Normal human breast epithelial cells (HBL100) are immortalized by endogenous SV40 genome and are not tumorigenic in nude mice if injected in the first 50 passages in culture. This cell line also depends on the expression of basic fibroblast growth factor (bFGF) for its viability in culture. The present study was designed to transform these cells with chemical carcinogens to establish malignant HBL100 cell lines in order to evaluate the eventual modulation in the expression of bFGF. In the first experiment, HBL100 cells were treated with 2 mu g/ml of 7, 12, dimethylbenz(a)anthracene (DMBA) for two 24 h periods. In a second experiment, HBL100 cells were transformed by exposing them to 20 mu g/ml of N-methyl-N-nitrosourea (MNU) twice a day for two days. Unlike the HBL100 parental cell line, the two newly derived cell lines (named respectively HBLTDMBA and HBLTMNU) were tumorigenic when injected in nude mice. The expression of bFGF, as measured by immunocytochemistry and Western blot analysis, was higher in the DMBA-transformed cell line than in the parental HBL100 and MNU-transformed lines. These results differentiate between transformation selectivity by two different carcinogens. The direct-acting carcinogen MNU, which induces point mutation, does not require enhanced expression of bFGF, whereas bFGF may be necessary for early events leading towards transformation by carcinogens requiring metabolism for their action, such as DMBA.

In the present study we pinpoint the time of nm23 down-regulation during the chemical transformation of the human breast epithelial cell line HBL100. The non-malignant HBL100 was transformed by exposing it to N-methyl-N-nitrosourea (MNU). We subsequently injected the transformed cells (HBL-T-MNU) into nude mice, resulting in tumor growth. With a second passage of these tumors in mice we observed lung and extra-regional node metastases. The expression of nm23 in the non-tumorigenic HBL100 cells was compared to the tumorigenic KBL-T-MNU cells as well as to the metastatic cell line HBL-T2(MNU) derived from the tumor induced by HBL-T-MNU cells. By using immunohistochemistry and Western blot analysis we documented the downregulation of nm23 expression in the tumorigenic HBL-T-MNU and metastatic HBL-T2(MNU) cell lines, as compared to the parental line HBL100. Once downregulated, nm23 expression in this model remains constant during the subsequent progression. These results suggest that nm23 down-regulation may indeed be associated with early neoplastic transformation and is maintained throughout neoplastic progression and metastatic stage.

Background: Monoclonal Antibody (MAb) 29-13 reacts with the human sarcoma-associated antigen (SAA) p200. We report here engineering and primary characterization of a single chain antibody (scFV2913). Materials and Methods: The scFV2913 recombinant gene, consisting of VH-linker-VK, was constructed with RT-PCR. This gene was cloned and expressed in E. coli. The renatured scFV2913 was used in the immunostaining study. Results: Consistent with its parent MAb 29-13, purified and renatured scFV2913 showed affinity and specificity to the SAA p200 according to the immunohistochemical staining study of 99 specimens of human sarcomas and other tissues. Conclusions: Due to its retained specificity and affinity, scFV2913 may be useful in immunodiagnosis and immunotherapy for sarcoma patients.

The aim of this study was to assess the translational value of the quantitative assay of mutant p53 protein expression as both a prognostic indicator and a tool to determine appropriate therapy in a group of relatively innocuous and morphologically similar soft tissue sarcomas (STSs). Using a quantitative ELISA, we analyzed mutant p53 protein expression in 47 well-differentiated (grade I) STSs from patients treated in our Department of Surgical Oncology. Sixteen of 47 tumors expressed up to 42.6 ng mutant p53 protein/mg total protein. After a mean follow-up of 112 months, 63% of the patients with mutant p53+ tumors but only 16% of the patients with mutant p53- tumors had died (P < 0.01). Mutant p53 expression of >/=4.5 ng predicted even greater reduction in survival. These data show that mutant p53 expression identifies biologically aggressive grade I STSs. This molecular marker should have translational value as a tool to select those patients likely to benefit from aggressive multimodal therapy and intense surveillance.

