A randomised, controlled, crossover study to investigate the safety and response of 1R,2S-methoxamine hydrochloride (NRL001) on anal function in healthy volunteers.

AIMS: This study aimed to assess the dose and volume effects of suppository preparations and safety of NRL001 (one of four possible stereoisomers of methoxamine hydrochloride) on anal sphincter tone using rectal suppositories in healthy adult volunteers. METHODS: This was a Phase I, single-centre, randomised, double-blind, three-way crossover study during which subjects received three single doses of 1 g rectal suppositories (containing 5 or 10 mg NRL001 or matching placebo) or 2 g rectal suppositories (containing 10 or 15 mg NRL001 or matching placebo) on three separate dosing days. The outcome measures were mean anal resting pressure (MARP) variables (monitored continuously for 20-30 min before and up to 6 h after dosing), pharmacokinetics (PK) and safety assessments. RESULTS: Twenty-six subjects were dosed with study medication. Two subjects were withdrawn prematurely and were not included in the main analysis. There was a dose-dependent increase in anal sphincter tone (MARP) when comparing the 5 and 10 mg doses of NRL001; however, the 15 mg dose did not have a significantly greater effect than the 10 mg dose. Suppository size (1 or 2 g) did not appear to have an effect on sphincter tone. There was no evidence against dose proportionality for the PK variables, but the mean maximum plasma concentration (Cmax ) for the 1 g suppository group was higher than for the 2 g group. Twenty-one adverse events were reported in 8 (30.8%) subjects. A dose dependent decrease in heart rate was noted; however, there were no adverse events reported that were related to this reduction in heart rate. CONCLUSIONS: The increase in anal sphincter tone supports the potential therapeutic use of NRL001 in treating faecal incontinence, with further studies in patients required. NRL001 was well tolerated in single doses of up to 15 mg.

A randomised, controlled, crossover study to investigate the pharmacodynamics, pharmacokinetics and safety of 1R,2S-methoxamine hydrochloride (NRL001) in healthy elderly subjects.

AIMS: This study aimed to assess the effects of a single dose of 10 mg NRL001 (the 1R,2S stereoisomer of methoxamine hydrochloride) in a 2 g suppository on pharmacodynamic and pharmacokinetic (PK) variables, and safety, in a healthy elderly population. METHODS: This was a Phase I, single-centre, randomised, double-blind, placebo-controlled crossover study during which subjects received a single 2 g suppository of 10 mg NRL001 and a matching placebo in two separate treatment periods. The main outcome measures were Holter-, vital signs- and electrocardiogram-derived cardiovascular variables; plasma PK analysis; and safety assessments. RESULTS: Twenty-six subjects were dosed with study medication. Statistically significant reductions in Holter-derived heart rate (HR), vital signs-derived HR and diastolic blood pressure (BP) were observed comparing NRL001 with placebo treatment, and also with increasing NRL001 plasma concentration. No statistically significant relationships were observed between NRL001 concentration and systolic BP, mean arterial pressure or QTC interval (both Bazett's and Fridericia's correction). Thirty-nine adverse events were reported in 20 (76.9%) subjects, mostly after dosing with NRL001. CONCLUSION: Administration of NRL001 suppositories led to decreases in HR when compared with placebo data. NRL001 was well tolerated with a good safety profile during the study. Healthy elderly subjects did not show significantly different biological responses to NRL001 suppositories compared with younger healthy volunteers in previous studies.

A double-blind, placebo-controlled, randomised, parallel-group, dose-escalating, repeat dose study in healthy volunteers to evaluate the safety, tolerability, pharmacodynamic effects and pharmacokinetics of the once daily rectal application of NRL001 suppositories for 14 days.

