IRF-1 responsiveness to IFN-γ predicts different cancer immune phenotypes.

BACKGROUND: Several lines of evidence suggest a dichotomy between immune active and quiescent cancers, with the former associated with a good prognostic phenotype and better responsiveness to immunotherapy. Central to such dichotomy is the master regulator of the acute inflammatory process interferon regulatory factor (IRF)-1. However, it remains unknown whether the responsiveness of IRF-1 to cytokines is able to differentiate cancer immune phenotypes. METHODS: IRF-1 activation was measured in 15 melanoma cell lines at basal level and after treatment with IFN-γ, TNF-α and a combination of both. Microarray analysis was used to compare transcriptional patterns between cell lines characterised by high or low IRF-1 activation. RESULTS: We observed a strong positive correlation between IRF-1 activation at basal level and after IFN-γ and TNF-α treatment. Microarray demonstrated that three cell lines with low and three with high IRF-1 inducible translocation scores differed in the expression of 597 transcripts. Functional interpretation analysis showed mTOR and Wnt/ß-cathenin as the top downregulated pathways in the cell lines with low inducible IRF-1 activation, suggesting that a low IRF-1 inducibility recapitulates a cancer phenotype already described in literature characterised by poor prognosis. CONCLUSION: Our findings support the central role of IRF-1 in influencing different tumour phenotypes.

Generation and genetic characterization of immortal human prostate epithelial cell lines derived from primary cancer specimens.

Difficulty in establishing long-term human prostate epithelial cell lines has impeded efforts to understand prostate tumorigenesis and to develop alternative therapies for prostate cancer. In the current study, we describe a method that was successful in generating 14 immortal benign or malignant prostate epithelial cell cultures from primary adenocarcinomas of the prostate resected from six successive patients. Immortalization with the E6 and E7 transforming proteins of human papilloma virus serotype 16 was necessary to establish long-term cultures. Microscopic examination of fresh tumor specimens exhibited a variable mixture of benign and malignant epithelium. Thus, single-cell cloning of tumor-derived cell cultures was essential for defining tumor cell lines. Efforts to characterize these cultures using traditional criteria such as karyotype, growth in nude mice, and prostate-specific antigen expression were noninformative. However, allelic loss of heterozygosity (LOH) represents a powerful alternative method for characterizing tumor cell lines originating from primary adenocarcinomas of the prostate. Microdissected fresh tumors from four of six patients revealed LOH at multiple loci on chromosome 8p, as assessed by PCR. LOH on chromosome 8p matching the patterns found in microdissected tumors was also observed in a tumor-derived cell line and its clones, as well as in one clone from a tumor-derived cell line from a second patient. LOH was not observed in immortal lines generated from autologous benign prostatic epithelium, seminal vesicle epithelium, or fibroblasts. The multifocal nature of prostate cancer, as well as the presence of an entire spectrum of malignant transformation within individual prostate glands, necessitates this type of careful analysis of derivative cell cultures for their validation as in vitro models that accurately reflect the primary cancers from which they are derived.

Phase I crossover study of paclitaxel with r-verapamil in patients with metastatic breast cancer.

PURPOSE: We conducted a phase I crossover study of escalating doses of both paclitaxel (Taxol; Bristol-Myers, Squibb, Princeton, NJ) and r-verapamil, the less cardiotoxic stereoisomer, in heavily pretreated patients with metastatic breast cancer. PATIENTS AND METHODS: Twenty-nine patients refractory to paclitaxel by 3-hour infusion were treated orally with r-verapamil every 4 hours starting 24 hours before the same-dose 3-hour paclitaxel infusion and continuing for a total of 12 doses. Once the maximum-tolerated dose (MTD) of the combination was determined, seven additional patients who had not been treated with either drug were evaluated to determine whether the addition of r-verapamil altered the pharmacokinetics of paclitaxel. Consenting patients had tumor biopsies for P-glycoprotein (Pgp) expression before receiving paclitaxel and after becoming refractory to paclitaxel therapy. RESULTS: The MTD of the combination was 225 mg/m2 of r-verapamil every 4 hours with paclitaxel 200 mg/m2 by 3-hour infusion. Dose-limiting hypotension and bradycardia were observed in three of five patients treated at 250 mg/m2 r-verapamil. Fourteen patients received 32 cycles of r-verapamil at the MTD as outpatient therapy without developing cardiac toxicity. The median peak and trough serum verapamil concentrations at the MTD were 5.1 micromol/L (range, 1.9 to 6.3), respectively, which are within the range necessary for in vitro modulation of Pgp-mediated multidrug resistance (MDR). Increased serum verapamil concentrations and cardiac toxicity were observed more frequently in patients with elevated hepatic transaminases and bilirubin levels. Hematologic toxicity from combined paclitaxel and r-verapamil was significantly worse compared with the previous cycle of paclitxel without r-verapamil. In the pharmacokinetic analysis, r-verapamil delayed mean paclitaxel clearance and increased mean peak paclitaxel concentrations. CONCLUSION: r-Verapamil at 225 mg/m2 orally every 4 hours can be given safely with paclitaxel 200 mg/m2 by 3-hour infusion as outpatient therapy and is associated with serum levels considered active for Pgp inhibition. The addition of r-verapamil significantly alters the toxicity and pharmacokinetics of paclitaxel.

