Paroxysmal nocturnal hemoglobinuria testing in patients with myelodysplastic syndrome in clinical practice-frequency and indications.

Background: Myelodysplastic syndrome (mds) is characterized by peripheral blood cytopenias, with most patients developing significant anemia and dependence on red blood cell (rbc) transfusion. In paroxysmal nocturnal hemoglobinuria (pnh), mutations in the PIGA gene lead to lack of cell-surface glycosylphosphatidylinositol, allowing complement-mediated lysis to occur. Paroxysmal nocturnal hemoglobinuria results in direct antiglobulin test-negative hemolysis and cytopenias, and up to 50% of patients with mds test positive for pnh cells. We wanted to determine whether pnh is considered to be a contributor to anemia in mds. Methods: Patients with a diagnosis of mds confirmed by bone-marrow biopsy since 2009 were reviewed. High-resolution pnh testing by flow cytometry examined flaer (fluorescein-labeled proaerolysin) binding and expression of CD14, CD15, CD24, CD45, CD59, CD64, and CD235 on neutrophils, monocytes, and rbcs. Results: In 152 patients with mds diagnosed in 2009 or later, the mds diagnosis included subtypes associated with pnh positivity (refractory anemia, n = 7, and hypoplastic mds, n = 4). Of 11 patients who underwent pnh testing, 1 was positive (9.0%). Reasons for pnh testing were anemia (n = 3), new mds diagnosis (n = 2), hypoplastic mds (n = 2), decreased haptoglobin (n= 1), increased rbc transfusion requirement (n= 1), and unexplained iron deficiency (n= 1). Conclusions: Testing for pnh was infrequent in mds patients, and the criteria for testing were heterogeneous. Clinical indicators prompted pnh testing in 6 of 11 patients. Given that effective treatment is now available for pnh and that patients with pnh-positive mds can respond to immunosuppressive therapy, pnh testing in mds should be considered. Prospective analyses to clarify the clinical significance of pnh positivity in mds are warranted.

Intra-cerebral relapse following prolonged remission after autologous stem cell transplantation for multiple myeloma.

Central nervous system (CNS) myeloma is a rare phenomenon, especially so after high-dose therapy (HDT) and stem cell transplantation. We describe a case of isolated CNS relapse of myeloma post autologous transplantation that followed a prolonged progression-free interval. Issues regarding the pathophysiology and management of this unusual complication are discussed.

Myelodysplastic syndrome with hypereosinophilia and a nonrandom chromosomal abnormality dic(1;7): confirmation of eosinophil clonal involvement by fluorescence in situ hybridization.

We report a case of de novo myelodysplastic syndrome (MDS) with hypereosinophilia and dic(1;7) in which eosinophil clonal involvement was confirmed by fluorescence in situ hybridization. There have been two previous reports in the literature of eosinophilic MDS with dic(1;7) or t(1;7) in which eosinophil clonality was demonstrated. The specific breakpoints on chromosomes 1 and 7 differ in the three cases, making it difficult to implicate disruption of a single gene as causative; nevertheless, the nonrandom occurrence of t(1;7) or dic(1;7) with malignant eosinophilic proliferations suggests that this chromosomal rearrangement is involved in the etiology of the disease.

Induction failure in de novo acute myelogenous leukemia is associated with expression of high levels of CD34 antigen by the leukemic blasts.

The prognostic significance of CD34 antigen expression in acute myelogenous leukemia (AML) is controversial. Most studies to date have reported on CD34 positivity and not the level of antigen present. In this study of 62 patients with de novo AML, 48 (77%) patients were CD34+ in varying levels (0-85 mean channels of fluorescence (MCF)). Forty seven of 62 were treated with combination chemotherapy and 39 (83%) of them achieved complete remission (CR). Patients with CD34- blasts were more likely to achieve CR; however, this trend was not statistically significant (p = .11). On the other hand, patients with higher levels of CD34 antigen on the blasts were less likely to attain CR (p < 0.001, multivariate analysis). The patients who achieved CR expressed lower levels of CD34 (0-57; median 9 MCF) as compared to those who did not achieve CR (15-85; median 30 MCF). Of the other antigens tested, partial or complete absence of CD33 (CD33 absent in > or =25% blasts) correlated with failure to achieve CR (p = 0.0029). These results are in keeping with the hypothesis that more primitive AML blasts with high levels of CD34 are chemoresistant.

