Intracranial Hemorrhage in Childhood Acute Leukemia: Incidence, Characteristics, and Contributing Factors.

BACKGROUND: Among all cancers, hematologic malignancy has the highest rate of intracranial hemorrhage. However, there are limited data on intracranial hemorrhage in childhood acute leukemia. We aimed to determine the incidence, characteristics, and factors associated with intracranial hemorrhage in children with acute leukemia. METHODS: We reviewed a database of patients aged one month to 15 years diagnosed with acute leukemia during 2003 to 2016 at a hospital in Thailand. Characteristics of patients with intracranial hemorrhage were compared with those of patients without intracranial hemorrhage. Multiple logistic regression was used to determine the associated factors. We performed survival analyses to compare survival and hazard ratios between groups. RESULTS: There were 494 children with acute leukemia (acute lymphoblastic leukemia 367, acute myelogenous leukemia 127). Median age was 4.9 years (interquartile range 3.0 to 9.2). Follow-up duration was 2.1 years. Intracranial hemorrhage occurred in 12 patients whose median age was 12.5 years (interquartile range 7.5 to 13.3). Incidence rate of intracranial hemorrhage was 6.2 (acute lymphoblastic leukemia 5.1, acute myelogenous leukemia 12.9) per 1000 person-years. Case fatality rate of intracranial hemorrhage was 75%. Patients with early intracranial hemorrhage had prolonged international normalized ratio and higher white blood cell count, whereas patients with late intracranial hemorrhage had more concurrent systemic infections. Most cases of intracranial hemorrhage were intraparenchymal with perihematomal edema. Median survival was 24 days in the intracranial hemorrhage group compared with four years in the non-intracranial hemorrhage group. Risk of death from intracranial hemorrhage was 3.2 times higher than that of the non-intracranial hemorrhage group. Age at diagnosis, initial white blood cell count, and lactate dehydrogenase were associated with increased risk of intracranial hemorrhage. CONCLUSIONS: Intracranial hemorrhage was common and often fatal in children with acute leukemia. Potential contributing factors differed by intracranial hemorrhage timing. Older age, white blood cell count, and lactate dehydrogenase were associated with high risk of intracranial hemorrhage.

Immature reticulocyte fraction is an early predictor of bone marrow recovery post chemotherapy in patients with acute leukemia.

OBJECTIVE: To establish the benefits of immature reticulocyte fraction (IRF) measurement using an automated hematology cells analyzer over absolute neutrophil count (ANC) in predicting bone marrow recovery post induction chemotherapy. METHODS: A prospective observational study was carried out in the Departments of Pathology, Medicine, and Pediatrics, Universiti Kebangsaan Malaysia, Medical Center (UKMMC), Kuala Lumpur, Malaysia during a period of 19 months from April 2009 to December 2010 to assess the bone marrow recovery in patients with acute leukemia. A total of 22 patients in remission induction phases were enrolled in this study. The blood specimens were collected from day zero after chemotherapy, and every 3 days until patients recovered hematologically. All blood samples were measured for ANC and IRF using an automated hematology analyzer (Beckman-Coulter LH750). RESULTS: The percentage of patients showing IRF recovery earlier than ANC recovery was 63.6% (14 out of 22 patients). There was a significant difference in the mean number of days for IRF recovery as compared with ANC recovery (14.05 and 17.18 days), p=0.005. CONCLUSION: This study proved that IRF was more useful in predicting bone marrow recovery in a patient with acute leukemia post induction chemotherapy compared with ANC. The IRF is not affected by infection, is easily measured, and inexpensive; thus, it is a reliable parameter to evaluate bone marrow reconstitution.

Analysis of class I and II aberrations in Iraqi childhood acute myeloid leukemia using filter paper cards.

