Biphenotypic Acute Leukemia Research Articles

Prognostic Implications of the Immunophenotype in Biphenotypic Acute Leukemia.

Biphenotypic acute leukemia (BAL) is a rare yet defined type of acute leukemia. We investigated the incidence, clinicopathologic characteristics, and clinical outcomes of BAL in Korean adults. The present study retrospectively analyzed clinicopathological and clinical data from 43 adult patients with BAL, defined using the EGIL scoring system, from 11 Korean institutes. The incidence of BAL was 2.1% among acute leukemias; 2.3% in male and 1.9% in female. Median age of 43 BAL patients, 25 males and 18 females, was 38 years (range, 16–74). The immunophenotype was myeloid/B-lymphoid (M+B) in 31 (72.1%), myeloid/T-lymphoid (M+T) in 10 (23.3%), and B/T-lymphoid (B+T) and myeloid/B/T-lymphoid (M+B+T) in one (2.3%) each. 37 patients had the results of cytogenetic analyses and Ph chromosome (n=14, 37.8%) was the single most common abnormal finding. Intensive induction chemotherapy was given in 36 of 43 patients: AML-type in 13 (36.1%), ALL-type in 8 (22.2%), and combined type in 15 (41.7%). Complete remission (CR) was induced in 29 patients (80.6%). The CR rate was significantly lower in M+T (5 of 9, 55.6%) than M+B (22 of 25, 88.0%) (P=0.039). Other unfavorable factors for CR were absence of CD19 (P=0.029) or CD20 (P=0.046), and presence of CD2 (P=0.040). The CR rates were not significantly different according to cytogenetic finding or type of induction chemotherapy. After median follow-up duration of 712 days among surviving patients, 11 patients relapsed with median relapse-free survival (RFS) of 3.08 years and 4-y RFS of 38.3% and 18 patients died with median overall survival (OS) of 2.49 years and 4-y OS of 30.1%. Multivariate analysis showed that high leukocyte counts (≥ 30,000/μl) at diagnosis (OR, 5.922; 95% CI, 1.379–25.429; P=0.017) was an independent unfavorable prognostic factor for RFS, and high leukocyte counts at diagnosis (OR, 9.348; 95% CI, 3.014–28.993; P<0.001) and M+T phenotype (OR, 3.259; 95% CI, 1.136–9.344; P=0.028) were independent unfavorable prognostic factors for OS. In summary, BAL was found in 2.1% of adult acute leukemias and M+T phenotype showed significantly poorer response to induction chemotherapy and inferior overall survival compared to M+B phenotype.

Identification of a Subgroup of Biphenotypic Myeloid/T-Cell Acute Leukemias with a CEBPA-Mutant Gene Expression Profile, Frequent CEBPA Promoter Hypermethylation and NOTCH1 Mutations.

Recently, gene expression profiling has allowed the identification of specific molecular signatures of established as well as novel subgroups of acute myeloid leukemia (AML). CEBPA mutations are observed in ~10% of AML. In a gene expression profiling study of 285 AML cases, we previously found two distinct clusters consisting of almost all specimens with CEBPA mutations, i.e. cluster #15 and cluster #4. In cluster #4, only 9 out of 15 cases were CEBPA mutant. Thus, this cluster contained 6 patients with a highly similar expression signature, but without CEBPA mutations. We wished to investigate whether these cases represent a previously unidentified subgroup of leukemias. All 6 cases appeared morphologically immature (FAB-M0 or M1) and did not carry any of the common genetic aberrations. Supervised array expression analysis revealed CEBPA to be among the most highly downregulated genes in these 6 cases, which was associated with CEBPA CpG promoter hypermethylation in 4/6. None of the CEBPA mutant cases or a random selection of 10 AML cases from other clusters exhibited CEBPA promoter methylation. Strikingly, several T-cell related genes, e.g. CD7, CD3D, IL7R and T-cell receptor delta locus, appeared significantly overexpressed in these cases. Multicolor flowcytometry demonstrated high expression of CD34 and combinations of myeloid (e.g. CD13, CD33, CD117 and/or myeloperoxidase) as well as T-lymphoid (CD7, CD3, TdT) lineage markers on all six. Expression of T-cell markers correlated with T-cell receptor rearrangements in 4/6 cases. Other highly expressed genes in these biphenotypic acute leukemias included NOTCH1 and its recently identified target gene TRIB2. Sequencing analysis demonstrated putatively activating NOTCH1 mutations in three out of six samples, as described previously in T-cell acute lymphoblastic leukemia (T-ALL). The remaining 3 cases and 25 control AMLs, including all CEBPA mutant cases, did not demonstrate any NOTCH1 mutation. Finally, based on this first cohort of patients, we determined the minimal number of probe sets accurately predicting cluster #4 biphenotypic cases, and identified eight probe sets, including those for T-cell receptor delta/alpha locus. Applying this predictor set to an independent new cohort of 272 AML cases, an identical subgroup of another 6 AML patients with frequent NOTCH1 mutations and CEBPA promoter hypermethylation could be identified. Available clinical data suggested an adverse prognosis of this type of leukemia. In conclusion, we identified a subgroup of biphenotypic acute leukemias characterized by a CEBPA mutant gene expression profile, frequent CEBPA promoter hypermethylation, and frequent NOTCH1 mutations. To our knowledge, this is the first report to describe NOTCH1 mutations in leukemia other than T-ALL.

