Costimulation of cutaneous T-cell lymphoma cells by interleukin-7 and interleukin-2: potential autocrine or paracrine effectors in the Sézary syndrome.

PURPOSE: To examine interleukin-7 (IL7)- and interleukin-2 (IL2)-induced proliferation of Sézary lymphoma cells and to consider if an autocrine or paracrine growth-stimulatory circuit involving IL7 exists in the Sézary syndrome (SS). MATERIALS AND METHODS: Fresh Sézary lymphoma cells were maintained in short-term culture in the presence of cytokines, and growth was measured by incorporation of (3H)-thymidine (TdR). Expression of IL7 and IL7 and IL2 receptors (IL7-R and IL2-R, respectively) was assessed by polymerase chain amplification of first-strand complementary DNA (RT-PCR), by affinity cross-linking of radioactive iodine-125-IL7, and by dual-color fluorescence-activated cell analysis. IL-7 production was measured by immunoassay. RESULTS: Sézary lymphoma cells from seven patients showed synergistic (five of seven) or additive (two of seven) proliferation when cultured in the presence of IL2 and IL7, as compared with culture with either cytokine alone. Two patients with evidence of synergistic stimulation of [3H]-TdR incorporation showed IL7-R gene expression by RT-PCR and IL7 affinity cross-linking. Incubation of all seven patients' cells with IL7 induced coexpression to varying degrees of IL7-R and IL2-R. Sézary lymphoma cells from at least three of five patients studied expressed IL7 mRNA, and skin from three of five patients studied, as well as normal skin, expressed IL7 mRNA by RT-PCR. CONCLUSION: Sézary lymphoma cells respond by proliferation to IL7 plus IL2, and in some instances produce IL7. Therapeutic maneuvers should be pursued to take advantage of this potential autocrine or paracrine growth-stimulatory mechanism.

Decreases in levels of serum fibronectin predict the severity of vascular leak syndrome in patients treated with ricin A chain-containing immunotoxins.

The major dose-limiting adverse effect of ricin A chain-containing immunotoxin (IT) therapy is vascular leak syndrome (VLS). Since plasma fibronectin (Fn) plays a role in maintaining microcirculatory integrity and since the gradient between plasma and tissue Fn can be altered in various pathological situations, we determined whether the administration of IT-ricin A chain to patients resulted in changes in the levels of serum Fn and, if so, whether these changes correlated with the severity of VLS. We also measured the serum levels of tumor necrosis factor alpha (TNFalpha), a proinflammatory cytokine which has been implicated in tissue damage and in interleukin 2-mediated VLS. Our results indicate that the most severe manifestations of VLS were associated with the highest pretreatment levels of Fn, the largest decreases in Fn immediately after starting IT therapy, increases in the levels of serum TNFalpha, higher concentrations of circulating IT, and the lowest numbers of circulating tumor cells. These parameters should, therefore, be useful for predicting which patients will have severe VLS.

An approach to the elucidation of metabolic breakdown products of the luteinizing hormone-releasing hormone.

Metabolic breakdown of the luteinizing hormone-releasing hormone (LH-RH) could lead to the following fragments containing pyroglutamic acid: pyroglutamic acid (1), pGlu-His (2), pGLu-His-Trp (3), pGlu-His-Trp-Ser (4), etc., and finally pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly (10). We have synthesized fragments 2-10 and successfully separated all ten metabolites and LH-RH by high-performance liquid chromatography (HPLC) with a muBondapak C18 column. In a test of the viability of the method, cochromatography of fragments 1-10 and LH-RH with the products of chymotryptic digestion of tritiated LH-RH showed radioactive peaks corresponding to the expected products, fragments 3 and 5. Analysis of the products of incubation of a rat kidney homogenate supernatant with LH-RH showed fragments 1-4 and LH-RH. The finding of breakdown at position 4 uncovers a new site of LH-RH breakdown and points the way to the design of potential LH-RH antagonists and agonists where the 4 position would be substituted with unnatural amino acids to prevent breakdown.