The lack of molecular diagnosis in the field of cancer in Iraq has motivated us to perform a genetic analysis of pediatric acute myelogenous leukemia (AML), including class I and II aberrations. Peripheral blood or bone marrow cells were collected from 134 AML children aged ≤15 years. Flinders Technology Associates (FTA) filter paper cards were used to transfer dried blood samples from five Iraqi hospitals to Japan. DNA sequencing was performed to identify class I mutations. Nested RT-PCR was used to detect class II aberrations, except that MLL rearrangement was detected according to long distance inverse-PCR. NPM1 and FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations were analyzed by GeneScan using DNA template. Among 134 Iraqi pediatric AML samples, the most prevalent FAB subtype was M2 (33.6 %) followed by M3 (17.9 %). Class I mutations: 20 (14.9 %), 8 (6.0 %), and 8 (6.0 %) patients had FLT3-ITD, FLT3-TKD, and KIT mutations, respectively. Class II mutations: 24 (17.9 %), 19 (14.2 %), and 9 (6.7 %) children had PML-RARA, RUNX1-RUNX1T1, and CBFB-MYH11 transcripts, respectively. MLL rearrangements were detected in 25 (18.7 %) patients. NPM1 mutation was detected in seven (5.2 %) cases. Collectively, approximately 30 % of AML children were proved to carry favorable prognostic genetic abnormalities, whereas approximately 10 % had high FLT3-ITD allelic burden and needed a special treatment plan including allogeneic hematopoietic stem cell transplantation. Acute promyelocytic leukemia (APL) was frequent among Iraqi pediatric AML. It is likely that molecular diagnosis using FTA cards in underdeveloped countries could guide doctors towards an appropriate treatment strategy.

The need for continuing chemotherapy for leukemic cell lysis pneumopathy in patients with acute myelomonocytic/monocytic leukemia.

Although fatal pulmonary complications frequently occur during the course of acute leukemia, a minor proportion of the complications are due to leukemia itself. Infections, drug reactions and concomitant medical conditions are the major causes of respiratory distress in leukemic patients. We treated four patients with acute myeloid leukemia complicated by leukemic cell lysis pneumopathy (LCLP). All of the patients had leukemia of monocytoid origin and their respiratory function deteriorated soon after chemotherapy initiation. Although two of the patients required mechanical ventilation, all four improved after continued chemotherapy. Our experience indicates that, in cases of LCLP, chemotherapy should be continued with maximal respiratory support.

Disease-stabilizing treatment with all-trans retinoic acid and valproic acid in acute myeloid leukemia: serum hsp70 and hsp90 levels and serum cytokine profiles are determined by the disease, patient age, and anti-leukemic treatment.

Heat shock protein (HSP) 70 and HSP90 are released by primary human acute myeloid leukemia (AML) cells during stress-induced spontaneous in vitro apoptosis. The AML cells also show constitutive release of several cytokines and the systemic serum levels of several soluble mediators are altered in patients with untreated AML. In the present study, we have investigated serum levels of HSP70/HSP90 and the serum cytokine profiles of patients with untreated AML and patients receiving AML-stabilizing palliative treatment based on all-trans retinoic acid (ATRA) plus valproic acid. Patients with untreated AML showed increased HSP90 levels and a distinct serum cytokine profile when compared with healthy controls, and low pre-therapy HSP90 levels were associated with a prolonged survival during treatment with ATRA + valproic acid + theophyllin. Hierarchical cluster analysis showed a close association between HSP70, HSP90, IL-1 receptor antagonist (IL-1ra), and hepatocyte growth factor (HGF) levels. Furthermore, disease-stabilizing therapy altered the serum-cytokine profile, but the correlations between HSP70/HSP90/IL-1ra/HGF were maintained only when ATRA + valproic acid were combined with theophyllin but not when combined with cytarabine. We conclude that both HSP levels and serum cytokine profiles are altered and may represent possible therapeutic targets or prognostic markers in human AML.

A multicenter retrospective study defining the clinical and hematological manifestations of brucellosis and pancytopenia in a large series: Hematological malignancies, the unusual cause of pancytopenia in patients with brucellosis.

The aim of the study is to review the clinical manifestations and the hematological findings of brucellosis and pancytopenia, with or without hematological malignancies. The records of 202 patients with brucellosis were evaluated retrospectively. Among these cases of brucellosis seen in a 6 year period between April 1999 and June 2005, 30 patients with pancytopenia were identified. The most common manifestation was fever, followed by weight loss, anorexia, malaise, arthralgia, and hepatosplenomegaly. Bone marrow biopsies revealed hypercellularity or normocellularity. The most common findings in the bone marrow evaluation were histiocytic hemophagocytosis and granulomas. Among all cases, we diagnosed 5 hematological malignancies (1 acute myelogenous leukemia, 2 acute lymphoblastic leukemia, and 2 multiple myeloma) concurrently with brucellosis. The clinical symptoms and findings were similar in patients with and without malignancies. In cases with malignancies, the bone marrow biopsy revealed predominant primary disease involvement. Significant increases in ESR and CRP, severe anemia and thrombocytopenia were observed in patients with malignancies. Peripheral blood counts in patients without malignancies returned to normal after antibiotic treatment for brucellosis. However, pancytopenia in two patients with malignancies did not recover because of primary resistant disease. We conclude that while histiocytic hemophagocytosis may be considered as a major cause of pancytopenia, leukemic infiltration can also be an extreme and unusual cause of pancytopenia in patients in whom brucellosis was concurrently diagnosed with hematological malignancies.