Testicular relapse of acute promyelocytic leukemia after allogeneic BMT.

After treatment of acute leukemia (typically ALL and the monocytic variants of AML), relapse may occur at sites other than the marrow. Isolated extramedullary relapse of acute promyelocytic leukemia (APL) however, is rare. We describe such an event in a man who underwent allogeneic BMT for APL in second relapse and 4 years later presented with testicular relapse. The marrow was morphologically and cytogenetically normal, but RT-PCR analysis revealed the specific PML/RAR chimeric RNA transcript.

Case Report: a patient with primary biliary cirrhosis and autoimmune hemolytic anemia.

Diseases of an autoimmune nature are well recognized in association with primary biliary cirrhosis. Although autoimmune thyroiditis and many rheumatological conditions are well described in primary biliary cirrhosis, autoimmune haematological diseases have been less well reported. We report on a 66 year old North American Indian man with coincident primary biliary cirrhosis and warm antibody haemolytic anaemia. This case report supports the suggestion of an association between autoimmune haemolytic anaemia and primary biliary cirrhosis.

Criteria for marrow engraftment: comparison of reticulocyte maturity index with conventional parameters.

Reticulocyte maturity index (RMI) has recently been proposed as an early indicator of marrow engraftment. We compared the RMI with conventional bone marrow engraftment criteria including total leukocyte count (WBC), absolute neutrophil count (ANC), reticulocyte count (RC) and the day of last platelet transfusion required to maintain the platelet count (PC) > or = 20 x 10(9)/l in 37 patients undergoing allo- or autologous BMT. There was no discrepancy in predicting engraftment between RMI, ANC, WBC and RC. RMI indicated engraftment earlier (median day 17, range 10-63 days) than the ANC (median day 19, range 8-63 days), WBC (median day 19, 9-71), RC (median day 19, 11-125) or PC (median day 29, 11-237). RMI heralded engraftment preceded ANC, WBC, RC or PC in 22, 21, 34 and 32 patients, respectively. RMI signal occurred 6 days prior to the rise in ANC in patients who engrafted later than 25 days (n = 7). Trend analysis showed that ANC fluctuated more frequently (6/37 patients) than RMI (1/37). Combined use of ANC and RMI (whichever increased first) predicted engraftment earlier (median 15 days) and more confidently (no false starts) than either used alone.

Lymphocytosis of large granular lymphocytes in patients with Hodgkin's disease.

Clonal disorders of large granular lymphocytes (LGL) of either CD3- (NK cell) or CD 3+ (T-cell) phenotype have been described. B-cell malignancies such as hairy cell leukemia and non-Hodgkin's lymphoma have been observed in association with the T-cell type of LGL leukemia. Here we report the occurrence of LGL lymphocytosis in four patients with Hodgkin's disease. Immunophenotyping studies showed that these LGL were CD 3- in three patients and CD3+ in the other. LGL were polyclonally expanded in both patients in whom clonality could be assessed.

Cytokine profile in acute myelofibrosis associated with aggressive large granular lymphocyte leukemia.

We report a patient with acute large granular lymphocyte (LGL) leukemia, presenting as acute myelofibrosis (AMF). The leukemic cells were immature T-cells (CD5+, CD7+, CD16-, CD56-, CD57-, and CD41-), had monosomy 7, and secreted large amounts of Transforming Growth Factor-beta 1(TGF-beta 1). The serum levels of interleukins (IL)-2, -2R, -6 and -8 were elevated, while the IL-1 beta, IL-4, and tumor necrosis factor-alpha were normal.

Deposition of transforming growth factor-beta in the marrow in myelofibrosis, and the intracellular localization and secretion of TGF-beta by leukemic cells.