Malignant Cells in the Marrow and Blood of Patients with Hematopoietic Malignancies Have Higher Mitochondrial Membrane Potential as Measured with the Potentiometric Dye JC-1.

Background: Immunophenotyping is an important method to define hematopoietic malignancies, but used alone, its ability to provide functional information about the malignant cells is limited. JC-1 is a fluorescent dye used to measure mitochondrial membrane potential in cells. We have developed a flow cytometric assay using JC-1 in conjunction with standard cell surface markers to characterize clinical samples from patients with hematologic malignancies.Methods: Ficoll-purified cells derived from the blood or marrow were stained with JC-1, and analyzed at 530 nm and 590nm to assess mitochondrial mass and mitochondrial membrane potential, respectively. Malignant and normal cell populations were assigned and gated by analysis of forward scatter and side scatter. Gating in this manner identified energetically discrete cell populations confirmed by back gating analysis.Results: To date we have measured the mitochondrial membrane potential from the bone marrow or peripheral blood of 3 normal individuals and 22 patients with acute and chronic leukemias and lymphoma involving the marrow. Samples have been analyzed from AML (6), ALL (3), CML- chronic phase (2), CLL (5), mantle cell lymphoma (3), diffuse large B-cell lymphoma (1) and acute biphenotypic leukemia (2). Compared with samples derived from normal individuals, malignant cells have a different energetic signature, and in nearly all instances, a substantially higher mitochondrial membrane potential. The mitochondrial membrane potential of malignant cells was 5.5 times higher (range 2.15–12) than normal cells within the same samples in 19 of 20 specimens (given the overlap in cell populations, CML samples were excluded and 1 CLL sample had a mitochondrial membrane potential that was the same as the normal population). Mitochondrial membrane potential is also higher in malignant cells compared with peripheral blood and marrow cells from normal individuals. While cells derived from patients with acute leukemias appear to be energetically homogeneous, cells from both patients with CML and 4 of 5 patients with CLL had at least 2 energetically discrete subpopulations despite being cytogenetically homogeneous (in the case of the CML samples) or immunophenotypically homogenous (in the case of the CLL samples).Conclusions: JC-1 staining can be performed reliably in concert with immunocytochemistry and provides insight into the metabolism of malignant hematopoietic cells. Preliminary data indicate that malignant cells from patients with leukemia and lymphoma have a high mitochondrial membrane potential which may reflect altered oxidative metabolism. This may serve as a metabolic signature for monitoring minimal residual disease or assessing the effect of therapeutic agents on functional status of malignant populations.

28 ORAL Imidazole derivatives with vascular disrupting activity

The FMS-like tyrosine kinase 3 (FLT3) is a cell surface receptor tyrosine kinase. Activating mutations of this gene occur in nearly 30% of acute myelogenous leukemia (AML) patients. These mutations, in part, result in activation of mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways. In this study, we found that AZD6244 (ARRY-142886), a novel inhibitor of MEK1/2 kinases, effectively inhibited the proliferation of acute biphenotypic leukemia MV4-11 and acute monocytic leukemia MOLM13 cells. The concentrations that inhibited 50% growth were approximately 0.3 and 1.2 μM, respectively, as measured by thymidine uptake on day 2 of culture. AZD6244 potently down-regulated the levels of phospho-ERK1/2 and its downstream effector, p-p70S6K, in the MV4-11 and MOLM13 cells as measured by Western blot analysis. Interestingly, when AZD6244 was combined with sunitinib, a FLT3 kinase inhibitor, growth inhibition and apoptosis of both MV4-11 and MOLM13 cells were synergistically enhanced in association with further down-regulation of phospho-ERK1/2 and p-p70S6K in these cells. Taken together, concomitant blockade of FLT3 and MEK signaling represents a promising treatment strategy for individuals with leukemia who possess activating mutations of FLT3.