Previous studies have suggested that differential expression and/or activation of integrins facilitates metastatic progression in murine and human melanoma. While recent data show that the integrin αvβ3 is involved in tumor angiogenesis and that tumor growth may be abrogated by αvβ3 inhibitorsin vitro,the clinical significance of β3 integrin expression in human malignant melanoma is not known. To assess the prognostic value of β3 integrin expression, we examined primary cutaneous melanomas from 160 patients followed for a mean of 98 months or until death. We quantified the percentage of tumor area stained with β3 integrin Ab CD-61 using an image analyzer. β3 integrin expression was detected in 107/160 primary melanomas (69%). β3-integrin-positive (β3+) tumors were thicker (mean 2.98 ± 0.3 mm) than β3-integrin-negative (β3−) melanomas (mean 1.64 ± 0.2 mm) (P= 0.002). Patients with β3+ melanomas were more likely to relapse (57/107, 53%) and to die from disease (45/107, 42%) than those with β3− tumors (6/53, 11%; and 4/53, 8%, respectively) (P< 0.001). Overall survival was greater for β3− than for β3+ patients (mean 102 ± 9 vs 69 ± 6 months) (P= 0.001). These data show that β3 integrin expression in primary cutaneous melanoma predicts subsequent metastatic progression. Further study of β3 integrins in the development of melanoma metastases may yield new therapeutic strategies, as well as prognostic information, for the treatment of this cancer.

BACKGROUND:Experimental evidence suggests that integrins are key regulators of the development of melanoma metastases, influencing both the likelihood and site of metastases. Whereas effective treatment of cutaneous melanoma remains surgical, elective lymph node dissection (ELND) is controversial. The present study was designed to investigate the relationship between integrin expression by a given primary melanoma and occult regional lymph node metastases.MATERIALS AND METHODS:We studied beta 1 integrin expression, by quantitative immunohistochemistry using an image analyzer, in the primary melanomas of 90 ELND patients.RESULTS:beta 1 integrin was expressed in > or = 10% of the primary tumor in 92% of cases eith lymph node involvement versus 9% of node negative cases (p < 0.001).CONCLUSIONS:Our data demonstrate that quantitative immunohistochemistry for beta 1 integrin expression in primary melanomas can identify patients likely to have occult lymph node metastases. This suggests that beta 1 integrins play a role in the lymphatic dissemination of cutaneous melanoma.

BACKGROUND:Understanding tumor antigen expression and its correlation with the cell cycle may help in designing immunotherapy by monoclonal antibodies. Therefore, we studied the in vitro expression of sarcoma-associated antigens p102 and p200 in the G, S, and G2/M phases of sarcoma cell lines.METHODS:The expression of human cell surface sarcoma-associated antigens p102 and p200 was studied in 13 human sarcoma cell lines, using flow cytometry.RESULTS:p102 was detected by monoclonal antibody 19-24-6 in all 13 sarcoma cell lines, and p200 was detected by monoclonal antibody 29-13-17 in five of 13. p102 antigen expression was 1.4- to 3.4-fold higher (p < 0.001) than p200 expression. Although sarcoma cell lines showed a wide range of p102/p200 antigen expression, over 99% of the entire in vitro and in vivo cell population was found to be p102- and/or p200-positive. In three cell lines, p102 expression was cell cycle-dependent, with relative fluorescence intensity ranging from 13.8% to 23.9% higher at the G1 phase than at the G2/M phase. In three cell lines, the expression of p200 at the GI phase was 22.4% to 40.9% percent higher than at the G2/M phase.CONCLUSIONS:The heterogeneity and cell cycle dependence of p102/p200 antigen expression in sarcoma cells suggest that monoclonal antibodies 19-24-6 and 29-13-17 might be applied to the immunotherapy of sarcoma.