AIMS: The 1R,2S stereoisomer of methoxamine hydrochloride, NRL001, is a highly selective α1-adrenoceptor agonist being developed for the local treatment of non-structural faecal incontinence caused by weak internal anal sphincter tone. This study investigated the steady state pharmacokinetics (PK) and safety of 2 g rectal suppositories containing NRL001 in different strengths (7.5, 10, 12.5 or 15 mg). METHODS: Healthy volunteers aged 18-45 years received 14 daily doses of NRL001 2 g suppositories or matching placebo. In each dose group nine participants received NRL001 and three received placebo. Blood samples to determine NRL001 concentrations were taken on Days 1, 7 and 14. Cardiovascular parameters were collected via electrocardiograms, Holter monitoring (three lead Holter monitor) and vital signs. RESULTS: Forty-eight volunteers were enrolled; 43 completed the study and were included in the PK analysis population. AUC and Cmax broadly increased with increasing dose, Tmax generally occurred between 4.0 and 5.0 h. Although the data did not appear strongly dose proportional, dose proportionality analysis did not provide evidence against dose proportionality as the log(dose) coefficients were not significantly < 1. NRL001 did not accumulate over time for any dose. Increasing NRL001 concentrations were related to changes in vital sign variables, most notably decreased heart rate. The most commonly reported adverse events (AEs) in the active treatment groups were paraesthesia and piloerection. CONCLUSIONS: Treatment with NRL001 was generally well tolerated over 14 days once daily dosing and plasma NRL001 did not accumulate over time. Treatment was associated with changes in vital sign variables, most notably decreased heart rate. AEs commonly reported with NRL001 treatment were events indicative of a systemic α-adrenergic effect.

Meta-analysis for cardiovascular effects of NRL001 after rectal application in healthy volunteers.

AIMS: NRL001 is a highly specific α1 -adrenoceptor agonist currently under evaluation for the treatment of faecal incontinence caused by a weak internal anal sphincter. The aim of this meta-analysis was to quantify the effect of NRL001 on cardiovascular parameters including heart rate, blood pressure and QT interval. METHODS: Data from the four Phase I healthy volunteer studies SUM (NCT00857467), SURD (NCT01099670), SUSD (NCT00850590) and SAGE (NCT01099683) were pooled and analyses were performed on individual subject data. Mixed effects regression analysis was used to determine the effect of NRL001 on heart rate, blood pressure and QT intervals. A multivariate statistical model was used to determine the effect of covariates on heart rate. RESULTS: Subjects given NRL001 experienced a dose related decrease in heart rate of up to 9.48 bpm compared with subjects in the placebo arms. No statistically significant evidence for a threshold effect was found. There was no clear evidence of dose effect of NRL001 on blood pressure. QT interval increased in all NRL001 subject as expected; QTC F also showed a statistically significant increase. However, QTC B was shortened with no significant treatment effect. CONCLUSIONS: NRL001 was found to have a dose-dependent effect on heart rate; however clinically-relevant bradycardia was not reported, indicating the decrease in heart rate was not of clinical significance. Furthermore, no clinically-significant drug effect on blood pressure or mean arterial pressure was observed. QT intervals were affected by changes in heart rate. However, trends were dependant on the correction factor used. No consistent QT effect was observed, but a thorough QTC study will be required to confirm the effects of rectally applied NRL001.

Randomised clinical trial: macrogol/PEG 3350+electrolytes versus prucalopride in the treatment of chronic constipation -- a comparison in a controlled environment.

BACKGROUND: Constipation is a common condition for which PEG 3350 is an established treatment and prucalopride has recently been approved for this indication. AIM: To compare the efficacy, safety and impact on quality of life (QoL) of PEG 3350 plus electrolytes (PEG 3350+E) vs. prucalopride in females with chronic constipation (CC) in whom laxatives have previously failed to provide adequate relief. METHODS: In this single-centre, randomised, double-blind, double-dummy study, patients with CC [<3 spontaneous complete bowel movements (SCBM)/week] remained in a controlled environment and received either a 26 g split dose of PEG 3350+E (N = 120) or 1-2 mg prucalopride (N = 120) daily for 28 days following a 14-day run-in period. The primary endpoint was the proportion of patients having ≥3 SCBMs during the last treatment week. RESULTS: Non-inferiority of PEG 3350+E to prucalopride was demonstrated in the per-protocol population [difference, 10.1% (66.67% vs. 56.52%), 97.5% lower confidence interval (CI) -2.7%, above the preset margin of -20%] and approached superiority in the modified intent-to-treat population (difference, 9.8%, 97.5% lower CI, -3.1%). Statistically significant differences in favour of PEG 3350+E were observed for most secondary variables (bowel movements, stool weight, consistency, time to next SCBM, patient perception of straining and completeness of defecation). Colonic transit time was dramatically reduced in both arms. Both treatments were well tolerated. CONCLUSION: PEG 3350+E was at least as effective as and generally better tolerated than prucalopride as a treatment for chronic constipation in this study population (NCT01251822; http://www.clinicaltrials.gov).

