Aberrations In Acute Leukemia Research Articles

A novel class of ZNF384 aberrations in acute leukemia

AbstractFusion of the ZNF384 gene as the 3' partner to several different 5' partner genes occurs recurrently in B-cell precursor acute lymphoblastic and mixed phenotype B/myeloid leukemia. These canonical fusions (ZNF384r) contain the complete ZNF384 coding sequence and are associated with a specific gene expression signature. Cases with this signature, but without canonical ZNF384 fusions (ZNF384r-like cases), have been described previously. Although some have been shown to harbor ZNF362 fusions, the primary aberrations remain unknown in a major proportion. We studied 3 patients with the ZNF384r signature and unknown primary genetic background and identified a previously unknown class of genetic aberration affecting the last exon of ZNF384 and resulting in disruption of the C-terminal portion of the ZNF384 protein. Importantly, in 2 cases, the ZNF384 aberration, indel, was missed during the bioinformatic analysis but revealed by the manual, targeted reanalysis. Two cases with the novel aberrations had a mixed (B/myeloid) immunophenotype commonly associated with canonical ZNF384 fusions. In conclusion, we present leukemia cases with a novel class of ZNF384 aberrations that phenocopy leukemia with ZNF384r. Therefore, we show that part of the so-called ZNF384r-like cases represent the same genetic subtype as leukemia with canonical ZNF384 fusions.

The Oncogenic Fusion Protein CBFB-SMMHC Downregulates CD48 to Evade NK Cell Recognition

Chromosomal translocations are a common cytogenetic aberration in acute leukemia, which frequently generate fusion proteins with oncogenic properties. We have previously studied the immune evasion mechanisms of two common oncogenic fusion proteins in acute myeloid leukemia (AML), PML-RARA and AML1-ETO. We investigated, in particular, the activity of natural killer (NK) cells, which are part of the innate immune system and play a significant role in eliminating leukemic cells. We found that both oncogenic fusion proteins downregulate the expression of CD48, a ligand of the NK cell activating receptor 2B4, to escape NK cell-mediated recognition of the tumor cells.In the current study we tested the possibility that other oncogenic fusion proteins are also able to evade NK cell recognition. To this aim we cloned several leukemic fusion proteins into lentiviral vectors and expressed them in U937 cells, which are commonly used to express leukemic fusion proteins. The leukemic fusion proteins included MLL-AF4, NUP98-HOXA9, DEK-NUP98 and CBFB-SMMHC. By using flow cytometry, we examined the effect of these fusion proteins on the expression of several NK cell ligands. CBFB-SMMHC was the only fusion protein which downregulated the expression of CD48.Next, we tested the functional significance of the downregulation of CD48 by CBFB-SMMHC by performing cytotoxicity assays with NK cells. We first used the NK cell line YTS eco since the cytotoxicity of these cells is mainly dependent on the 2B4-CD48 interaction. We found that cells which express CBFB-SMMHC were killed significantly less than the control cells, and that this reduced killing was due to CD48 downregulation. We also tested the killing of CBFB-SMMHC expressing cells by primary bulk NK cells and found that cells which express this fusion protein were killed significantly less.Mechanistically we show that overexpression of CBFB-SMMHC reduces the mRNA levels of CD48. Additionally, we tested whether treatment with HDAC inhibitors (HDACi) can reverse the downregulation of CD48 by CBFB-SMMHC. We treated cells which express CBFB-SMMHC with various HDACi and examined the effect of this treatment on the expression of CD48. Two specific class I HDACi, mocetinostat and entinostat, upregulated the expression of CD48 in cells which express this fusion protein.Taken together we show that modulation of CD48 expression is an immune evasion mechanism, which is specific to core binding factor (CBF) proteins (AML1-ETO and CBFB-SMMHC) and PML-RARA. Although the three fusion proteins which we found to down-regulate the expression of CD48 are associated with better prognosis in AML, about 30% of CBF-AML patients eventually relapse. Based on our findings we suggest that down-regulation of CD48 by these fusion proteins, which leads to NK cell immune evasion, contributes to the persistence of residual disease in these subtypes of leukemia and eventually to a clinical relapse. Finally, we propose that NK cell based therapies (i.e., NK cell infusion) or class I HDACi could be potential adjunctive therapies for CBF-AML or APL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CLINICOHEMATOLOGICAL, IMMUNOPHENOTYPING, MOLECULAR PROFILE, AND OVERALL SURVIVAL IMPACT IN ACUTE LYMPHOID LEUKEMIA PATIENTS FROM NORTH INDIA

