The simultaneous expression of both lymphoid and myeloid phenotypic features in acute leukaemia is rare. We report 3 cases of biphenotypic hybrid acute leukaemia seen in our institution. All 3 patients achieved remission with treatment for acute lymphoblastic leukaemia but two subsequently relapsed while on treatment. The hybrid acute leukaemias are important areas for further research both for delineation of basic biology and choice of optimal treatment.
In vitro deoxyribonucleic acid (DNA) amplification by the polymerase chain reaction (PCR) followed by hybridization with oligonucleotide probes were used to study ras gene mutations in acute myeloid leukemia (AML). The DNA of 30 AML patients at presentation of the disease at the University of Malaya Hospital, Kuala Lumpur were screened for ras gene mutations in codons 12, 13 and 61 of the N-ras, K-ras and H-ras genes. Four patients (13.3%) had ras gene mutations. They were all below their early thirties in age. Of the four patients with ras gene mutations, three were M3 and one was M4 according to the French American British (FAB) classification of AML.
The haematological findings and case history of 3 patients with the association of acute myeloid leukemia and translocation involving the long arm of chromosome no. 11 are presented. The recipient chromosome for the translocated material from chromosome 11 differs in all the three cases being namely chromosomes 1, 10 and 17.
Immunophenotypic studies using immunofluorescent flow cytometry were performed on the blast cells of 36 patients with acute leukaemia using a panel of eight monoclonal antibodies. Six patients had blasts which co-expressed markers for lymphoid and myeloid differentiation, and which were therefore defined as biphenotypic hybrid acute leukaemia. Of the six, three patients were in the paediatric age group (below 12 years old) while the other three were more than 12 years old. Peripheral blood counts were variable; however, bone marrow infiltration was extensive (blasts > or = 75% in all). At the time of study, remission was achieved in only two patients. The authors' data show that biphenotypic hybrid acute leukaemia is not rare in Malaysia. This represents a subgroup of acute leukaemia identifiable by immunophenotyping but not by the French-American-British classification based on morphological and basic cytochemical studies alone. The recognition of this subgroup is important for both practical and theoretical reasons. There are implications for treatment of the individual patient because treatment directed at a single lineage may not be effective. The two colour flow cytometry proved to be a useful tool for diagnosis and classification of acute leukaemia.
Clostridium septicum infection has been shown to have a strikingly high association with either bowel or blood malignancies. The infection may be fatal if unrecognised. We report a case of C. septicum bacteremia in a man diagnosed with acute myeloid leukaemia.
A 45-year-old Malay lady developed brisk vesicular, plaque-like reaction to a Mantoux test concomitant with a diagnosis of acute myeloid leukaemia (AML). The lesion resolved one month after chemotherapy. Similar lesions developed later after she was bitten by mosquitoes on the forearms. She also had the lesions over her cheek. A skin biopsy showed infiltration of the dermis with neutrophils and some monocytoid cells. The lesion resolved one week after prednisolone therapy.
This study reviewed the immunophenotyping results of children with acute leukemia in Kelantan, Malaysia. In the 3.5-year period (January 1994 to June 1997), 45 cases were identified. All children were under the age of 12 years and the predominant ethnic group was Malay. Thirty-six cases (80%) were acute lymphoblastic leukemia (ALL) and 9 cases (20%) were acute myeloblastic leukemia (AML). Of the ALL cases, 3% were of B-cell and 22% of T-cell origin, and 96% of the B-lineage ALL were CD10 positive. All the AML cases expressed CD33 and 78% were positive for CD13. The incidence of mixed-lineage leukemias was 13.8% for My+ ALL and 11.1% for Ly+ AML.
Fusarium species is an emerging genus of fungal pathogens which until recently were rare causes of human disease apart from localized infection of the skin and nails. Two cases of fungaemia due to Fusarium sp. in children are described. The first child, an 8-year old girl with acute myeloid leukaemia developed character-sitic pyoderma gangrenosum-like skin lesions before succumbing to disseminated Fusarium infection and acute respiratory distress syndrome. The second child, a 5-month old boy, developed pneumonia associated with a transient erythematous skin rash while on chemother-apy for congenital leukaemia. Both patients had Fusarium isolated from blood. The second child improved after six weeks of treatment with ampho-tericin B and granulocyte-macrophage colony stimulat-ing factor but ultimately she died of the disease follow-ing discharge. Fusarium spp should be recognised as an opportunistic pathogen in immunocompromised patients. Current literature suggests that liposomal amphotericin B in conjunction with leukocyte growth factors are the treatment of choice in this potentially fatal infection.
The aim of this study was to determine the incidence and outcome of herpes zoster hospitalised children with cancer in Kota Baru. It was a retrospective review from January 1994 to December 1998. The diagnosis of herpes zoster was a clinical one. Herpes zoster was diagnosed in 10 of 188 (5%) children with malignancy. The most common malignancy was leukaemia. Nine children were treated with acyclovir. No child developed visceral dissemination and there were no deaths.
