 pT3 (12.8%). The overall positive surgical-margin rate was 15.7%. Of those patients with pT2 disease; 8 specimens (13.3%) had a positive margin, whereas 33% of patients with pT3 disease had a positive surgical margin. Parenchymal exposure of benign glands on the inked surface was recognized in 8 patients (13.3%). Data from PSA assays 1 month postoperatively were available in 69 patients. Serum PSA was undetectable (< or =0.1 ng/mL) in 67 men (97%). CONCLUSION: An LRP can offer surgical margins comparable to those of open procedures series reported in the literature. Long-term progression and survival outcome data are necessary before this procedure should be offered as a replacement for open prostatectomy.

Aged↗
PubMed2002

Urokinase receptor antibody can reduce tumor volume and detect the presence of occult tumor metastases in vivo.

The serine protease urinary plasminogen activator or urokinase (uPA), produced in abundance by many malignancies, plays a key role in tumor cell invasion and metastasis. uPA is localized within the malignant cell milieu via its cell surface receptor [uPA receptor (uPAR)], which is expressed by tumor and tumor-associated cells. In the present study, we have used a syngeneic model of rat breast cancer to directly evaluate the role of uPAR as a diagnostic and therapeutic target in metastatic breast cancer. A polyclonal antibody against the ligand-binding NH(2)-terminal domain of rat uPAR (ruPAR) was developed. This antibody recognizes ruPAR by both immunofluorescence and Western blot analysis. Recombinant ruPAR and ruPAR IgG displaced the binding of (125)I-labeled ruPAR IgG to rat prostate cancer cells (Dunning R3227 Mat Ly Lu) and breast cancer cells (Mat B-III) overexpressing ruPAR (Mat B-III-uPAR). ruPAR IgG also blocked the invasive capacity of these tumor cells in a dose-dependent manner. Mat B-III-uPAR cells were inoculated s.c. into the mammary fat pad of syngeneic female Fischer rats. On day 10 after tumor cell inoculation, animals were injected with (125)I-labeled preimmune or ruPAR IgG and then sacrificed at timed intervals. Maximum (125)I uptake was observed in primary tumors and in tissues commonly affected by tumor metastases (liver, spleen, lungs, and lymph nodes) at 12 h. Injection of (125)I-labeled preimmune or ruPAR IgG into normal non-tumor-bearing animals resulted in minimal basal levels of uPAR expression and established the specificity of the ruPAR IgG. Similar results were obtained by Northern blot and PCR analysis of mRNA isolated from tissues of normal and tumor-bearing animals. To evaluate the effectiveness of this antibody in tumor progression, ruPAR IgG (50-100 microg/day) was injected s.c. for 7 days (day 1-7) at the site of tumor cell inoculation (mammary fat pad), and animals were sacrificed at various time points for evaluation of tumor growth and metastases. Animals receiving ruPAR IgG showed a marked decrease in tumor growth and metastases as compared with control tumor-bearing animals receiving the same dose of preimmune rabbit IgG. Histological analysis of experimental primary tumors showed marked tumor necrosis that was due to increased tumor cell apoptosis as determined by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Together, these studies demonstrate the ability of anti-uPAR antibody to decrease tumor volume and detect the presence of microscopic occult tumor metastases in malignancies where uPA/uPAR play a key role in tumor progression.

Animals↗
PubMed2003

Humoral immune response to a therapeutic polyvalent cancer vaccine after complete resection of thick primary melanoma and sentinel lymphadenectomy.