Flow cytometric detection of neoplastic T cells in patients with mycosis fungoides based on levels of T-cell receptor expression.

The authors report the flow cytometric detection of neoplastic T cells in the peripheral blood of four out of five (80%) patients with peripheral blood involvement with mycosis fungoides (Sezary syndrome) based on the levels of T-cell receptor expression as measured by CD3 and TCR-alpha beta staining. Antigen receptor expression was abnormal in terms of increased density of surface CD3 or TCR-alpha beta per cell. Other immunophenotypic abnormalities were present in three of these patients. However, in one patient abnormal T-cell receptor expression was the only immunophenotypic evidence of neoplasia, although morphologically abnormal lymphocytes were present and a T-cell clone was detected by polymerase chain reaction (PCR). In another patient, the authors were able to detect development of a new, more aggressive neoplastic T-cell population based on levels of T-cell receptor expression. Levels of T-cell receptor expression may be of diagnostic utility in the evaluation of peripheral blood for the presence of neoplastic T-cell populations.

B-cell monoclonal lymphocytosis and B-cell abnormalities in the setting of familial B-cell chronic lymphocytic leukemia.

BACKGROUND: Among all hematologic malignancies, B-cell chronic lymphocytic leukemia (BCLL) has the highest familial clustering (three- to sevenfold increase), strongly suggesting a genetic component to its etiology. Familial BCLL can be used as a model to study the early pathogenesis of this disease. METHODS: We examined nine kindreds from the National Cancer Institute's Familial BCLL Registry, consisting of 19 affected members with BCLL and 33 clinically unaffected first-degree relatives. Flow cytometric immunophenotyping to detect a B-cell monoclonal lymphocytosis (BCML) was performed. Monoclonality was confirmed by polymerase chain reaction analysis of whole blood DNA. Cell cycle analysis for aneuploidy was conducted. RESULTS: In all affected individuals, we observed the classic BCLL CD5/CD19/CD20/CD23 immunophenotypic patterns. Six of the 33 unaffected individuals (18%) had evidence of BCML. Additional individuals (13/33, 39%) showed some other abnormality, whereas 14 individuals (42%) were normal. Based on an estimated prevalence of 0.7% for BCML in the general population, the finding of six subjects (18%) with clonal abnormalities in this relatively modest sample was significantly greater than expected (i.e., 18% vs. 0.7%, P < 5.7 x 10(-9)). CONCLUSIONS: Individual components of BCML and other B-cell abnormalities were observed in almost half of the apparently unaffected individuals. Our findings suggested that BCML may be an early detectable abnormality in BCLL. The spectrum of some of these observed abnormalities suggested the involvement of different B-cell subpopulations or different pathways in clonal evolution. Population-based, longitudinal studies will be required to determine the incidence of BCML and other B-cell abnormalities and their relation to disease progression in BCLL and other closely related B-cell lymphoproliferative disorders.

Bcl-2 level as a biomarker for 13q14 deletion in CLL.

BACKGROUND: Deletion 13q14.3 is the most common cytogenetic abnormality in chronic lymphocytic leukemia (CLL). Previously it was reported that miR-15/16 is the target of 13q14 deletions and plays a tumor suppressor role by suppressing Bcl-2. Therefore, Bcl-2 expression was examined more closely to determine whether it would predict 13q14 deletion status. METHODS: A multi-color flow panel consisting of anti-Bcl-2/anti-lambda/anti-kappa/CD19/CD5/CD3/CD20 was performed. The ability of Bcl-2 to predict 13q14 deletion was tested using the conventional Bcl-2 index (c-index): mean fluorescence intensity (MFI) of CLL clone/MFI of residual T cells. Fifty-four untreated CLL/MBL patients were studied. Bimodal Bcl-2 expression was evaluated to test the ability of Bcl-2 to detect intraclonal heterogeneity. Other CLL prognostic markers including CD38, CD49d, CD26, and CD69 were evaluated. FISH was performed on selected sorted populations. RESULTS: The Bcl-2 c-index strongly predicts del13q14 P < 0.0001. A statistically significant association was observed between the percentage of cells carrying the deletion and the level of Bcl-2 expression P < 0.05. Cells sorted based on Bcl-2 expression showed enrichment of both hemizygous and homozygous del 13q14 cells. Also, we observed that an alteration in Bcl-2 level over time predicts changes in 13q14 deletion status. And a statistically significant correlation between the bimodal pattern of CD69 expression and the presence of 13q14 deletion was found P < 0.0001. CONCLUSION: Bcl-2 expression using the c-index strongly predicts 13q14 deletion and can be used to distinguish homozygous, heterozygous, and diploid CLL clonal cells. Further systematic studies of this biomarker are needed for confirmation and expansion of these findings.

