Cheong Sk

Epithelial membrane antigen (EMA) or MUC1 expression in monocytes and monoblasts

Aims: Epithelial membrane antigen (EMA) or MUC1 belongs to a heterogeneous group of heavily glycosylated proteins and is expressed in most normal and epithelial neoplastic cells. EMA is also expressed in plasma cells, anaplastic large cell lymphoma (Ki‐1 antigen), malignant histiocytosis and erythroleukaemia. In 1996, Cheong et al. (Hematology 1996; 1: 223) demonstrated the positive expression of EMA in monoblasts. Since there were very few useful markers for differentiating subtypes of acute myeloid leukaemia with a monocytic component from the those without, a study was conducted to evaluate the prevalence of EMA expression and its relationship with known markers for monocytic‐macrophage lineage (CD11c, CD14 and intracellular CD68) in monocytes and monoblasts. Methods: EMA detection was performed by flow cytometry in monocytes and monoblasts. EMA expression was compared with other known markers of monocytic‐macrophage lineage (CD11c, CD14 and intracellular CD68). Samples of purified monocytes were obtained from 20 healthy volunteers. Twenty‐two cases of monocytic AML (M4 and M5) were studied and controls were selected from 20 cases of acute lymphoblastic leukaemia (ALL) and 18 cases of non‐monocytic AML (M0, M1, M2, M3, and M7). Results: EMA was shown to be expressed strongly on the surface of all purified monocytes. EMA expression was observed on blast cells in 18/22 (81.8%) cases of AML M4 and M5, but not in that of non‐monocytic AML or ALL. In this study EMA monoclonal antibody has demonstrated a strong association (P<0.001) with all the other known markers of monocytic‐macrophage lineage in acute leukaemia subtypes. EMA had also shown 100% specificity and 81.8% sensitivity in the diagnosis of AML M4 and M5. Conclusions: The monoclonal antibody EMA (clone E29) is a useful marker in the classification of acute myeloid leukaemia and can be used as a supplementary analysis for the diagnosis of acute leukemia with monocytic involvement.

Cell viability of acute myeloid leukaemia blasts in culture correlates with treatment outcome.

Abstract Despite the advances in understanding the pathophysiology of acute myeloid leukaemia (AML), the cure rate for acute myeloid leukaemia patients remains low. Cytogenetic abnormalities and age are the prognostic factors that guide treatment decisions. However, many AML patients still die. The biological factors that influence treatment outcome are largely unknown. Thus, the objective of our study was to use the in vitro viability test to correlate with treatment outcome. Acute myeloid leukaemia blasts demonstrated differing ability to survive in culture. Our examination of blast phenotype at various days in culture showed two possible growth directions. First, cells underwent maturation by increased expression of CD16 and down-regulated CD34 (a haemopoietic stem cell marker). These cells also appeared to have undergone apoptosis. Alternatively, cells continued to survive in culture and maintained high expression of CD34. An MTT assay was carried out to determine viability after three days of culture. Lower optical density values were obtained for samples that underwent apoptosis and higher values were obtained for samples that survived in culture. Apoptosis was measured by Annexin V/propidium iodide staining. A comparison between results of MTT assay and duration of disease free survival revealed that a higher viability in vitro correlated significantly with shorter survival duration in the patient (R −0·761, p=0·002, n=13). Thus, this study further supports the hypothesis that AML patients with poor survival may be related to having blasts with a biologically more immature or stem cell-like nature.

A Prospective Randomized Study of Chop Versus Chop Plus Alpha-2B Interferon in Patients with Intermediate and High Grade Non-Hodgkin's Lymphoma: The International Oncology Study Group NHL1 Study

The addition of a brief alpha interferon regimen to each CHOP induction cycle, plus one year of alpha interferon thrice weekly maintenance therapy, has no early effect on response rates or survival in patients with Intermediate or High grade cell NHL. Background: The CHOP (Cyclophosphamide, Adriamycin, Vincristine, Prednisone) regimen is the most widely used first-line therapy for patients with Intermediate or High Grade (IG/HG) non-Hodgkins lymphoma (NHL). Alpha 2b interferon (INF) enhances response rates and improves survival in low-grade NHL. The International Oncology Study Group (IOSG) conducted a prospective randomized study comparing CHOP alone or combined with INF in patients with IG/HG-NHL. The primary study aim was to compare the objective response rates in these patient cohorts. Patients and Methods: Patients with a confirmed diagnosis of measurable NHL of International Working Formulation (IWF) groups D to H histology were randomized to receive CHOP alone or CHOP with 5Mu INF SC for 5 days on days 22 to 26 of each 28 day cycle with INF 5 million units (Mu) given three times per week sub-cutaneously for 52 weeks in those patients who responded to CHOP plus INF. Results: The overall response rates were equivalent in both groups: CHOP alone (214 patients) 81% (complete 55%, partial 26%); CHOP plus INF (221 patients) 80% (complete 54%, partial 26%). At 36 months, the actuarial survival rate was equivalent in both groups. Conclusions: There is no apparent early advantage in terms of response or survival conferred by adding the study INF regimen to CHOP therapy for patients with IG/HG-NHL.