Budget-impact model for colonoscopy cost calculation and comparison between 2 litre PEG+ASC and sodium picosulphate with magnesium citrate or sodium phosphate oral bowel cleansing agents.

OBJECTIVE: With the availability of several bowel cleansing agents, physicians and hospitals performing colonoscopies will often base their choice of cleansing agent purely on acquisition cost. Therefore, an easy to use budget impact model has been developed and established as a tool to compare total colon preparation costs between different established bowel cleansing agents. METHODS: The model was programmed in Excel and designed as a questionnaire evaluating information on treatment costs for a range of established bowel cleansing products. The sum of costs is based on National Health Service reference costs for bowel cleansing products. Estimations are made for savings achievable when using a 2-litre polyethylene glycol with ascorbate components solution (PEG+ASC) in place of other bowel cleansing solutions. Test data were entered into the model to confirm validity and sensitivity. The model was then applied to a set of audit cost data from a major hospital colonoscopy unit in the UK. RESULTS: Descriptive analysis of the test data showed that the main cost drivers in the colonoscopy process are the procedure costs and costs for bed days rather than drug acquisition costs, irrespective of the cleansing agent. Audit data from a colonoscopy unit in the UK confirmed the finding with a saving of £107,000 per year in favour of PEG+ASC when compared to sodium picosulphate with magnesium citrate solution (NaPic+MgCit). For every patient group the model calculated overall cost savings. This was irrespective of the higher drug expenditure associated with the use of PEG+ASC for bowel preparation. Savings were mainly realized through reduced costs for repeat colonoscopy procedures and associated costs, such as inpatient length of stay. CONCLUSIONS: The budget impact model demonstrated that the primary cost driver was the procedure cost for colonoscopy. Savings can be realized through the use of PEG+ASC despite higher drug acquisition costs relative to the comparator products. From a global hospital funding perspective, the acquisition costs of bowel preparations should not be used as the primary reason to select the preferred treatment agent, but should be part of the consideration, with an emphasis on the clinical outcome.

Intra-anal application of L-erythro methoxamine gel increases anal resting pressure in patients with incontinence.

BACKGROUND: This study examined the effects of a single dose of intra-anal L-erythro methoxamine on mean anal resting pressure (MARP) and cardiovascular variables in patients with faecal incontinence. METHODS: Patients had anorectal physiology tests and ultrasonography before participating. Six patients received 0.3 and 1 per cent gels on separate days, two patients received 0.3 per cent gel, and two patients received 1 per cent gel. MARP, blood pressure, pulse rate and plasma drug concentrations were measured for 6 h after application. RESULTS: Intra-anal 0.3 per cent gel caused a rapid, significant rise in MARP lasting 2 h (P = 0.036). In four of these patients, the response was sufficient to increase MARP to within the normal range at 2 h. Application of 1 per cent gel caused a significant rise in MARP for 4 h after application (P = 0.028). There was a significant decrease in pulse at 2 and 1 h respectively after application of 0.3 and 1 per cent gels. CONCLUSION: Intra-anal application of L-erythro methoxamine can be used to increase MARP in patients with faecal incontinence. Application of 1 per cent L-erythro methoxamine gel produced a rapid, sustained rise in MARP, which raises the possibility of therapeutic application.

Intra-anal and rectal application of L-erythro methoxamine gel increases anal resting pressure in healthy volunteers.

BACKGROUND: This study examined the effect of a single local application of L-erythro methoxamine, an alpha(1)-adrenoceptor agonist, on mean anal resting pressure (MARP) and cardiovascular variables in healthy volunteers. METHODS: L-Erythro methoxamine gel was administered in a single-blind manner; 0.3-3 per cent gels were applied perianally (n = 12), 1-3 per cent gels intra-anally (n = 16) and 1 per cent gel rectally (n = 8). MARP, systolic blood pressure, diastolic blood pressure and pulse rate were measured before application and for up to 6 h afterwards. Blood samples were taken to estimate plasma drug levels. RESULTS: Perianal gel produced no increase in MARP. Intra-anal 1 per cent and 3 per cent gel produced a significant rapid rise in MARP for 4 and 5 h respectively after application (P = 0.012 and P = 0.017 respectively). Rectal 1 per cent gel increased MARP for 2 h after application (P = 0.036). Intra-anal gel resulted in an increase in systolic blood pressure (1 per cent gel at 2 h, P = 0.042; 3 per cent gel at 4 h, P = 0.017). One per cent intra-anal and rectal gels caused a decrease in the pulse rate for 2 h after application (P = 0.012 and P = 0.018 respectively). Six subjects complained of nausea and three of headache after gel application. CONCLUSION: Intra-anal and rectal gel produced a sustained rise in MARP with rapid onset in volunteers. This raises the possibility of a therapeutic application for L-erythro methoxamine in patients with passive incontinence and internal anal sphincter dysfunction.