The marrows of 10 patients with hematologic malignancies were examined by immunohistochemistry using anti TGF-beta antibody, CC(1-30), which detects secreted TGF-beta, and compared with four normal marrows. TGF-beta was not demonstrated in marrows with a normal level of reticulin fibrosis; however, TGF-beta was observed within collagen in marrows having collagen fibrosis or increased reticulin fibrosis. The extent of TGF-beta deposition paralleled the severity of fibrosis (P < .0001), and occurred even with normal or reduced numbers of megakaryocytes. Using another TGF-beta antibody, LC(1-30), which detects intracellular TGF-beta, TGF-beta was detected by immunofluorescence in discrete sites in the cytoplasm of immature and mature myeloid and large granular lymphocytic leukemia cells. These sites colocalized with areas detected by an anti-granule antibody (D545) suggesting that TGF-beta was stored in granules. However, neither the TGF-beta mRNA content nor the degree of TGF-beta secretion by these leukemic cells correlated with the extent of TGF-beta deposition in the marrow. Thus, TGF-beta deposition in marrow may contribute to myelofibrosis, but the source of this cytokine in the absence of megakaryocytes requires further study.

Treatment of acute promyelocytic leukemia in patients presenting at Vancouver General Hospital from 1983 to 1992.

Between 6/83 and 8/92, 23 of 361 patients (6.4%) presenting at Vancouver General Hospital with acute myelogenous leukemia had acute promyelocytic leukemia (APL). Treatment plan was: 1) induction with high-dose cytosine arabinoside and an intercalator; and 2) consolidation with allogeneic bone marrow transplantation (BMT) for those aged < or = 50 years with a sibling donor or repeat of induction for the the others. Complete remission (CR) was achieved in 20 patients (87%). Eleven patients in CR were eligible for allogeneic BMT; 4 were considered unsuitable, 2 refused, and 5 underwent this treatment--1 died of acute graft-versus-host disease, 1 relapsed and 3 are leukemia-free and well 1.6, 3.3 and 3.9 years after diagnosis. Fifteen patients did not undergo allogeneic BMT in CR; 4 received no further treatment and all died, 2 relapsed before consolidation therapy and both died, 1 underwent autologous BMT and died of complications, and 8 received consolidation treatment as planned--1 died of sepsis, 2 relapsed and 5 are leukemia-free and well 1.0, 3.8, 4.5, 4.9 and 8.5 years after diagnosis. The actuarial overall survival for all 23 patients was 38% (95% confidence interval [CI] 18-57%). The actuarial 2-year leukemia-free survival was 60% (95% CI 20-85%) for the 8 patients who underwent consolidation chemotherapy as planned and 53% (95% CI 68-86%) for the 5 patients who underwent allogeneic BMT in CR. These results suggest that patients with APL who are able to undergo consolidation chemotherapy have a relatively good prognosis and allogeneic BMT may reasonably be held in reserve for salvage therapy.

Transforming growth factor beta 1 gene expression in human airways.

BACKGROUND: Asthmatic airways have a characteristic deposition of connective tissue under the epithelial basement membrane, but the mediators involved in this alteration are unknown. Several authors have postulated that transforming growth factor beta 1 (TGF-beta 1) could be overexpressed in asthmatic airways. METHODS: Lung samples from 16 asthmatic patients, six patients with chronic obstructive pulmonary disease (COPD), and six non-obstructed smokers were analysed. RNA was extracted from these tissues to measure expression of TGF-beta 1 by Northern blot analysis using a cDNA probe for TGF-beta 1. The level of expression was quantitated by densitometry using glyceraldehyde 3-phosphate dehydrogenase mRNA as a control. TGF-beta 1 was localised to specific cell types in these lungs by immunohistochemical analysis using polyclonal antibodies specific for intracellular and extracellular TGF-beta 1. RESULTS: The 2.5 kb TGF-beta 1 mRNA was seen in all 18 samples analysed by Northern blotting and densitometric analysis showed no difference between the asthmatic group (mean (SD) 108% (43%)), the group with COPD (122% (33%)), and the non-obstructed group (100% (49%)). The TGF-beta 1 precursor was immunolocalised throughout the airway wall including the epithelium and in alveolar macrophages. The mature TGF-beta 1 was localised primarily within the connective tissue of the airway wall. These patterns of expression of both forms of TGF-beta 1 were similar in lungs from asthmatic patients, those with COPD, and controls. CONCLUSIONS: While TGF-beta 1 mRNA and protein are abundantly expressed in human lungs, there is no clear difference in expression between the airways of asthmatic subjects and those of smokers with and without COPD.