Allogeneic haemopoietic stem cell transplantation was initially considered as a means of delivering supralethal doses of chemotherapy with or without total body irradiation for the treatment of malignancy. However, it has become clear that this mode of therapy does not eradicate the malignancy in many patients and its benefit is largely due to the immune mediated graft versus malignancy effect. This has led to development of alternative strategy to utilize a less intensive preparative regimen pre-transplantation that provides sufficient immunosuppression to achieve engraftment of an allogeneic stem cell graft, thus allowing the evolution of a graft versus malignancy effect post-transplantation. Since September 1999, we had carried out 10 cases of allogeneic peripheral blood stem cell transplantation: one case of aplastic anaemia, four cases of acute myeloid leukemia (AML) in first remission, and five cases of chronic myeloid leukemia (CML) in chronic phase. The preparative regimen was non-myeloablative comprising Fludarabine with Cyclophosphamide or Busulphan. Recovery from transplantation was rapid with no or brief period of neutropenia or thrombocytopenia. Engraftment was established by determining donor's short tandem repeats in the recipient's bone marrow at day 30, 60 and 100 post-transplantation. Seven cases (70%) show partial or complete donor's chimerism by day 30 indicating successful engraftment. No treatment mortality was noted at day 100. Graft versus host disease was generally limited. Up to the date of reporting, two patients with CML had graft failure, one was successfully re-transplanted later. Two patients with AML had since relapsed and passed away. The others remain alive and well. The cost of transplantation on average was estimated to be about a quarter of that using a myeloablative regimen. It appears that this treatment strategy is a promising approach for the management of blood disorders.
Dendritic cells (DC) are efficient and potent antigen-presenting cells. Pilot clinical trials indicated that DC loaded with tumour antigen could induce tumour-specific immune responses in various cancers including B-cell lymphoma, melanoma and prostate cancer. Owing to extensively low number of DC in the blood circulation, a variety of sources have been used to generate DC including monocytes, CD34+ stem cells and even with leukaemic blast cells. We demonstrate here a simple method to generate DC from acute myeloid leukaemia (AML) cells and monocytes from healthy donor or remission samples. AML cells or monocytes were cultured in RPMI 1640 media supplemented with foetal bovine serum or autologous serum where possible and different combinations of cytokines GM-CSF, IL-4 and TNF-alpha. The generated DC were evaluated for their morphology by phase contrast microscopy and May Grunwald Giemsa staining. Viability of cells was determined by trypan blue dye exclusion. Percentage of yields and immunophenotypes were carried out by flow cytometry. We found that cultured AML cells and monocytes developed morphological and immuno-phenotypic characteristics of DC. Monocytes are better than AML blast in generating DC and serve as a ready source for dendritic cell vaccine development.
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.
Dendritic cells (DC) are professional antigen presenting cells of the immune system. Through the use of DC vaccines (DC after exposure to tumour antigens), cryopreserved in single-use aliquots, an attractive and novel immunotherapeutic strategy is available as an option for treatment. In this paper we describe an in vitro attempt to scale-up production of clinical-grade DC vaccines from leukemic cells. Blast cells of two relapsed AML patients were harvested for DC generation in serum-free culture medium containing clinical-grade cytokines GM-CSF, IL-4 and TNF-alpha. Cells from patient 1 were cultured in a bag and those from patient 2 were cultured in a flask. The numbers of seeding cells were 2.24 x 10(8) and 0.8 x 10(8), respectively. DC yields were 10 x 10(6) and 29.8 x 10(6) cells, giving a conversion rate of 4.7% and 37%, respectively. These DC vaccines were then cryopreserved in approximately one million cells per vial with 20% fresh frozen group AB plasma and 10% DMSO. At 12 months and 21 months post cryopreservation, these DC vaccines were thawed, and their sterility, viability, phenotype and functionality were studied. DC vaccines remained sterile up to 21 months of storage. Viability of the cryopreserved DC in the culture bag and flask was found to be 50% and 70% at 12 months post cryopreservation respectively; and 48% and 67% at 21 months post cryopreservation respectively. These DC vaccines exhibited mature DC surface phenotypic markers of CD83, CD86 and HLA-DR, and negative for haemopoietic markers. Mixed lymphocyte reaction (MLR) study showed functional DC vaccines. These experiments demonstrated that it is possible to produce clinical-grade DC vaccines in vitro from blast cells of leukemic patients, which could be cryopreserved up to 21 months for use if repeated vaccinations are required in the course of therapy.
Multidrug resistance (MDR) is believed to be responsible for poor response of patients towards chemotherapy particularly patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). The best-characterized resistance mechanism is the one mediated by permeability-glycoprotein (P-gp) encoded by MDR1 gene, which is responsible for drug efflux. We studied P-gp and multidrug resistance-associated protein 1 (MRP1) expression and functional activities in 43 newly diagnosed acute leukemia cases (19 paediatric ALL cases and 24 adult AML cases). The expression and functional activities were examined using flow cytometry and MultiDrugQuant assay kit (involving calcein AM uptake and efflux). P-gp and MRP1 expression and its functional activities were observed in 68.4% of paediatric ALL. In adult AML cases, all cases expressed MRP1 and its functional activities but only 58.3% were positive for P-gp and its functional activities. We were able to show a significant correlation between the expression of the multidrug resistant protein (P-gp and MRP1) and their functional activity in adult AML and paediatric ALL samples.
Invasive aspergillosis predominantly occurs in immunocompromised patients and is often resistant to different therapeutically strategies. However, mortality significantly increases if the central nervous system is affected. In this report we describe two cases of invasive aspergilosis, one with kidney involvement with a successful treatment while the other with pulmonary and cerebral involvement with a grave outcome.