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On the basis of somatic hypermutation status of their B-cell antigen receptor (BCR) genes, chronic lymphocytic leukemia (CLL) patients can be divided into unmutated CLL (U-CLL) or mutated CLL (M-CLL). Approximately 30% of CLL patients express a stereotypic BCR, which may indicate that specific antigenic stimulation is driving CLL pathogenesis. Recently, it was reported that BCRs from CLL cells are capable of antigen-independent, cell-autonomous signaling, through recognition of an internal framework 2 (FR2) BCR epitope. We hypothesized that the level of cell-autonomous signaling may differ between CLL subgroups. Therefore, we analyzed Ca(2+) signaling in a series of primary stereotypic or heterogeneous U-CLL and M-CLL (n=68) and healthy controls (n=14). We confirmed that basal Ca(2+) signaling in CLL cells is higher than in normal B cells. Interestingly, we found that basal signaling was particularly increased in M-CLL. The degree of basal signaling did not correlate with membrane immunoglobulin levels, HCDR3 characteristics or FR2/FR3 sequence. We conclude that the level of basal Ca(2+) signaling is not uniformly enhanced in CLL B cells, but is associated with CLL immunoglobulin heavy chain V mutational status, reflecting a distinct cellular origin and possibly a different anergic state induced by repetitive or continuous antigen binding in vivo. [ABSTRACT FROM AUTHOR]

On the basis of somatic hypermutation status of their B-cell antigen receptor (BCR) genes, chronic lymphocytic leukemia (CLL) patients can be divided into unmutated CLL (U-CLL) or mutated CLL (M-CLL). Approximately 30% of CLL patients express a stereotypic BCR, which may indicate that specific antigenic stimulation is driving CLL pathogenesis. Recently, it was reported that BCRs from CLL cells are capable of antigen-independent, cell-autonomous signaling, through recognition of an internal framework 2 (FR2) BCR epitope. We hypothesized that the level of cell-autonomous signaling may differ between CLL subgroups. Therefore, we analyzed Ca2+ signaling in a series of primary stereotypic or heterogeneous U-CLL and M-CLL (n=68) and healthy controls (n=14). We confirmed that basal Ca2+ signaling in CLL cells is higher than in normal B cells. Interestingly, we found that basal signaling was particularly increased in M-CLL. The degree of basal signaling did not correlate with membrane immunoglobulin levels, HCDR3 characteristics or FR2/FR3 sequence. We conclude that the level of basal Ca2+ signaling is not uniformly enhanced in CLL B cells, but is associated with CLL immunoglobulin heavy chain V mutational status, reflecting a distinct cellular origin and possibly a different anergic state induced by repetitive or continuous antigen binding in vivo. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented which discusses the examination of the presence of myelodysplastic syndrome (MDS)-associated phenotypic alterrations (MDS-PAs) in bone marrow (BM) samples from newly diagnosed patients referred immunophenotypic investigation of plasma cell (PC) neoplasia.

The article discusses the nature of persistent polyclonal B-cell lymphocytosis (PPBL), a rare disease associated with smoking and mostly affects middle-aged women. It states that PPBL is characterized by persistent expansion of CD2+IgM+IgD+B cells, the increase IgM serum levels and the presence of circulating binucleated lymphocytes. It notes the extensive proliferation of such cells in PPBL. It also emphasizes the difference pathophysiology of the PPBL with mature B-cell malignancies.

The article presents a study which assesses the frequency of paroxysmal nocturnal hemoglobinuria (PNH) clones in refractory cytopenia of childhood (RCC) with high-sensitivity flow cytometry, and correlated the presence of PNH clones with clinical characteristics and response to immunosuppressive therapy (IST). It notes the attainment of peripheral blood samples for PNH analysis from 87 primary RCC patients. The study shows that PNH clones are frequently present in RCC.

Most current treatment protocols for acute lymphoblastic leukemia (ALL) include minimal residual disease (MRD) diagnostics, generally based on PCR analysis of rearranged antigen receptor genes. Although flow cytometry (FCM) can be used for MRD detection as well, discordant FCM and PCR results are obtained in 5-20% of samples. We evaluated whether 6-color FCM, including additional markers and new marker combinations, improved the results. Bone marrow samples were obtained from 363 ALL patients at day 15, 33 and 78 and MRD was analyzed using 6-color (218 patients) or 4-color (145 patients) FCM in parallel to routine PCR-based MRD diagnostics. Compared with 4-color FCM, 6-color FCM significantly improved the concordance with PCR-based MRD data (88% versus 96%); particularly the specificity of the MRD analysis improved. However, PCR remained more sensitive at levels <0.01%. MRD-based risk groups were similar between 6-color FCM and PCR in 68% of patients, most discrepancies being medium risk by PCR and standard risk by FCM. Alternative interpretation of the PCR data, aimed at prevention of false-positive MRD results, changed the risk group to standard risk in half (52%) of these discordant cases. In conclusion, 6-color FCM significantly improves MRD analysis in ALL but remains less sensitive than PCR-based MRD-diagnostics. [ABSTRACT FROM AUTHOR]

The authors reflect on the EuroFlow Consortium, which is supported by the European Union. They cite the main objectives of the project such as the development and testing of new antibodies, introduction of immunobead technology and flow cytometry software development. They state that the Consortium has achieved most of its goals with the development of an eight-color antibody protocols for hematological diagnosis.

Most consensus leukemia & lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2-7 sequential design-evaluation-redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies. [ABSTRACT FROM AUTHOR]

The EU-supported EuroFlow Consortium aimed at innovation and standardization of immunophenotyping for diagnosis and classification of hematological malignancies by introducing 8-color flow cytometry with fully standardized laboratory procedures and antibody panels in order to achieve maximally comparable results among different laboratories. This required the selection of optimal combinations of compatible fluorochromes and the design and evaluation of adequate standard operating procedures (SOPs) for instrument setup, fluorescence compensation and sample preparation. Additionally, we developed software tools for the evaluation of individual antibody reagents and antibody panels. Each section describes what has been evaluated experimentally versus adopted based on existing data and experience. Multicentric evaluation demonstrated high levels of reproducibility based on strict implementation of the EuroFlow SOPs and antibody panels. Overall, the 6 years of extensive collaborative experiments and the analysis of hundreds of cell samples of patients and healthy controls in the EuroFlow centers have provided for the first time laboratory protocols and software tools for fully standardized 8-color flow cytometric immunophenotyping of normal and malignant leukocytes in bone marrow and blood; this has yielded highly comparable data sets, which can be integrated in a single database. [ABSTRACT FROM AUTHOR]