Treatment of chronic anal fissure by application of L-arginine gel: a phase II study in 15 patients.

PURPOSE: Local application of exogenous nitric oxide donors, such as isosorbide dinitrate and glyceryl trinitrate, promotes fissure healing by reducing anal resting pressure and improving anodermal blood flow. The major drawback of these nitric oxide donors is headache. The overall incidence of this side effect is approximately 40 percent. Recently we have shown in healthy volunteers that L-arginine, being an intrinsic precursor of nitric oxide, reduces anal resting pressure without headache as a side effect. The aim of the present study was to evaluate the effect of L-arginine on anal resting pressure, anodermal blood flow, and fissure healing in patients with chronic anal fissure. METHODS: Fifteen patients with a chronic anal fissure were included in the present study. Before entering the study 10 patients were unsuccessfully treated by local application of isosorbide dinitrate. Six of these patients experienced severe headache during treatment with isosorbide dinitrate. All patients were treated for at least 12 weeks by local application of a gel containing L-arginine 400 mg/ml five times a day. In patients with a persistent fissure, treatment was continued until 18 weeks. Anal manometry and laser Doppler flowmetry of the anoderm were performed before treatment, 20 minutes after local application of the first dose, and after 12 weeks of treatment. A visual analog scale was used to assess fissure-related pain and headache. RESULTS: One patient dropped out after one day of treatment, and one was excluded because of violation of the study protocol. After 12 weeks of treatment complete fissure healing was observed in 3 of 13 (23 percent) patients, and after 18 weeks the healing rate was 8 of 13 (62 percent) patients. None of the 13 patients experienced typical nitric oxide-induced headache. The pressure recordings showed a significant reduction of maximum anal resting pressure (mean +/- SD): pretreatment 89 +/- 17 mmHg; 20 minutes after application of the first dose 67 +/- 17 mmHg; 12 weeks after treatment 74 +/- 14 mmHg (P < 0.005). Recordings of anodermal blood flow showed a significant increase in flow: pretreatment 0.36 +/- 0.25 volts; 20 minutes after application of the first dose 0.59 +/- 0.27; 12 weeks after treatment 0.64 +/- 0.33 (P < 0.005). CONCLUSIONS: Local application of L-arginine promotes fissure healing without headache as a side effect, and L-arginine is effective even in patients not responding to isosorbide dinitrate treatment.

Topical L-arginine gel lowers resting anal pressure: possible treatment for anal fissure.

PURPOSE: Exogenous nitric oxide donors, such as glyceryl trinitrate, have been used as treatment for anal fissures; however, headaches develop in 60 percent of patients. Nitric oxide produced from the cellular metabolism of L-arginine mediates relaxation of the internal anal sphincter. This study investigated whether topical L-arginine gel reduces maximum anal resting pressure in volunteers. METHOD: In a two-center study, volunteers received a single topical dose of L-arginine or placebo (Aquagel ). Anal manometry was performed for two hours after application of 400 mg of L-arginine gel or placebo gel to the anal verge in 25 volunteers. Side effects were recorded after single application and also after repeated dosing for three days. RESULTS: L-Arginine reduced maximum anal resting pressure by 46 percent from a median of 65 cm of water to a minimal value of 35 cm of water ( P< 0.001, Wilcoxon's signed-rank test). The difference between L-arginine and placebo using repeated-measures testing was significant at P< 0.005. No side effects occurred with either gel; in particular, no episodes of headache were recorded. CONCLUSION: Topical L-arginine gel significantly lowers maximum anal resting pressure; its onset of action is rapid, and duration is at least two hours ( P< 0.01). L-arginine may have therapeutic potential, but further evaluation is needed before it can be used as a possible alternative treatment for chronic anal fissure.

