Flow cytometric DNA analysis of carcinoid tumors of the ileum and appendix.

Although all carcinoid tumors are considered potentially malignant, the biologic behaviors of appendiceal and ileal carcinoids are distinctly different. Appendiceal carcinoids often behave in a benign fashion, whereas ileal carcinoids pursue an aggressive course with frequent metastasis. Whether differences in DNA ploidy are related to this disparity in tumor behavior was addressed in this study. Flow cytometric DNA analyses were performed on paraffin blocks from 11 ileal and seven appendiceal carcinoid tumor cases. The mean coefficient of variation for all samples was 3.4 +/- 0.7. DNA aneuploidy was seen in two of the appendiceal cases and in six of the ileal cases. Metastases were seen in one of the appendiceal carcinoid cases, and that tumor was aneuploid. In six cases of carcinoid of the ileum, metastases were seen; of these, five tumors were aneuploid. In the ileal cases, despite the low number of cases examined, the correlation between DNA aneuploidy and metastases nearly reached statistical significance (P = .07) and showed a much stronger correlation than tumor size and metastases (P = .4). Although no statistical significance was reached in this study, the results are highly suggestive of DNA aneuploidy being an important predictor of malignant behavior in carcinoids of the ileum.

DNA flow cytometric analysis of hemangiopericytoma.

The biologic behavior of hemangiopericytoma is difficult to predict using clinical or histologic criteria. The authors studied 22 hemangiopericytomas (including "angioblastic meningiomas") from 16 patients. Included in the study were six recurrent tumors and one metastatic tumor. DNA flow cytometric analysis was performed on 21 tumors for which paraffin-embedded material was available. All of the tumors were DNA diploid. However, among patients with adequate follow-up information, all tumors that exhibited aggressive behavior (local recurrence, metastasis, death due to invasive disease) had S-phase fractions of greater than 9% and proliferative indices (S-phase plus G2M phase) of greater than 11%. There was also a trend toward aggressive behavior in tumors with necrotic foci. Tumors arising in the central nervous system behaved more aggressively than primary tumors at other sites. This study showed a trend toward more aggressive behavior in hemangiopericytomas with higher proliferative indices. DNA ploidy, however, was not a useful indicator of biologic behavior in these tumors.

Enhanced detection of malignant lymphoma in cerebrospinal fluid by multiparameter flow cytometry.

Immunophenotyping by flow cytometry has not been widely applied to cerebrospinal fluid (CSF) analysis. We attempted to optimize flow cytometric detection of malignant lymphoma in CSF samples by the routine use of 3- and 4-color flow cytometry, with specific selection of lymphoid cells by fluorescence vs 90 degrees light scatter gating. Thirty-six consecutive CSF samples were immunophenotyped by flow cytometry, and the results were compared with those of standard microscopic examination. Lymphoid events were adequate for analysis in 27 of the 36 samples. Each of the 9 unsuccessful samples was more than 24 hours old at analysis or contained fewer than 1 x 10(4) total cells (< or =1 cell/microL). Lymphoma was detected in 10 of the remaining 27 cases. Six lymphomas were detected by morphology and flow cytometry, 1 only by morphologic examination, and 3 only by flow cytometry. Therefore, the combination of flow cytometry and morphologic examination enhanced the detection by 43% over morphologic examination alone. Flow cytometry permitted the detection of lymphoid clones totaling less than 1% of total cells. Multicolor flow cytometry is a rapid and sensitive technique that enhances detection of lymphoma in paucicellular CSF samples. Given the great sensitivity of flow cytometry, future studies will be necessary to assess the significance of detecting small lymphoid clones in this setting.

CD5- small B-cell leukemias are rarely classifiable as chronic lymphocytic leukemia.