Immune selection after antigen-specific immunotherapy of melanoma.

BACKGROUND: Melanoma antigen (MA)-specific vaccination strongly enhances antitumor reactivity in vivo and is capable of producing strong cytotoxic T lymphocyte responses in vitro. Furthermore, specific human leukocyte antigen-restricted T cell activation is hypothesized to occur in response to peptide-based immunotherapy, which may lead to the preferential killing of tumor cells bearing the relevant MA. The development of melanoma antigen-loss variants may subsequently occur in vivo. METHODS: Analysis of 532 melanoma lesions from 204 patients was performed on fine-needle aspiration biopsy specimens. Lesions were graded for the expression of the MAs gp100 and MART-1 with use of immunocytochemistry. A total of 351 melanoma lesions were divided into cohorts on the basis of the treatment received. The pretreatment group (n = 175) consisted of lesions obtained before any form of gp100 immunotherapy, with the posttreatment group (n = 176) consisting of lesions obtained after vaccination with a modified gp100 epitope, gp209-2M +/- interleukin 2 (IL-2). RESULTS: The percentage of lesions not expressing the gp100 antigen is greater than the percentage not expressing MART-1 (26% vs 14%). The frequency of lesions with high expression (> 75%) of gp100 significantly decreased with therapy (47% vs 34%) and conversely negative lesions increased (18% vs 29%). Treatment of lesions with peptide alone (no IL-2) revealed a significant decrease in gp100 expression (47% vs 32%), enhanced with the addition of IL-2 to therapy (47% vs 35%). The expression of MART-1 remained essentially unchanged unless IL-2 was added (54% vs 54%, MART-1 peptide alone, 54% vs 43%, MART-1 peptide + IL-2). Of 94 patients (181 lesions) assessed for gp100 expression before treatment, 10 patients responded to therapy. Pretreatment lesions in responding patients expressed some level of gp100 in all cases compared with 27% of nonresponding lesions, which were negative for gp100 expression. CONCLUSIONS. This study indirectly demonstrates that vaccination with an MA-derived peptide can result in immune selection in vivo. Furthermore, it provides strong immunologic evidence for the specificity of MA vaccines and to the relevance of MA expression in predicting the response to vaccination.

Characteristics of nine newly derived mesothelioma cell lines.

This report characterizes nine new cell lines derived from patients with malignant pleural mesothelioma. The lines were initiated between July 1990 and July 1992 from solid tumors (5 lines) or effusions (4 lines) and had proliferated for a period of at least 2 months without senescence. They were characterized by cell size, doubling time, immunohistochemical analyses, electron microscopy, and chromosomal karyotyping. Growth factor/cytokine elaboration was determined using enzyme-linked immunoassays. The established lines were similar in morphology to their parent tumor (ie, epithelial or sarcomatoid). Cell sizes ranged from 59 to 81 microns, and the doubling times varied from 31 to 65 hours. The lines stained with cytokeratin and showed expected negative staining for adenomarkers including B72.3 and carcinoembryonic antigen. All cell lines exhibited aneuploidy, with modal chromosome numbers between 40 and 81 and had multiple chromosomal aberrations. Significant production of granulocyte-monocyte colony-stimulating factor, leukemia inhibitory factor, platelet-derived growth factor, and interleukin-6 was seen. These new cell lines derived from human mesotheliomas can now be used to aid in the design of innovative treatment strategies.

If cells could talk. The application of new techniques to cytopathology.

Cytopathology is no longer simply a screening modality limited to the "Pap mills" of yore. The news in cervicovaginal cytology is automation. The news in FNA cytology is the application of molecular techniques. Whether it is the detection of specific proteins/antigens for definitive diagnoses/treatment guidance in immunotherapy, or it is "reading nucleic acids," the cytopathologist of the future will be called upon to gather and report more detailed and precise information. As we develop methods for extrapolating the secrets previously locked within the individual cells, it becomes evident that the cells were talking all along, we just did not know how to listen.

Comparison of antibodies to HBME-1 and calretinin for the detection of mesothelial cells in effusion cytology.