Objective: Cytogenetic plays an inevitable role in predicting the diagnosis of acute leukemia. The recurrent chromosomal aberrations in acute leukemia have provided critical insights into the pathophysiological mechanism of leukemogenesis. Cytogenetics findings at diagnostics provide important information for decision-making in both childhood and adult acute lymphoblastic leukemia (ALL). The cure rate for ALL is >80% in children and 35% in adults. Despite the therapeutic advances in ALL, several important biological and pathophysiological questions remain to be answered to achieve an accurate diagnosis, timely prognosis, and maximum therapeutic benefit.
 Methods: The present study was carried out at tertiary care hospital, New Delhi, India. A total of 144 newly diagnosed ALL patients were analyzed for clinicohematological profile, immunophenotyping, conventional, and molecular cytogenetics.
 Results: The study population was found to have normal karyotypes in most of the cases; however, abnormalities also reported. Our study clearly indicates that the application of fluorescence in situ hybridization has increased sensitivity and accuracy for detecting various chromosomal abnormalities, more so with the cryptic rearrangements.
 Conclusion: We observed that the prevalence of the molecular subgroup of leukemia with a potential for a favorable clinical outcome (ETV6-RUNX1 and hyperdiploidy) in precursor B-ALL is higher in the North India.

Identification of CHD4 As a Potential Therapeutic Target of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by impaired myeloid differentiation of hematopoietic progenitors, causing uncontrolled proliferation and accumulation of immature myeloid cells in the bone marrow. Rearrangements of the mixed lineage leukemia (MLL) gene are common aberrations in acute leukemia and occur in over 70% in childhood leukemia and 5-10% in leukemia of adults. MLL rearrangements encode a fusion oncogenic H3K4 methytransferase protein, which is sufficient to transform hematopoietic cells and give rise to an aggressive subtype of AML. Leukemia where the MLL fusion oncogene is expressed is characterized by dismal prognosis and 30-60% of 5-years overall survival rate.The current standard treatment for AML is chemotherapy and in certain cases bone marrow transplantation. However, chemotherapy causes severe side effects on normal cells and an increased risk of relapse. Consequently, discovery of novel drug targets with better efficacy and low toxicity are needed to improve treatment of AML.In this study, we aimed to identify genes that are required for growth of AML cells and that encode proteins that potentially could be used as therapeutic targets. To do this, we performed high-throughput RNAi screening covering all annotated human genes and the homologous genes in mice, using barcoded lentiviral-based shRNA vectors. Stable loss-of-function screening was done in three AML cell lines (two human and one murine AML cell lines) as well as in a non-transformed hematopoietic control cell line. The candidate genes were selected based on that shRNA-mediated knockdown caused at least a 5-fold growth inhibition of leukemic cells and that the individual candidates were targeted by multiple shRNAs.The chromodomain Helicase DNA binding protein 4 (CHD4), a chromatin remodeler ATPase, displayed the most significant effect in reduced AML cell proliferation upon inhibition among the overlapping candidate genes in all three AML cell lines. CHD4 is a main subunit of the Nucleosome Remodeling Deacetylase (NuRD) complex and has been associated with epigenetic transcriptional repression. A recent study has shown that inhibition of CHD4 sensitized AML cells to genotoxic drugs by chromatin relaxation, which increases rate of double-stranded break (DSB) in leukemic cells.To verify whether CHD4 is exclusively essential for AML with MLL rearrangements, we inhibited CHD4 expression with two independent shRNAs in various AML cell lines with and without MLL translocations. In vitro monitoring of growth and viability indicated that knockdown of CHD4 efficiently suppressed growth in all tested cell lines, suggesting that CHD4 is required in general for growth of leukemic cells. To test the effect of CHD4 inhibition in normal hematopoiesis, we pursued knockdown of CHD4 and monitored effects in hematopoiesis using colony formation assays of human CD34+ cells. The results demonstrated that CHD4 knockdown had minor effects in colony formation as well as growth and survival of normal hematopoietic cells.Furthermore, to explore whether inhibition of CHD4 can prevent AML tumor growth and disease progression in vivo, we have generated a mouse model for AML. By transplanting AML cells transduced with shRNA against CHD4 into recipient mice, we showed that shRNA-mediated targeting of CHD4 not only significantly prolonged survival of AML transplanted mice but also in some cases completely rescued some mice from development of the disease. Collectively, these data suggested that CHD4 is required for AML maintenance in vivo.Next, to determine whether suppression of CHD4 can inhibit cell growth of different subpopulations and subtypes of AML, we performed loss of function studies of CHD4 on patient-derived AML cells ex vivo. Loss of CHD4 expression significantly decreased the frequency of leukemic initiating cells in different subtypes AML patient samples. In further in vivo studies using a xeno-tranplantation model for AML, we demonstrated that shRNA-mediated inhibition of CHD4 significantly reduced the frequency of leukemic cells in the marrow 6 weeks after transplantation.Taken together our results demonstrated the critical and selective role of CHD4 in propagation of patient-derived AML cells as well as in disease progression in mouse models for AML. We believe that CHD4 represents a novel potential therapeutic target that can be used to battle AML. DisclosuresNo relevant conflicts of interest to declare.