Biphenotypic Acute Leukemia withBCR-ABLmRNA Transcript b3a2 Type: A Case Report with Review of the Literature

Biphenotypic acute leukemia (BAL) is a subtype of leukemia of ambiguous lineage in the World Health Organization classification system. About one third of the cases have the Philadelphia chromosome, and some cases are associated with other structural abnormalities involving 11q23. BAL is known to have a poor prognosis in both children and adults. According to the previously reported BAL cases with positive BCR-ABL fusion gene, most of the BCR-ABL mRNA transcript type was e1a2. So, we describe here a 30-year-old adult BAL case with the karyotype 46,XY,t(9;22)(q34;q11.2) resulting in a very rare b3a2 type of BCR-ABL mRNA transcript.

Diagnóstico laboratorial das leucemias mielóides agudas

As leucemias agudas caracterizam-se pela proliferação clonal e pelo bloqueio maturativo das células hematopoéticas, com substituição difusa da medula óssea por células neoplásicas. A leucemia mielóide aguda (LMA) é um grupo heterogêneo de doenças clonais do tecido hematopoético, que acomete predominantemente idosos acima de 60 anos de idade. A LMA apresenta oito subtipos distintos morfologicamente: LMA M0 a M7. Os métodos diagnósticos para identificação da LMA e classificação dos subtipos são baseados em critérios morfológicos, citoquímicos e de imunofenotipagem, acrescidos de análise genética. Além de ser importante para a diferenciação do tipo da linhagem da leucemia, se mielóide (LMA) ou linfóide (LLA), o diagnóstico é também de grande importância para identificar a leucemia bifenotípica aguda (BAL). O objetivo deste trabalho foi realizar uma revisão bibliográfica sobre LMA, dando ênfase aos métodos laboratoriais utilizados para a sua identificação e diferenciação.

Methylenetetrahydrofolate reductase A1298C genotypes are associated with the risks of acute lymphoblastic leukaemia and chronic myelogenous leukaemia in the Korean population

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme involved in folate metabolism, DNA methylation and synthesis. We investigated the association between MTHFR polymorphisms and the risks of acute and chronic leukaemias. MTHFR C677T and A1298C were genotyped in 396 Korean individuals using multiplex polymerase chain reaction/restriction fragment-length polymorphism. They were acute lymphoblastic leukaemia (ALL, n = 89), acute myeloid leukaemia (AML, n = 55), biphenotypic acute leukaemia (n = 12), chronic myelogenous leukaemia (CML, n = 40), and normal controls (n = 200). C677T genotypes were not associated with the risk of each disease. A1298C variants, however, significantly decreased the risks of ALL and CML compared with 1298AA. Odds ratios and 95% confidence intervals of 1298AC and 1298AC + CC were 0.53 (0.31-0.93) and 0.54 (0.31-0.93) in ALL, and 0.34 (0.14-0.80) and 0.40 (0.18-0.89) in CML, respectively, compared with 1298AA. These findings demonstrate that the development of ALL and CML is more dependent on folate status, and more susceptible to DNA instability than that of AML. In addition, A1298C rather than C677T may be a more important genetic risk modifier in leukaemogenesis at least in the Korean population.

Early Stem Cell Transplantation for Refractory Acute Leukemia after Salvage Therapy with High-Dose Etoposide and Cyclophosphamide