PURPOSE: A therapeutic polyvalent cancer vaccine (Canvaxin vaccine; CancerVax Corp, Carlsbad, CA) induces antibodies to a glycoprotein tumor-associated antigen (TA90). However, endogenous immune responses to TA90 have also been reported. This study examined anti-TA90 antibody responses with respect to the survival of patients who received adjuvant vaccine immunotherapy after resection of thick (> or = 4 mm) primary cutaneous melanoma. PATIENTS AND METHODS: Serum specimens were obtained from 54 patients immediately before and then 1, 2, 4, and 6 months after wide local excision of thick primary cutaneous melanoma and sentinel lymphadenectomy. All patients were offered adjuvant therapies with the vaccine, high-dose interferon, or other agents. An enzyme-linked immunosorbent assay was used to determine serial serum titers of immunoglobulin G (IgG) and IgM antibodies against TA90. These titers were correlated with clinical course. RESULTS: Forty-three patients chose vaccine therapy, and 11 patients chose postoperative observation. Preoperative anti-TA90 IgG and IgM titers were similar for vaccine and observation groups (P =.184). At a median follow-up of 26 months, univariate analysis of Cox regression showed that disease-free survival and overall survival of vaccine patients were significantly correlated with maximal IgM response (P =.0006 and.006, respectively) but not with maximal IgG response (P =.73 and.95, respectively). Neither response predicted survival in the observation group. CONCLUSION: Postoperative vaccine therapy may enhance IgG and IgM immune responses to TA90 after surgical resection, but only the IgM response is correlated with improved survival. These findings may become useful to guide selection of patients for postoperative adjuvant therapy of high-risk melanoma.

Adult↗
PubMed2005

Detection of occult cancer cells in peripheral blood and bone marrow by quantitative RT-PCR assay for cytokeratin-7 in breast cancer patients.

The clinical significance of occult micrometastasis (O.M) remains unknown. We investigated it in peripheral blood (P.B.) and bone marrow (B.M.) in breast cancer patients with surgery. First, we investigated the expression levels of 7 representative molecular markers for detecting O.M (CEA, CK-7, CK-18, CK-19, CK-20, MAM and MUC-1) in 27 cancer and 8 non-epithelial cell lines using quantitative RT-PCR (QRT-PCR), and showed that the expression level of CK-7 was higher in every cancer cell line than in the non-epithelial cell lines. Next, we studied the clinical significance of O.M in P.B. and B.M. by QRT-PCR for CK-7 in breast cancer patients with surgery. Based on comparison with 17 non-cancer controls, 37 (18.0%) and 100 (48.5%) of the 206 patients were positive for CK-7 in P.B. and B.M., respectively. In 98 cases observed over 24 months after surgery, the CK-7-positive group in P.B. had poorer disease-free survival (DFS) than the negative group (p<0.01). The CK-7-positive group in P.B. showed poorer DFS than the negative group in 132 lymph node-negative cases (p=0.01), and moreover, in 61 lymph node-negative cases observed over 24 months after surgery, the CK-7-positive group in P.B. showed poorer DFS than the negative group (p<0.0001). In B.M., no significant difference in DFS was found between the CK-7-positive and CK-7-negative groups. QRT-PCR for CK-7 could be a useful and universal method for detecting O.M, and the quantitative detection of CK-7 in P.B. would have a prognostic value as a marker of early recurrence in breast cancer patients with surgery.

Adult↗
PubMed2003

In vivo distribution of receptor for ecotropic murine leukemia virus and binding of envelope protein of Friend Murine leukemia virus.

Ecotropic infection by Murine leukemia virus (MuLV) infection is initiated by the interaction between the receptor-binding domain of the viral surface glycoprotein (SU) and the cell-surface receptor, mCAT-1. To study the in vivo localization of viral binding site in mice, green fluorescence protein (GFP)-tagged Friend SU (F-SU/GFP) was incubated with tissue sections. Lymphohematopoietic organs and a part of the glandular tissues of C3H as well as C57BL/6 mice revealed positive signals for F-SU/GFP binding on the cell surface. In contrast, C4W mice, which is a partial congenic mouse strain carrying the Fv-4 (r) gene on a BALB/c genetic background, exhibited negative signals in most of the organs except for a very weak binding in the pancreas. The expression of mCAT-1 mRNA determined by reverse transcriptase (RT)-polymerase chain reaction (PCR) revealed a similar distribution in C3H, C57BL/6 and C4W mice. Most of the organs including lymphohematopoietic organs and glandular organs revealed significant expression of mRNA for mCAT-1 gene, while the liver, heart and muscle did not. The results from binding assay were consistent with the fact that Friend MuLV-induced pathogenesis was usually associated with lymphohematopoietic systems, although mRNA expression for mCAT-1 was rather ubiquitous. The discrepancy between F-SU/GFP binding and mRNA expression for mCAT-1 in lymphohematopoietic organs of C4W mice would support the receptor interference effect by the Fv-4 (r) gene causing the resistance of C4W mouse to Friend MuLV infection.