Effect of ionizing radiation on cellular procoagulability and co-ordinated gene alterations.

BACKGROUND AND OBJECTIVES: Ionizing radiation (IR) is associated with thrombotic vascular occlusion predicting a poor clinical outcome. Our study examined whether IR induced tissue factor (TF) expression and procoagulability. We further investigated coordinated gene alterations associated with TF upregulation in the myelomonocytic leukemia THP-1 cells. DESIGN AND METHODS: TF expression was determined by quantitative Reverse Transcriptase (TaqMan) PCR, TF ELISA and TF activity by a two stage chromogenic assay in the time course of days 1, 3, 7, 10, and 17 post IR. To detect IR-induced alterations in gene expression, Affymetrix HG U133 Plus 2.0 microarrays were used. RESULTS IR induced a significant increase in TF/GAPDH mRNA ratios and cellular TF protein on days 3 and 7 post IR (20 Gy [p>or=0.01] and 40 Gy [p or=0.001] vs. control respectively), suggesting IR immediately alters the cellular thrombogenicity. TF upregulation post IR was confirmed in PBMNCs. Gene expression profiling showed IR increased the expression of inflammatory and apoptosis-related pathways known to be involved in the regulation of TF expression. INTERPRETATION AND CONCLUSIONS: TF upregulation together with inflammation and apoptosis may increase the thrombogenicity of tissues. The demonstrated upregulation of TF might play a pivotal role in radiation associated thrombosis.

(-)-Menthol inhibits WEHI-3 leukemia cells in vitro and in vivo.

(-)-Menthol ([1-alpha]-5-methyl-2-[1-methylethyl]-cyclohexanol), is a widely used flavoring ingredient in mouthwash, foods, toothpaste and cigarettes. The studies reported here revealed that (-)-menthol induced cytotoxicity against murine leukemia WEHI-3 cells in vitro in a dose-dependent manner. The effects of (-)-menthol on WEHI-3 cells in vivo (BALBIc mice) were also examined, and it was observed that the Mac-3 and CD11b markers were decreased, indicating inhibition of differentiation of the precursor of macrophage and granulocyte. The weights of liver and spleen samples from mice treated with (-)-menthol were found to be decreased compared to untreated animals.

[Haematological characteristics, FAB and WHO classification of 153 cases of myeloid acute leukaemia in Tunisia]. / Caractéristiques hématologiques et classification FAB et OMS de 153 cas de leucémies aiguës myéloïdes en Tunisie.

A complete blood analysis with a careful morphologic examination of peripheral blood and bone morrow smears completed by cytochemical reaction will help to classify the most acute myeloid leukaemia (AML). Actually, the study of other cytogenetis and immunophenotypic markers are now necessary to confirm diagnosis. The World Health Organisation WHO classification (2001) incorporates theses approaches. The purpose of this study is a bio-clinical review according to the WHO recommendations in 153 cases of LAM diagnosed between January 1998 and December 2003. The patients were aged 2 months to 90 years with sex ratio (M/F) of 1,22. The morphologic conclusion was difficult in 12% cases. Presence of dysplasia is noted in 50% of cases with multilineage dysplasia in 42% of cases. Our results showed cloned chromosomal abnormalities in 57% of cases (t(8;21): 12%, t(15;17) : 10%, Inv16: 1,3%, 11q23: 2,6% et complex karyotype: 14,3%). In 69% of cases with multilineage dysplasia, the karyotype was normal. 3 cases of LAM were noted at patients treated for breast cancer with chirurgic chemotherapy and radiotherapy 3, 4 et 5 years after treatment (LAM3 with t(15;17), LAM4 with genetic abnormalities of chromosomes 3, 5, 7, 8, 9, 14 et 16 et LAM 6 with genetic abnormalities of chromosomes 4, 7, 12, 14, 19 et 21). In WHO classification, cytology is essential in diagnosis of LAM even if the karytype have an important prognostic value. Research of signs of dysplasia lineage after lineage constitutes an important microscopic work and it is difficult to quantify dysplasia when the lineage is poor.