Primary refractory acute leukemia (AL) has a poor prognosis, although some patients can be salvaged with allogeneic stem cell transplantation (SCT). Induction of complete remission (CR) with conventional chemotherapy before SCT may improve outcome in this patient population. Between March 1991 and October 2003, 59 adults with primary refractory AL were treated with continuous-infusion etoposide (VP) 2.4 to 3.0 g/m 2 followed by cyclophosphamide (Cy) 6.0-7.2 g/m 2 intravenously over 3 to 4 days with the intention of proceeding to SCT in CR1. Forty-two patients had acute myelogenous leukemia (AML), 13 patients had acute lymphoblastic leukemia (ALL), and 4 patients had acute biphenotypic leukemia. The most frequent nonhematologic toxicities were oral mucosal, gastrointestinal, and hepatic toxicities (44%, 20%, and 15% of patients, respectively). Thirty-two (57%) of 56 evaluable patients entered CR1 with a median time to platelet and neutrophil recovery of 22 and 26 days, respectively. CR1 rates were similar in AML (54%) and ALL/acute biphenotypic leukemia (67%; P = .52), and analysis of baseline characteristics did not reveal any predictors of response to VP/Cy. Twenty-nine of 32 CR1 patients subsequently underwent SCT (24 allogeneic and 5 autologous). Estimated 5-year event-free survival (EFS) and overall survival for the entire cohort are 23% and 26%, respectively. In the allogeneic SCT group, 5-year EFS was 52% for AML patients and 14% for ALL patients ( P = .04), and only male sex was predictive of a favorable outcome ( P = .03). VP/Cy is able to induce CR1 in most patients with primary refractory AL with an acceptable toxicity profile. Subsequent allogeneic SCT can lead to long-term EFS in a significant proportion of patients.

Cytogenetics, molecular and ultrastructural characteristics of biphenotypic acute leukemia identified by the EGIL scoring system

Biphenotypic acute leukemia (BAL) is a rare, difficult to diagnose entity. Its identification is important for risk stratification in acute leukemia (AL). The scoring proposal of the European Group for the Classification of Acute Leukemia (EGIL) is useful for this purpose, but its performance against objective benchmarks is unclear. Using the EGIL system, we identified 23 (3.4%) BAL from among 676 newly diagnosed AL patients. Mixed, small and large blast cells predominated, with FAB M2 and L1 constituting the majority. All patients were positive for myeloid (M) markers and either B cell (B) (17 or 74%) or T cell (T) (8 or 34%) markers with two exceptional patients demonstrating trilineage phenotype. Six (50%) of studied M-B cases were positive for both IGH and TCR. In six (26%) patients myeloid lineage commitment was also demonstrable by electron cytochemistry. Abnormal findings were present in 19 (83%) patients by cytogenetics/FISH/molecular analysis as follows: t(9;22) (17%); MLL gene rearrangement (26%); deletion(6q) (13%); 12p11.2 (9%); numerical abnormalities (13%), and three (13%) new, previously unreported translocations t(X;6)(p22.3;q21); t(2;6)(q37;p21.3); and t(8;14)(p21;q32). In conclusion, the EGIL criteria for BAL appear robust when compared against molecular techniques that, if applied routinely, could aid in detecting BAL and help in risk stratification.

Acute biphenotypic leukemia arising in a patient with essential thrombocythemia

Acute leukemia is an uncommon complication of patients with essential thrombocythemia (ET). We describe a patient with ET, who transformed to acute biphenotypic leukemia 4 and 1/2 years after initial ET diagnosis. She had received hydroxyurea, anagrelide, and interferon, in different combinations and varying doses, before leukemic transformation. Acute biphenotypic leukemia was confirmed on bone marrow studies and immunophenotyping. Complete remission (CR) was achieved with induction chemotherapy for acute leukemia. This was followed with consolidation chemotherapy and the patient has remained in CR 9 months after initial induction chemotherapy. To our knowledge, this is a rare event of acute biphenotypic leukemic transformation of a patient with ET.

IgH and TCRgamma Gene Rearrangements, Cyclin A1 and HOXA9 Gene Expression in Biphenotypic Acute Leukemias.