Expression of the CD5 antigen by neoplastic cells often is considered a diagnostic criterion for B-cell chronic lymphocytic leukemia (B-CLL). However, published series frequently include a number of CD5- cases. We studied the spectrum of CD5- B-cell lymphoproliferative disorders presenting with leukemia involvement and reassessed the prevalence of CD5- B-CLL. We immunophenotyped 192 cases of clonal, small lymphocytic, B-cell disorders involving peripheral blood or bone marrow. Of these, 41 CD5- cases were further analyzed, correlating the immunophenotypic findings with pathologic material and clinical data. Only 3 CD5- cases were classified as CD5- B-CLL. These 3 cases had features unusual for B-CLL, including bright surface immunoglobulin expression, bright CD20 expression, and absence of CD23 expression (2 cases) or Richter syndrome (1 case). The remainder of the CD5- cases consisted of hairy cell leukemia, hairy cell variant, prolymphocytic leukemia, follicular center cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma (SMZL), small lymphocytic lymphoma with marrow fibrosis, and lymphoma, not further classified. Eight cases remained unclassified, but some displayed features of SMZL. CD5- lymphoproliferative disorders of peripheral blood or bone marrow are unlikely to be CLL and often are classified more appropriately as non-Hodgkin lymphoma in the leukemia phase.

Lymphoid aggregates in bone marrow mimic residual lymphoma after rituximab therapy for non-Hodgkin lymphoma.

Rituximab is a novel anti-CD20 monoclonal antibody used in the treatment of relapsed low-grade non-Hodgkin lymphoma. To determine the impact of this therapy on the interpretation of posttherapy specimens, we reviewed the pretherapy and posttherapy bone marrow and peripheral blood morphologic and flow cytometric findings for 20 patients who received rituximab. Nine patients had a total of 13 posttherapy bone marrow specimens; all were positive for lymphoma before therapy. After therapy, 11 of 13 posttherapy bone marrow specimens were interpreted as positive or suggestive of lymphoma based on routine H&E-stained sections. However, immunohistochemical and/or flow cytometric immunophenotyping showed that 6 of the 11 cases were negative for lymphoma; the lymphoid infiltrates were composed entirely of T cells without B cells. We report that posttherapy bone marrow specimens from patients treated with rituximab may mimic residual lymphoma if examined by morphologic features alone. Familiarity with this finding and the use of ancillary immunophenotypic studies will aid in the accurate interpretation of posttherapy specimens.

Karyotype correlates with peripheral blood morphology and immunophenotype in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is recognized as a distinct entity. However, morphologic and immunophenotypic heterogeneity exist. Twenty-six patients with CLL were studied to investigate whether an association exists among peripheral blood karyotype, morphology and immunophenotype. Clonal cytogenetic abnormalities were detected in 14 patients (53%), using conventional karyotyping techniques in addition to fluorescence in situ hybridization (FISH) for chromosome 12. By FAB guidelines, 7 of the 8 patients (88%) with trisomy 12 had mixed cell morphology compared to only 3 of 18 (17%) without trisomy 12 (P = .004). One patient (12%) with trisomy 12 had lymphocyte morphology typical for CLL. Six of the eight (75%) with trisomy 12 had atypical immunophenotype including one or more of the following: strong CD20 expression, strong surface light chain expression, or absence of CD23 expression. Only 2 of the 18 patients (11%) without trisomy 12 had atypical immunophenotype (P = .005). None of the three patients with clonal structural abnormalities of chromosome 13q14 had mixed cell morphology or atypical immunophenotype. One of the 12 patients (8%) without clonal cytogenetic abnormalities had mixed cell morphology and one had atypical immunophenotype. This study suggests that a correlation exists among karyotype, morphology, and immunophenotype in CLL, and that CLL subgroups can be identified based on laboratory parameters. Although normal karyotypes or clonal structural abnormalities of 13q14 are associated with morphology and immunophenotype considered typical for CLL, trisomy 12 is associated with mixed cell morphology and atypical immunophenotype. These findings may have implications for evaluating variation in both disease course and response to emerging therapies.

Karyotype evolution in a simian virus 40-transformed tumorigenic human cell line.