Animals↗
PubMed2003

Treatment of patients with progressive unresectable metastatic melanoma with a heterologous polyvalent melanoma whole cell vaccine.

Unresectable metastatic melanoma has no elective treatment. Neither chemotherapy, intravenous IL-2 nor biochemotherapy clearly improves the overall survival. Recent assays with therapeutic vaccines have been recently yielded promising results. Here, we describe the application, clinical tolerance and antitumoural activity of a heterologous polyvalent melanoma whole cell vaccine in patients with metastatic melanoma. Twenty-eight AJCC stage III/IV melanoma patients with progressive unresectable metastatic disease were treated with our heterologous polyvalent melanoma whole cell vaccine between July 1, 1998 and July 1, 2002. All patients had already been unsuccessfully treated with high doses of IFN-alpha2 and/or polychemotherapy and/or biochemotherapy and/or perfusion of extremities, or could not receive other treatments due to their age or underlying illness. Twenty-three were assessable. The vaccine was constituted by 10 melanoma cell lines, derived from primary, lymph node and metastatic melanomas. Prior to intradermal inoculation, the cells were irradiated and mixed with BCG, and 50% were treated with DNFB. After a median follow-up of 19 months, 26% of patients responded: 3 CR (18, 16+, and 26+ months), 2 PR (8 and 22 months) and 1 MR (36+ months). The median survival of the whole group was 20.2 months. None of the 28 patients initially included in the study presented significant toxicity. This vaccination program had specific antitumoural activity in advanced metastatic melanoma patients and was well tolerated. The clinical responses and the median survival of our group of patients, together with the low toxicity of our polyvalent vaccine, suggest that this approach could be applied to earlier metastatic melanoma patients.

Adult↗
PubMed1983

Malignant rabbit fibroma virus causes secondary immunosuppression in rabbits.

Shope fibroma virus (SFV) causes a localized, self-limited, fibroblastic proliferation in adult rabbits. Extracts of Shope fibroma tumors were found to contain a second virus that induces a rapidly progressive disseminated tumor. Dissemination of this malignant fibroma is associated with activation of commensal mucosal infection with Pasteurella multocida, causing purulent conjunctivitis and rhinitis and resulting in death from nasal obstruction. We have isolated this new agent by two cycles of plaque purification. It is a poxvirus that is antigenically virtually identical to SFV as measured by a plaque reduction assay, but behaves differently both in vivo and in vitro. We have called this virus malignant rabbit fibroma virus (MV). Electrophoresis of restriction digests made with HIND III indicates that despite the antigenic similarity of SFV and MV, the locations of HIND III sites in the two viral genomes are quite different. These experiments have enabled us to determine that MV was present in small quantities in our initial uncloned stock of Patuxent strain SFV. Lymphocytes from rabbits bearing MV-induced tumors responded poorly to both B and T lymphocyte mitogens. This nonspecific immunologic dysfunction is evident at or before the time when metastases and Gram-negative infection develop, and it becomes more profound as the disease progresses. MV-induced tumors may provide a model for Gram-negative infection and decreased immunologic responsiveness associated with malignancies.

Animals↗
PubMed1989

In vitro clonogenic growth and metastatic potential of human operable breast cancer.