Biphenotypic acute leukemia (BAL) represents a rare type of acute leukemia (AL) with the reported incidence of 4.0–8.5% and poor response to treatment. In adult BAL, poor prognosis is related to unfavorable cytogenetics and P-glycoprotein over-expression (Legrand et al, Br J Haematol 1998; 100:147–55). It is presumed that better knowledge of its biological features could improve the classification and clinical management of this rare type of acute leukemia. Due to paucity of data regarding biological parameters of BAL, in this study we have analyzed the rearrangements of immunoglobulin heavy chain (IgH) and T-cell receptor g (TCRg) genes, expression of cyclin A1 (CycA1) and HOXA9 genes as well as in vitro growth of 10 de novo BALs and compared them with 15 AML and 15 ALL, respectively. The diagnosis of BAL was established according to EGIL criteria. BAL represented 4.3% of adult and 3.0% of pediatric patients with de novo AL referred to our institution during the 1999–2003 period. Of 10 BALs studied, lymphoid BAL (L2) was identified in 3 children, myeloid BAL (M1 and M5) was identified in 3 adults and one child, BAL of undifferentiated cytomorphology (AUL) in 2 adult patients, whereas one 5-month-old child presented with two separate blast cell populations (L1+M5b, bilineal BAL). With regard to immunophenotype, the group of BAL patients was also heterogeneous: 8/10 expressed the combination of myeloid and B-lymphoid antigens and 2/10 expressed myeloid and T-lymphoid antigens. The majority of BAL expressed CD34 (89%). Cytogenetic analysis was obtained in 7 BAL patients, of whom 3 showed chromosome 11 aberrations and 1 had Ph+ chromosome. Our results indicate that IgH and TCRγ gene rearrangements correlated well with lymphoid BAL morphology. Interestingly, the majority of lymphoid BAL showed oligoclonal amplification of antigen-receptor genes, indicating the coexistence of several malignant clones at the time of presentation. The expression of CycA1 correlated well with myeloid and undifferentiated BAL morphology. In contrast, the expression of HOXA9, a marker associated with myeloid cell lineage, showed no strong correlation with BAL morphology. BAL showed almost equal in vitro growth in three different serum-free culture conditions (no addition of GF, addition of IL-7 and FL or GM-CSF and FL), indicating that their growth was less dependent on the addition of exogenous GFs. Finally, in vitro growth of blasts during a 7-day culture showed autonomous cell growth in 3/10 AML and 3/8 myeloid BAL samples tested, but not in any of the AL with lymphoid features. Finally, further studies are needed to confirm these findings and to extend research to a broader spectrum of cell markers aiming to give a better definition of this rare group of AL.

A LDI-PCR Based Method Allows the Identification of Any MLL Rearrangement.

Chromosomal rearrangements of the MLL gene are responsible for 5–10% of all acute leukemias, biphenotypic leukemias and myelodysplastic syndromes. 5–10% of these MLL aberrations are therapy-related leukemias with the frequent fusion partners ELL, MLLT3, MLLT10 and MLLT2. The large number of known and unknown MLL fusions (>80) renders a precise diagnosis a demanding task. Even though all MLL rearrangements are associated with high risk acute leukemias, the outcome (favorable or poor) depends on the partner gene. Thus, the identification of MLL gene fusions is necessary for rapid clinical de-ci-sions resulting in specific therapy regimens. After their cytogenetic identification, only the most common MLL fusions MLLT2, MLLT3, MLLT1, MLLT4, ELL and MLLT10 (80%) are investigated by RT-PCR analysis, whereas infrequent or unknown MLL rearrangements (20%) are more or less excluded from further analysis. Therefore we established a LDI-PCR (long-distance inverse PCR) based method that uses small amounts of genomic DNA to determine any type of MLL associated chromosomal abnormality. With this diagnostic tool more than 400 prescreened und unscreened adult and pediatric samples (AML/ALL) from different European diagnostic centers have been analyzed. During this study more than 200 MLL rearrangements were characterized for their precise localization of genomic break-points. The identified MLL rearrangements consist of 25 different fusion genes including the following eight novel MLL partner genes: ACACA, ARGHEF17, BCL9L, MAML2, SELB, SMAP1, and TIRAP. The identified MLL aberrations are basically reciprocal translocations, but also deletions, inversions, insertions and interstitial duplications have been determined. Combining the data of our study and data retrieved from the literature, a total of 88 partner genes can now be annotated. 52 fusion partners (60%) have been characterized on the molecular level whereas at least 36 (40%) remain to be identified. These results demonstrate that this new diagnostic tool in combination with split-signal FISH is qualified for the rapid analysis of known as well as unknown MLL partner genes. Furthermore, the determined patient specific fusion sequences are useful for minimal residual disease (MRD) monitoring of MLL associated acute leukemias. The use of these MRD markers will contribute to improve the treatment and outcome of acute leukemia patients. A first prospective study was already initiated by the German ALL study group and verified the reliability of these genomic markers for MRD monitoring.

Use of Transmission Electron Microscopy in Diagnosis of Acute Leukemias: A Prospective Study of Fifty Cases.