Background. Elective lymph node dissection for malignant melanoma is still controversial. Experimental studies suggest that differential expression, activation, or both of PI integrins facilitate melanoma metastases. However, the clinical significance of beta(1) integrin expression in human melanoma is unclear.Methods. We examined primary cutaneous melanomas from 76 patients undergoing elective lymph node dissection. We quantified the percentage of tumor area stained by pr integrin antibody with an image analyzer.Results. beta(1) integrin was expressed in all 23 primary tumors from patients with pathologically positive lymph nodes (LNs) but in only 14 (26%) of 53 cases with pathologically negative nodes (p < 0.001). No patients with beta(1) integrin-negative tumors had LN involvement, whereas 23 (62%) of 37 patients with beta(1) integrin-positive tumors had LN metastases (p < 0.001). Furthermore, 21 (91%) of 23 cases with LN metastases but only 4 (8%) of 53 cases without had beta(1) integrin staining of 10% or more of tumor area (p < 0.001).Conclusions. Our study is the first to show a correlation between expression of a molecular marker in the primacy cutaneous melanoma and likelihood of regional LN metastases. beta(1) immunostaining of 10% or more of tumor area reliably predicts patients most likely to harbor occult LN metastases and likely to benefit from ELND.

Nucleolar organizer regions (NORs) are loops of ribosomal DNA (rDNA) in the nucleolus and are associated with acidic proteins. They are seen in routinely processed paraffin sections by using a one-step colloidal silver (Ag) staining method; they appear as black dots termed "AgNORs". The quantitative assay of AgNORs has been used to differentiate benign from malignant neoplasms. Melanocytic lesions differ significantly in AgNOR counts between malignant melanoma and nevi. However, conflicting results have been reported as to AgNORs' prognostic value in melanoma. A recent study showed AgNOR counts to be a more accurate prognostic indicator than Breslow's thickness. In this study, we counted the AgNORs in 26 patients with primary cutaneous melanomas (CMM) between 2.0 mm and 2.5 mm thick. Of these, 14 are alive without disease (AN) at 5 years after diagnosis (group 1) and 12 are dead of disease (DD) in less than 5 years (group 2). The AgNORs were scored in 30 nuclei per tumor, and the means were calculated. For group 1, the mean number of AgNORs per nucleus was 6.88, ranging from 3.73 to 12.70. For group 2, the mean number was 6.97, ranging from 3.63 to 11.67. Statistical analysis using analysis of variance (ANOVA) showed no significant difference between the groups (p = 0.33). In our study, AgNOR counts did not prove to be of prognostic value in malignant melanoma.

Sarcoma and normal tissue plasma membrane lectin-reactive glycoproteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Two peanut agglutinin-reactive N-acetylgalactosamine-containing glycoproteins of 1.05 x 10(6) and 1.25 x 10(5) Da and one lentil agglutinin-reactive mannose/N-acetylglucosamine(-fucose)/sialic acid-containing glycoprotein of 1.7 x 10(5) Da (Gp170) were detected in osteosarcoma and malignant fibrous histiocytoma (MFH), respectively. However, these glycoproteins were not detected in normal tissue plasma membranes. Concanavalin A, wheat germ and Ulex europaeus Type I agglutinins did not reveal any unique sarcoma-associated membrane glycoproteins. Preliminary studies on monoclonal antibodies (mAbs) generated against Gp170 (mAb 64-35-84) and against lentil-reactive glycoproteins from MFH (mAbs 67-34 and 67-117) revealed high specific binding to a number of membranes isolated from MFH and osteosarcoma tissues, with no crossreactivity to normal human tissues tested (liver, spleen and skin). Detailed analysis of mAb 67-102, which was generated against lentil-reactive glycoproteins isolated from MFH plasma membranes, exhibited significant binding to membranes isolated from osteosarcoma, liposarcoma and MFH; moderate binding to synovial sarcoma, aggressive fibromatosis and fibrosarcoma; and minimal to no binding to other soft tissue sarcoma plasma membranes. No binding was observed to twenty normal tissue specimens, with the exception of low positive binding to two of five fat and two of three colon specimens.