Cytogenetic Profile in 7209 Indian Patients with <i>de novo</i> Acute Leukemia: A Single Centre Study from India

Background: Cytogenetics is one of the most important diagnostic parameters in the classification of acute leukemia. Recurrent chromosomal aberrations in acute leukemia have provided insights into the molecular mechanism of leukemogenesis. The variable frequencies of recurrent cytogenetic markers due to ethical/racial differences have been reported from Western and some Asian countries. Objective: We report cytogenetic data of largest cohort of 7209 adult and pediatric patients with de novo acute leukemia (AL) to determine the prevalence of various cytogenetic sub groups and compare with the Western and Asian population. Material & Methods: The AL patients included 2609 AML (adult: 2042, pediatric: 567), 3708 B-cell-precursor (BCP)-ALL (adult: 1300, pediatric: 2408) and 892 cases of T-ALL (adult: 480, pediatric: 412). Cytogenetic studies included conventional karyotyping and FISH using panel of probes. Results: The incidence of t(8;21) was high, comparable to other Asian countries. In comparison to our series and Western population, t(15;17) was more prevalent in Chinese population. Cytogenetic profiling of BCP-ALL revealed low prevalence of ETV6/RUNX1 in ours as well as other Asian population. The MLL aberrations in BCP-ALL and TLX1 & TLX3 aberrations in T-ALL occurred less frequently in our series as compared with Western population. Conclusion: The present study with a large cohort showed the heterogeneity of AL that involved various factors, such as age, gender and prevalence of distinct cytogenetic subgroups. Our data in comparison with other population based studies revealed differential distribution of some cytogenetic sub-groups indicating geographic heterogeneity due to differential environmental exposure which probably influenced underlying genetic susceptibility.

Diagnostic Utility of Multiprobe Fluorescence in situ Hybridization Assay for Detecting Cytogenetic Aberrations in Acute Leukemia

BackgroundSpecific cytogenetic aberrations detected by conventional karyotyping or FISH play a major role in the diagnosis, prognosis, and treatment of patients with acute leukemia. The FISH technique enhances the capacity of conventional karyotyping to detect subtle chromosomal aberrations. Multiprobe FISH assay (Cytocell, UK) can hybridize multiple probes to a single slide, thereby increasing the detection rate of cytogenetic aberrations. This study aimed to evaluate multiprobe FISH in detecting cytogenetic abnormalities in acute leukemia.MethodsThirty newly diagnosed acute leukemia patients who attended the hematology clinic at Dong-A University Hospital from October 2008 to October 2012 were enrolled in the study. The multiprobe FISH results were compared with those of G-banding.ResultsMultiprobe FISH detected the chromosomal aberrations identified by G-banding, as well as additional aberrations in 6 of 30 (20.0%) cases, which included ETV6/RUNX1 translocation, p16 deletion, TP53 deletion, and IGH break-apart.ConclusionsThe multiprobe FISH assay was a more sensitive and reliable technique compared with G-banding. It was also more cost-effective and yielded faster results.

