Phase II study of theophylline in chronic lymphocytic leukemia: a study of the Eastern Cooperative Oncology Group (E4998).

The Eastern Cooperative Oncology Group (ECOG) performed a phase 2 study in B-cell chronic lymphocytic leukemia (CLL) of oral theophylline, a methylxanthine that inhibits cyclic nucleotide phosphodiesterases, thereby inducing the intracellular accumulation of cyclic adenosine monophosphate (cAMP). In 25 patients with Rai stages 0-I, theophylline, 200 mg given orally every 12 h was well tolerated. There was one complete response after 22.5 months of treatment, which continues at 27+ months, and 18 other patients had stable disease. In vitro exposure of patients' lymphocytes to aminophylline (75-250 microg/ml), the soluble form of theophylline, resulted in dose- and time-dependent induction of apoptosis in 9/20 patients studied. Apoptosis was documented flow-cytometrically by monitoring the expression of bcl-2 and bax, forward light scatter, fluorescence intensity of binding of CD45 antibody, and the binding of annexin. Patients whose leukemic lymphocytes were susceptible to apoptosis induction by aminophylline in vitro experienced a significantly longer progression-free survival than patients whose cells were resistant to the drug in culture (P=0.025). This suggests that in a CLL population treated with theophylline, induction of an apoptotic response to the drug in vitro is prognostic for absence of clinical progression.

Cellular changes and induction of apoptosis in human promyelocytic HL-60 cells infected with the agent of human granulocytic ehrlichiosis (HGE).

Human granulocytic ehrlichiosis (HGE) is an emerging and occasionally fatal human infectious disease whose pathogenesis is largely unknown. Goodman et al. (1) recently described the successful cultivation of the HGE infectious agent in human promyelocytic HL-60 leukemic cells. It was reported in the same study that infectivity invariably led to host cell death, although the mechanism by which HGE infection triggers cellular self-destruction is as yet undetermined. In this communication, we show that in vitro passage of HGE pathogen-infected blood elicits a significantly dysfunctional G1-to-S transition. Moreover, we provide evidence that the cytopathic properties of the HGE pathogen are attributed to its ability to induce apoptosis in host HL-60 cells. Determination of specific protein expression changes by Western blot analysis showed that HGE infection resulted in reduced expression of PCNA and pRB, both of which play a role in cell cycling. Moreover, the steady state level of bcl-2, which protects eukaryotic cells against apoptosis, is suppressed by exposure to the HGE agent. These results suggest that this pathogen HGE induces apoptosis in HL-60 cells by a mechanism involving the shut-off of multiple cell cycle and apoptosis regulatory events.

Aberrations of chromosomes 9 and 17 in bilharzial bladder cancer as detected by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH), using repetitive alpha satellite probes to chromosomes 9 and 17, was performed on 27 paraffinembedded bladder specimens from 18 Egyptian patients affected with bilharzial carcinoma. Fluorescence in situ hybridization results in the carcinomas and benign mucosa of patients with schistosomiasis were compared with flow cytometric DNA ploidy and cell cycle assays. These results demonstrate the feasibility of this technique for studies of these archival specimens, and suggest early onset of chromosomal aberrations in histologically benign mucosa of schistosomal cystitis with progression during the development of bladder cancer. On the basis of these data, monosomy 9 may be an early chromosomal change in urothelium of the bilharzia-infested bladder, and a predictor of incipient carcinoma in patients with bilharzial cystitis.

Third-color CD45 staining of paraformaldehyde-fixed peripheral blood lymphocytes.

Third-color CD45 gating is a useful procedure in various clinical applications (Borowitz et al.: Am J Clin Pathol 100:534-540, 1993; Steltzer et al.: Ann NY Acad Sci 677:267-280, 1993) allowing for the selective immunophenotyping of abnormal cell populations. In laboratories where dual-color protocols are in place, the need for CD45 gating may not become evident until after cells have been exposed to fixatives. This brief study demonstrates that third-color CD45 staining and gating is possible with paraformaldehyde fixed lymphocytes, eliminating the need to restain fresh cells with the entire antibody panel, and with a significant saving of time and money. Gates must be modified from those obtained by fresh cell staining in order to include both bright and dim CD45 populations. In a standard protocol, third-color CD45 staining intensity was decreased 10-fold post-fixation with paraformaldehyde except, unexpectedly, for CD8+ cells. The latter phenomenon could be explained if the spatial arrangement of CD45 and CD8 is such that the CD45 epitope is protected from cross-linking by paraformaldehyde fixation in CD8+ cells, but not others.