Combined normal donor and CLL: Single tube ZAP-70 analysis.

INTRODUCTION: Zeta-chain-associated protein kinase 70 (ZAP-70) has been identified as an independent prognostic marker in chronic lymphocytic leukemia (CLL). Based on our previous studies, we have developed a combined one-tube technology with multiple internal controls to optimize ZAP-70 assessment. METHODS: Forty-eight untreated CLL cases were examined for ZAP-70 expression using a modified 7-color one-tube assay. Normal donor (ND) whole blood is stained with CD3 APC-Cy7 and CD19 APC. In a second tube, patient whole blood is stained with CD5 PE-Cy7, CD19 PerCP-Cy5.5, and CD20 eFluor450. After surface staining and fixation, these two tubes are combined. After saponin permeabilization, the cells were stained with two anti-ZAP-70 clones (1E7.2/AF488 and SBZAP/PE). The results obtained from this modified tube were compared with those obtained concurrently using the non-mixed single sample tubes. Five different methods of ZAP-70 expression analysis were evaluated: percentage positive cells using ND T-cells as a reference; the internal patient T-cell/clone ratio; ND T-cell/clone ratio; clone/ND B-cell ratio; and modified Z-index. RESULT: Overall, the combined patient and ND mix tube performed better than the non-mixed single sample tube. The strongest correlations between ZAP-70 expression and immunoglobulin heavy chain variable (IGHV) mutational status were seen with percentage positive ND T-cell, ND T-cell/clone ratio, and clone/ND B-cell ratio for both 1E7.2 and SBZAP clone (P < 0.0001). CONCLUSION: The modified one tube method combining the ND and patient sample provides highly reliable results that correlate with the IGHV mutational status. This method should be considered as part of the next step in standardization of the ZAP-70 assay in CLL.

Methodological comparison of two anti-ZAP-70 antibodies.

BACKGROUND: ZAP-70 expression is a stage independent prognostic marker in CLL. However, interlaboratory variation is large, and there is neither a consensus nor a regulatory approved methodology. METHODS: Two anti-ZAP70 clones (1E7.2 and SBZAP) were compared in 45 untreated CLL patients. Nine different methods for ZAP-70 expression analysis were evaluated: M1, isotype control to determine negative; M2, internal residual T-cell to determine positive; M3, normal donor (ND) T-cell to determine positive; M4, internal T-cell/clone ratio; M5, ND residual T-cell/clone ratio; M6, clone/normal remaining B-cell ratio; M7, clone/ND B- cell ratio; M8, CLL-Z score; M9, modified CLL-Z score. A scoring system was designed integrating both 1E7.2 and SBZAP clones to assign ZAP-70 expression. RESULTS: The correlation coefficients for the four selected highest statistically significant methods were as follows (M1 = 0.71, M3 = 0.72, M7 = 0.67, and M9 = 0.64). These four methods were used to generate a combined score. The two reagents showed agreement using the designed scoring system for 37/45 samples (82%), and 8/45 (18%) showed equivocal result with one of the two clones. Seven of the eight equivocal samples were resolved using the scoring system. CONCLUSIONS: Four of the nine methods of analysis were compared for each reagent. The use of two independent ZAP-70 reagents increases analytical certitude and the scoring method aids in the resolution of equivocal results. The combined use of two reagents, four methods of analysis, and a scoring method allowed for assignment of ZAP-70 expression in 44/45 samples (98%) tested and improved performance of this important prognostic assay.