Normal human foreskin fibroblasts (HSF4) were transfected using the pSV3-neo plasmid. A pool of 10 G418-resistant colonies, HSF4-T12, showed a progressive increase in the expression of a number of in vitro transformation markers with passage in culture and became immortalized. Although no tumors were formed when cells were injected subcutaneously into nude mice, this cell line produced progressive tumors when cells were injected into preimplanted Gelfoam sponges in the mice. When these tumors were cultured in vitro and subsequently injected subcutaneously, progressive tumors were produced with median latency periods as short as 4 weeks. Three phases of cytogenetic change could be distinguished. At early passages after transfection. HSF4-T12 exhibited many random chromosomal changes. At a time just after immortalization, both flow karyotype and G-banded analyses showed the appearance of balanced clonal rearrangements. These included t(2;4), t(2;14), t(3;?), 6p-, i(6p), 8p-, t(14;15), i(15), and t(18;?). These clonal rearrangements were stable with passage in culture, and less variability from cell to cell was noted. The only consistent chromosomal loss observed was -Y. Analysis of three independent tumors showed characteristic loss of chromosomal material rather than balanced chromosomal rearrangements. Frequent loss of 6q and chromosomes #13, 15, 20, and Y was noted.

In situ hybridization in flow cytometry.

Molecular approaches to diagnostic questions in clinical medicine are greatly impacting the way researchers and clinicians investigate and treat disease. By combining molecular techniques with classical immunologic tools such as flow cytometry (FCM; 1-3), one can begin to more fully understand and appreciate the role of cellular heterogeneity in disease processes. The marriage of these two powerful techniques, termed molecular cytometry, will, in one instance, allow investigators to explore expression of nucleic acid sequences in subpopulations of cells defined by immunologic phenotype while, conversely, making it possible to examine the heterogeneity of cellular characteristics within populations identified by the presence of specific nucleic acid sequences or gene expression. Future developments may result in several advantages for the patient that may include, but are not limited to, earlier detection of viral infection, earlier and more sensitive detection of malignancy, and higher sensitivity and resolution of small populations of infected or aberrant cells. These developments may also assist in the identification of therapeutically resistant populations within a neoplasia, more effective and specific monitoring of therapy, and possibly the identification of new and disease-specific targeted therapies based on genetic information. The characterization and assessment of cellular heterogeneity is clearly key to understanding disease onset, progression, and therapeutic response in both infectious disease and in human malignancies.

Atypical hairy cell leukemia.

The morphologic differential diagnosis of mature B-cell neoplasms with cytoplasmic projections includes splenic lymphoma with villous lymphocytes and hairy cell leukemia. Although the classification of hairy cell leukemia is not universally recognized, 3 variants have been described, namely, classic, variant, and Japanese variant, each of which has different clinical and immunophenotypic features. Classic hairy cell leukemia is virtually always CD11c(+), CD25(+), and CD103(+). Variant and Japanese variant hairy cell leukemias are usually CD11c(+), always CD25(-), and occasionally CD103(+). Each variant is characteristically CD10(-). We present a case of hairy cell leukemia with a unique immunoprofile in that the cells were CD10(+), CD25(+), and CD103(-), and we review the criteria helpful in differentiating "hairy" B-cell neoplasms. This case emphasizes the variability of hairy cell leukemia and the need to correlate all clinical and pathologic data in reaching a diagnosis.

Cell bioprocessing in space: applications of analytical cytology.

Cell bioprocessing in space consists of the preparation, cultivation, purification and investigation of cells and their products in the microgravity environment of orbital space flight. Inertial acceleration is used as an independent variable to explore the limits of specific bioprocessing functions, such as cell growth and secretion, gravity-dependent phenomena in cell bioreactors, cell fusion, the influence of thermal convection on processes at cellular dimensions, the electrophoretic separation of cell subpopulations and subcellular particles, and two-phase partitioning of cells, bioparticles, and macromolecules. Analytical cytology techniques are under development for on-orbit application to future cell growth and separation experiments, such as those anticipated in the Space Station era.

Flow cytometric analysis of neoplastic nodules and hepatocellular carcinomas induced by ciprofibrate in the rat.