The distinction of mesothelial cells in cytologic samples is often a diagnostic challenge. This is particularly true in potentially malignant effusions in which reactive mesothelial cells may simulate adenocarcinoma (ACA) cells, and in the differentiation of ACA vs. mesothelioma. We sought to determine the superior antibody for the positive identification of mesothelial cells in these circumstances. Cell block sections of 25 reactive and 8 malignant mesothelioma effusions were immunostained with an avidin-biotin procedure, using antibodies to HBME-1 and calretinin. No pretreatment of samples was necessary for the HBME-1-stained slides; microwave antigen retrieval was performed on all slides stained for calretinin. A negative control was performed on each sample. The staining intensity of tumor cells was scored on a scale of 0-3+, with the proportion of immunoreactive cells categorized as <25%, 25-50%, 50-75%, and >75%. The predominant staining pattern for HBME-1 was surface, with rare samples also exhibiting cytoplasmic staining as well. The calretinin-staining pattern was cytoplasmic, with peripheral condensation/prominence and accompanying nuclear staining. All samples were immunoreactive with both antibodies. Fifty-five percent (18/33) of samples showed significantly stronger immunoreactivity with calretinin than with HBME-1; 45% (15/33) of samples showed equivalent staining with the two markers. None of the samples in this study showed stronger immunoreactivity with HBME-1 than with calretinin. Sixty-one percent (20/33) of samples stained with HBME-1 at a moderate (2+) intensity. Fifty-five percent (18/33) of samples stained with calretinin at a strong (3+) intensity. While only 12% of samples showed >75% immunoreactivity for HBME-1, 58% of samples showed >75% of cells immunoreactive for calretinin. Calretinin is the preferred marker in identifying mesothelial cells in cytologic samples, showing the highest sensitivity for mesothelial cells, as evidenced by a more intense staining reaction in a higher percentage of cells than with HBME-1. Published 2001 Wiley-Liss, Inc.

Immunophenotypic analysis of non-Hodgkin's lymphomas in cytologic specimens: a correlative study of immunocytochemical and flow cytometric techniques.

Most Non-Hodgkin's lymphomas(NHL) can be accurately diagnosed and classified based on morphologic and immunophenotypic findings on cytologic specimens. Immunophenotyping can be accomplished via immunocytochemistry (IC) or flow cytometry (FC). We reviewed our experience with 98 cytology specimens (70 fine-needle aspirates [FNA] and 28 effusions) that were submitted for immunophenotyping utilizing both IC and FC between January 1992 and December 1997 for the diagnosis of NHL. Eighty-five percent of the cases were immunophenotyped by both techniques. Among these there were only two discrepancies between IC and FC, yielding a 98% correlation rate. Of the 98 cases, 11% could not be immunophenotyped by FC and 4% could not be immunophenotyped by IC. The advantage of IC is the preservation of cytomorphology, which results in the requirement for a lower number of neoplastic cells and a limited, targeted panel of antibodies. This is especially useful in predominantly necrotic lymphomas in which only a few well-preserved neoplastic cells may be present, rendering the specimen inadequate for immunophenotyping by FC. The advantages of FC are in the detection of a small population of monoclonal cells in a background of reactive cells (particularly useful in effusion samples in which the predominant cell population is often reactive T lymphocytes), increased diagnostic precision through evaluation of objective parameters, and the use of multiple markers with dual labelling. We conclude that IC and FC are both excellent methods for immunophenotyping of cytology specimens and can be used interchangeably depending on the institutional expertise and availability.

E-cadherin, N-cadherin, and calretinin in pleural effusions: the good, the bad, the worthless.

The distinction between reactive mesothelial cells (RMC), malignant mesothelioma (MM), and metastatic adenocarcinoma (ACA) in pleural effusions may be impossible based on morphology alone. E-cadherin, N-cadherin, and calretinin are newly described immunocytochemical markers which can potentially be utilized for facilitating this distinction. E-cadherin and N-cadherin are calcium-dependent intercellular adhesion molecules expressed in epithelial cells and mesenchymal/mesothelial cells, respectively. The differential expression of E-cadherins in epithelial cells and N-cadherins in mesothelial cells has been utilized to differentiate reactive mesothelial cells, MMs and ACAs. Calretinin is a calcium-binding protein within the family of EF-hand proteins. It is abundantly expressed in peripheral and central nervous tissues, and has been shown to consistently immunoreact with mesothelial cells. We studied cell block sections from 77 pleural effusions (22 RMC, 26 MM, and 29 ACA) to investigate the potential immunocytochemical use of anti-E-cadherin, anti-N-cadherin, and anti-calretinin antibodies for differentiating between RMC, MM, and ACA in pleural effusions. A modified avidin-biotin peroxidase complex (ABC) method was used. E-cadherin immunostaining was observed in 14% of RMC, 46% of MMs, and 97% of ACAs. A distinct membrane staining pattern was seen in ACAs. The pattern of staining was cytoplasmic in all reactive RMC and varied from membrane to cytoplasmic in MMs. Anti-N-cadherin immunoreacted with 77% of RMC, 35% of MMs, and 48% of ACAs. Twenty-seven percent of RMC, 58% of MMs, and 31% of ACAs immunoreacted with anti-calretinin. Based on these results, we conclude that anti-E-cadherin is a potentially useful marker in the distinction of ACA cells from RMC. However, it is not as useful for the distinction of ACA and MM. Anti-N-cadherin and anti-calretinin did not reliably distinguish between reactive mesothelial, MM, and ACA cells in pleural effusions.