Down-regulation of the RUNX1-target gene NR4A3 contributes to hematopoiesis deregulation in familial platelet disorder/acute myelogenous leukemia

AbstractRUNX1 encodes a DNA-binding α subunit of the core-binding factor, a heterodimeric transcription factor. RUNX1 is a master regulatory gene in hematopoiesis and its disruption is one of the most common aberrations in acute leukemia. Inactivating or dominant-negative mutations in the RUNX1 gene have been also identified in pedigrees of familial platelet disorders with a variable propensity to develop acute myeloid leukemia (FPD/AML). We performed analysis of hematopoiesis from 2 FPD/AML pedigrees with 2 distinct RUNX1 germline mutations, that is, the R139X in a pedigree without AML and the R174Q mutation in a pedigree with AML. Both mutations induced a marked increase in the clonogenic potential of immature CD34+CD38− progenitors, with some self-renewal capacities observed only for R174Q mutation. This increased proliferation correlated with reduction in the expression of NR4A3, a gene previously implicated in leukemia development. We demonstrated that NR4A3 was a direct target of RUNX1 and that restoration of NR4A3 expression partially reduced the clonogenic potential of patient progenitors. We propose that the down-regulation of NR4A3 in RUNX1-mutated hematopoietic progenitors leads to an increase in the pool of cells susceptible to be hit by secondary leukemic genetic events.

T(12;17)(p11;q11) in AML

Sun G, Koeffler HP, Gale RP, Sparkes RS, Schreck RR. Use of conditioned media in cell culture can mask cytogenetic abnormalities in acute leukaemia. Cancer Genet Cytogenet 1990;46:107-113. UKCCG (United Kingdom Cancer Cytogenetics Group). Translocations involving 12p in acute myeloid leukemia: association with prior myelodysplasia and exposure to mutagenic agents. Genes Chromosom Cancer 1992;5:252254. Kerndrup GB, Kjeldsen E. Acute leukemia cytogenetics: an evaluation of combining G-band karyotyping with multi-color spectral karyotyping. Cancer Genet Cytogenet 2001;124:7-11. Brozek I, Babinska M, Kardaś I, Woźniak A, Balcerska A, Hellmann A, Limon J. Cytogenetic analysis and clinical significance of chromosome 7 aberrations in acute leukaemia. J Appl Genet 2003;44:401-412.

The Spectra of Chromosomal Aberrations In Acute Leukemia and Implications for the Development of Optimal Profiles of Leukemic Fusion Genes In Multiplex RT-PCR System.

AbstractAbstract 3362 Background:Cytogenetic aberrations have been identified as important prognostic parameters for acute leukemia, in addition to diagnosis and treatment. The aim of the current study was to investigate the spectrum of chromosomal abnormalities in East Asian (Korean) patients with acute leukemia. Based on the spectrum of genetic aberrations found in our study, we proposed a revised list of chromosomal aberrations for the development of optimal profiles of leukemic fusion genes in multiplex RT-PCR system. Patients and Method:We prospectively analyzed blood or bone marrow samples from 348 patients (260 adults, 88 children) with leukemia from 2006 to 2009 at the Chonnam National University Hwasun Hospital (Hwasun, Korea). Simultaneous analyses of conventional cytogenetics, FISH and a commercially available multiplex RT-PCR system (HemaVision) were performed. Result:Thirty-six types of chromosomal abnormalities were found in the total patient population. Twenty types of chromosomal abnormalities were detected in 42% of all patients by multiplex RT-PCR system and in 33% of all patients by conventional cytogenetics including FISH. Within the group of 213 AML patients, the common cytogenetic abnormalities were t(15;17)(q21;q22) (n=35), t(8;21)(q22;q22) (n=31), 11q23 abnormalities (n=7), inv(16)(p13;q22) (n=3), t(9;9)(q34:q34) (n=2), and the abnormalities t(9;22)(q34;q11), t(9;12)(q34;p13), t(3;21)(q26;q22), t(11;17)(q23;q21), t(12;22)(p13;q11) and t(16;21)(p11.2;q22.3) were each involved in one case. In the group of 104 ALL patients, the following fusion transcripts were detected: 21 t(9;22)(q34;q11), 15 t(12;21)(p13;q22), nine 11q23 abnormalities, four t(1;19)(q23;p13), with t(9;9)(q34:q34), t(9;12)(q34;p13) and del(1p34) each involved in one case. In addition, cryptic translocations were detected in 9% of patients with normal karyotypes or numerical aberrations by conventional cytogenetics. Among 53 patients showing negative results for multiplex RT-PCR, one child patient with early pre-B-ALL harbored MLL1/AF4 fusion genes and 5 patients had 3 types of clinically significant chromosomal aberrations such as t(3;3)(q21;q26.2), t(8;14)(p24.1;q32) and i(17)(q10). Conclusion:The current study demonstrated the spectrum and frequency of chromosomal abnormalities in Korean leukemia patients, which were slightly differed from previous studies including western and some Asian countries. These results may offer important implications in understanding characteristics of molecular pathophysiology, the development of a new RT-PCR system as well as revision of the current commercial available RT-PCR system. Disclosures:No relevant conflicts of interest to declare.