Correlation between expression of CD56/NCAM and severe leukostasis in hyperleukocytic acute myelomonocytic leukaemia.

OBJECTIVE: The possible contribution of surface molecules to the development of leukostasis syndrome in hyperleukocytic acute myeloid leukaemia (AML) was assessed by routine immunophenotyping and grading of the probability of clinical leukostasis. METHODS: Fifty-three patients (23 women, 30 men, median age 59 yr) with hyperleukocytic AML [white blood count (WBC) above 50 x 10(9)/L] were graded for the probability of clinical leukostasis according to the severity of neurologic, pulmonary and other symptoms possibly caused by leukostasis using a recently published scoring system. Age, WBC, absolute blast count, haemoglobin, cytogenetic risk group, infection, relative CD56 expression and absolute count of CD56 positive blasts were analyzed in multivariate stepwise backward logistic regression analysis. RESULTS: In patients with acute monocytic leukaemia (AML M4/M5) the absolute count of leukaemic blasts expressing CD56/NCAM was highly associated with the development of symptoms graded as highly probable leukostasis and all three patients succumbing to early death were CD56 positive. Only the absolute count of CD56 positive blasts was a significant predictor of risk of severe leukostasis (P = 0.020). This was not found in AML without monocytic involvement (AML M1, M2, M3v). CONCLUSIONS: The expression of CD56/NCAM, a surface marker used in routine immunophenotyping of AML, may help to predict severe and potentially fatal leukostasis in hyperleukocytic acute myelomonocytic leukaemia. These results emphasize the usefulness of this four-stage clinical grading scale for analysing the factors, which lead to severe leukostasis in hyperleukocytic patients. We extend previous findings that the mechanisms of leukostasis are different depending on the involvement of the monocytic lineage.

Cooperative antitumor effects of vitamin D3 derivatives and rosemary preparations in a mouse model of myeloid leukemia.

1alpha,25-dihydroxyvitamin D(3) (1,25D(3)) is a powerful differentiation agent, which has potential for treatment of myeloid leukemias and other types of cancer, but the calcemia produced by pharmacologically active doses precludes the use of this agent in the clinic. We have shown that carnosic acid, the major rosemary polyphenol, enhances the differentiating and antiproliferative effects of low concentrations of 1,25D(3) in human myeloid leukemia cell lines (HL60, U937). Here we translated these findings to in vivo conditions using a syngeneic mouse leukemia tumor model. To this end, we first demonstrated that as in HL60 cells, differentiation of WEHI-3B D(-) murine myelomonocytic leukemia cells induced by 1 nM 1,25D(3) or its low-calcemic analog, 1,25-dihydroxy-16-ene-5,6-trans-cholecalciferol (Ro25-4020), can be synergistically potentiated by carnosic acid (10 microM) or the carnosic acid-rich ethanolic extract of rosemary leaves. This effect was accompanied by cell cycle arrest in G0 + G1 phase and a marked inhibition of cell growth. In the in vivo studies, i.p. injections of 2 microg Ro25-4020 in Balb/c mice bearing WEHI-3B D(-) tumors produced a significant delay in tumor appearance and reduction in tumor size, without significant toxicity. Another analog, 1,25-dihydroxy-16,23Z-diene-20-epi-26,27-hexafluoro-19-nor-cholecalciferol (Ro26-3884) administered at the same dose was less effective than Ro25-4020 and profoundly toxic. Importantly, combined treatment with 1% dry rosemary extract (mixed with food) and 1 microg Ro25-4020 resulted in a strong cooperative antitumor effect, without inducing hypercalcemia. These results indicate for the first time that a plant polyphenolic preparation and a vitamin D derivative can cooperate not only in inducing leukemia cell differentiation in vitro, but also in the antileukemic activity in vivo. These data may suggest novel protocols for chemoprevention or differentiation therapy of myeloid leukemia.

An in vitro study on the mechanisms of coagulation activation in acute myelogenous leukemia (AML): role of tissue factor regulation by cytotoxic drugs and GM-CSF.