Detection of circulating tumor cells and micrometastases in stage II, III, and IV breast cancer patients utilizing cytology and immunocytochemistry.

Evaluation for circulating tumor cells and bone marrow micrometastases has generated considerable interest due to a potential association with disease recurrence and poor prognosis. In this study, we examined bone marrow and apheresis samples from Stage II, III, and IV patients (n 120) enrolled in various clinical breast cancer trials at the National Institutes of Health/National Cancer Institute. For each patient sample, two Diff-Quik-stained cytospins were reviewed for morphology, and approximately 1 x 10(6) cells were analyzed for the expression of cytokeratins using an avidin-biotin immunoperoxidase method. Keratin-positive malignant cells appearing as single cells or in small clusters were detected in bone marrow samples from Stage IV patients only (9/68, 13%) and detected in apheresis samples from both Stage III and IV patients (13/245, 5%). These findings indicate that the combination of cytomorphology with immunocytochemistry can be utilized for the investigation of circulating tumor cells and bone marrow micrometastases, and that positive results appear to correlate with high tumor stage/burden.

Absence of SV-40 large T antigen (Tag) in malignant mesothelioma effusions: an immunocytochemical study.

Simian Virus 40 (SV 40) was recently implicated in the pathogenesis of malignant mesothelioma. The oncogenic capacity of SV-40 is a function of a nuclear protein, the large T antigen (Tag). SV-40 Tag DNA sequences are detected by the polymerase chain reaction in 40-80% of malignant mesothelial proliferations. However, the role of immunohistochemistry (IHC) in demonstrating the nuclear localization of Tag is controversial. We sought to determine the clinical utility of SV-40 Tag IHC in pleural effusion cytology as an ancillary tool in the cytologic diagnosis of malignant mesothelioma (MM). Formalin-fixed, paraffin-embedded cell block sections from 100 pleural effusions (32 MMs, 25 benign reactive, 43 metastatic adenocarcinomas) were immunostained for the SV-40 anti-Tag, using two primary monoclonal SV-40 Tag antibodies: clone Pab 416 and clone Pab 101. Despite strong and consistent immunoreactivity in positive controls, no nuclear immunostaining was observed in any case. We believe the small sample size in cytology cell block sections, the low viral copy number in infected cells, and the effect of formalin fixation may have resulted in absence of immunoreactivity. The role of SV-40 Tag IHC in diagnostic cytopathology remains unclear unless further studies reliably show its detection.

Fluorescence in situ hybridization (FISH): a user's guide to optimal preparation of cytologic specimens.

Fluorescence in situ hybridization (FISH) is a reliable method for tagging centromeric regions of specific chromosomes in interphase nuclei. Not only is FISH useful for chromosome enumeration, but as region-specific chromosome probes are developed, the clinical applications and potentials for use by pathologists are extensive. This technique lends itself particularly to use in cytology preparations because the cells are disaggregated and monolayer preparations yield excellent technical hybridization results. Over a 7-mo period we processed cytologic samples in an attempt to define and outline a method for optimal specimen processing for FISH use in cell suspensions, techniques applicable to all fresh cytology specimens which can also be used for the processing of surgical pathology aspirates and other material. All samples should be promptly processed to ensure specimen viability, and triaged on an individual basis to ensure preparation of moderately cellular monolayered cytospins. Equivalent nuclear probe signals have been obtained with several sample fixation methods: air-drying, 95% ethanol, methanol (Diff-Quik fixative), and Carnoy's solution. No difference was noted in the nuclear probe signals or specimen adhesion on positively charged or noncharged slides. After initial fixation our slides remained at room temperature until FISH was performed, without any adverse effects. A short digestion with proteinase K and subsequent rehybridization yielded positive results on samples that originally yielded poor nuclear probe signals.