Clinical Utility of Fluorescence in-situ Hybridization Profile Test in Detecting Genetic Aberrations in Acute Leukemia

Cytogenetic abnormalities are one of the most reliable prognostic factors in acute leukemia. Combination of conventional chromosome analysis (CCA) and FISH provides higher sensitivity in detecting these genetic abnormalities, and it is effective to apply several FISH probes as a profile test. The objective of this study was to investigate the utility of FISH profile analyses in the initial diagnosis of acute leukemia. Two hundred and forty one de novo acute leukemia patients diagnosed from January, 2002 to November, 2007 were included. For acute lymphoblastic leukemia profile test, FISH probes for BCR/ABL, TEL/AML1, MLL gene rearrangement and CDKN2A deletion were used. For acute myeloid leukemia profile test, probes for AML1/ETO, MLL and CBFbeta gene rearrangement were used. The results of CCA and FISH profile tests were collected, and the positive rates were compared. ALL FISH profile tests revealed additional genetic aberrations not detected by chromosome analysis in 48.6% (67/138) of cases, including those with normal karyotypes or no mitotic cells (37%, 51/138). Among these 51 cases, TEL/AML1 abnormalities were detected in 44.3%, followed by the abnormal CDKN2A signal (24.6%) and hyperdiploidy (18.0%). AML FISH profile tests revealed additional genetic abnormalities in 7.8% (8/103) of cases. FISH analysis as a profile test detected additional genetic aberrations in a significant proportion of acute leukemia, and was effective especially in detecting cryptic translocations, submicroscopic deletions and complex karyotypes. Our study supports the need to incorporate FISH profile test at initial work up in acute leukemia.

Comparison of Clinical Outcomes of Patients with Relapsed Acute Promyelocytic Leukemia Induced with Arsenic Trioxide and Consolidated with Either an Autologous Stem Cell Transplant or an Arsenic Trioxide–Based Regimen

In patients with relapsed acute promyelocytic leukemia (APL), the best consolidation regimen following induction of remission with arsenic trioxide (ATO) remains to be defined. Since January 2000, 37 patients with relapsed APL were treated at our center. The median age was 34 years (range, 6-57 years), and there were 20 males (54.1%). The median duration of first remission was 20.3 months (range, 2.9-81.2 months). Relapse was treated with single-agent ATO in 22 patients (59.5%), ATO+ATRA in 5 patients (13.5%), and ATO+ATRA + anthracycline in 10 patients (27%). Thirty-three patients (89%) achieved molecular remission after induction and a consolidation course. Fourteen patients opted to undergo autologous stem cell transplantation (SCT), and the remaining 19 patients received monthly cycles of ATO as a single agent (n=13) or ATO+ATRA (n=6) for 6 months. At a median follow-up of 32 months, the 5-year Kaplan-Meier estimate of event-free survival (EFS) was 83.33% +/- 15.21% in those who underwent autologous SCT versus 34.45% +/- 11.24% in those who did not (P=.001; log-rank test). Following remission induction with ATO-based regimens in patients with relapsed APL, consolidation with autologous SCT is associated with a significantly superior clinical outcome compared with ATO- and ATO+ATRA-based maintenance regimens.