Alterations in DNA ploidy accompany hepatocellular carcinoma (HCC). However, changes in DNA content are also seen in regenerating liver and with increasing age. Thus, to investigate the role of DNA ploidy changes in development of HCC, flow cytometric DNA content determinations were done in a rat model system of peroxisome proliferator-induced HCC. Paraffin blocks of liver isolated from 18 Fisher 344 male rats fed ciprofibrate for 20 weeks (4), 40 weeks (4) or 20 months (10) were examined. Livers from age-matched control rats were also examined. From the 20 month ciprofibrate group, nine neoplastic nodules (NNs), 27 HCCs and four non-tumorous surrounding tissue controls (NTCs) were examined. Significant DNA tetraploid populations were seen in both the NNs and NTCs. A significant increase in the percentage of DNA diploid cells was observed in the NN samples. No significant difference in the percentage S-phase cells was seen. Emergence of cell populations with new DNA ploidy classes (8c or DNA aneuploid) as compared with NTCs was only seen in HCCs (7 of 27), and five of these seven were DNA aneuploid, as distinct from DNA tetraploid, populations. A total of 16 of 24 HCC samples that were adequate for cell cycle analysis had average percent S-phase greater than the mean of the NTCs plus three standard deviations. Although a direct role cannot be inferred, these results support the hypothesis that increases in the fraction of diploid cells is an important early event in the development of rat HCC and that further alterations in DNA ploidy and increased proliferative fraction accompany the development of HCC.

TIMP-2, a growth-stimulatory protein from SV40-transformed human fibroblasts.

The mechanisms by which the early genes of simian virus 40 (SV40) transform human cells are unclear; however, this is clearly a multistep process involving a number of cellular and genetic changes. An early change following expression of the SV40 genes is growth under reduced serum conditions, which could be consistent with the production of autocrine/paracrine growth factors. HSF4-T12 is a human fibroblast cell line produced by transfection of primary cells with the genes for large T and small t antigens. A progressive stepwise transformation was observed with in vitro culture, eventually resulting in a tumorigenic cell line. Serum-free defined medium conditioned by HSF4-T12 was able to stimulate growth of normal human fibroblasts as determined by growth curve and [3H]-thymidine incorporation assays. Purification of this activity by heparin affinity chromatography and nondenaturing polyacrylamide gel electrophoresis resulted in a single band of approximately 21 kDa on a nonreducing, denaturing gel. A partial 14-amino acid sequence was found to share 100% homology with a region of tissue inhibitor of metalloproteinases-2 (TIMP-2). Western blot analysis with anti-TIMP-2 antiserum confirmed this identification, and addition of this same antiserum to HSF4-T12-conditioned medium resulted in inhibition of stimulatory activity.

Viral and cellular oncogene expression during progressive malignant transformation of SV40 transformed human fibroblasts.

In vitro investigation of the multistep neoplastic progression which occurs during transformation of human cells has been hindered by resistance of human cells to both immortalization and tumorigenicity (Mut. Res. 199; 273, 1988). Previously our laboratory established a cell line, HSF4-T12, by transfection of normal human foreskin fibroblasts with the plasmid pSV3-neo which contains the early genes of simian virus 40 (SV40). A multistep progression in karyotypic alterations and transformed phenotype occurred resulting in a neoplastic cell line that was immortal, transformed, and tumorigenic. We have examined changes in the SV40 proteins, large T (T-antigen) and small t (t-antigen) antigens, and in the cellular protein, p53, during progressive transformation of these cells. Total viral protein expression relative to total cellular protein increased following immortalization of HSF4-T12 as did the ratio of T-antigen to t-antigen. Interestingly, no significant change in DNA content accompanied immortalization. However, during the progressive in vitro transformation of HSF4-T12 which occurred primarily post-immortalization, DNA index increased to 1.6 but only small additional increases in T-antigen expression were seen. No consistent or critical role for t-antigen in development of the tumorigenic phenotype was found in this system.

Cell cycle kinetics in rat prostatic epithelia: nuclear migration during G2 phase.