Establishment of a cytokine-producing anaplastic large-cell lymphoma cell line containing the t(2;5) translocation: potential role of cytokines in clinical manifestations.

A permanent cell line, HSC-M1, was established from a child with advanced CD30 (Ki-1)+ anaplastic large-cell lymphoma (ALCL). Clinical features included irritability, fever, weight loss, tender lymphadenopathy, pneumonitis, neutrophilia, and bone marrow erythrophagocytosis. While HSC-M1 cells exhibited an immunophenotype characteristic of ALCL of T-cell lineage, the cell line also demonstrated features of monocyte-macrophage lineage. Cytogenetic and polymerase chain reaction (PCR) analysis of the HSC-M1 cell line and involved bone marrow demonstrated the characteristic non-random chromosomal translocation t(2:5)(p23:q35). Reverse transcriptase PCR for mRNA expression of cytokines and cytokine receptors showed that HSC-M1 cells expressed the message for multiple cytokines and their receptors. Measurement of cytokine levels in serum samples using enzyme-linked immunosorbent assays showed increased concentrations of several cytokines. The increased levels of some cytokines correlated with disease activity and clinical symptoms. Although spontaneous production by HSC-M1 cells of some of these cytokines was demonstrated, the production of others was only detectable after stimulation with exogenous CD30 ligand. With few exceptions, there was good correlation between serum cytokine levels and cytokines produced by HSC-M1 cells. These findings indicate that cytokine production is a feature of ALCL cells and that some of the clinical manifestations in ALCL may result from cytokines produced by either the malignant or accessory cells.

Hodgkin's disease: a disorder of dysregulated cellular cross-talk.

Hodgkin's disease (HD) is a peculiar type of human malignant lymphoma characterized by a very low frequency of tumor cells, the so called Hodgkin and Reed-Sternberg (H-RS) cells, embedded in a hyperplastic background of non-neoplastic (reactive) cells recruited and activated by H-RS cells-derived cytokines. H-RS cells can be functionally regarded as antigen-presenting cells (APC) able to elicit an intense, but anergic and ineffective, T-cell mediated immune response along with a hyperplastic inflammatory reaction which involves several cell types including T- and B-cells, neutrophils, eosinophils, plasma cells, fibroblasts and stromal cells. In tissues involved by HD, malignant H-RS cells and their reactive neighboring cells are able to cross-talk via a complex network of cytokine- and cell contact-dependent interactions. As a result of such interactions, mediated by specific surface receptors and adhesion molecules on both tumor and non-neoplastic cells, H-RS cells may receive several proliferative and anti-apoptotic signals favoring the cellular expansion and tumor cell survival in HD. The ineffective T-cell immune response elicited by the abnormal APC function of H-RS cells may further contribute to the biologic and clinical progression of HD. Innovative therapeutic strategies aimed at blocking the pathways of dysregulated cellular cross-talk among H-RS cells and bystander reactive cell populations might be beneficial in the treatment of HD patients.

CD30 ligand in lymphoma patients with CD30+ tumors.

PURPOSE: CD30 ligand (CD30L), which is expressed on resting B and activated T lymphocytes, can induce cell death in several CD30+ cell lines. Patients with CD30+ tumors (Hodgkin's disease and Ki-1+ non-Hodgkin's lymphoma) frequently have elevated soluble CD30 (sCD30) levels in their serum, which correlates with a poor prognosis. The role of sCD30 in protecting tumor cells from CD30L-mediated cell death and the pattern of CD30L expression on human peripheral-blood lymphocytes (PBLs) of normal donors and patients with CD30+ tumors are investigated. MATERIALS AND METHODS: CD30L surface protein expression was determined by two-color flow cytometry on PBLs of patients with CD30+ tumors and normal individuals. CD30L levels were determined on subsets of PBLs before and after stimulation with phytohemagglutinin (PHA), anti-CD3 antibody, or CD40L. sCD30 was measured by enzyme-linked immunosorbent assay (ELISA). The apoptotic activity of membrane-bound CD30L was tested in a CD30+ cell line by the annexin V-binding method. RESULTS: Unstimulated T lymphocytes of normal donors and patients with lymphoma rarely expressed CD30L surface protein, but were able to express it after stimulation with PHA or anti-CD3 antibody. Resting B cells of patients with CD30+ tumors had lower levels of detectable surface CD30L compared with normal donors (mean, 55% and 80.6%, respectively; P = .0008). Patients with high levels of serum sCD30 had lower detectable levels of CD30L on their PBLs (R2 = .72, P = .0008) and exogenous sCD30 blocked membrane-bound CD30L-mediated apoptosis in a CD30+ cell line. CONCLUSION: In patients with CD30+ tumors, sCD30 can decrease the availability of CD30L on PBLs. Blocking the apoptosis-inducing activity of CD30L by its soluble receptor may explain how CD30+ tumors escape immunosurveillance and may be related to the reported poor prognosis of patients who have elevated sCD30 levels.