Ultrastructural studies have contributed substantially to the understanding of the cellular morphology of the acute leukemias. We present here 50 cases (aged 3-55 years, M: F-32: 18) of acute leukemias, which were studied for morphology, conventional cyto chemistry, immunophenotyping, and transmission electron microscopy (TEM) including ultrastructural cytochemistry using myeloperoxidase (MPO) and platelet peroxidase activity. TEM morphology using ultrastructural cytochemistry of MPO helped diagnose three cases of acute myeloid leukemia with minimal myeloid differentiation (AML M0). The blasts showed large round nuclei, 1–2 nucleoli, chromatin with peripheral condensation and abundant mitochondria. Of total of 5 cases of acute promyelocytic leukemias (APML), all had strong Sudan Black, MPO, dual esterase positivity; one case was non-specific esterase positive and sensitive to fluoride. On TEM, this unusual case was identified to be microgranular variant of APML. TEM morphology and ultrastructural cytochemistry using platelet peroxidase helped diagnose 3 cases of AML M7 (acute megakaryocytic leukemia), 2 cases of acute biphenotypic leukemias and also in differentiating one case of acute proerythroblastic leukemia (AML M6b) from 3 cases of AML M6a or acute erythroleukemia. Thus, TEM is helpful in differentiating further the subgroups of AML-M5 and AML-M6, in identifying the microgranular variant of APML, and in confirming the diagnosis of AML-M0 and biphenotypic leukemia. Also, in cases with very hypercellular marrow and with associated myelofibrosis, where the bone marrow aspiration gives low cell count, TEM and ultrastructural cytochemistry are a valuable aid to arriving at a accurate diagnosis.Characteristics of acute leukemia cases (n=50)FAB SubtypeMorphology, Conventional Cytochemistry and ImmunophenotypingTransmission Electron MicroscopyAML-M03 (?)3AML-M1and M21111AML-M34, 1(?)5: Hypergranular-4, hypogranular-1AML-M455AML-M555a-4, 5b-1AML-M646a-3 (erythroleukemia), 6b-1AML-M733Biphenotypic2 (?)2ALL-L188ALL-L244(?) = doubt in diagnosis

Pediatric Acute Biphenotypic Leukemia (aBL): A Clinico-Pathologic Analysis of a Single Institution Experience.

Current classification of childhood leukemia relies on morphologic, immunophenotypic, and molecular characteristics. aBL is rare and poorly understood. We retrospectively evaluated 7 patients diagnosed with aBL according to European Group for Immunological Classification of Leukemias (EGIL) criteria(2 or more lineages with a score greater than 2). [Display omitted] Among 269 leukemia patients diagnosed in 2001–2004, 7 (2.6%) were classified as having aBL. Clinical characteristics/course and laboratory data are listed below in Tables1&2.TABLE 1:CLINICAL CHARACTERISTICS & COURSEPATIENTAGESEXRACEINITIAL WBCTREATMENTCOURSE112FAA5,600CCG1961-> CCG2891, HDARAC, HSCTIND. FAILURE; R1:CNS,BM R2:BM R3:CNS,BM DOD, 24M217FC81,600CCG2891R1:CNS,BM DOD, 10M32MC40,000CCG1961RER; CR1, 16M42MH5,630CCG1991-> CCG1961, HSCTSER; CR1, 14M56FH38,000CCG1961RER; CR1, 12M616MAA150,000CCG1961-> AAML03P1IND. FAILURE; R1:BM DOD, 9M711MC77,100AAML03P1, HSCTRER; CR1, 7MTable 2:MORPHOLOGY, IMMUNOPHENOTYPE & CYTOGENETICSPATIENTFAB MORPHOLOGYFLOW CYTOMETRYEGIL SCORE(T/B/M)CYTOGENETICS/FISH1*L1, M5CD3, CD7, pCD5, CD33,MPO3.5/0/36,XX,add(1)(p22);t(3;12)(p24;p13),del(6)(q21q22),del(8)(p21),-9,del(13)(q13q14), +222*L1, M2P1: CD19, CD22, CD33, Tdt P2: CD2,CD14,CD15, CD33, MPO2/3.5/446,XX,der(8)t(8;?;17)(q11.2;?;q21),der(9)t(8;9)(q21.2;p12),del(15)q21), der(17)t(15;?;17)(q21;?;q11.2)3L2P1: CD33, CD34 P2: CD13, CD15, CD10, CD19, CD220/4/2.546, XY, del12p-/TEL deletion4L1CD19,CD22, CD13, CD15, CD330/3/2.546, XY5L1, M5CD19, pCD20, CD22, CD13, CD15, pCD333.5/0/3.546, XY, del(5)(q15q33), dic(7;9)(p11.2;p12),inv(9)c,t(10,11)(p13;q21)6L2cCD3, pCD5, CD7, CD13, CD15, CD33,pCD1170/4/3.546, XX/Trisomy 107*L1, M5CD2,CD3,CD7, TdT, pCD22,CD13, CD15, pMPO, CD1174/1/4.546, XY* =Peripheral Blood5 patients had abnormal karyotypes and/or FISH studies; none had MLL rearrangements or t(9;22). Induction treatment consisted of AML-type therapy (CCG 2891 /COG AAML03P1) in 2 patients who achieved CR by day 28. Two patients received ALL-type therapy (CCG 1961) and entered CR by Day 28. Three patients required dose intensification due to induction failure: 2 switched from ALL to AML-type therapy. Post induction chemotherapy was given in 4 cases; allogeneic HSCT in 3 cases (2 in CR1; 1 in 2nd relapse). 2/4 and 2/3, respectively, remain alive in 1st CR after chemotherapy or HSCT. All 3 deaths were from relapse of aBL within 2 yrs from diagnosis & occured in adolescents with complex karyotypes. In our experience, pediatric aBL represents a high risk population, especially in adolescents. Intensive induction chemotherapy is necessary in the majority of cases to achieve CR, although it is unclear whether ALL or AML-type therapy is superior. MLL & t(9;22) may not be as common in pediatric aBL as previously described. Collaborative trials and biologic studies are clearly needed for better understanding and outcomes.