Flow cytometric characterization of proliferation-associated nuclear antigen (p105) during the cell cycle in normal lymphocytes and promyelocytic leukemia cells (HL-60).

Bivariate flow cytometric analysis of p105 expression and DNA content was performed in human lymphocytes and promyelocytic leukemia cells (HL-60). We also employed a new method of detecting DNA strand breaks associated with apoptosis by labeling the 3'-OH termini in the breaks with biotinylated dUTP in a reaction employing exogenous terminal deoxynucleotidyl transferase (TdT). Phytohemagglutin-stimulated proliferating lymphocytes express p105 maximally after 48 hours, similar to HL-60 cells in exponential growth phase. Antigen expression in G1 was notably heterogeneous in G1 phase of both cell types and highest in M-phase cells treated for 6 hours with vinblastine. However, the p105-DNA ratio changed very little. Cycloheximide did not affect P105 expression. Methotrexate decreased p105 expression. Camptothecin and teniposide induced apoptosis, but apoptotic cells still expressed p105. Dual-parameter measurement also demonstrated that TdT-positive apoptotic cells expressed p105 at a higher level than apoptotic TdT negative cells. The data on drug treatment suggest that expression of p105 may be useful in monitoring chemotherapeutic effects but not as a marker of cell death from apoptosis.

Comparison of fluorescence in situ hybridization and flow cytometric DNA ploidy analysis in paraffin-embedded prostatic adenocarcinoma specimens.

Cancer cell nuclei extracted from 15 paraffin-embedded radical prostatectomy specimens were examined with fluorescent in situ hybridization (FISH) and flow cytometry (FCM) for DNA ploidy. Repetitive chromosome-specific alpha-satellite DNA probes to centromeres 7 and 10 were used in the FISH assay. Results from FISH and FCM were compared and viewed in relation to clinical experience. Of the 15 tumors examined, 9 were hyperdiploid for at least one chromosome by FISH assay. Seven were aneuploid by FCM, showing a good correlation between the two methods (P = .006). Using either evidence of clinical progression or a postoperative prostate-specific antigen (PSA) level > 0.5 ng/mL by Hybritech assay to indicate the risk of clinical progression, 56% of FISH hyperdiploid tumors had a risk of progression of carcinoma versus 17% of FISH diploid tumors. A preoperative PSA level > 30 ng/mL had a marginal correlation with risk of progression (P = .05). We demonstrate that FISH is a useful tool for chromosomal analysis in paraffin-embedded tumor specimens.

Effects of suramin on expression of proliferation associated nuclear antigens in DU-145 prostate carcinoma cells.

Suramin is the first putative growth factor inhibitor in clinical trial that has demonstrated antitumor activity. Bivariate flow cytometric analysis of the effects of suramin on the cell cycle was performed on DU-145 prostate carcinoma cell line. The effects of suramin on the expression of proliferation associated antigens (p145, p120, PCNA, Ki-67, and cyclin A) were also studied. At concentrations of 50-500 micrograms/ml, suramin arrested cells in G1 and decreased S-phase moderately. The expression of p145, p120, PCNA, Ki-67 and cyclin A in these cells was reduced. The data suggest that suramin not only inhibits proliferation, but also reduced the expression of proliferation associated antigens. The expression of these antigens may be considered a means to monitor suramin treatment in vivo and in vitro.

Bivariate flow cytometric analysis of p53 and DNA content in hepatocellular carcinoma.

Bivariate flow cytometric analysis of p53 protein and DNA content was studied in archival specimens of hepatocellular carcinoma (HCC) from Chinese patients and corresponding benign liver tissues from a series of 51 patients at Sun Yat-sen University of Medical Sciences. Extracted nuclei were stained with the fluoresceinated monoclonal antibody PAb 1801, which recognizes human p53 protein (mutant and wild types). The nuclei were counterstained with the DNA stain propidium iodide. They were measured on an Ortho FC-200 flow cytometer and the data acquired and analyzed with an IBM 386 personal computer using Kusuda's Get Simple and List Simple software. Of the 51 hepatomas studied, 26 (51%) were p53 positive as compared with 4 (16%) of 24 samples of benign liver tissue from the same patients (P < .0257). The S-phase fraction of p53-positive HCC (12.3 +/- 8.8%) (SD) was significantly greater (P < .05) than for p53-negative HCC (7.4 +/- 7.2%). p53 Expression did not correlate with age, sex, alpha-fetoprotein, hepatitis B surface antigen, tumor size, tumor grade or survival rate. List Simple software permitted analysis of each specimen together with its isotype control (IgG1) on the same cytogram so that p53 expression could be determined separately for the diploid and aneuploid populations of aneuploid tumors and for tumor cells of diploid tumors in the various phases of the cell cycle. Since p53 (PAb 1801) expression can withstand formalin fixation and pepsin treatment of paraffin-embedded tissues, flow cytometric analysis of archival specimens is feasible, and clinical correlations such as these may be carried out in retrospective studies of other tumors.