Hematopoietic stem cell transplantation is becoming an increasingly important approach to treatment of different malignant and non-malignant disorders. There is thus growing demand for diagnostic assays permitting the surveillance of donor/recipient chimerism posttransplant. Current techniques are heterogeneous, rendering uniform evaluation and comparison of diagnostic results between centers difficult. Leading laboratories from 10 European countries have therefore performed a collaborative study supported by a European grant, the EuroChimerism Concerted Action, with the aim to develop a standardized diagnostic methodology for the detection and monitoring of chimerism in patients undergoing allogeneic stem cell transplantation. Following extensive analysis of a large set of microsatellite/short tandem repeat (STR) loci, the EuroChimerism (EUC) panel comprising 13 STR markers was established with the aim to optimally meet the specific requirements of quantitative chimerism analysis. Based on highly stringent selection criteria, the EUC panel provides multiple informative markers in any transplant setting. The standardized STR-PCR tests permit detection of donor- or recipient-derived cells at a sensitivity ranging between 0.8 and 1.6%. Moreover, the EUC assay facilitates accurate and reproducible quantification of donor and recipient hematopoietic cells. Wide use of the European-harmonized protocol for chimerism analysis presented will provide a basis for optimal diagnostic support and timely treatment decisions. [ABSTRACT FROM AUTHOR]

We investigated whether the newly developed antibody (Ab) -targeted therapy inotuzumab ozogamicin (CMC-544), consisting of a humanized CD22 Ab linked to calicheamicin, is effective in pediatric primary B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells in vitro, and analyzed which parameters determine its efficacy. CMC-544 induced dose-dependent cell kill in the majority of BCP-ALL cells, although IC50 values varied substantially (median 4.8 ng/ml, range 0.1-1000 ng/ml at 48 h). The efficacy of CMC-544 was highly dependent on calicheamicin sensitivity and CD22/CMC-544 internalization capacity of BCP-ALL cells, but hardly on basal and renewed CD22 expression. Although CD22 expression was essential for uptake of CMC-544, a repetitive loop of CD22 saturation, CD22/CMC-544 internalization and renewed CD22 expression was not required to achieve intracellular threshold levels of calicheamicin sufficient for efficient CMC-544-induced apoptosis in BCP-ALL cells. This is in contrast to studies with the comparable CD33 immunotoxin gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukemia (AML) patients, in which complete and prolonged CD33 saturation was required for apoptosis induction. These data suggest that CMC-544 treatment may result in higher response rates in ALL compared with response rates obtained in AML with Mylotarg, and that therefore clinical studies in ALL, preferably with multiple low CMC-544 dosages, are warranted. [ABSTRACT FROM AUTHOR]

Severe combined immunodeficiency (SCID) patients with an inactivating mutation in recombination activation gene 1 (RAG1) lack B and T cells due to the inability to rearrange immunoglobulin (Ig) and T-cell receptor (TCR) genes. Gene therapy is a valid treatment option for RAG-SCID patients, especially for patients lacking a suitable bone marrow donor, but developing such therapy has proven challenging. As a preclinical model for RAG-SCID, we used Rag1-/- mice and lentiviral self-inactivating (SIN) vectors harboring different internal elements to deliver native or codon-optimized human RAG1 sequences. Treatment resulted in the appearance of B and T cells in peripheral blood and developing B and T cells were detected in central lymphoid organs. Serum Ig levels and Ig and TCR Vβ gene segment usage was comparable to wild-type (WT) controls, indicating that RAG-mediated rearrangement took place. Remarkably, relatively low frequencies of B cells produced WT levels of serum immunoglobulins. Upon stimulation of the TCR, corrected spleen cells proliferated and produced cytokines. In vivo challenge resulted in production of antigen-specific antibodies. No leukemia development as consequence of insertional mutagenesis was observed. The functional reconstitution of the B- as well as the T-cell compartment provides proof-of-principle for therapeutic RAG1 gene transfer in Rag1-/- mice using lentiviral SIN vectors. [ABSTRACT FROM AUTHOR]

Analysis of short tandem repeats (STR) by PCR analysis is routinely used in chimerism diagnostics to monitor donor engraftment and to diagnose relapse. Some applications require chimerism analysis of low cell numbers, but no standardized protocol is available for DNA isolation from 1000 to 30 000 cells. The EU-supported EuroChimerism Consortium (project QLRT-2001-01485) selected four different protocols for 'small-scale' DNA isolation, which were tested by six laboratories for their ability to recover reproducible amounts of good quality DNA, suited for PCR-based STR analysis. The protocols included two direct lysis methods with and without detergents and proteinase K, and two commercial column-based kits. The direct lysis method using detergents and proteinase K showed the highest DNA recovery and the best performance in the multiplex PowerPlex16 STR assay. DNA isolated with this method also showed the highest sensitivity in chimerism analysis using singleplex PCR reactions of EuroChimerism STR markers. Sensitivity was reached ranging from 1 to 20% of recipient cells in a donor background. In conclusion, the direct lysis method using detergents and proteinase K is a standardized DNA isolation method well suited for chimerism studies on low cell numbers. [ABSTRACT FROM AUTHOR]

Response to therapy as determined by minimal residual disease (MRD) is currently used for stratification in treatment protocols for pediatric acute lymphoblastic leukemia (ALL). However, the large MRD-based medium risk group (MRD-M; 50-60% of the patients) harbors many relapses. We analyzed MRD in 131 uniformly treated precursor-B-ALL patients and evaluated whether combined MRD and IKZF1 (Ikaros zinc finger-1) alteration status can improve risk stratification. We confirmed the strong prognostic significance of MRD classification, which was independent of IKZF1 alterations. Notably, 8 of the 11 relapsed cases in the large MRD-M group (n=81; 62%) harbored an IKZF1 alteration. Integration of both MRD and IKZF1 status resulted in a favorable outcome group (n=104; 5 relapses) and a poor outcome group (n=27; 19 relapses), and showed a stronger prognostic value than each of the established risk factors alone (hazard ratio (95%CI): 24.98 (8.29-75.31)). Importantly, whereas MRD and IKZF1 status alone identified only 46 and 54% of the relapses, respectively, their integrated use allowed prediction of 79% of all the relapses with 93% specificity. Because of the unprecedented sensitivity in upfront relapse prediction, the combined parameters have high potential for future risk stratification, particularly for patients originally classified as non-high risk, such as the large group of MRD-M patients. [ABSTRACT FROM AUTHOR]