Proliferative events in luminal epithelial cells of the rat ventral prostate were studied in 2 experiments. In experiment 1 a longitudinal survey of the rat prostate was conducted between 22 and 88 days of age. The pattern of temporal changes in the weight of the ventral prostate paralleled that of the testis, an observation consistent with the knowledge that the prostate is an androgen target organ. However, the total number of mitotic cells per prostate showed a biphasic pattern of distribution. The count was high (82 per prostate) in the prostate of 22-day-old rats and it decreased to a low value (25 per prostate) by age 30 days, a period corresponding to the onset of puberty in rats. The total mitotic count per prostate increased gradually and reached a peak (85 per prostate) around age 60 days when it started to decline gradually despite a continuous increase in prostatic weight. In experiment 2 cell cycle parameters of these cells were determined in young adult rats (age 70 days or 250 gm. body weight) by an in vivo 3H-thymidine pulse-chase approach. The apparent duration of various phases of the cell cycle was estimated as G1--11.5 hours, S--6.5 hours, G2--3.0 hours, M--2.0 hours and total cell cycle--23 hours. The fraction of cells in the S phase was 2.1%, which was extrapolated to give rise to a total growth fraction of 7.4%. In addition, it was noted that there was a migration of the nucleus from the base toward the apex of the epithelial cells during the G2 phase of the cell cycle in preparation for cell mitosis. Following mitosis nuclei of the 2 daughter cells returned to the usual basal position. These parameters will be used as basis of our future kinetic studies in the rat prostate.

Reducing cellular autofluorescence in flow cytometry: an in situ method.

Cellular autofluorescence affects the sensitivity of flow cytometric assays by interfering with detection of low level specific fluorescence. These detection limits increase with use of protocols, such as thermocycling and fluorescent in-situ hybridization (FISH), that can increase intrinsic cellular fluorescence to 5,000-20,000 fluorescein isothiocyanate (FITC) equivalents. In order to improve signal to noise ratios when using FITC labeled probes in these procedures, we employed a method using the polyanionic azo dye, trypan blue, to reduce intracellular autofluorescence. Dyes such as these are commonly used in immunofluorescent microscopy to reduce background fluorescence. By using this method, we realized an approximately 5-fold increase in signal to noise ratio (S/N) in the direct detection of RNA target probes using flow cytometry. Trypan blue aided in the resolution of dim surface antibodies, internal markers and probes, and functions to reduce background autofluorescence after thermocycling and hybridization. This technique is rapid and easily applicable for reducing intracellular autofluorescence, and can be used in single and dual color applications.

Lymphocyte subsets and activation markers in patients with acute episodes of idiopathic anaphylaxis.

BACKGROUND: Idiopathic anaphylaxis (IA), a type of anaphylaxis in which no external allergen can be identified, is a corticosteroid-responsive disease, that suggests that it may have an immunologic pathogenesis. OBJECTIVE: The objective of this study is to compare patients with acute episodes of IA with normals, patients with chronic idiopathic urticaria, and patients with IA in remission relative to lymphocyte subsets and activation markers. METHODS: This is a prospective cohort study of 38 adults: 5 normals, 4 idiopathic urticaria, 11 IA patients in remission, 9 IA patients with acute attacks who had not yet received prednisone, and 9 IA patients who had received prednisone. The main outcome measures were lymphocyte subset and activation markers determined by two and three color flow cytometry (CD2, CD3, CD4, CD5, CD8, CD16, CD19, CD23, CD25, CD56, and HLA-DR). RESULTS: Comparing patients with acute IA with those in remission, the only significant difference was that the acute IA patients had a significantly higher percentage of CD3+HLA-DR+ cells. Normals had a significantly lower percentage of CD3+ HLA-DR+ cells than all other groups. Patients with acute IA on prednisone as well as IA patients in remission had a significantly higher percentage of CD 19+ CD23+ cells than normals. CONCLUSIONS: These results suggest that there are more activated T cells in patients with acute episodes of IA than in patients in remission. Perhaps, these activated T cells have a role in the pathogenesis of IA.

TIMP-2 growth-stimulatory activity: a concentration- and cell type-specific response in the presence of insulin.