CD79a expression in acute myeloid leukemia t(8;21) and the importance of cytogenetics in the diagnosis of leukemias with immunophenotypic ambiguity

Acute leukemias that express antigens associated with more than one lineage have been classified as acute lymphocytic leukemia with myeloid markers, acute myeloid leukemia with lymphoid markers, or biphenotypic acute leukemia (BAL). Antibody to cytoplasmic CD79a has been recently introduced to flow cytometry. CD79a functions in and has a high degree of specificity for B-cell differentiation. It has only recently begun to be reported in biphenotypic acute leukemias. Cases of acute leukemia submitted to the flow cytometry laboratory were retrospectively reviewed beginning from the time analysis for cytoplasmic CD79a was added to leukemia and lymphoma panels. Among 89 cases of AML, 2 showed strong coexpression of CD79a. Both cases were differentiated FAB AML-M2 and demonstrated the t(8;21) with cytogenetics and the AML1/ETO rearrangement with fluorescence in situ hybridization (FISH). These are recurring abnormalities in FAB AML-M2. The immunophenotyping met proposed scoring criteria for a diagnosis of BAL. Nevertheless, the cytogenetic and FISH findings indicate that CD79a, despite its specificity for B-cell differentiation, represented the aberrant presence of a B-cell antigen in leukemias of distinct myeloid linage. It is doubtful that, in this setting, CD79a expression should be considered a manifestation of lineage ambiguity.

Pediatric Invasive Fungal Rhinosinusitis in Immunocompromised Children With Cancer

1) To determine the factors that predispose towards invasive fungal rhinosinusitis (FS) in immunocompromised children with cancer, and 2) to propose practice guidelines for management of pediatric FS in immunocompromised patients. Retrospective chart review of 9 patients who developed invasive FS compared to 8 patients who did not develop invasive FS during the study period. Presenting signs and symptoms, nasal endoscopic findings, radiographs, laboratory studies, histologic and microscopic samples, and outcomes were compared. Seventeen consecutive pediatric immunocompromised patients with hematologic and lymphoid neoplasms underwent nasal endoscopy and biopsy for possible FS. Nine patients were diagnosed with 10 episodes of FS; 1 patient developed FS with different organisms on 2 separate occasions separated by 6 months; 8 patients were not diagnosed with FS. Eight patients had acute myelogenous leukemia (AML); 6 patients had acute lymphoblastic leukemia (ALL); 1 patient had Burkitt's lymphoma, 1 patient had undifferentiated leukemia; and 1 patient had biphenotypic acute leukemia. All patients with FS had an absolute neutrophil count (ANC) 600 or less (range 0-600). All patients with FS had either persistent fevers or sinus symptoms (facial pain, nasal congestion, rhinorrhea). Sinus CT scans were abnormal in all patients with FS and without FS. Two patients with FS had maxillary sinus retention cysts. Operative endoscopic findings were helpful diagnostically when necrosis or ulceration was found. All patients in the non-FS group normalized their ANCs; 2 of the 9 patients in the FS group did not normalize their ANC. These 2 patients died from disseminated FS or from complications due to their immunosuppression. All patients with FS had either persistent fevers or symptoms localized to the sinuses (facial pain, nasal congestion, or rhinorrhea). Endoscopic examination was helpful when necrosis was detected. We recommend directed biopsies of suspicious lesions, the middle and inferior turbinate, in immunocompromised, neutropenic pediatric patients with cancer who present with either persistent fevers or localizing symptoms to the sinuses. We favor the use of "rush" biopsies over frozen sections because of the better-quality sections and ability to perform appropriate stains.