In light of the reported cytotoxic effects of the antifungal agent ketoconazole on several malignant cell lines, we investigated the in vitro and in vivo effects of this drug on the human fibrosarcoma cell line HT-1080. Utilizing an anchorage dependent in vitro assay, we demonstrated an IC90 of 10.0 micrograms/ml at 72 hours and a decreasing IC90 with increasing exposure (IC90 = 6.2 micrograms/ml at 12 days). In vivo investigation utilizing the subcutaneous growth of this tumor in athymic mice and the oral, subcutaneous or intraperitoneal administration of ketoconazole at doses of 5-50 mg/kg/day demonstrated no consistent activity. Ketoconazole appears to have significant in vitro activity for HT-1080 but this does not translate into significant in vivo activity and its clinical relevance in sarcoma remains uncertain.

METHOD:A new neoplastic cell line, UISO-GCT-1, was established from a giant cell tumor of the right tibia in an 18-year-old man. Immunohistochemical, cytogenetic, ultrastructural, and growth studies were performed.RESULTS:Multinucleated giant cells (> 4-6 nuclei/cell) persisted in a culture relatively late in passage (passage 17), which is unique in cell lines of giant cell tumor of bone. Mononuclear and multinucleated cells in monolayer culture expressed vimentin and tartrate-resistant acid phosphatase and reacted with monoclonal antibodies to CD13 and CD68, suggesting a monocytic-macrophage origin of these cells. Mononuclear and multinuclear cells also selectively expressed high molecular weight cell membrane antigens specifically associated with soft tissue sarcomas and osteosarcomas. Karyotypically, UISO-GCT-1 cells were hypodiploid, hypotetraploid, and multiploid, with more than 200 chromosomes per mitosis present in some cells. Other chromosomal aberrations observed included ring chromosomes, double minutes, translocations, multiple fragments, and multiradials.CONCLUSION:Collectively, observations of this study suggest that karyotypically abnormal giant cell tumors of bone arise from a monocyte-macrophage lineage and subsequently express an antigenic profile similar to malignant mesenchymal tumors.

BACKGROUND:Few studies exist that describe Merkel cell carcinoma (MCC) growth characteristics in vitro, in vivo, or both.OBJECTIVE:Our purpose was to evaluate the pathologic features of MCC implanted into athymic mice and to determine cytogenetic abnormalities in the established cell line.METHODS:Tumor tissues from a patient with MCC were grown in culture. Cultured cells were karyotyped and inoculated subcutaneously into athymic mice. Nude mouse tumors were re-implanted into other athymic mice. Tissues from the primary skin tumor and the nude mouse tumor were processed for light and electron microscopy and immunocytochemistry.RESULTS:The cell line showed a doubling time of 64.8 hours. Xenografts of 4 x 10(6) cells produced tumors in athymic mice with a doubling time of 16.1 days. The nude mouse tumors showed pathologic features similar to those of the primary skin tumor. Cytogenetic studies showed a t(1;17) (p36;q21) translocation in 100% of the cells.CONCLUSION:MCC implanted into athymic mice retained the pathologic features of the primary skin tumor and behaved aggressively. The t(1;17) (p36;q21) translocation may be a marker of an aggressive phenotype.

Malignant melanoma is extremely rare in patients with albinism. To date, in the English language literature, there have been only sixteen documented cases of malignant melanoma in albino patients. These cases include thirteen cutaneous, one oral, one ocular, and one anal; only one of these was in a child. Here, we present the case of the youngest known albino patient to have cutaneous malignant melanoma.

Monoclonal antibody (MoAb) 19-24, which recognizes a cell surface sarcoma-associated antigen p102, was linked via a biotin-avidin-biotin bridge to adriamycin (ADR). The molar ratios of ADR: total protein ranged from 2 to 7.5. Significant differences were not observed between the binding ability of the ADR-MoAb conjugate and of the unconjugated MoAb 19-24 to fresh sarcoma tissue membranes. The ADR-MoAb conjugate also retained the ability to compete with MoAb 19-24 for binding to sarcoma-associated antigen p102. In vitro cytotoxicity studies using human fibrosarcoma cells showed that the ADR-MoAb conjugate maintained 40% of the efficacy of free ADR. However, the conjugate was not cytotoxic to p102 antigen negative rat fibrosarcoma SP-24 cells. These data support the suggestion that MoAb-drug conjugates might be helpful in developing highly specific antisarcoma therapeutic agents.