AML1/RUNX1, One of the Most Common Targets of Aberration in Acute Myeloid Leukemia as a Transcriptional Regulator of Vascular Endothelial Growth Factor (VEGFA).

Cellular VEGFA is an independent adverse prognostic factor related to worse outcome in AML and is significantly upregulated in leukemic blasts (de bont ES. et al., 2002). Transcriptional activation of the VEGFA promoter represents the core mechanism through which expression of VEGFA can be regulated, but is still not identified in AML (Carmeliet P. and Jaine RK., 2000; Kolch W. et al., 1995; Yancoupolus GD. et al., 2000). One of the most common targets of aberrations in acute leukemia is Runt domain transcription factor (AML1/RUNX1), due to point mutations (Roumier C. et al., 2003) or disruption of chromosome 21, which occurs in about 10%-15% of all de novo AML patients. In the current study, functional 5′ deletion analysis of the VEGFA promoter was performed to identify VEGFA promoter regions involved in VEGFA transcriptional regulation in AML. Loss of the region spanning −2274/−507 resulted in a significant increase of promoter activity in two AML cell lines, whereas the promoter activity dropped after deletion of the promoter region −286 to −52. Computer-assisted sequence analysis of the −2247/−507 fragment of the VEGFA promoter, revealed three perfect AML1/RUNX1-binding sites in the first three deletion regions. The loss of each AML1/RUNX1-binding site corresponded to an (further) increase in VEGFA promoter activity. siRNA mediated AML1/RUNX1 depletion caused a 24% to 36% increase in VEGFA mRNA expression in two different AML cell lines (HL-60 and TF-1), whereas VEGFA promoter activity was two-fold increased in HL-60 cells treated with AML1/RUNX1 siRNA compared to the non-silencing control siRNA. In addition, mutation of all three AML1/RUNX1 sites resulted in a 22 fold increase of VEGFA promoter activity in HL-60 cells. VEGFA mRNA expression was found to be higher in AML patients with a t(8;21) translocation compared to patients without this translocation. To investigate whether blocking of the fusion protein AML1-ETO, generated by the t(8;21) translocation, would have an effect on VEGFA mRNA we have used a siRNA against AML-ETO in Kasumi-1. siRNA mediated depletion of AML1-ETO caused a 40% decrease in VEGFA mRNA expression. AML-ETO has the potential to interact with AML1/RUNX1 co-factors such as histone deacetylase (HDAC) which results in a dominant negative effect on AML1/RUNX1 transcriptional regulation. To test whether AML1/RUNX1 transcription repression of VEGFA is histone deacetylase (HDAC) dependent HL-60 cells were treated with 200 nM Trichostatin A (TSA), a potent inhibitor of HDAC, this resulted in an 75% increase of luciferase activity after 24h. In contrast, TSA treatment of Kasumi-1 cells containing the AML-ETO fusion protein had no effect on VEGFA expression. These results indicate that VEGFA is transcriptionally regulated by AML1/RUNX1, one of the most common targets of aberrations in AML, through three AML1/RUNX1 repressor elements located in the promoter of VEGFA. Furthermore, prognostically unfavorable high VEGFA expression in AML is caused by a dominant negative effect of the fusion protein AML-ETO on AML1/RUNX1 mediated repression of VEGFA transcription. These results underscore the importance of AML1/RUNX1 aberrations in AML development and progression and better understanding the molecular basis for aberrant AML/RUNX1 signaling pathway may help design more effective treatment strategies.

Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia.