AML patients may suffer from a disseminated coagulopathy, which can aggravate a pre-existing bleeding tendency due to thrombocytopenia and platelet dysfunction. The cellular and molecular mechanisms underlying this coagulopathy, however, are not completely understood. Indeed, the broad and increasing therapeutic use of cytotoxic drugs and growth factors is likely to contribute to the complexity of hemostatic abnormalities encountered in this hematologic malignancy. The nature of coagulation activation in AML was therefore investigated in vitro using the human leukemic cell line, HL60. Tissue factor (TF) was almost entirely located on the cell surface and bound factor VIIa, but only 15-25% of this TF was primarily functionally active. Treatment with increasing concentrations of daunorubicin or cytosine-beta-D-arabinofuranoside, two cytotoxic drugs commonly used in AML therapy, induced apoptosis and secondary necrosis of HL60 cells and resulted in marked decryption of TF PCA independent of de novo protein synthesis. This PCA-modulating effect was concomitant with and functionally dependent on the exposure of phosphatidylserine on the outer membrane leaflet. Similar observations were made in analogous ex vivo studies on patient-derived myeloblasts. Incubation of HL60 cells with GM-CSF, a cytokine expressed in the bone marrow microenvironment and used as an adjunct to AML treatment, evoked a cellular response, which included both enhanced TF production and release of VEGF-A and uPA into the culture medium. We conclude that both decryption of pre-formed TF PCA by chemotherapeutic drugs and de novo induction of TF by cytokines such as GM-CSF can regulate the pro-coagulant phenotype of HL60 cells in vitro.

Genome-wide analysis of acute myeloid leukemia with normal karyotype reveals a unique pattern of homeobox gene expression distinct from those with translocation-mediated fusion events.

Gene expression profiles were determined from presentation peripheral blood and bone marrow samples of 28 patients with acute myeloid leukemia (AML). Hierarchical clustering sorted the profiles into separate groups, each representing one of the major cytogenetic classes in AML [i.e., t(8;21), t(15;17), inv(16), 11q23, and normal karyotype]. Statistical group comparison identified genes whose expression was strongly correlated with these chromosomal classes. Moreover, the normal karyotype AMLs were characterized by distinctive up-regulation of certain members of the class I homeobox A and B gene families, implying a common underlying genetic lesion. These data reveal novel diagnostic and therapeutic targets and demonstrate the potential of microarray-based dissection of AML.

Transient hematologic and clinical effect of E21R in a child with end-stage juvenile myelomonocytic leukemia.

E21R is a modified granulocyte macrophage-colony-stimulating factor (GM-CSF) protein which results in antagonism of GM-CSF function via selective binding to the GM-CSF receptor complex. Juvenile chronic myelomonocytic leukemia (JMML) is a rare leukemia where spontaneous proliferation of myeloid and monocytic precursors in patients' bone marrow cultures is dependent on GM-CSF. For patients who progress after systemic chemotherapy, there are no effective therapies. In vitro and in vivo studies in an animal model demonstrating that E21R exerts an antileukemic action prompted us to consider its potential utility in a child with end-stage JMML. E21R was well-tolerated during the 3 courses of subcutaneous treatment. A clear in vivo efficacy was observed after 2 courses of E21R but the disease appeared completely refractory during the third course. This novel therapeutic approach clearly deserves further evaluation in JMML.

Identification of the hemoglobin scavenger receptor/CD163 as a natural soluble protein in plasma.

The hemoglobin scavenger receptor (HbSR/CD163) is an interleukin-6- and glucocorticoid-regulated macrophage/monocyte receptor for uptake of haptoglobin-hemoglobin complexes. Moreover, there are strong indications that HbSR serves an anti-inflammatory function. Immunoprecipitation and immunoblotting enabled identification of a soluble plasma form of HbSR (sHbSR) having an electrophoretic mobility equal to that of recombinant HbSR consisting of the extracellular domain (scavenger receptor cysteine-rich 1-9). A sandwich enzyme-linked immunosorbent assay was established and used to measure the sHbSR level in 130 healthy subjects (median, 1.87 mg/L; range, 0.73-4.69 mg/L). To evaluate the sHbSR levels in conditions with increased leukocyte stimulation and proliferation, 140 patients admitted to a hematological department were screened. Several patients, with a broad spectrum of diagnoses, had a level of sHbSR above the range of healthy persons. Patients with myelomonocytic leukemias and pneumonia/sepsis exhibited the highest levels (up to 67.3 mg/L). In conclusion, sHbSR is an abundant plasma protein potentially valuable in monitoring patients with infections and myelomonocytic leukemia.