Establishment and characterization of a human leukemic cell line (SR-91) with features suggestive of early hematopoietic progenitor cell origin.

The characterization of a cell line (designated SR-91) from a patient with clinical and morphological diagnosis of acute lymphoblastic leukemia (CD3-, CD2+, CD7+, germline TCR genes) who relapsed early after allogeneic bone marrow transplantation, is reported. The line was established from blood cells obtained at diagnosis and placed in suspension culture with medium conditioned by 5637 cells. SR-91 cells are negative for lymphoid surface markers (CD3-, CD2-, CD7-) but positive for markers indicative of myeloid progenitor cells, such as CD33 and CD34. It is likely that the conditioned medium has induced myeloid differentiation from a lymphohematopoietic progenitor cell. After establishment, cells proliferated in response to GM-CSF stimulation but they are not factor-dependent and do not produce GM-CSF. No proliferative response to IL-1, IL-2, IL-3, IL-4, IL-6 or M-CSF was observed. Cells were completely resistant to anti-proliferative effects of tumor necrosis factor-alpha and interferon-alpha or -gamma, and showed no lysis after incubation with freshly isolated natural killer cells or IL-2-activated natural killer cells.

Therapy-related acute myelogenous leukemia associated with 11q23 chromosomal abnormalities and topoisomerase II inhibitors: report of four additional cases and brief commentary.

We report 4 additional cases of therapy-related acute myelogenous leukemia (t-AML) with the translocation t(9;11)(p22q23). Chemotherapy for the primary malignancy (breast carcinoma in 2, non-Hodgkin's lymphoma in 2) included agents with topoisomerase II inhibitory activity (doxorubicin in 2; doxorubicin and etoposide in 1; doxorubicin, etoposide and mitoxantrone in 1) as well as alkylators. In agreement with previous reports, the leukemia was monoblastic (FAB M5 subtype) in all 4 patients, with only 1 having prior myelodysplasia, and the latency period from primary therapy was relatively short (24-48 months). All patients received potentially curative treatment for the leukemia which included allogeneic bone marrow transplantation in 3; however, all died (3 of t-AML and 1 of lymphoma). Therapy-related AML associated with exposure to agents with topoisomerase II inhibitory activity (epipodophyllotoxins and anthracyclines) is a distinct entity, the genetic basis and optimal treatment of which remain to be determined.

Immunocytochemical localization of secreted transforming growth factor-beta 1 to the advancing edges of primary tumors and to lymph node metastases of human mammary carcinoma.

The level of expression and localization of transforming growth factor-beta 1 (TGF-beta 1) were analyzed by immunocytochemistry using antibodies that distinguished the sites of intracellular synthesis and extracellular secretion of TGF-beta 1 in 28 cases of infiltrating duct carcinoma of breast, 12 of which had lymph node metastases. Twenty-seven of 28 primary tumors and all 12 lymph node metastases showed extracellular deposition of TGF-beta 1. The extracellular TGF-beta 1 staining was either confined to or more strongly expressed at the advancing edges of the tumor than in the center of the primary tumor. By contrast, 19 of 28 primary tumors and 11 of 12 metastases contained intracellular TGF-beta 1, and no variation in the intensity was seen. The metastatic tumors were significantly more intensely stained for both intra- and extracellular TGF-beta 1 than the primary tumor tissues. The preferential expression of secreted TGF-beta 1 at the advancing tumor edges and in lymph node metastases suggests a role for TGF-beta 1 in the malignant progression of breast carcinoma.

Wide distribution of granulophysin epitopes in granules of human tissues.