p145 expression during the cell cycle in HL-60 cell line and normal human lymphocytes: effects of camptothecin, vinblastine, cycloheximide, actinomycin D, retinoic acid and DMSO.

Bivariate flow cytometric analysis of nucleolar antigen p145 was performed on quiescent and phytohemagglutinin-stimulated human lymphocytes and on a promyelocytic cell line (HL-60). Data were acquired on a FACScan flow cytometer and analyzed using LYSYS II. Quiescent lymphocytes did not express p145. PHA-stimulated proliferating lymphocytes expressed p145 maximally after 48 h, similarly to HL-60 cells in exponential growth. Antigen expression in G1 was notably heterogeneous in both cell types. The ratio of p145/DNA was highest in early S and decreased during mid and late S and remained low in G2M. p145 expression was lowest in M-phase cells treated for 6 h with vinblastine. Cycloheximide and actinomycin D had similar effects on p145 in HL-60 cells: expression of p145 gradually decreased from 1 to 6 h incubation in all phases of the cell cycle. Camptothecin did not decrease p145 expression and apoptotic cells from CAM-treated cultures still expressed p145. Retinoic acid and DMSO induced differentiation in HL-60 cells, and as this process progressed, p145 levels gradually fell until they approached isotype antibody control levels at 9 and 6 days, respectively. However, after 5 days treatment with 2 nM retinoic acid apoptotic cells appeared which still expressed p145. The data on drug treatment suggest that p145 exists in undifferentiated and proliferating cells and may not be a specific marker for malignancy, but may prove useful as a monitor of chemotherapeutic effects in cancer treatment.

Increase in acridine orange (AO) fluorescence intensity of monocytes cultured in plastic tissue culture plates as measured by flow cytometry.

Although the green-red fluorescence of AO is an accepted measure of DNA-RNA content, respectively, it is actually a measure of the fluorescence of dye bound to nucleic acids, and may vary with changes in accessibility to the dye. It has been shown for example that extraction of nuclear proteins results in a marked increase in DNA stainability. Moreover, in certain cell systems the binding of fluorochromes correlates with structural modifications in chromatin that accompany cell differentiation. We report here that changes in green & red fluorescence intensity also occur in long-term monocyte cultures. The increased red fluorescence intensity observed in cultured monocytes may reflect ribosomal RNA synthesis and the increased green fluorescence enhanced AO accessibility to DNA due to changes in chromatin organization. We compared cultured monocytes from bladder cancer patients and healthy donors. The results indicate a small but statistically significantly greater increase in mean green & red fluorescence of cultured monocytes from the cancer patients. These fluorescence variations may indicate differences in the immunologic status of cancer patients and/or be related to disease state.

Lack of specificity in the mechanisms involved in the enhancement of the concanavalin A driven human T lymphocyte stimulation by beta-endorphin: studies on activation marker expression, cell cycle and interleukin release.

We report on the effects of a physiological concentration of Beta-Endorphin (BE) (10(-12)M) on Concanavalin A (ConA) stimulated human peripheral blood T-lymphocytes and monocytes. We evaluated the effect of timing of BE addition to the culture medium on thymidine uptake, the kinetics of expression of activation markers (CD69, CD25 and CD71) on CD4+ and CD8+ lymphocytes, and of class II MHC antigens on CD14+ cells (monocytes), the kinetics of interleukin-1 (IL-1), interleukin-2 (IL-2) and interferon gamma (IFN-gamma) release, and the cell cycle. Data show that BE is able to influence T lymphocyte only when added together with ConA at the beginning of culture, suggesting its major activity is on the early phases of the T cell response. BE did not increase the amount of class II MHC antigens on monocytes and did not preferentially stimulate CD69, CD25 and CD71 antigen expression on either CD4+ or CD8+ lymphocytes. After 24 hours, the relative proportions of CD4+ and CD8+ lymphocyte in S and G2-M phases were not affected by BE, although the opioid did augment the number of cells in the proliferative compartments of the cell cycle, S and G2-M, indicating an actual increase in the number of cells committed to proliferation. BE did not consistently influence the amount of IL-1, IL-2 and IFN-gamma found in the supernatant of ConA stimulated cultures. The mechanism of the enhancing effect on the proliferative response of normal human lymphocytes to ConA by BE, does not seem to be selective for or unique to specific lymphocyte subsets.