In addition to proteinase-inhibitory activities, growth-stimulatory activities have been described for all three known members of the tissue inhibitors of the metalloproteinase (TIMP) family, TIMP-1, TIMP-2, and ChIMP-3, believed to be the chicken homologue of TIMP-3. However, the mechanism by which the TIMPs stimulate cell growth is unclear. In this report we have demonstrated that rTIMP-2 was growth-stimulatory for human foreskin fibroblasts (HSF4, HSF43, HS68), lung adenocarcinoma cells (A549), human melanoma cells (WM115), and the Burkitt's lymphoma cell line RAMOS, and this stimulatory response was concentration-dependent, with the greatest stimulation occurring a 10-30 pM rTIMP-2 in [3H]thymidine incorporation assays and at 20-100 pM in cell growth assays. Normal human colon (18Co) and lung (37Lu) fibroblasts showed no response to rTIMP-2. [3H]Thymidine incorporation was inhibited by rTIMP-2 treatment in the nonadherent cell line HL60. These studies also demonstrated that for the cell types tested, TIMP-2 alone was insufficient for a growth stimulatory response requiring, at a minimum, the presence of insulin. In the absence of any "co-factor(s)," such as insulin, TIMP-2 treatment was inhibitory.

Expansions of clonal and oligoclonal T cells in B-cell chronic lymphocytic leukemia are primarily restricted to the CD3(+)CD8(+) T-cell population.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of mature-appearing clonal B cells exhibiting coexpression of CD5 and CD23. In addition to the accumulation of neoplastic B cells, numerous T-cell abnormalities also occur in B-CLL patients. In this study, the presence, and distribution within the T-cell subsets, of clonal/oligoclonal T cells was studied. Multicolor flow cytometric techniques were employed using combinations of anti-CD3, anti-CD4, and anti-CD8 antibodies coupled with antibodies specific for V(alpha) and V(beta) T-cell receptor (TCR) epitopes. Molecular studies of TCR gene sequences were done to confirm the presence of clonal/oligoclonal T-cell populations. In the flow cytometric studies, examination of V(alpha)/V(beta)expression found evidence of clonal/oligoclonal expansion in 9 of 19 patients studied. In eight of the nine patients, the expansions were restricted to the CD3(+)CD8(+) cell population. Molecular analyses were performed in 16 patients, 12 of whom showed a clonal or oligoclonal pattern. Of the four patients who were negative in the molecular analyses, all demonstrated flow cytometric evidence of clonal/oligoclonal expansions. Thus, when the flow cytometric and molecular analyses were considered together, all 16 patients for whom parallel analyses were done showed evidence of clonal/oligoclonal expansions. These results confirm previous work demonstrating that the majority of B-CLL patients harbor clonal/oligoclonal expansions within the T-cell population. Additionally, based on the relative numbers of cells expressing specific V(alpha) or V(beta)epitopes, these results show that these expansions occur primarily within the CD3(+)CD8(+) T-cell population.

Flow cytometric assessment of the cellular pharmacokinetics of fluorescent drugs.

Development of multidrug resistance (MDR) in cancer cells decrease net doxorubicin uptake as a result of either increased efflux, or decreased intracellular sequestration, or decreased membrane permeability. Kinetic parameters of drug uptake can distinguish among these forms of altered transport. Cellular uptake of fluorescent drugs was monitored by a flow cytometric assay using a rapid-injection system and analyzed with a three-compartment model in which rapid diffusion from extracellular fluid into the cell was followed by uptake into a nonexchangeable pool. In agreement with our recent studies of 14C-doxorubicin distribution (Dordal et al.: J Pharmacol Exp Ther 271:1286-1290, 1994), sequestration of doxorubicin was decreased 2.7-fold in P-glycoprotein-expressing SU-4R lymphoma cells compared to drug-sensitive SU-4 cells (14.0 +/- 4.8 vs. 5.0 +/- 0.9 nl s-1) without a change in membrane permeability or evidence of active efflux. In contrast, sequestration of the highly fluorescent dye rhodamine 123 was decreased 20-fold (17.1 +/- 8.3 vs. 0.9 +/- 0.8 nl s-1). Resistant cells were significantly less permeable to rhodamine than sensitive cells (3.8 +/- 1.2 vs. 10.2 +/- 2.6 x 10(5) cm2 s-1), and rhodamine efflux was increased by 24%. Thus, SU-4R cells exhibit multiple alterations that cause decreased intracellular drug concentrations, of which decreased sequestration is quantitatively the most significant.