BACKGROUND: The identification and characterization of granule membrane proteins are becoming increasingly important in understanding the packaging and secretory function of granules and characterizing diseases involving granules. A granule membrane protein, granulophysin, has recently been identified in the membranes of platelet dense granules, organelles that contain stored ADP, ATP, serotonin, and calcium. Antibodies that recognize granulophysin also stain granules of monocytes, neutrophils, and lymphokine activated killer cells. EXPERIMENTAL DESIGN: In the present study, the distribution of epitopes recognized by antigranulophysin monoclonal antibodies in human tissues was investigated using immunohistochemistry on paraffin sections. Quantitation of the protein was also performed by enzyme-linked immunosorbent assay. The protein was also analyzed in various tissues using Western blotting. RESULTS: Granulophysin was localized to the granules of skin melanocytes, neurons, endocrine gland cells, exocrine glands (except mucin producing cells), and surface lining cells. Analysis by Western blots revealed a typical staining pattern for granulophysin in lung, adrenal gland, liver, brain, prostate, and pituitary. Atypical bands were present in the pancreas head (47 kDa) and skeletal muscle (34 kDa). A clear distinction was demonstrated between granulophysin and synaptophysin through both immunochemistry and blotting, despite the known cross-reactivity of these two proteins. CONCLUSIONS: The findings demonstrate that granulophysin is a widely distributed protein that is frequently associated with granules. We speculate that it may be critical in granule function.

Local and systemic eosinophilia in patients with carcinoma of the uterine cervix undergoing radiation therapy: correlation with radiation response.

Forty-nine patients with squamous carcinoma of the uterine cervix undergoing supervoltage 60Co radiotherapy were investigated to determine the significance of quantitation of eosinophils in the tumour tissue as well as in blood. The blood absolute eosinophil count (AEC) at presentation was higher in patients with Stages III and IV disease (0.52 +/- 0.09 x 10(9)/l, mean +/- SEM) when compared with early stages (Stages I and II, 0.35 +/- 0.07 x 10(9)/l). Forty-four patients (90%) showed a steady rise in AEC during the course of therapy. The patients showing good radiation response (n = 13, greater than or equal to 50% regression), displayed a more pronounced rise in AEC during the course of radiotherapy (0.66 +/- 0.24 x 10(9)/l) and had more eosinophils in the tumour tissue (7.4 +/- 0.66/oil immersion field) than the poor responders (n = 36, less than 50% regression, rise in eosinophil count 0.22 +/- 0.04 x 10(9)/l, eosinophils in tumour tissue 2.9 +/- 0.55/oil emersion field, P less than 0.0001 for both parameters). We conclude that quantitation of eosinophils in blood and tumour tissue before and during radiotherapy in patients with squamous carcinoma of the uterine cervix is useful in predicting radiation response.

Leukophysin: a 28-kDa granule membrane protein of leukocytes.

A membrane glycoprotein of human platelet dense granules, called granulophysin, with serologic homology to synaptophysin has recently been identified. To determine if this protein was present in granulated leukocytes, we examined several cell types for the presence of the protein by indirect immunofluorescence. Antigranulophysin mAb staining was detected in a granular pattern in the cytoplasm of permeabilized IL-2-stimulated CD3+ peripheral lymphocytes, neutrophils, U937 monocytes, and mast cells. Immunohistochemistry of human lymph nodes showed cytoplasmic staining of macrophages, neutrophils, and some dendritic cells. Induction of granule exocytosis in granulated CD3+ lymphocytes after stimulation with PMA and calcium ionophore A23187 resulted in a redistribution of the reactive epitope from the cytoplasm to the plasma membrane. Subcellular fractions contained two peaks of reactivity; the first peak coincided with N-benzyloxycarbonyl-L-lysine thiobenzyl ester-esterase activity in dense granules whereas the second peak was present in lighter fractions. The affinity purified protein from both peaks was identical in Western blot analysis and had a molecular mass of 28 kDa under reducing conditions. The protein could only be solubilized in detergent suggesting that it was an integral membrane protein. We have named this protein leukophysin to differentiate it from the 40-kDa granulophysin of platelets. Monocytes contained a protein with identical m.w. to leukophysin, whereas a protein of a slightly higher m.w. was detected in neutrophils. We propose that leukophysin is a common granule membrane protein of leukocytes.