Expression of multidrug resistance (MDR) proteins and in vitro drug resistance in acute leukemias

Abstract The expression of the multi drug resistance (MDR) proteins may influence the outcome of treatment in patients with acute leukemia. The aim of this study was to determine the IC50 of cytotoxic drugs (cytosine arabinoside, ara-C and daunorubicin, dnr) using the in vitro 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-suljophenyl)2H-tetrazolium, inner salt (MTS) assay method. A total of 82 newly diagnosed acute leukemia cases (43 adult myeloid leukaemia, AML cases and 39 acute lymphoblastic leukaemia, ALL cases) and 16 relapsed cases (8 AML cases and 8 ALL cases) were studied. The MTS assay was performed using two cytotoxic drugs, dnr and ara-C. Cells were incubated with different concentrations of drugs for 4 days and the IC50 was extrapolated from the viability curve. In newly diagnosed cases, we found that childhood ALL samples showed higher IC50 values of dnr (0.040 ± 2.320) compared to adult AML samples (0.021 ± 0.158). In contrast, newly diagnosed adult AML samples showed higher IC50 values of ara-C (0.157 ± 0.529) compared to childhood ALL samples (0.100 ± 2.350). In relapsed cases, two samples of childhood ALL showed IC50 values of dnr (0.910 ± 1.760) and ara-C (1.310 ± 2.390), which was higher compared to childhood AML samples (0.129 ± 0.214 and 0.210 ± 0.003, respectively). However, there was no correlation between IC50 values of these drugs tested with clinical outcome. In conclusion, we found that MTS assay is an easy, rapid and non laborious method to study in vitro drug resistance in acute leukaemia cases.

G6PD deficiency with hemolytic anemia due to a rare gene deletion—A report of the first case in Malaysia

Abstract A 2-year-old Chinese boy was referred to Hospital UKM for investigation of recurrent episodes of dark-coloured urine and pallor since birth. He was born prematurely at 34 weeks gestation and developed severe early-onset neonatal jaundice requiring exchange blood transfusion. Screening at birth showed Glucose-6-phosphate dehydrogenase (G6PD) deficiency. On admission, physical examination revealed pallor, jaundice and mild hepatomegaly. Results of laboratory investigations showed a hemoglobin level of 11.0 g/dl with a hemolytic blood picture, reticulocytosis of 20% and red cell G6PD activity reported as undetectable. The patients DNA was analysed for G6PD mutations by PCR-based techniques and DNA sequencing and results showed a 24 bp deletion of nucleotide 953–976 in the exon 9 of the G6PD gene. DNA analysis was also performed on blood samples of the patients mother and female sibling confirming their heterozygous status, although both showed normal red cell G6PD activity levels. The patient was discharged well and his parents were appropriately advised on the condition and the importance of taking folic acid regularly. This is a first case report in Malaysia of G6PD deficiency causing chronic-hemolytic anemia. The rare 24 bp deletion causes the G6PD Nara variant, previously reported only in two other unrelated males, a Japanese and a Portuguese both with chronic hemolytic anemia.

In vivo Evidence of All-Trans Retinoic Acid Inducing Maturation in Acute Promyelocytic Leukaemia

All-trans retinoic acid therapy induces maturation in acute promyelocytic leukaemia. We document in vivo evidence of differentiation by all-trans retinoic acid in a case of acute promyelocytic leukaemia which was characterized by cytoplasmic vacuolations.