Immunophenotypic characterization of B-cell chronic lymphoproliferative disorders (B-CLPD) is becoming increasingly complex due to usage of progressively larger panels of reagents and a high number of World Health Organization (WHO) entities. Typically, data analysis is performed separately for each stained aliquot of a sample; subsequently, an expert interprets the overall immunophenotypic profile (IP) of neoplastic B-cells and assigns it to specific diagnostic categories. We constructed a principal component analysis (PCA)-based tool to guide immunophenotypic classification of B-CLPD. Three reference groups of immunophenotypic data files-B-cell chronic lymphocytic leukemias (B-CLL; n = 10), mantle cell (MCL; n = 10) and follicular lymphomas (FL; n = 10)--were built. Subsequently, each of the 175 cases studied was evaluated and assigned to either one of the three reference groups or to none of them (other B-CLPD). Most cases (89%) were correctly assigned to their corresponding WHO diagnostic group with overall positive and negative predictive values of 89 and 96%, respectively. The efficiency of the PCA-based approach was particularly high among typical B-CLL, MCL and FL vs other B-CLPD cases. In summary, PCA-guided immunophenotypic classification of B-CLPD is a promising tool for standardized interpretation of tumor IP, their classification into well-defined entities and comprehensive evaluation of antibody panels. [ABSTRACT FROM AUTHOR]

Analysis of minimal residual disease (MRD) in childhood acute myeloid leukemia (AML) may predict for clinical outcome. MRD levels were assessed by flowcytometric immunophenotyping in 94 children with AML enrolled into a single trial (United Kingdom Medical Research Council AML12 and similar Dutch Childhood Oncology Group ANLL97). An aberrant immunophenotype could be detected in 94% of patients. MRD levels after the first course of chemotherapy predicted for clinical outcome: 3-year relapse-free survival was 85%+/-8% (s.e.) for MRD-negative patients (MRD<0.1%), 64%+/-10% for MRD-low-positive patients (0.1%or=0.5%; P<0.001), whereas overall survival was 95%+/-5%, 70%+/-10% and 40%+/-13%, respectively, (P<0.001). Multivariate analysis allowing for age, karyotype, FLT3-internal tandem duplications and white blood cell count at diagnosis showed that MRD after the first course of chemotherapy was an independent prognostic factor. Although comparison of paired diagnosis-relapse samples (n=23) showed immunophenotypic shifts in 91% of cases, this did not hamper MRD analysis. In conclusion, flowcytometric MRD detection is possible in children with AML. The level of MRD after the first course of chemotherapy provides prognostic information that may be used to guide therapy. [ABSTRACT FROM AUTHOR]

X-linked agammaglobulinemia (XLA) is the most common primary immunodeficiency (PID) in man and caused by mutations in the Bruton’s tyrosine kinase (BTK) gene. XLA is characterized by a B-cell differentiation arrest in bone marrow, absence of mature B cells and immunoglobulins (Igs), and recurrent bacterial infections. We used self-inactivating lentiviral vectors expressing codon-optimized human BTK under the control of three different ubiquitous or B cell-specific promoters. Btk−/− mice engrafted with transduced cells showed correction of both precursor B-cell and peripheral B-cell development. Lentiviral vectors containing the wildtype BTK sequence did not correct the phenotype. All treated mice with codon-optimized BTK exhibited the recovery of B1 cells in the peritoneal cavity, and of serum IgM and IgG3 levels. Calcium mobilization responses upon B-cell receptor stimulation as well as in vivo responses to T cell-independent antigens were restored. Viral promoters overexpressing BTK >100-fold above normal resulted in erythro-myeloid proliferations independent of insertional mutagenesis. However, transplantation into secondary Btk−/− recipients using cellular promoters resulted in functional restoration of peripheral B cells and IgM levels, without any adverse effects. In conclusion, transduction of human BTK corrects B-cell development and antigen-specific antibody responses in Btk−/− mice, thus indicating the feasibility of lentiviral gene therapy for XLA, provided that BTK expression does not vastly exceed normal levels. [ABSTRACT FROM AUTHOR]

Relapse of pediatric acute lymphoblastic leukemia (ALL) remains the main cause of treatment failure after allogeneic stem cell transplantation (alloSCT). A high level of minimal residual disease (MRD) before alloSCT has been shown to predict these relapses. Patients at risk might benefit from a preemptive alloimmune intervention. In this first prospective, MRD-guided intervention study, 48 patients were stratified according to pre-SCT MRD level. Eighteen children with MRD level 1 × 10−4 were eligible for intervention, consisting of early cyclosporine A tapering followed by consecutive, incremental donor lymphocyte infusions (n=1–4). The intervention was associated with graft versus host disease grade II in only 23% of patients. Event-free survival in the intervention group was 19%. However, in contrast with the usual early recurrence of leukemia, relapses were delayed up to 3 years after SCT. In addition, several relapses presented at unusual extramedullary sites suggesting that the immune intervention may have altered the pattern of leukemia recurrence. In 8 out of 11 evaluable patients, relapse was preceded by MRD recurrence (median 9 weeks, range 0–30). We conclude that in children with high-risk ALL, immunotherapy-based regimens after SCT are feasible and may need to be further intensified to achieve total eradication of residual leukemic cells. [ABSTRACT FROM AUTHOR]

Relapse is the most common cause of treatment failure in pediatric acute lymphoblastic leukemia (ALL) and is often difficult to predict. To explore the prognostic impact of recurrent DNA copy number abnormalities on relapse, we performed high-resolution genomic profiling of 34 paired diagnosis and relapse ALL samples. Recurrent lesions detected at diagnosis, including PAX5, CDKN2A and EBF1, were frequently absent at relapse, indicating that they represent secondary events that may be absent in the relapse-prone therapy-resistant progenitor cell. In contrast, deletions and nonsense mutations in IKZF1 (IKAROS) were highly enriched and consistently preserved at the time of relapse. A targeted copy number screen in an unselected cohort of 131 precursor B-ALL cases, enrolled in the dexamethasone-based Dutch Childhood Oncology Group treatment protocol ALL9, revealed that IKZF1 deletions are significantly associated with poor relapse-free and overall survival rates. Separate analysis of ALL9-treatment subgroups revealed that non-high-risk (NHR) patients with IKZF1 deletions exhibited a ∼12-fold higher relative relapse rate than those without IKZF1 deletions. Consequently, IKZF1 deletion status allowed the prospective identification of 53% of the relapse-prone NHR-classified patients within this subgroup and, therefore, serves as one of the strongest predictors of relapse at the time of diagnosis with high potential for future risk stratification. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented related to the treatment of acute lymphoblastic leukemia in children.