Simultaneous detection of fluorescent in situ hybridization and in vivo incorporated BrdU in a human bladder tumour.

We have used fluorescent in situ hybridization and simultaneous in vivo bromodeoxyuridine labelling of a solid bladder cancer to examine tumour cell subsets for possible proliferative growth differences. In this dual-labelled preparation, most tumour cell nuclei exhibited monosomy 9, consistent with reported karyotypes of bladder cancer. Incorporated bromodeoxyuridine was visualized with a fluoresceinated antibody in 5-6 per cent of the tumour cells, concordant with S-phase estimates by cell cycle analysis of the flow cytometric DNA histogram. A majority of the bromodeoxyuridine-positive cells also carried the monosomy 9 chromosome abnormality. This is the first report to demonstrate the feasibility of combined in situ hybridization and detection of bromodeoxyuridine incorporated in vivo in human tumour cells in order to provide information on the growth rate of specific subsets of tumour cells identified by chromosomal constitution.

Targeted cytogenetic analysis of gastric tumors by in situ hybridization with a set of chromosome-specific DNA probes.

Fluorescent in situ hybridization (FISH) with biotinated chromosome-specific repetitive DNA probes was used for the cytogenetic study of ten gastric adenocarcinomas. All tumors (eight male, two female patients) were histologically moderately or poorly differentiated and nine of ten had metastasized to regional lymph nodes. The authors applied a set of satellite DNA probes, specific for chromosomes 1, 7, 17, X, and Y in order to detect numerical chromosome aberrations in freshly isolated tumor cell nuclei. Normal diploid human lymphocyte nuclei and, in a number of cases, normal gastric mucosa served as controls. Parallel with the hybridization experiments DNA flow cytometric study of acridine orange (AO)-stained tumor cells was carried out. By means of FISH the authors found seven cases to be aneuploid, the other three cases appeared diploid. This was confirmed by DNA flow cytometric analysis with AO. Furthermore, loss of the Y chromosome in a high percentage of cells was seen by FISH in six of eight tumors from male patients. In the other two male samples a possible loss was observed in a small proportion of cells (15%). In three patients from whom the authors had normal gastric mucosa the Y loss was restricted to the tumor cells. These data indicate that in situ hybridization with chromosome-specific repetitive DNA probes can serve as a cytogenetic tool for the analysis of interphase nuclei of solid human tumors, at least with respect to the detection of numerical chromosome abnormalities.

The use of Phadiatop in mass-screening programmes of inhalant allergies: advantages and limitations.

The validity of Phadiatop as a tool in the mass screening for inhalant allergies was investigated. Seventy-nine out of 600 recruits (13.2%) were classified as allergic to inhalant allergens (68 oculorhinitis; 11 asthma) on the basis of positive history, confirmed by skin testing and/or RAST for the seven most common aero-allergens in Italy. Another 74 subjects had positive RAST and/or skin tests, yet had never experienced allergic symptoms. Phadiatop was positive in 145/600: in 78 out of 79 allergics and in 67 RAST-positive non-allergics. Correlation between Phadiatop and RAST was statistically significant, though higher for grass pollens (r = 0.85, P less than 0.001) than for Dermatophagoides pteronyssinus (r = 0.68, P less than 0.01). PRIST (cut-off 220 IU/ml) identified 36/79 allergics (45.6% sensitivity, whereas that of Phadiatop was 98.7%). As the high percentage of cases in the unselected population had specific serum IgE against inhalant allergens and no allergic symptomatology, the Phadiatop-positive predictive value falls to 53.7%, thus creating the need for Phadiatop-positive subjects to undergo further investigation for an appropriate diagnosis. Given this limitation, Phadiatop appears to be an important step forward in the field of mass-screening programmes for inhalant allergies.

Cytogenetic analysis of human solid tumors by in situ hybridization with a set of 12 chromosome-specific DNA probes.