Differential CD95 expression and function in T and B lineage acute lymphoblastic leukemia cells.

CD95 (Fas/APO-1) is a cell surface receptor able to trigger apoptosis in a variety of cell types. The expression and function of the CD95 antigen on leukemic blasts from 42 patients with B lineage and 53 patients with T lineage acute lymphoblastic leukemia (ALL) were investigated using immunofluorescence staining and apoptosis assays. The CD95 surface antigen was expressed in most ALL cases, with the T lineage ALL usually showing a higher intensity of surface CD95 expression as compared with the B lineage ALL cells (relative fluorescence intensity, RFI: 4.8 +/- 0.47 vs 2.2 +/- 0.23, respectively, P < 0.01). Functional studies disclosed that upon oligomerization by anti-CD95 monoclonal antibodies the CD95 protein was either not able to initiate apoptosis of leukemic cells (75% of cases) or induced low rates of apoptosis (20% of cases). Only in 5% of cases did the apoptosis rate exceed the 20% level of the CD95-specific apoptosis. Most of the CD95-sensitive cases were found among T lineage ALLs (38% of T lineage vs 10% of B lineage ALLs). Overall, the extent of CD95-induced apoptosis did not correlate with the expression level of CD95. Similarly, no significant correlation between expression level and functionality of CD95 in human leukemia cell lines of B and T cell origin could be observed. Bcl-2 protein has been associated with prolonged cell survival and has been shown to block partially CD95-mediated apoptosis, but for ALL cells no correlation between bcl-2 expression and spontaneous or CD95-mediated apoptosis could be found. The results obtained in this study indicate that, despite constitutive expression of CD95, the ALL cells are mainly resistant to CD95-triggering. More detailed investigations of the molecular mechanisms involved in the intracellular apoptotic signal transduction, such as interactions of the bcl-2 and the other members of the bcl-2 family, and functionality of the interleukin-1beta converting enzyme (ICE) like-proteases, may give new insights into key events responsible for the resistance or sensitivity to the induction of apoptosis in acute leukemia.

The abundant presence of Söderström bodies in cytology smears of fine-needle aspirates contributes to distinguishing high-grade non-Hodgkin's lymphoma from carcinoma and sarcoma.

Söderström bodies, also termed lymphoglandular bodies (LGB) and detectable in fine-needle aspiration cytology smears, have long been accepted as indicative of lymphoid tissues. To investigate the validity of this association, we examined 588 cytologic smears from high-grade non-Hodgkin's lymphoma (NHL), carcinoma, and sarcoma. Slides with lymphocytes in the vicinity of carcinoma and sarcoma cells had been excluded. Two independent observers scored smears to number, size, color, form, and smear background of the LGB. In 68 of 359 (19%) nonlymphoid malignancies rare (defined as < 1 LGB per high-power field) or occasional LGB (defined as 1-20 LGB per high-power field) were detectable. Half of these tumors consisted of melanomas, small cell lung carcinomas, and teratomas; the other half encompassed undifferentiated sarcomas. However, none of the smears obtained from carcinoma or sarcoma tissue had abundant LGB (defined as > 20 LGB per high-power field). When number of LGB was estimated to be abundant, the sensitivity for diagnosing a lymphoma was 54%; however, specificity was 100%. The difference in showing LGB between high-grade NHL and carcinoma/sarcoma was highly significant (p = 0.0001). The presence of abundant LGB in cytologic smears strongly suggests the diagnosis of lymphoma, while the absence of LGB nearly excludes this diagnosis. No trends were observed with the other criteria which were tested. LGB in aspiration cytology smears from malignant tumors thus represent a useful tool to distinguish high-grade NHL from carcinoma and sarcoma.