Almost a quarter of pediatric patients with acute lymphoblastic leukemia (ALL) suffer from relapses. The biological mechanisms underlying therapy response and development of relapses have remained unclear. In an attempt to better understand this phenomenon, we have analyzed 41 matched diagnosis–relapse pairs of ALL patients using genome-wide expression arrays (82 arrays) on purified leukemic cells. In roughly half of the patients, very few differences between diagnosis and relapse samples were found (‘stable group’), suggesting that mostly extra-leukemic factors (for example, drug distribution, drug metabolism, compliance) contributed to the relapse. Therefore, we focused our further analysis on 20 sample pairs with clear differences in gene expression (‘skewed group’), reasoning that these would allow us to better study the biological mechanisms underlying relapsed ALL. After finding the differences between diagnosis and relapse pairs in this group, we identified four major gene clusters corresponding to several pathways associated with changes in cell cycle, DNA replication, recombination and repair, as well as B-cell developmental genes. We also identified cancer genes commonly associated with colon carcinomas and ubiquitination to be upregulated in relapsed ALL. Thus, about half of the relapses are due to the selection or emergence of a clone with deregulated expression of genes involved in pathways that regulate B-cell signaling, development, cell cycle, cellular division and replication. [ABSTRACT FROM AUTHOR]

Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements. [ABSTRACT FROM AUTHOR]

Acute lymphoblastic leukemia (ALL) in infants younger than 1 year is a rare but relatively homogeneous disease ( approximately 80% MLL gene rearranged, approximately 70% CD10-negative) when compared with childhood and adult ALL. Several studies in children and adults with ALL have shown that minimal residual disease (MRD) status is a strong and independent prognostic factor. We therefore evaluated the prognostic significance of MRD in infant ALL. Ninety-nine infant patients treated according to the Interfant-99 protocol were included in this study. MRD was analyzed by real-time quantitative PCR analysis of rearranged immunoglobulin genes, T-cell receptor genes and MLL genes at various time points (TP) during therapy. Higher MRD levels at the end of induction (TP2) and consolidation (TP3) were significantly associated with lower disease-free survival. Combined MRD information at TP2 and TP3 allowed recognition of three patients groups that significantly differed in outcome. All MRD-high-risk patients (MRD levels > or =10(-4) at TP3; 26% of patients) relapsed. MRD-low-risk patients (MRD level <10(-4) at both TP2 and TP3) constituted 44% of patients and showed a relapse-rate of only 13%, whereas remaining patients (MRD-medium-risk patients; 30% of patients) had a relapse rate of 31%. Comparison between the current Interfant-06 stratification at diagnosis and the here presented MRD-based stratification showed that both stratifications recognized different subgroups of patients. These data indicate that MRD diagnostics has added value for recognition of risk groups in infant ALL and that MRD diagnostics can be used for treatment intervention in infant ALL as well. [ABSTRACT FROM AUTHOR]

Childhood B-cell precursor acute lymphoblastic leukemia (BCP ALL) is generally a clonal disease in which the number of IGH rearrangements per cell does not exceed the number of the IGH alleles on chromosome 14. Consequently, monoclonal high hyperdiploid (HeH) cases with a trisomy 14 can harbor three rearrangements, a pattern that otherwise may be misinterpreted to be oligoclonal. Oligoclonal IGH rearrangements, on the other hand, may be instable at relapse and should therefore not be used for minimal residual disease analysis. We thus investigated the association between IGH allele copy numbers and the IGH rearrangement patterns in 90 HeH BCP ALL with either two (13%) or three copies (87%) of chromosome 14. HeH cases (44%) had an oligoclonal IGH rearrangement pattern, but true oligoclonality—after correction for the respective copy number of IGH alleles—was only 16%. Monoclonal and oligoclonal HeH cases had predominantly VH to preexisting DJH recombinations, a finding that contrasts with oligoclonal cases of other major genetic BCP ALL subgroups in which VH replacements prevail. We conclude that for the precise assessment and correct interpretation of clonality patterns in BCP ALL, the IGH allele copy number has to be taken into consideration.Leukemia (2009) 23, 870–876; doi:10.1038/leu.2008.390; published online 15 January 2009 [ABSTRACT FROM AUTHOR]

In clinical gene therapy trials for X-linked severe combined immunodeficiency, the development of leukemia has come up as a severe adverse effect. In all five cases, T-cell acute lymphoblastic leukemia (T-ALL) occurred as a direct consequence of insertional mutagenesis by the retrovirus used to deliver the therapeutic gene. Here, we review the mechanisms of insertional mutagenesis, the function of the Il2RG gene and the future developments in the field. New lentiviral and gamma retroviral vectors can significantly improve the safety profile of the tools used but still carry the risk of insertional mutagenesis, as shown in this issue of Leukemia. Finally, the unfortunate side effects of gene therapy have given more insight into the development of human T-ALL. [ABSTRACT FROM AUTHOR]

Detection of minimal residual disease (MRD) is the most sensitive method to evaluate treatment response and one of the strongest predictors of outcome in childhood acute lymphoblastic leukemia (ALL). The 10-year update on the I-BFM-SG MRD study 91 demonstrates stable results (event-free survival), that is, standard risk group (MRD-SR) 93%, intermediate risk group (MRD-IR) 74%, and high risk group (MRD-HR) 16%. In multicenter trial AIEOP-BFM ALL 2000, patients were stratified by MRD detection using quantitative PCR after induction (TP1) and consolidation treatment (TP2). From 1 July 2000 to 31 October 2004, PCR target identification was performed in 3341 patients: 2365 (71%) patients had two or more sensitive targets (10−4), 671 (20%) patients revealed only one sensitive target, 217 (6%) patients had targets with lower sensitivity, and 88 (3%) patients had no targets. MRD-based risk group assignment was feasible in 2594 (78%) patients: 40% were classified as MRD-SR (two sensitive targets, MRD negativity at both time points), 8% as MRD-HR (MRD 10−3 at TP2), and 52% as MRD-IR. The remaining 823 patients were stratified according to clinical risk features: HR (n=108) and IR (n=715). In conclusion, MRD-PCR-based stratification using stringent criteria is feasible in almost 80% of patients in an international multicenter trial.Leukemia (2008) 22, 771–782; doi:10.1038/leu.2008.5; published online 31 January 2008 [ABSTRACT FROM AUTHOR]

A letter to the editor is presented regarding the implications of time dependence in immunoglobulin and T-cell receptor amplification, on the monitoring of residual diseases.