We have applied fluorescent in situ hybridization (FISH) to assess the presence of numerical chromosome aberrations in fresh specimens of human solid tumors of varying histology. For this purpose, a set of 12 biotinylated chromosome-specific, repetitive alpha-satellite DNA probes (for chromosomes 1, 6, 7, 9, 10, 11, 15, 16, 17, 18, X and Y) were hybridized directly to isolated interphase nuclei. Utilizing this approach, we found numerical chromosome changes in all tumors. FISH ploidy profiles were in accordance with flow cytometric DNA histograms of these tumor cells.

New, simple flow cytometry technique to discriminate between internalized and membrane-bound particles in phagocytosis.

This report describes a new flow cytometry technique to measure phagocytic activity and discriminate simultaneously between internalized and membrane-bound particles. Fluorescein-conjugated heat-killed Candida albicans (F-Ca) are opsonized with purified antibodies or normal human serum and used as targets for human polymorphonuclear granulocytes (PMN). The procedure is based on the observation that F-Ca lose their green fluorescence and acquire red fluorescence upon incubation with ethidium bromide (EB) through the resonance energy-transfer phenomenon occurring between the two fluorochromes. PMN are incubated with opsonized F-Ca particles for 20 min at 37 degrees C or, as a control, at 4 degrees C and in the presence of cytochalasin B, an inhibitor of the phagocytic process that does not affect membrane binding of F-Ca. EB is added, and green and red fluorescence associated with PMN is evaluated using a mercury-lamp-powered instrument. Because EB does not penetrate intact cell membranes, internalized particles are not affected by EB and remain green, whereas membrane-bound particles assume an intense red stain. By means of contour plot analysis, the number of PMN containing and/or binding F-Ca particles can be readily assessed. The method described here allows precise quantitative analysis of the phagocytic process on the part of human PMN in a single, one-step assay that does not require sophisticated instrumentation or reagents and should prove to become a test suitable for clinical purposes.

Hereditary angioneurotic edema: clinical and laboratory findings in 58 subjects.

An in-depth analysis of clinical and laboratory findings in 58 patients affected by hereditary angioneurotic edema (HANE) is reported with special focus on problems related to the diagnosis of the disorder. The functional C1 inhibitor (C1INH) assay is the method of choice in the diagnosis of HANE, as it is capable of revealing the disorder with 100% accuracy. The immunochemical assay of C1INH detected HANE in 84.5% of the cases, i.e., immunochemical deficiency of C1INH (type I HANE). C4 was markedly reduced in both type I and type II HANE; thus, C4 levels can be particularly useful when C1INH functional tests are not available. CH50 testing is of little diagnostic value since total hemolytic complement activity is reduced in a variety of other congenital or acquired pathologies involving the complement system. The CH50 assay after incubation in low ionic strength buffer may be utilized in mass screening programs for qualitative evaluation. However, the test has the drawback of not being applicable in cases of frank hypocomplmentemia. While a depletion of the complement classical pathway was detected in most cases, no alteration in the complement alternative pathway was recorded, nor there was any reduction in immunoglobulin levels. Family history was positive in 100% of the cases. Attacks were almost always brought on by stress and/or trauma, though the causes were sometimes unknown. Edema could be cutaneous (non-pitting and non-pruritic) in 94.2%, laryngeal (often life-threatening) in 48% and abdominal (almost always painful) in 88.4% of patients. Associated pathologies were found in 2 patients, i.e., lupus rash and C3NeF-positive chronic membranoproliferative glomerulonephritis, respectively.

Recombinant interferon alpha-2 modulates hairy cell phenotype "in vitro".

The "in vitro" effect of IFN-alpha on the phenotypic profile of atypical cells from 5 hairy cell leukemia patients was investigated in a 72 hr culture assay. Cytochemical investigations revealed a dramatic decrease in the cytoplasmic content of acid phosphatase and tartrate resistant acid phosphatase in the absence of any apparent morphological modification. Flow cytometry showed that IFN-alpha markedly reduced the density of surface Ig without modifying the original isotype pattern. The expression of the receptor for the Fc fragment of IgG was also reduced. The class II MHC antigen recognized by the monoclonal antibody 12 remained essentially unchanged. Hairy cells were negative for OKT10 and PCA-1 and remained so after IFN-alpha incubation. Present data indicate that IFN-alpha is able to consistently and selectively affect membrane and cytoplasmic features of hairy cells in a short term period. The possibility is envisaged that these changes may be related to the therapeutic efficacy of IFN-alpha.