In 254 patients with operable [International Union against Cancer (1972) Stages I, II, and III] breast cancer, the relations between in vitro clonogenic growth in soft agar of primary breast cancer tumors and their metastatic potential expressed by the relapse-free survivals (RFS) as well as overall survivals were studied. Sixty-four % (163 of 254) of cancers formed distinct colonies (30 or more colonies in a single dish, or 10 or more colonies in plural dishes). Other breast cancers (36%, 91 of 254) were designated to be negative for the clonogenicity. There was no correlation between the positive or negative clonogenicity and clinicopathological characteristics in breast cancer patients, including the age of patients, menopausal status, tumor size, T classification, International Union against Cancer stage, histological type (Japanese Breast Cancer Society), histologically proved axillary lymph node metastasis, and estrogen receptor (ER). At the time of median follow-up of 43 mo (range, 25 to 61 mo) after mastectomy, a recurrence of malignancy occurred in 19.0% (31 of 163) of the patients with positive clonogenic tumors, and in 8.8% (8 of 91) of those with negative clonogenic tumors (P = 0.03). There also was a significant difference (P less than 0.03 by log rank test, P less than 0.05 by generalized Wilcoxon test) in RFS curves between positive and negative clonogenicity groups. These differences in RFS were also noted in Stage II patients in favor of the negative colony formation group. In ER-negative cancer patients, the RFS of patients with positive clonogenic cancers was shown to be worse (P less than 0.03 by log rank test, P less than 0.05 by generalized Wilcoxon test) than patients with negative clonogenic cancers. There was no difference in RFS in ER-positive cancer patients. There was a trend (P = 0.09 by log rank test) of worse overall survival rate in patients with positive clonogenic growth. In a multivariate comparison using the principal component analysis composed of factors including positive node, T classification, histological type, age, ER, and colony formation, the clonogenicity showed a significant effect on the recurrence of malignancy and also on the survival of the patients after mastectomy. In conclusion, in vitro clonogenic growth of the primary tumor of breast cancer was shown to be one of the independent factors of metastatic potential in operable breast cancer patients after mastectomy.

Breast Neoplasms↗
PubMed2005

Targeting tomoregulin for radioimmunotherapy of prostate cancer.

Radiotherapy is an effective approach for the treatment of local prostate cancer. However, once prostate cancer metastasizes, radiotherapy cannot be used due to the distribution of multiple metastases to lymph nodes and bones. In contrast, radioimmunotherapy should still be efficacious in metastatic prostate cancer as radioisotopes are brought to tumor cells by targeting antibodies. Here we identify and validate a prostate-expressed molecule, tomoregulin, as a target for radioimmunotherapy of prostate cancer. Tomoregulin is a transmembrane protein selectively expressed in the brain, prostate, and prostate cancer, but not expressed in other normal tissues. Immunohistochemical studies of tomoregulin protein in clinical samples show its location in the luminal epithelium of normal prostate, benign prostatic hyperplasia, and prostatic intraepithelial neoplasia. More importantly, the tomoregulin protein is expressed in primary prostate tumors and in their lymph node and bone metastases. The nature of tomoregulin as a transmembrane protein and its tissue-specific expression make tomoregulin an attractive target for radioimmunotherapy, in which tomoregulin-specific antibodies will deliver a radioisotope to prostate tumor cells and metastases. Indeed, biodistribution studies using a prostate tumor xenograft model showed that the (111)In-labeled anti-tomoregulin antibody 2H8 specifically recognizes tomoregulin protein in vivo, leading to a strong tumor-specific accumulation of the antibody. In efficacy studies, a single i.p. dose of 150 microCi (163 microg) (90)Y-labeled 2H8 substantially inhibits the growth rate of established LNCaP human prostate tumor xenograft in nude mice but produces no overt toxicity despite cross-reactivity of 2H8 with mouse tomoregulin. Our data clearly validate tomoregulin as a target for radioimmunotherapy of prostate cancer.

Animals↗
PubMed2005

Molecular staging of the sentinel lymph node in melanoma patients: correlation with clinical outcome.