Data on secondary acute lymphoblastic leukaemia (sALL) following ALL treatment are very rare. However, the incidence might be underestimated as sALLs without a significant lineage shift might automatically be diagnosed as relapses. Examination of immunoglobulin and T-cell receptor gene rearrangements brought a new tool that can help in discrimination between relapse and sALL. We focused on the recurrences of childhood ALL to discover the real frequency of the sALL after ALL treatment. We compared clonal markers in matched presentation and recurrence samples of 366 patients treated according to the Berlin–Frankfurt–Munster (BFM)-based protocols. We found two cases of sALL and another three, where the recurrence is suspicious of being sALL rather than relapse. Our proposal for the ‘secondary ALL after ALL’ diagnostic criteria is as follows: (A) No clonal relationship between diagnosis and recurrence; (B) significant immunophenotypic shift – significant cytogenetic shift – gain/loss of a fusion gene. For the sALL (A) plus at least one (B) criterion should be fulfilled. With these criteria, the estimated frequency of the sALL after ALL is according to our data 0.5–1.5% of ALL recurrences on BFM-based protocols. Finally, we propose a treatment strategy for the patients with secondary disease.Leukemia (2007) 21, 1431–1435; doi:10.1038/sj.leu.2404718; published online 26 April 2007 [ABSTRACT FROM AUTHOR]

Minimal residual disease (MRD) diagnostics is used for treatment stratification in childhood acute lymphoblastic leukemia. We aimed to identify and solve potential problems in multicenter MRD studies to achieve and maintain consistent results between the AIEOP/BFM ALL-2000 MRD laboratories. As the dot-blot hybridization method was replaced by the real-time quantitative polymerase chain reaction (RQ-PCR) method during the treatment protocol, special attention was given to the comparison of MRD data obtained by both methods and to the reproducibility of RQ-PCR data. Evaluation of all key steps in molecular MRD diagnostics identified several pitfalls that resulted in discordant MRD results. In particular, guidelines for RQ-PCR data interpretation appeared to be crucial for obtaining concordant MRD results. The experimental variation of the RQ-PCR was generally less than three-fold, but logically became larger at low MRD levels below the reproducible sensitivity of the assay (<10−4). Finally, MRD data obtained by dot-blot hybridization were comparable to those obtained by RQ-PCR analysis (r2=0.74). In conclusion, MRD diagnostics using RQ-PCR analysis of immunoglobulin/T-cell receptor gene rearrangements is feasible in multicenter studies but requires standardization; particularly strict guidelines for interpretation of RQ-PCR data are required. We further recommend regular quality control for laboratories performing MRD diagnostics in international treatment protocols.Leukemia (2007) 21, 706–713. doi:10.1038/sj.leu.2404535; published online 8 February 2007 [ABSTRACT FROM AUTHOR]

The occurrence of leukemia in a gene therapy trial for SCID-X1 has highlighted insertional mutagenesis as an adverse effect. Although retroviral integration near the T-cell acute lymphoblastic leukemia (T-ALL) oncogene LIM-only protein 2 (LMO2) appears to be a common event, it is unclear why LMO2 was preferentially targeted. We show that of classical T-ALL oncogenes, LMO2 is most highly transcribed in CD34+ progenitor cells. Upon stimulation with growth factors typically used in gene therapy protocols transcription of LMO2, LYL1, TAL1 and TAN1 is most prominent. Therefore, these oncogenes may be susceptible to viral integration. The interleukin-2 receptor gamma chain (IL2Rγ), which is mutated in SCID-X1, has been proposed as a cooperating oncogene to LMO2. However, we found that overexpressing IL2Rγ had no effect on T-cell development. In contrast, retroviral overexpression of LMO2 in CD34+ cells caused severe abnormalities in T-cell development, but B-cell and myeloid development remained unaffected. Our data help explain why LMO2 was preferentially targeted over many of the other known T-ALL oncogenes. Furthermore, during T-cell development retrovirus-mediated expression of IL2Rγ may not be directly oncogenic. Instead, restoration of normal IL7-receptor signaling may allow progression of T-cell development to stages where ectopic LMO2 expression causes aberrant thymocyte growth.Leukemia (2007) 21, 754–763. doi:10.1038/sj.leu.2404563; published online 1 February 2007 [ABSTRACT FROM AUTHOR]

The t(4;11)-positive acute lymphoblastic leukemia (ALL) is a rare disease in children above the age of 1 year. We studied the clinical and biological characteristics in 32 consecutively diagnosed childhood cases (median age 10.0 years, range 1.0–17.1 years). Immunophenotyping revealed a pro-B and a pre-B stage in 24 and eight cases, respectively. IGH genes were rearranged in 84% of leukemias with a predominance of incomplete DJH joints. Whereas IGK-Kde and TCRD rearrangements were rare, TCRG rearrangements were present in 50% of cases and involved mainly Vγ11 or Vγ9 together with a Jγ1.3./2.3 gene segment, an unusual combination among t(4;11)-negative B-cell precursor ALL. Oligoclonality was found in about 30% as assessed by heterogeneous IGH and TCRG rearrangements. Our data are in line with transformation of a precursor cell at an early stage of B-cell development but retaining the potential to differentiate to the pre-B cell stage in vivo. Although a distinct difference between infant and older childhood cases with t(4;11) became evident, no age-related biological features were found within the childhood age group. In contrast to infants with t(4;11)-positive ALL, childhood cases had a relatively low cumulative incidence of relapse of 25% at 3.5 years with BFM-based high-risk protocols.Leukemia (2007) 21, 642–646. doi:10.1038/sj.leu.2404577; published online 8 February 2007 [ABSTRACT FROM AUTHOR]

The aim of this study was to identify immunobiological subgroups in 133 infant acute lymphoblastic leukemia (ALL) cases as assessed by their immunophenotype, immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement pattern, and the presence of mixed lineage leukemia (MLL) rearrangements. About 70% of cases showed the pro-B-ALL immunophenotype, whereas the remaining cases were common ALL and pre-B-ALL. MLL translocations were found in 79% of infants, involving MLL-AF4 (41%), MLL-ENL (18%), MLL-AF9 (11%) or another MLL partner gene (10%). Detailed analysis of Ig/TCR rearrangement patterns revealed IGH, IGK and IGL rearrangements in 91, 21 and 13% of infants, respectively. Cross-lineage TCRD, TCRG and TCRB rearrangements were found in 46, 17 and 10% of cases, respectively. As compared to childhood precursor-B-ALL, Ig/TCR rearrangements in infant ALL were less frequent and more oligoclonal. MLL-AF4 and MLL-ENL-positive infants demonstrated immature rearrangements, whereas in MLL-AF9-positive leukemias more mature rearrangements predominated. The immature Ig/TCR pattern in infant ALL correlated with young age at diagnosis, CD10 negativity and predominantly with the presence and the type of MLL translocation. The high frequency of immature and oligoclonal Ig/TCR rearrangements is probably caused by early (prenatal) oncogenic transformation in immature B-lineage progenitor cells with germline Ig/TCR genes combined with a short latency period. [ABSTRACT FROM AUTHOR]