CD30 ligand is frequently expressed in human hematopoietic malignancies of myeloid and lymphoid origin.

CD30 ligand (CD30L) is a type-II membrane glycoprotein capable of transducing signals leading to either cell death or proliferation through its specific counterstructure CD30. Although several lines of evidence indicate that CD30L plays a key role as a paracrine- or autocrine-acting surface molecule in the deregulated cytokine cascade of Hodgkin's disease, little is known regarding its distribution and biologic significance in other human hematopoietic malignancies. By analyzing tumor cells from 181 patients with RNA studies and immunostaining by the anti-CD30L monoclonal antibody M80, we were able to show that human hematopoietic malignancies of different lineage and maturation stage display a frequent and broad expression of the ligand. CD30L mRNA and surface protein were detected in 60% of acute myeloid leukemias (AMLs), 54% of B-lineage acute lymphoblastic leukemias (ALLs), and in a consistent fraction (68%) of B-cell lymphoproliferative disorders. In this latter group, hairy cell leukemia and high-grade B-cell non-Hodgkin's lymphoma (B-NHL) expressed a higher surface density of CD30L as compared with B-cell chronic lymphocytic leukemia and low-grade B-NHL. Purified plasmacells from a fraction of multiple myeloma patients also displayed CD30L mRNA and protein. A more restricted expression of CD30L was found in T-cell tumors that was mainly confined to neoplasms with an activated peripheral T-cell phenotype, such as T-cell prolymphocytic leukemia, peripheral T-NHL, and adult T-cell leukemia/lymphoma. In contrast, none of the T-lineage ALLs analyzed expressed the ligand. In AML, a high cellular density of CD30L was detected in French-American-British M3, M4, and M5 phenotypes, which are directly associated with the presence on tumor cells of certain surface structures, including the p55 interleukin-2 receptor alpha-chain, the alpha(M) (CD11b) chain of beta2 integrins, and the intercellular adhesion molecule-1 (CD54). Analysis of normal hematopoietic cells evidenced that, in addition to circulating and tonsil B cells, a fraction of bone marrow myeloid precursors, erythroblasts, and subsets of megakaryocytes also express CD30L. Finally, we have shown that native CD30L expressed on primary leukemic cells is functionally active by triggering both mitogenic and antiproliferative signals on CD30+ target cells. As opposed to CD30L, only 10 of 181 primary tumors expressed CD30 mRNA or protein, rendering therefore unlikely a CD30-CD30L autocrine loop in human hematopoietic neoplasms. Taken together, our data indicate that CD30L is widely expressed from early to late stages of human hematopoiesis and suggest a regulatory role for this molecule in the interactions of normal and malignant hematopoietic cells with CD30+ immune effectors and/or microenvironmental accessory cells.

Recent progress on the role of Axl, a receptor tyrosine kinase, in malignant transformation of myeloid leukemias.

Receptor tyrosine kinases (RTK) play an important role in the signal transduction of normal and malignant cells. There are different families of RTKs which are mainly characterized by differences in the ligang-binding extracellular domains. Axl (or UFO/Ark) is the first member of a new class of RTK with two fibronectin type III domains and two immunoglobulin-like domains present at the extracellular domain. The axl-gene has been isolated by means of gene transfection studies using DNA of patients with chronic myelogeneous leukemia. For a previous and the present study, we used a sensitive reverse-transcriptase polymerase chain reaction assay to detect axl's mRNA in cells from normal and malignant hematopoietic tissue. Axl's mRNA expression was mainly detected in myelo-monocytic cells, whereas much weaker transcription was seen in lymphatic cells and in lymphatic leukemias. In normal bone marrow, axl was heavily transcribed in marrow stromal cells. Further, we analysed Axl protein expression using monoclonal antibody M50 in peripheral stem cell harvests; in most harvests, no co-expression of CD34 and Axl was detected. However, in one patient with AML in complete remission, Axl was co-expressed on 80% of the CD34-positive population. These data show that axl is preferentially expressed in monocytes and stromal cells. Furthermore, a fraction of CD34-positive progenitor cells may express Axl. The exact mechanism for transformation of myeloid progenitor cells through Axl, however, remains to be determined.