BACKGROUND: This study was designed to determine the debated prognostic significance of reverse transcriptase-polymerase chain reaction (RT-PCR) positivity in melanoma patients' sentinel lymph node (SLN) negative by conventional histopathology (PATH). PATIENTS AND METHODS: Patients with primary stage I-II cutaneous melanoma underwent radioguided sentinel lymphadenectomy. Their SLNs were assessed for tyrosinase (Tyr) and melanoma antigens recognized by T-cells (MART-1) mRNA expression using RT-PCR, in parallel with hematoxylin and eosin staining and immunohistochemistry. Tyr and MART-1 expression in the SLNs were correlated with PATH assay results, standard prognostic factors, time to progression and overall survival. RESULTS: Twenty-three of the 124 patients (18.5%) had positive SLNs by both PATH and RT-PCR (PATH+/PCR+). Sixteen patients (13%) were negative by PATH and positive by RT-PCR (PATH-/PCR+). Eighty-five patients (68.5%) had SLNs that were negative by both PATH and RT-PCR (PATH-/PCR-). At a median follow-up of 30 months, recurrence rates among the three cohorts were statistically different (PATH+/PCR+, 60%; PATH-/PCR+, 31%; PATH-/PCR-, 9.4%). Seven of 23 (30%) and two of 16 (12.5%) patients died in the PATH+/PCR+ and PATH-/PCR+ SLN groups, respectively, whereas no patient died in the PATH-/PCR- SLN group. CONCLUSIONS: RT-PCR is more sensitive than PATH to detect SLN metastases and it is a reliable predictor of disease relapse in stage I-II melanoma patients.

Adult↗
PubMed2005

In vivo cutaneous interferon-gamma gene delivery using novel dicationic (gemini) surfactant-plasmid complexes.

BACKGROUND: Localized scleroderma (morphea and linear scleroderma) is a connective tissue disease, accompanied by excessive proliferation and deposition of collagen within the skin, inflammation, vasculopathy and a deranged immune system. Interferon gamma (IFNgamma), an inhibitor of collagen synthesis and an immunomodulator, could be a potential therapeutic agent if it could be delivered into or expressed locally in affected skin in a non-invasive manner. In this study, the feasibility of topical delivery of the IFNgamma gene and expression of IFNgamma were investigated in mice. METHODS: Novel dicationic (gemini) surfactant (spacer length n=2-16; alkyl chain m=12 or 16)-DNA complexes were formulated and characterized by circular dichroism and atomic force microscopy to select gemini analogues with the highest transfection efficiency (TE). Transfection and cellular expression of IFNgamma from the bicistronic pGTmCMV.IFN-GFP plasmid were evaluated in PAM 212 keratinocyte culture by ELISA and fluorescence microscopy. Topical delivery of plasmid using liposomal and nanoemulsion systems, based on gemini surfactant 16-3-16, was evaluated in mice by IFNgamma expression analysis. RESULTS: In vitro TE was found to be dependent on the spacer length of the gemini surfactant, with the C3 spacer showing the highest activity (both 12-3-12 and 16-3-16). Both gemini cationic liposomes and gemini nanoemulsion (3x25 microg DNA/animal) produced significantly higher levels of IFNgamma in the skin (359.4 and 607.24 pg/cm2) compared to naked DNA (135.69 pg/cm2) or a liposomal Dc-chol formulation (82.15 pg/cm2). IFNgamma expression in the lymph nodes was higher in the animals treated with gemini liposomes (422.74 pg/animal) compared to the nanoemulsion formulation (131.27 pg/animal) or the Dc-chol formulation (82pg/animal). CONCLUSIONS: The feasibility of topical delivery of pGTmCMV.IFN-GFP plasmid in mice using gemini cationic surfactant based delivery systems was demonstrated. IFNgamma expression after treatment with gemini-DNA formulations in the skin was 3-5-fold higher compared to the treatment with naked DNA (p<0.05), and 4-6-fold higher than the Dc-chol-DNA complex, indicating a significant advance in topical DNA delivery across intact skin in vivo.