Most modern treatment protocols for acute lymphoblastic leukaemia (ALL) include the analysis of minimal residual disease (MRD). To ensure comparable MRD results between different MRD-polymerase chain reaction (PCR) laboratories, standardization and quality control are essential. The European Study Group on MRD detection in ALL (ESG-MRD-ALL), consisting of 30 MRD-PCR laboratories worldwide, has developed guidelines for the interpretation of real-time quantitative PCR-based MRD data. The application of these guidelines ensures identical interpretation of MRD data between different laboratories of the same MRD-based clinical protocol. Furthermore, the ESG-MRD-ALL guidelines will facilitate the comparison of MRD data obtained in different treatment protocols, including those with new drugs.Leukemia (2007) 21, 604–611. doi:10.1038/sj.leu.2404586; published online 8 February 2007 [ABSTRACT FROM AUTHOR]

Lymphoproliferations are generally diagnosed via histomorphology and immunohistochemistry. Although mostly conclusive, occasionally the differential diagnosis between reactive lesions and malignant lymphomas is difficult. In such cases molecular clonality studies of immunoglobulin (Ig)/T-cell receptor (TCR) rearrangements can be useful. Here we address the issue of clonality assessment in 106 histologically defined reactive lesions, using the standardized BIOMED-2 Ig/TCR multiplex polymerase chain reaction (PCR) heteroduplex and GeneScan assays. Samples were reviewed nationally, except 10% random cases and cases with clonal results selected for additional international panel review. In total 75% (79/106) only showed polyclonal Ig/TCR targets (type I), whereas another 15% (16/106) represent probably polyclonal cases, with weak Ig/TCR (oligo)clonality in an otherwise polyclonal background (type II). Interestingly, in 10% (11/106) clear monoclonal Ig/TCR products were observed (types III/IV), which prompted further pathological review. Clonal cases included two missed lymphomas in national review and nine cases that could be explained as diagnostically difficult cases or probable lymphomas upon additional review. Our data show that the BIOMED-2 Ig/TCR multiplex PCR assays are very helpful in confirming the polyclonal character in the vast majority of reactive lesions. However, clonality detection in a minority should lead to detailed pathological review, including close interaction between pathologist and molecular biologist.Leukemia (2007) 21, 222–229. doi:10.1038/sj.leu.2404482; published online 14 December 2006 [ABSTRACT FROM AUTHOR]

The BIOMED-2 multiplex polymerase chain reaction (PCR) tubes for analysis of immunoglobulin and T-cell receptor (TCR) gene rearrangements have recently been introduced as a reliable and easy tool for clonality diagnostics in suspected lymphoproliferations. Quality and performance assessment of PCR-based clonality diagnostics is generally performed using human leukemia/lymphoma cell lines as controls. We evaluated the utility of 30 well-defined human T-cell lines for quality performance testing of the BIOMED-2 PCR primers and protocols. The PCR analyses of the TCR loci were backed up by Southern blot analysis. The clonal TCRB, TCRG and TCRD gene rearrangements were analyzed for gene segment usage and for the size and composition of their junctional regions. In 29 out of 30 cell lines, unique clonal TCR gene rearrangements could be easily detected. Besides their usefulness in molecular clonality diagnostics, these cell lines can now be authenticated based on their TCR gene rearrangement profile. This enables their correct use in molecular clonality diagnostics and in other cancer research studies.Leukemia (2007) 21, 230–237. doi:10.1038/sj.leu.2404486; published online 14 December 2006 [ABSTRACT FROM AUTHOR]

Deregulated Notch signaling occurs in the majority of human T-ALL. During normal lymphoid development, activation of the Notch signaling pathway poses a T-cell fate on hematopoietic progenitors. However, the transcriptional targets of the Notch pathway are largely unknown. We sought to identify Notch target genes by inducing Notch signaling in human hematopoietic progenitors using two different methods: an intracellular signal through transfection of activated Notch and a Notch-receptor dependent signal by interaction with its ligand Delta1. Gene expression profiles were generated and evaluated with respect to expression profiles of immature thymic subpopulations. We confirmed HES1, NOTCH1 and NRARP as Notch target genes, but other reported Notch targets, including the genes for Deltex1, pre-T-cell receptor alpha and E2A, were not found to be differentially expressed. Remarkably, no induction of T-cell receptor gene rearrangements or transcription of known T-cell specific genes was found after activation of the Notch pathway. A number of novel Notch target genes, including the transcription factor TCFL5 and the HOXA cluster, were identified and functionally tested. Apparently, Notch signaling is essential to open the T-cell pathway, but does not initiate the T-cell program itself. [ABSTRACT FROM AUTHOR]

A plethora of studies have documented that gene expression profiling using DNA microarrays for various types of hematological malignancies provides novel information, which may have diagnostic and prognostic implications. However, to successfully use microarrays for this purpose, the quality and reproducibility of the whole procedure need to be guaranteed. Critical steps of the method are handling, processing and storage of the leukemic sample, purification of tumor cells (or lack thereof), RNA extraction methods, quality control of RNA, labeling techniques, hybridization, washing, scanning, spot filtering, normalization and initial interpretation, and finally the biostatistical analysis. These items have been extensively discussed and evaluated in different multi-center quality rounds within the three networks, that is, I-BFM-SG, the German Competence Network ‘Acute and Chronic Leukemias’ and the European LeukemiaNet. Based on the exchange of knowledge and experience between the three networks over the last few years, we have formulated guidelines for performing microarray experiments in leukemia. We confine ourselves to leukemias, but many of these requirements also apply to lymphomas or other clinical samples, including solid tumors.Leukemia (2006) 20, 1385–1392. doi:10.1038/sj.leu.2404274; published online 8 June 2006 [ABSTRACT FROM AUTHOR]

Many acute lymphoblastic leukemias can be considered as malignant counterparts of cells in the various stages of normal lymphoid development in bone marrow and thymus. T-cell development in the thymus is an ordered and tightly controlled process. Two evolutionary conserved signaling pathways, which were first discovered in Drosophila, control the earliest steps of T-cell development. These are the Notch and Wnt-signaling routes, which both are deregulated in several types of leukemias. In this review we discuss both pathways, with respect to their signaling mechanisms, functions during T-cell development and their roles in development of leukemias, especially T-cell acute lymphoblastic leukemia.Leukemia (2006) 20, 1197–1205. doi:10.1038/sj.leu.2404255; published online 11 May 2006 [ABSTRACT FROM AUTHOR]