To evaluate whether the increase in disease-free survival and survival previously reported for women with melanomas with estrogen receptors (ER) was a reflection of the histopathology of the primary melanoma, the interrelationship of histopathologic characteristics of 143 patients with such tumors was examined. The ER was assayed in the primary tumor from 44 patients and in 99 metastatic deposits from the other patients. Tumor thickness, level of invasion, prognostic index, mitoses/mm2, ulceration, vascular invasion, necrosis, histologic grade, preexisting nevus, and predominant malignant cell type were examined. There was no relationship between ER presence and any histopathologic characteristic examined, irrespective of the tumor source (primary or metastatic). Examination of histopathologic characteristics as a function of sex and receptor status showed a slight but insignificant predominance of more well-differentiated, thinner tumors in women whose lesions were positive for the ER. These results suggest that the increased disease-free survival in patients with ER-positive lesions is not attributable to the pathologic characteristics of the primary tumor examined during this study.

Clinical and pathologic variables were compared between "older" (greater than or equal to 70 years) and "younger" (30 to 39 years) patients with primary invasive cutaneous melanoma. Older patients had more nodular melanomas and acral lentiginous melanomas (58%); superficial spreading melanomas predominated in younger patients (74%). Mean tumor thickness was greater in the older patients (3.95 vs 2.02 mm). Invasive levels 2 and 3 occurred more often in younger patients (41.1% vs 13%); level 5 occurred more often in older patients (30.4% vs 5.3%). Microscopic ulceration occurred more often in older (46.4%) than in younger patients (19.4%). Older patients classified as clinical stage I at presentation or with primary lesions 1.50- to 3.00-mm thick had poorer survival. Younger women survived longer than younger men; this was not true of older patients. The elderly patients with cutaneous melanoma were more likely to have poor prognostic features and thus more likely to die from melanoma than the younger patients.

Fifteen women with genital malignant melanoma were studied. Their ages ranged from 19 to 66 years (mean 49.1 years); 12 were white, and three were black. The sites of involvement were the mons pubis (one patient), perineal body (one), labium majus (three), labium minus (three), and vagina (seven). Ten patients (66.6%) died of their disease, one is alive with disease, and four are alive without evidence of disease. For the living patients the duration of follow-up was 20 to 118 months (mean 63.6 months). Of those who died, survival ranged from 3 to 76 months (mean 25.1 months). The predominant type of malignancy was superficial spreading melanoma 50%. Nodular melanoma represented 22%, and the nodular polypoidal variant 14%. Melanoma of the squamous mucosa, also referred to as lentiginous melanoma, constituted 14%. By using Chung's method of determining levels of invasion, we found that no lesion was in situ (level I), two were level II (less than 1.0 mm thick), one was level III (between 1.0 and 2.0 mm) and the remaining 11 patients had lesions that were greater than 2.0 mm (levels IV and V). Because the subcutaneous fat is not consistently present in all sites of the female genitalia, all tumors thicker than 2.0 mm were included in level IV, and no level V tumors were classified in our study. Using Breslow's microstaging method, we found the thickness to range from 0.65 to 9.5 mm (mean 4.75 mm). When we correlated survival with level and thickness of tumor in nine patients who died, one tumor was level III and eight were level IV; thickness ranged from 1.65 to 9.0 mm (mean 5.64 mm).(ABSTRACT TRUNCATED AT 250 WORDS)

Pigmented tumors resembling cutaneous melanoma were first reported in Sinclair miniature swine in 1967. Since that time, carefully planned breeding has established that this is an inherited malignancy the natural history of which mimics human cutaneous melanoma in a number of ways. Because of these characteristics, miniature swine melanoma appears to be an effective model with which to investigate the mechanisms influencing initiation, growth, and progression of human melanoma. This investigation characterized histologically the cutaneous melanoma in miniature swine and compared the findings with human neoplasm. Primary cutaneous melanoma in swine has been reclassified and standardized according to the classifications currently in vogue in human melanoma. Our results suggest that the condition in miniature swine is histologically similar to that in humans. These observations will provide a basis for interpretation of the results derived in the biologic studies performed in this model.