Improved ZAP-70 assay using two clones, multiple methods of analysis and clinical correlation.

INTRODUCTION: In a companion methodological study, we compared two anti-ZAP-70 clones (1E7.2 AF 488 and SBZAP PE) and four selected methods of analysis. Clinical correlations are required for validation. METHODS: Multicolor flow-cytometric evaluation of ZAP-70, CD38, CD69, CD26, CD49d, and CD27 was tested in 45 untreated-CLL patients. Four methods of ZAP-70 expression analysis and a scoring system were designed. A correlation analysis between ZAP-70 score, immunoglobulin heavy chain variable (IGHV) mutational status, fluorescence in situ hybridization, and these biomarkers was undertaken. RESULTS: There is a strong correlation between ZAP-70 expression and IGHV mutational status. The scoring system for a single reagent (P = 0.0006 or 0.0002) favors the use of multiple methods of analysis. The combined score was substantially equivalent (P = 0.0003). There was also a correlation with del 13q14 (P = 0.017) and trisomy12 (P = 0.011). A correlation for CD38 and ZAP-70 score was seen using both 1E7.2 AF488 and SBZAP PE when ≥20% or ≥7% cutoff was used. A positive correlation was seen for CD49d expression using both reagents. CD26 showed a correlation with ZAP-70 expression, but it was dependent upon the method of analysis. CD69 and CD27 showed no statistically significant correlation. CONCLUSION: In our study population, ZAP-70 expression is the better predictor of the IGHV mutational status. The correlation analysis confirms that the use of four methods of analysis with a single reagent or both reagents is superior to the use of a single method of analysis. The routine use of CD38, CD49d, and CD26 will require standardization.

Handling of luteinizing hormone-releasing hormone by renal proximal tubular segments in vitro.

[pyroglutamyl-3,4-3H]Luteinizing hormone-releasing hormone (LHRH) was microperfused through isolated segments of rabbit proximal straight tubules and incubated with isolated brush border microvilli from rabbit renal tubules. About 4.8% of perfused 3H label was reabsorbed into the bathing medium per millimeter of tubule length per minute, and 1% or less of perfused label was sequestered per millimeter of nephron segment. The 3H label content of the bathing medium varied linearly with perfusion time (30 min), suggesting a constant rate of reabsorption. Analysis by high performance liquid chromatography showed that the collection fluid and brush border incubation medium contained significant amounts of labeled pGlu-His, pGlu-His-Trp, and pGlu-His-Trp-Ser, as well as LHRH, while the bathing medium contained pGlu, pGlu-His, pGlu-His-Trp-Ser, a very small amount of pGlu-His-Trp, and no LHRH. These data suggest that the partial hydrolysis of [3H]LHRH to these peptide metabolites takes place in proximal tubules through contact digestion by brush border enzymes. The metabolites and/or hormone are probably reabsorbed and broken down further within the cell to produce pGlu, which becomes an additional metabolite found in the bathing medium.

Differences between in vitro and in vivo degradation of LHRH by rat brain and other organs.

Homogenates of brain, pituitary, liver, lung, ovary, and testes were incubated with [pyro Glu1-3,4-3H]luteinizing hormone-releasing hormone ([3H]LHRH), and the profiles of metabolites generated as a function of time were determined. After 5 min of incubation, 5 was the predominant metabolite in most homogenates. Although the profiles of metabolites varied at different time intervals, metabolites 2, 3, 4, and 5, and in some instances 7 and 9, appeared to form simultaneously and were detectable at 10 min. Neither metabolite 6 nor other larger metabolites formed initially as dominant degradation products. The findings suggest cleavage of LHRH by the simultaneous action of several endopeptidases. After a single vascular transit of [3H]LHRH, metabolites were determined in the venous blood of liver, lung, and brain of rats in vivo. There were no metabolites of [3H]LHRH in venous blood of liver and lung; however, metabolites 2-4 were present in venous blood of the brain. Incubation of rat anterior pituitary cells with [3H]LHRH yielded metabolites 1-4 but not metabolites 5 or 9 as in homogenates. Incubation of [3H]LHRH with porcine follicular granulosa cells resulted in the generation of metabolites 2-7 and 9, similar to the profile in homogenates. Thus, since homogenates contain enzymes of disrupted cells, they do not always reflect mechanisms for in vivo hydrolysis of circulating LHRH. Brain degraded 12.1% of LHRH during a single vascular transit and may account for substantial degradation of the circulating hormone.