Chromosomal rearrangements of the human MLL gene are a hallmark for aggressive (high-risk) pediatric, adult and therapy-associated acute leukemias. These patients need to be identified in order to subject these patients to appropriate therapy regimen. A recently developed long-distance inverse PCR method was applied to genomic DNA isolated from individual acute leukemia patients in order to identify chromosomal rearrangements of the human MLL gene. We present data of the molecular characterization of 414 samples obtained from 272 pediatric and 142 adult leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) was determined and several new TPGs were identified. The combined data of our study and published data revealed a total of 87 different MLL rearrangements of which 51 TPGs are now characterized at the molecular level. Interestingly, the four most frequently found TPGs (AF4, AF9, ENL and AF10) encode nuclear proteins that are part of a protein network involved in histone H3K79 methylation. Thus, translocations of the MLL gene, by itself coding for a histone H3K4 methyltransferase, are presumably not randomly chosen, rather functionally selected. [ABSTRACT FROM AUTHOR]

T-cell large granular lymphocytes (LGL) proliferations range from reactive expansions of activated T cells to T-cell leukemias and show variable clinical presentation and disease course. The vast majority of T-LGL proliferations express TCRαβ. Much less is known about the characteristics and pathogenesis of TCRγδ+ cases. We evaluated 44 patients with clonal TCRγδ+ T-LGL proliferations with respect to clinical data, immunophenotype and TCR gene rearrangement pattern. TCRγδ+ T-LGL leukemia patients had similar clinical presentations as TCRαβ+ T-LGL leukemia patients. Their course was indolent and 61% of patients were symptomatic. The most common clinical manifestations were chronic cytopenias – neutropenia (48%), anemia (23%), thrombocytopenia (9%), pancytopenia (2%) – and to a lesser extent splenomegaly (18%). Also multiple associated autoimmune (34%) and hematological (14%) disorders were found. Leukemic LGLs were predominantly positive for CD2, CD5, CD7, CD8, and CD57, whereas variable expression was seen for CD16, CD56, CD11b, and CD11c. The Vγ9/Vδ2 immunophenotype was found in 48% of cases and 43% of cases was positive for Vδ1, reflecting the TCR-spectrum of normal TCRγδ+ T-cells in adult PB. Identification of the well-defined post-thymic Vδ2-Jδ1 selection determinant in all evaluable Vγ9+/Vδ2+ patients, is suggestive of common (super)antigen involvement in the pathogenesis of these TCRγδ+ T-LGL leukemia patients.Leukemia (2006) 20, 505–513. doi:10.1038/sj.leu.2404112; published online 26 January 2006 [ABSTRACT FROM AUTHOR]

The t(10;11)(p13;q14-21) is found in T-ALL and acute myeloid leukemia and fuses CALM (Clathrin-Assembly protein-like Lymphoid-Myeloid leukaemia gene) to AF10. In order to gain insight into the transcriptional consequences of this fusion, microarray-based comparison of CALM-AF10+ vs CALM-AF10- T-ALL was performed. This analysis showed upregulation of HOXA5, HOXA9, HOXA10 and BMI1 in the CALM-AF10+ cases. Microarray results were validated by quantitative RT-PCR on an independent group of T-ALL and compared to mixed lineage leukemia-translocated acute leukemias (MLL-t AL). The overexpression of HOXA genes was associated with overexpression of its cofactor MEIS1 in CALM-AF10+ T-ALL, reaching levels of expression similar to those observed in MLL-t AL. Consequently, CALM-AF10+ T-ALL and MLL-t AL share a specific HOXA overexpression, indicating they activate common oncogenic pathways. In addition, BMI1, located close to AF10 breakpoint, was overexpressed only in CALM-AF10+ T-ALL and not in MLL-t AL. BMI1 controls cellular proliferation through suppression of the tumor suppressors encoded by the CDKN2A locus. This locus, often deleted in T-ALL, was conserved in CALM-AF10+ T-ALL. This suggests that decreased CDKN2A activity, as a result of BMI1 overexpression, contributes to leukemogenesis in CALM-AF10+ T-ALL. We propose to define a HOXA+ leukemia group composed of at least MLL-t, CALM-AF10 and HOXA-t AL, which may benefit from adapted management. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor about the identification of e19a2 BCR-ABL fusions at the DNA level by ligation-mediated polymerase chain reaction.

It is now well established that gene expression profiling using DNA microarrays can provide novel information about various types of hematological malignancies, which may lead to identification of novel diagnostic markers. However, to successfully use microarrays for this purpose, the quality and reproducibility of the procedure need to be guaranteed. The quality of microarray analyses may be severely reduced, if variable frequencies of nontarget cells are present in the starting material. To systematically investigate the influence of different types of impurity, we determined gene expression profiles of leukemic samples containing different percentages of nonleukemic leukocytes. Furthermore, we used computer simulations to study the effect of different kinds of impurity as an alternative to conducting hundreds of microarray experiments on samples with various levels of purity.As expected, the percentage of erroneously identified genes rose with the increase of contaminating nontarget cells in the samples. The simulations demonstrated that a tumor load of less than 75%can lead to up to 25%erroneously identified genes. A tumor load of at least 90%leads to identification of at most 5%false-positive genes. We therefore propose that in order to draw well-founded conclusions, the percentage of target cells in microarray experiment samples should be at least 90%.Leukemia (2005) 19, 618-627. doi:10.1038/sj.leu.2403685 Published online 3 March 2005 [ABSTRACT FROM AUTHOR]

T-cell acute lymphoblastic leukemia (T-ALL) is associated with chromosomal aberrations characterized by juxtaposition of proto-oncogenes to T-cell receptor gene loci (TCR), resulting in the deregulated transcription of these proto-oncogenes. Here, we describe the molecular characterization of a novel chromosomal aberration, inv(14)(q11.2q32.31), in a T-ALL sample, involving the recently described BCL11B gene and the TCRD locus. The inversion joined the 5'part of BCL11B, including exons 1-3, to the TRDD3 gene segment of the TCRD locus, whereas the reciprocal breakpoint fused the TRDV1 gene segment to the fourth exon of BCL11B. The TRDV1-BCL11B joining region was 1344?bp long and contained fragments derived from 20q11.22, 3p21.33 and from 11p12, indicating the complex character of this aberration. A strong expression of in-frame transcripts with truncated BCL11B and TCRD constant region (TRDC) were observed, but in contrast to normal T cells and other T-ALL samples, no wild-type BCL11B transcripts were detected in the T-ALL sample. Screening of 37 other T-ALLs revealed one additional case with expression of the BCL11B-TRDC fusion transcript. As BCL11B appears to play a key role in T-cell differentiation, BCL11B disruption and disturbed expression may contribute to the development of T-cell malignancies in man.Leukemia (2005) 19, 201-208. doi:10.1038/sj.leu.2403619 [ABSTRACT FROM AUTHOR]