A young male with bone pain

A 38-year-old male presented with a 6-month history of pain in the right thigh associated with weight loss. His full blood count was normal, as were his biochemistry, immunology, autoimmune screen and prostate-specific antigen. Inflammatory markers were elevated. All preliminary radiological investigations were normal. Bone scan and magnetic resonance imaging were highly suggestive of a leukaemic process. Bone marrow biopsy and peripheral blood film confirmed the diagnosis of an acute biphenotypic leukaemia. This case report highlights the fact that bone pain associated with a normal peripheral blood count may be the presentation of an acute haematological disorder in both adults and children.

IgH and TCRγ gene rearrangements, cyclin A1 and HOXA9 gene expression in biphenotypic acute leukemias

In this study we investigated IgH and TCRγ gene rearrangements, cyclin A1 and HOXA9 gene expression as well as the in vitro growth of biphenotypic acute leukemia (BAL) blasts in relation to acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The aim of the study was to correlate BAL morphology and its biological parameters in order to get information that might be used for additional stratification of BAL. This rare form of AL was identified in a total of 10 patients, comprising 4.3% of adult and 3.0% of pediatric patients with de novo AL referred to our institution during the 1999–2003 period. Our results indicate that IgH and TCRγ gene rearrangements correlated well with lymphoid BAL morphology, whereas the expression of cyclin A1 correlated with myeloid and undifferentiated BAL morphology. Surprisingly, HOXA9 expression, a marker associated with myeloid cell lineage, showed no strong correlation with BAL morphology. Finally, in vitro growth of blasts during a 7-day culture showed autonomous cell growth in 3/10 AML and 3/8 myeloid BAL samples tested, but not in any of the AL with lymphoid features. Further studies are needed to confirm these findings and to extend research to a broader spectrum of cell markers.

Biphenotypic Acute Leukemia with t(15;17)

Biphenotypic acute leukemias (BAL) represent 5% of all acute leukemias. The most frequent cytogenetic abnormalities described are Philadelphia chromosome and 11q23 involvement. Here we report a BAL case, with blasts showing lymphoblast morphology and positivity for myeloperoxidase (in 6% of the blast cells). Immunophenotype revealed the compromise of myeloid and B-lymphoid lineages. Cytogenetic analysis showed the t(15;17) and 8 trisomy. PML/RARa rearrangement was detected by fluorescent in situ hybridization (FISH) on interphase nuclei while PML/RARa fusion transcript was detected in the bone marrow and peripheral blood by molecular biology studies (RT-PCR). This report describes a BAL case with an unfrequent cytogenetic abnormality, and highlights the importance of correlating the results of multiple diagnostic methods in order to establish a correct diagnosis and treatment in BAL patients.

Dimerization of MLL fusion proteins and FLT3 activation synergize to induce multiple-lineage leukemogenesis

The mechanisms by which mixed-lineage leukemia (MLL) fusion products resulting from in utero translocations in 11q23 contribute to leukemogenesis and infant acute leukemia remain elusive. It is still controversial whether the MLL fusion protein is sufficient to induce acute leukemia without additional genetic alterations, although carcinogenesis in general is known to result from more than 1 genetic disorder accumulating during a lifetime. Here we demonstrate that the fusion partner–mediated homo-oligomerization of MLL-SEPT6 is essential to immortalize hematopoietic progenitors in vitro. MLL-SEPT6 induced myeloproliferative disease with long latency in mice, but not acute leukemia, implying that secondary genotoxic events are required to develop leukemia. We developed in vitro and in vivo model systems of leukemogenesis by MLL fusion proteins, where activated FMS-like receptor tyrosine kinase 3 (FLT3) together with MLL-SEPT6 not only transformed hematopoietic progenitors in vitro but also induced acute biphenotypic or myeloid leukemia with short latency in vivo. In these systems, MLL-ENL, another type of the fusion product that seems to act as a monomer, also induced the transformation in vitro and leukemogenesis in vivo in concert with activated FLT3. These findings show direct evidence for a multistep leukemogenesis mediated by MLL fusion proteins and may be applicable to development of direct MLL fusion–targeted therapy.