Animals↗
PubMed1990

Nuclear deoxyribonucleic acid ploidy and serum prostate specific antigen in operable prostatic adenocarcinoma.

A total of 71 patients with prostate carcinoma who underwent radical retropubic prostatectomy had preoperative measurement of serum prostate specific antigen levels and subsequent nuclear deoxyribonucleic acid ploidy analysis of the resected tumors. Prostate specific antigen levels were determined by a commercially available prostate specific antigen radioimmunometric assay (normal range 0 to 4.0 ng./ml.). The paraffin-embedded blocks of the prostate specimens were analyzed by flow cytometry for nuclear deoxyribonucleic acid content using Hedley's technique and propidium iodide staining. A strong association was found between nuclear deoxyribonucleic acid ploidy patterns and preoperative prostate specific antigen values. All patients with tetraploid and aneuploid tumors had elevated preoperative prostate specific antigen values. By contrast, all patients with preoperative antigen values of less than 4 ng./ml. had diploid tumors (p less than 0.0034). Prostate specific antigen levels were proportional to the estimated volume of the primary tumor (p less than 0.0065). However, even after statistical adjustment for tumor volume the preoperative prostate specific antigen value was still significantly and independently correlated with deoxyribonucleic acid ploidy pattern (p less than 0.007). However, only 35% of the patients with diploid tumors had antigen levels within the normal range. Conversely, 65% of the patients with diploid neoplasms had abnormally high antigen levels preoperatively. These results suggest that patients with localized biopsy proved prostate carcinoma and normal preoperative prostate specific antigen values are most likely to have diploid tumors; based on a 95% confidence level, the true proportion of patients with normal prostate specific antigen values who also have a deoxyribonucleic acid diploid tumor is 85 to 100%. Since localized deoxyribonucleic acid diploid tumors are known to be associated with a favorable prognosis for patients with prostate carcinoma treated by radical prostatectomy, a preoperative serum prostate specific antigen level within the normal range may help to predict disease outcome.

Adenocarcinoma↗
PubMed2001

Systemic and local immunosuppression in pancreatic cancer patients.

Pancreatic cancer is characterized by an extremely poor prognosis. For the development of more effective immunotherapies, the systemic and local immunological escape mechanisms need to be further elaborated. These mechanisms may include the secretion of immunosuppressive cytokines, the local hindrance of tumor-infiltrating lymphocytes (TILs), or the loss of the signal transducing CD3 zeta-chain of TILs. In this study, we have analyzed these parameters in 116 patients suffering from pancreatic ductal adenocarcinoma. Mean concentrations of interleukin (IL)-10 and transforming growth factor-beta1/2 were considerably higher than in control sera (P < 0.0001). Disseminated tumor cells were found in 16 of 39 cases. In 28 of 33 surgical specimens, TILs did not reach tumor cells in significant numbers, being "trapped" in the peritumoral tissues. We suggest this as a simple but highly effective tumor escape mechanism. In cases of a TIL/tumor cell contact, CD3 zeta was mostly lost. Overall, 27 of 33 surgical specimens, 9 of 19 peritumoral lymph nodes, and 13 of 25 peritoneal lavage specimens showed significant loss of CD3 zeta (P < 0.02). Elevated concentrations of IL-10/TGF-beta1/2 were, in all but one of three cases, correlated with a CD3 zeta loss in corresponding specimens. Patients with disseminated tumor cells also showed a CD3 zeta loss in all but two corresponding tumor specimens. These results present strong evidence for an active systemic immunosuppression in pancreatic cancer, as shown by elevated IL-10 and TGF-beta1/2 serum levels as well as the presence of disseminated tumor cells. Killing of tumor cells by potentially cytotoxic TILs is obviously suppressed by the prevention of a direct TIL/tumor cell contact and the inactivation of TILs, as shown by a severe loss of CD3 zeta. In addition to active immunization strategies, successful immunotherapies have to focus on restoring in vivo T-cell function to improve the almost always fatal prognosis of pancreatic cancer.

Adult↗