Using the multiplex PCR tubes of the BIOMED-2 Concerted Action, TCRB gene rearrangements were detected in 35%of childhood (n=161) and adult (n=172) precursor-B-ALL patients (Vß-(Dß)-Jßin 25%; Dß-Jßin 15%). The presence of TCRB rearrangements showed a significant relation with age (highest frequency of 46%between 5 and 10 years of age) and the presence of TEL-AML1 transcripts, and was associated with relatively high frequencies of IGK-Kde, TCRG, and Vd2-Jarearrangements. In 62 out of 65 patients with Southern blot-detected Vß-(Dß)-Jßand/or Dß-Jßrearrangements, at least one TCRB gene rearrangement was detected by PCR. Based on combined Southern blot and PCR analysis, oligoclonal TCRB gene rearrangements were observed in only 12%of patients. Analysis of paired diagnosis and relapse samples (n=26) showed that 20 out of 24 (83%) Vß-(Dß)-Jßrearrangements and eight out of 14 (57%) Dß-Jßrearrangements remained stable. Using real-time quantitative PCR, a quantitative range?10-4 was obtained in 64%of TCRB gene rearrangements and in 86%of cases a sensitivity?10-4 was obtained. In conclusion, TCRB gene rearrangements occur in 35%of precursor-B-ALL patients and are relatively stable and sensitive PCR targets for detection of minimal residual disease, particularly if this concerns complete Vß-(Dß)-Jßrearrangements.Leukemia (2004) 18, 1971-1980. doi:10.1038/sj.leu.2403505 Published online 7 October 2004 [ABSTRACT FROM AUTHOR]

Chromosome aberrations are frequently observed in precursor-B-acute lymphoblastic leukemias (ALL) and T-cell acute lymphoblastic leukemias (T-ALL). These translocations can form leukemia-specific chimeric fusion proteins or they can deregulate expression of an (onco)gene, resulting in aberrant expression or overexpression. Detection of chromosome aberrations is an important tool for risk classification. We developed rapid and sensitive split-signal fluorescent in situ hybridization (FISH) assays for six of the most frequent chromosome aberrations in precursor-B-ALL and T-ALL. The split-signal FISH approach uses two differentially labeled probes, located in one gene at opposite sites of the breakpoint region. Probe sets were developed for the genes TCF3 (E2A) at 19p13, MLL at 11q23, ETV6 at 12p13, BCR at 22q11, SIL-TAL1 at 1q32 and TLX3 (HOX11L2) at 5q35. In normal karyotypes, two colocalized green/red signals are visible, but a translocation results in a split of one of the colocalized signals. Split-signal FISH has three main advantages over the classical fusion-signal FISH approach, which uses two labeled probes located in two genes. First, the detection of a chromosome aberration is independent of the involved partner gene. Second, split-signal FISH allows the identification of the partner gene or chromosome region if metaphase spreads are present, and finally it reduces false-positivity. [ABSTRACT FROM AUTHOR]

Gemtuzumab ozogamicin (Mylotarg) induces remission in approximately 30% of relapsed AML patients. We previously demonstrated that gemtuzumab infusion results in near-complete CD33 saturation in peripheral blood, and that saturating gemtuzumab levels result in continuous binding and internalization of gemtuzumab due to renewed CD33 expression. We now demonstrate that a high CD33-antigen load in peripheral blood is an independent adverse prognostic factor, likely due to peripheral consumption of gemtuzumab. Indeed, CD33 saturation in bone marrow is significantly reduced (40-90% saturation) as compared with CD33 saturation in corresponding peripheral blood samples (>90%). In vitro, such reduced CD33 saturation levels were strongly related with reduced cell kill. Apparently, high CD33-antigen loads in blood consume gemtuzumab and thereby limit its penetration into bone marrow. Consequently, CD33 saturation in bone marrow is reduced, which hampers efficient cell kill. Therefore, gemtuzumab should be administered at higher or repeated doses, or, preferably, after reduction of the leukemic cell burden by classical chemotherapy. [ABSTRACT FROM AUTHOR]

Current MRD studies in T-cell acute lymphoblastic leukemia (T-ALL) mainly use T-cell receptor gamma, delta and SIL-TAL1 gene rearrangements as MRD-PCR targets. However, low frequency or limited diversity of these markers restricts the number of evaluable patients, particularly because two markers are recommended for MRD monitoring. Hence, we developed a new strategy implementing the TCR beta (TCRB) locus for MRD quantification. The frequency and characteristics of complete and incomplete TCRB rearrangements were investigated in 53 childhood and 100 adult T-ALL patients using the BIOMED-2 multiplex PCR assay. Clonal rearrangements were identified in 92% both childhood and adult T-ALL (Vβ--Dβ--Jβ rearrangements in 80%, Dβ--Jβ rearrangements in 53%). Comparative sequence analysis of 203 TCRB recombinations revealed preferential usage of the 'end-stage' segment Jβ2.7 in childhood T-ALL (27%), whereas Jβ2.3 was most frequently involved in adult T-ALL (24%). In complete rearrangements, three downstream Vβ segments (19--&frac120;--&frac121;--1) were preferentially used. Subsequently, a TCRB real-time quantitative PCR assay to quantify MRD with 13 germline Jβ primer/probe combinations and allele-specific oligonucleotides was developed and applied to 60 clonal TCRB rearrangements. The assay allowed the detection of one leukemic cell within at least 104 polyclonal cells in 93% of cases and will be of high value for future MRD studies. [ABSTRACT FROM AUTHOR]

In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCR?d+ T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.Leukemia (2003) 17, 2257-2317. doi:10.1038/sj.leu.2403202 [ABSTRACT FROM AUTHOR]

A total of 28 children and nine adults with relapsed T-ALL were analyzed for the configuration of their T-cell receptor (TCR) and TAL1 genes at diagnosis and relapse to evaluate their stability throughout the disease course. A total of 150 clonal TCR and TAL1 gene rearrangements were identified in the 37 patients at diagnosis. In 65% of cases all rearrangements and in 27% of cases most rearrangements found at diagnosis were preserved at relapse. Two children with unusually late T-ALL recurrences displayed completely different TCR gene rearrangement sequences between diagnosis and relapse. This indicates that a proportion of very late T-ALL recurrences might represent second T-ALL. Specifically, 88% of clonal rearrangements identified at diagnosis in truly relapsed T-ALL were preserved at relapse. This is significantly higher as compared to previously studied precursor-B-ALL (~70%). Thus, from biological point of view, immunogenotype of T-ALL is more stable as compared with precursor-B-ALL. The overall stability of TCR gene rearrangements was higher in adult T-ALL (97%) than in childhood T-ALL (86%). Based on the stability of TCR gene rearrangements, we propose a strategy for PCR target selection (TCRD+TAL1 ? TCRB ? TCRG), which probably allows reliable minimal residual disease detection in all T-ALL patients.Leukemia (2003) 17, 2149-2156. doi:10.1038/sj.leu.2403081 [ABSTRACT FROM AUTHOR]