In vivo metabolism of tritiated LHRH by the whole kidney and individual tubules of rats.

[pyroglutamyl-3,4-3H]Luteinizing hormone-releasing hormone ([3H]LHRH) and [14C]inulin were infused into individual nephrons in Inactin-anesthetized rats and the amount of radioactive label and the identity of the radioactively labeled material in urine were determined. The site of infusion was identified by latex injection and microdissection. [3H]LHRH was microinfused at 1.5 X 10(-5 M (concentration 10(6)-10(7) higher than in plasma) and analysis of urinary metabolites was performed by high-performance liquid chromatography. The urinary recovery of tritium label was 81% when proximal tubules were infused and 94% when distal tubules were infused. For proximal tubules 90% of the label recovered in urine appeared as pGlu-His (metabolite 2), pGlu-His-Trp (metabolite 3), and pGlu-His-Trp-Ser (metabolite 4), and 10% as LHRH. With distal tubules only LHRH was detected in the urine. [3H]LHRH was presented to the renal artery of the filtering rat kidney in vivo, and urine and renal venous blood were analyzed for breakdown products. The urine contained metabolites 2, 3, and 4 and no LHRH, whereas venous blood contained mainly pGlu, metabolite 4, and LHRH. When [3H]LHRH was perfused in vivo through the nonfiltering rat kidney or rat lower limb, renal or femoral venous blood was found to contain only LHRH. These studies suggest that [3H]LHRH undergoes glomerular filtration and contact digestion by brush border enzymes of the proximal tubule to produce metabolites 2, 3, and 4. These metabolites and possibly LHRH are partially reabsorbed and undergo further intracellular degradation to produce pGlu. Endothelial and interstitial cells in the kidney and leg do not appreciably metabolize [3H]LHRH.

Doublet discrimination in DNA cell-cycle analysis.

Differences in doublet analysis have the potential to alter DNA cell-cycle measurements. The techniques for doublet determination are often used interchangeably without regard for the complexity in cell shapes and sizes of biological specimens. G(0/1) doublets were identified and quantitated using fluorescence height versus area and fluorescence width versus area pulse measurements, by enumerating the proportion of G(2) + M cells that lack cyclin B1 immunoreactivity, and modeled in the DNA histograms by software algorithms. These techniques were tested on propidium iodide-stained whole epithelial cells or nuclei from asynchronous cultures, or after exposure to chemotherapeutic agents that induced cell-cycle arrest and were extended to human breast tumor specimens having DNA diploid patterns. G(0/1) doublets were easily discernible from G(2) + M singlets in cells or nuclei that are generally homogenous and spherical in shape. Doublet discrimination based on pulse processing or cyclin B1 measurements was nonconcordant in some nonspherical cell types and in cells following cell cycle arrest. Significant differences in G(0/1) doublet estimates were observed in breast tumor specimens (n = 50), with estimates based on pulse width twice those of pulse height and nearly five times greater than computer estimates. Differences between techniques are attributed to difficulties in the separation of the boundaries between G(0/1) doublets and G(2) + M singlet populations in biologically heterogeneous specimens. To improve reproducibility and enhance standardization among laboratories performing cell cycle analysis in experimental cell systems and in human breast tumors, doublet discrimination analysis should best be accomplished by computer modeling. Shape and size heterogeneity of tumor and arrested cells using pulse-processing can lead to errors and make interlaboratory comparison difficult.

Bilateral adrenal hemorrhage and adrenal insufficiency in a patient with lymphomatous adrenal infiltration following administration of a fusion toxin (DAB486 interleukin-2).