Prospective studies on the detection of minimal residual disease (MRD) in acute leukemia patients have shown that large-scale MRD studies are feasible and that clinically relevant MRD-based risk group classification can be achieved and can now be used for designing new treatment protocols. However, multicenter international treatment protocols with MRD-based stratification of treatment need careful standardization and quality control of the MRD techniques. This was the aim of the European BIOMED-1 Concerted Action 'Investigation of minimal residual disease in acute leukemia: international standardization and clinical evaluation' with participants of 14 laboratories in eight European countries (ES, NL, PT, IT, DE, FR, SE and AT). Standardization and quality control was performed for the three main types of MRD techniques, ie flow cytometric immunophenotyping, PCR analysis of antigen receptor genes, and RT-PCR analysis of well-defined chromosomal aberrations. This study focussed on the latter MRD technique. A total of nine well-defined chromosome aberrations with fusion gene transcripts were selected: t(1;19) with E2A-PBX1, t(4;11) with MLL-AF4, t(8;21) with AML1-ETO, t(9;22) with BCR-ABL p190 and BCR-ABL p210, t(12;21) with TEL-AML1, t(15;17) with PML-RARA, inv (16) with CBFB-MYH11, and microdeletion 1p32 with SIL-TAL1. PCR primers were designed according to predefined criteria for single PCR (external primers A <--> B) and nested PCR (internal primers C <--> D) as well as for 'shifted' PCR with a primer upstream (E5' primer) or downstream (E3' primer) of the external A <--> B primers. The 'shifted' E primers were designed for performing an independent PCR together with one of the internal primers for confirmation (or exclusion) of positive results. Various local RT and PCR protocols were compared and subsequently a common protocol was designed, tested and adapted, resulting in a standardized RT-PCR protocol. After initial testing (with adaptations whenever necessary) and approval by two or three laboratories, the primers were tested by all participating laboratories, using 17 cell lines and patient samples as positive controls. This testing included comparison with local protocols and primers as well as sensitivity testing via dilution experiments. The collaborative efforts resulted in standardized primer sets with a minimal target sensitivity of 10-2 for virtually all single PCR analyses, whereas the nested PCR analyses generally reached the minimal target sensitivity of 10-4. The standardized RT-PCR protocol and primer sets can now be used for molecular classification of acute leukemia at diagnosis and for MRD detection during follow-up to evaluate treatment effectiveness.

Trisomy 4 as the sole karyotypic anomaly in acute biphenotypic leukemia with B lineage markers and in acute minimally differentiated myeloid leukemia (MO)

Trisomy 4 is a recently defined chromosomal aberration in acute leukemia. The first reports suggested that this cytogenetic anomaly belongs to the M4 leukemia subtype of the FAB classification, but recent reports have described this alteration in a wider spectrum of leukemia subtypes. Here we report two cases of trisomy 4 as the sole chromosome anomaly: one was observed in a patient with acute biphenotypic leukemia with B-lineage markers and the second in a patient diagnosed with acute minimally differentiated myeloid leukemia (M0) with myelodysplastic features. To our knowledge these are, respectively, the first and second reports of trisomy 4 as the sole chromosomal anomaly in these leukemia subtypes.

Trisomy 4 may occur in a broad range of hematologic malignancies

Trisomy 4 is an uncommon numerical chromosomal aberration in acute leukemia. We describe three cases of trisomy 4, occurring in two patients with acute myeloid leukemia (M1 and M5a) and in one patient with T-cell acute lymphoblastic leukemia. Our results suggest that trisomy 4 may occur in a broader range of hematologic malignancies than previously described.

Trisomy 13 as a primary chromosome aberration in acute leukemia

Four patients with acute leukemia displayed trisomy 13 as the primary chromosome abnormality. The two patients with acute nonlymphocytic leukemia FAB-type M1 (ANLL-M1) had the karyotypes 47, XY,+13 48, XY,+13,+13 and 47,XY,+13, a patient with the hypogranular form of ANLL M3 had 47,XX,+13, and the fourth patient, who had acute undifferentiated leukemia (AUL), had the karyotype 47, XY,+13 48, XY,+8,+13 . Including these four cases, a total of 24 hematologic neoplasms with an extra chromosome 13 as the sole aberration have now been reported. Except for the AUL, all have been of myeloid origin—20 ANLL, one myelodysplastic syndrome, and two chronic myeloproliferative disorders. Trisomy 13 as the sole acquired karyotypic abnormality therefore seems to be strongly associated with myeloid differentiation of the neoplastic cells and with a differentiation block leading to acute leukemia.