Autoimmune Thrombocytopenia and Neutropenia after Autologous Peripheral Blood Stem Cell Transplantation

Accessible online at: www.karger.com/journals/aha Autoimmune thrombocytopenia (AIP) after autologous hematopoietic stem cell transplantation occurs infrequently, with less than 10 cases being reported in the literature. Autoimmune neutropenia (AIN) develops even less frequently after AHSCT. We describe a case of AIP and AIN that developed 76 days after transplant and responded well to corticosteroid therapy. A 19-year-old patient underwent autologous peripheral blood stem cell transplantation (APBSCT) for acute myeloid leukemia (AML). His full blood count pre-transplant showed a hemoglobin level of 12.3 g/dl, leukocytes of 5.6 ! 109/l and a platelet count (PC) of 248 ! 109/l. The conditioning regimen included busulphan and cyclophosphamide. Engraftment of neutrophils (absolute neutrophil count, ANC, 10.5 ! 109/l) and platelets (unsupported PC 120 ! 109/l) was documented on days 10 and 40 after transplantation, respectively. By day 68 the PC and ANC were 126 ! 109/l and 3.3 ! 109/l, respectively; however the PC and ANC fell to 4 ! 109/l and 0.56 ! 109/l, respectively, on day 76. The PC has remained less than 20 ! 109/l over the next 3 weeks. Infusions of random single-donor platelets in an attempt to maintain the PC above 10 ! 109/l were unsuccessful. There was gingival and cutaneous bleeding. Autoantibody screening including anticardiolipin antibody was negative. There was no evidence of viral or fungal infection. Bone marrow (BM) aspirate obtained on day 90 showed normal marrow with plentiful megakaryocytes and active granulopoiesis consistent with AIP and AIN. Peripheral blood lymphocyte subset study showed reduced CD4+ cells (4 cells/Ìl; normal ratio, 410–1,590), a low CD4:CD8 ratio (0.02; normal ratio 0.8–4.2) and normal CD8+ count. The patient received 60 mg prednisolone daily starting on day 96 that resulted in elevation of the PC and ANC to 99 ! 109/l and 3.8 ! 109/l, respectively, after 1 week of treatment. The PC and ANC remained above 150 ! 109/l and 4.5 ! 109/l during steroid therapy. On day 152 while receiving 30 mg prednisolone daily, he developed herpes zoster infection of the C2–C3 dermatomes. The skin lesions resolved with acyclovir and prednisolone was discontinued. When he was last seen on day 193, 20 days after discontinuation of prednisolone, the PC and ANC had decreased to 67 ! 109/l and 0.72 ! 109/l, respectively. This patient clearly had immune-mediated thrombocytopenia and neutropenia as demonstrated by an increase in megakaryocytes and granulopoiesis in the BM in the presence of a low PC and ANC and a dramatic response to steroid therapy. Other causes of platelet and granulocyte destruction were excluded. Seven cases of AIP after autologous BM transplant or APBSCT (3 patients) have been reported in patients with AML, lymphoblastic lymphoma and breast carcinoma [1–5]. Only 1 case of AIP associated with AIN after allogeneic BM

An unusual cause of tremor in an elderly man

Answers on p 277. A 70 year old man presented with a two day history of coarse tremors of the head and upper limbs. The tremor was evident at rest and it became more obvious on movement. There were no symptoms of hyperthyroidism or history of a similar problem in the past. Apart from slight low back pain, he had been …

Unexpected Epithelial Membrane Antigen (EMA) and Cytokeratin Expression in a Case of Infantile Acute Monoblastic Leukaemia.

A previously healthy eleven month old male Malay infant presented with fever, upper respiratory tract infection and right knee swelling. Pallor, bilateral proptosis, hepatosplenomegaly, multiple scalp swellings and a right cheek swelling were observed. Investigations revealed that he had acute monoblastic leukemia or FAB M5a. Immunophenotyping by flow cytometry showed that the blast cells were positive for CD45, CD13, CD33, HLA-DR, CDllc, CD71, EMA, and Cytokeratin. They were negative for CD34, CD19, CD10, CD22, CD2, CD3, CD4, CD7, CD8, CD61, NK, Glycophorin A, and CD14. The monoblasts were used to evaluate anti-EMA and anti-cytokeratin. They were unexpectedly found to be positive. Acute monoblastic leukaemias are well known to show extramedullary infiltration and this may be their primary mode of presentation. Thus, in immunochemostry, when using EMA and cytokeratin expression in the differential diagnosis of neoplastic diseases, it is important to consider that monoblasts may express these markers as illustrated by this case.

Acute myeloid leukaemia with tell-tale computed tomography scans

Answers on p 737. A 36 year old man with acute myeloid leukaemia presented with recurrent episodes of fever and a three day history of right sided pleuritic pain and left upper abdominal pain after …