DAB486IL-2 is a novel fusion toxin in which the ADP-ribosyltransferase and membrane-translocating domains of diphtheria toxin have been combined with the interleukin-2 (IL-2) gene, creating a recombinant protein capable of selectively intoxicating cells bearing the high-affinity IL-2 receptor. Clinical activity has been documented in Hodgkin disease and the non-Hodgkin lymphomas; toxicities have been minimal and include mild hepatic transaminitis, proteinuria, and hypersensitivity reactions. In this report, a patient with tumor-stage cutaneous T-cell lymphoma developed clinical adrenal failure with bilateral adrenal hemorrhage and necrosis 7 weeks after completing a 5-day course of treatment with DAB486IL-2. The relationship of fusion toxin therapy to the development of this unusual toxicity is discussed.

CD97 is a processed, seven-transmembrane, heterodimeric receptor associated with inflammation.

CD97 is a receptor that spans the membrane seven times, a defining feature of G protein-coupled receptors. CD97 is predominantly expressed in leukocytes, but the function and accurate protein structure of this receptor have not been described. We show here that CD97 has the novel property among G protein-coupled receptors characterized to date of being processed intracellularly in either the endoplasmic reticulum or early Golgi from a proprotein into a noncovalently associated two-subunit structure that becomes expressed on the cell surface and is composed of a large extracellular protein (CD97alpha) and a seven-membrane spanning protein (CD97beta). CD97beta is part of an evolutionarily conserved subfamily of four proteins, including two Caenorhabditis elegans proteins of as yet unknown function, which is distinct from but most closely related to the glucagon receptor family. CD97alpha exists in three alternatively spliced isoforms that contain between three and five epidermal growth factor (EGF)-like repeats that are related to the calcium binding EGF-like repeats in the microfibril protein fibrillin. Leukocytes strongly positive for CD97 are concentrated at sites of inflammation relative to CD97 expression in normal lymphoid tissues. Soluble CD97alpha was found in body fluids from inflamed tissues, suggesting that a functional consequence of the CD97 heterodimeric structure is the stable existence of CD97alpha in a cellfree form. CD97 appears to be a multifunctional protein that may play a signal transduction role associated with the establishment or development of an inflammatory process.

Effects of the tyrosine-kinase inhibitor geldanamycin on ligand-induced Her-2/neu activation, receptor expression and proliferation of Her-2-positive malignant cell lines.

Geldanamycin belongs to the family of benzoquinoid ansamycin tyrosine-kinase inhibitors. We have examined its effects on Her-2/neu kinase activity, protein expression level, and proliferation of Her-2+ malignant cells. In SK-BR-3 breast-cancer cells, short-time treatment with geldanamycin completely abrogated gp30-ligand-induced activation of Her-2 without a change of receptor-expression level. Longer treatment of intact cells with geldanamycin induced decreased steady-state Her-2 autophosphorylation activity, which correlated with reduction of Her-2 protein expression and phosphotyrosine content of several proteins. The decrease was time- and dose-dependent, starting after 1 hr at 100 nM concentration and reaching completion by 24 hr. The reduction of the Her-2 protein level probably resulted from increased degradation, since the Her-2 mRNA level remained constant. Geldanamycin effects were not specific for Her-2, since the non-receptor tyrosine-kinase fyn was inhibited equally. In contrast to these results, protein-kinase-C activity was not affected. In 3 other malignant cell lines expressing different amounts of Her-2 (SK-BR-3 > SK-OV-3 > OVCAR3 > MCF7), geldanamycin also effectively reduced Her-2-kinase activity proportionally to the decrease of protein expression. In contrast, in a [3H]-thymidine-uptake assay, cell growth was meaningfully inhibited by geldanamycin at nanomolar concentrations only in SK-BR-3 (IC50 2 nM) and MCF7 (IC50 20 nM), while OVCAR3 was only moderately sensitive (IC50 2 microM) and SK-OV-3 was clearly resistant to geldanamycin. In direct comparison with herbimycin A, another benzoquinoid ansamycin that has been more thoroughly characterized, the biologic effects of geldanamycin were more pronounced.