This research program focusses on the diagnosis and classification of leukemias and malignant lymphomas as well as on the evaluation of treatment effectiveness during follow-up via detection of low frequencies of malignant cells, i.e. detection of ‘minimal residual disease’ (MRD). The research program combines molecular and cellular studies on normal and malignant hematopoiesis, particularly focusing on immature lymphoid differentiation. The research program consists of three main projects:

Normal and aberrant V(D)J recombination in leukemias and malignant lymphomas: basic aspects and diagnostic applications. Project leader: A.W. Langerak.

V(D)J recombination of immunoglobulin (Ig) and T-cell receptor (TCR) genes is a key process during early lymphoid differentiation, which is required to establish a broad repertoire of antigen-recognizing receptors. Although the V(D)J recombination process is tightly regulated, aberrant V(D)J recombination occurs, resulting in the coupling of Ig/TCR loci to oncogenes. As a consequence, the involved oncogene is transcriptionally deregulated, eventually resulting in a block in lymphoid differentiation. This differentiation arrest is postulated to lead to a pre-leukemic cell population, whereas multiple additional genetic hits will result in overt (acute) leukemias or lymphomas. In mature leukemias the exact TCR and Ig receptors are considered to reflect antigenic stimulation. Insight into normal and oncogenic TCR/Ig recombination events will thus shed light on the pathogenic mechanisms underlying leukemia formation. This fundamental knowledge can be translated into better prognostic classification and improved treatment stratification of lymphoid malignancies. As a direct spin-off, these studies might contribute to the identification of novel therapeutic targets.

Immunobiology of acute leukemia and treatment evaluation. Project leader: V.H.J. van der Velden. 

Diagnosis and classification of acute leukemias form the basis for identification of prognostic subgroups, which need different treatment protocols or different treatment arms. Most molecular techniques for detection of oncogenetic events are relatively slow, taking a few days up to a week, whereas flow cytometry is fast and accurate. Therefore we aim at innovation of flow cytometry for fast and accurate diagnosis and classification of acute leukemias by designing new software, novel 8-color antbody panels, and new techniques for detection of oncogenic events at the protein level.

Although most patients with acute leukemia achieve remission according to cytomorphological and clinical criteria, many patients relapse and die from their disease. Therefore sensitive techniques are needed to monitor the patients for the presence of MRD during and after treatment in order to obtain better insight into the in vivo effectiveness of treatment, with flow cytometric immunophenotyping using aberrant marker expression and detection of tumor-associated proteins or with molecular MRD techniques via PCR analysis of Ig/TCR gene rearrangements and chromosome aberrations. To more selectively kill leukemic cells, drug-conjugated antibodies can be used. Our studies aim at the identification of parameters that affect the efficacy of antibody-based therapies, like CD22 and CD33 antibodies.

Unraveling oncogenic processes in leukemias. Project leaders: dr. R.W. Hendriks and prof. dr. F.J.T. Staal.

Several genetic events can predispose early lymphoid cells to transformation into malignant cells. Abnormal expression and overexpression of specific genes can deregulate normal activation, proliferation, and apoptosis processes, which can form the basis of uncontroled selective outgrowth of the involved cells. A typical example is the aberrant expression of signaling molecules (Btk, Slp65, Gata3, Notch, etc). Another example is the unwanted activation of the LM02 gene in retroviral gene therapy studies.

Normal and aberrant V(D)J recombination in leukemias and malignant lymphomas: basic aspects and diagnostic applications.

• Implementation of the Ig/TCR BIOMED-2 multiplex PCR clonality assays in our routine diagnostics at Immunology, Erasmus MC plus in many laboratories worldwide. Development of guidelines illustrating performance, limitations and pitfalls of the Ig/ TCR BIOMED-2 multiplex PCR assays (van Dongen et al, Leukemia 2003; Sandberg et al, J Mol Diagn 2004, Van Krieken et al, Leukemia 2007; Evans et al, Leukemia 2007; Bruggemann et al, Leukemia 2007; Langerak et al, Leukemia 2007; Sandberg et al, Leukemia 2007; Langerak et al, Exp Opin Med Diagn 2007; Langerak,  Leuk Res 2008, Groenen et al, J Hematopathol 2008).
• Dissection of TCR V(D)J recombination during normal thymus development and identification of candidate key regulators in this process (Dik et al, J Exp Med 2005) 
• Identification of several novel TCR-associated chromosome aberrations in human T-ALL, including BCL11B, NKX2-5/CSX, MYB and BM1 partner genes (Pzrzybylski et al, Leukemia 2004, Pzrzybylski et al, Haematologica 2005; Clappier et al, 2007; Larmonie et al, 2008).
• Unravelling of the mechanisms involved in illegitimate TCR recombination and LMO2 oncogene activation in t(11;14)+ T-ALL. (Dik et al, Blood 2007)
• Immunophenotypical and molecular characterization of large cohorts of the rare CD4+ (Garrido et al, Blood 2007) and TCRγδ T-LGL (Sandberg et al, Leukemia 2006) leukemia types, underlining antigenic stimulation as important driving force in the pathogenesis of these mature leukemia types; demonstration that human CD4+ T-LGL leukemias recognize hCMV (Rodriguez-Caballero et al, Blood 2008).
• Evaluation of novel prognostic markers in human B-CLL (Van ‘t Veer, Haematologica 2006; Boonstra, Cytometry B 2006; Dijkstra, Leukemia 2009).
• Identification of a unique morphological spectrum of lymphomas in Nijmegen breakage syndrome (NBS) patients with a high frequency of consecutive lymphoma formation (Gladkowska-Dura et al, J Pathol 2008).

Immunobiology and diagnosis of acute leukemia and treatment evaluation.

• Development of new software for fully-integrated analysis of flow cytometric data in diagnosis and classification of hematological malignancies (Pedreira et al, Cytometry A 2008).
• Invention, development and clinical testing of a novel immunobead assay for detection of fusion proteins in lysates of leukemic cells (Weerkamp et al, Leukemia 2009).
• Immunoglobulin and T cell receptor gene rearrangements have been investigated in detail with respect to type of leukemia, fusion gene transcripts, age of the patient, and stability, thereby providing more insight in acute leukemia and allowing better selection of MRD-PCR targets. Data on infant ALL, hyperdiploid childhood ALL, t(12;21)-positive childhood ALL and t(4;11)-positive childhood ALL have been published (Panzer-Grümayer et al, Clin Cancer Res 2005; Jansen et al, Leukemia 2007; Mann et al, Leukemia 2007; Csinady et al, Leukemia 2009).
• Within the International BFM Study Group, PCR-based minimal residual disease (MRD) diagnostics for childhood acute lymphoblastic leukemia in a multi-center setting have been optimized (Van der Velden et al, Leukemia 2007). Within the European Study Group for MRD analysis in acute lymphoblastic leukemia (an international consortium of over 30 laboratories), guidelines for the interpretation of real-time quantitative PCR-based MRD data have been developed (Van der Velden et al, Leukemia 2007). Such guidelines are crucial for uniform interpretation of MRD data and for comparing different studies.
• Within the international Interfant-99 study, it was proven that detection of minimal residual disease after the first and second course of chemotherapy allows recognition of patients with good, intermediate, and poor prognosis (Van der Velden et al, Leukemia 2009). This information will be used to apply MRD diagnostics in the Interfant-06 protocol.
• Minimal residual disease diagnostics have been implemented in the DCOG-ALL10 protocol.
• The efficacy of several new drugs (e.g. dasatinib) in acute leukemia has been evaluated by minimal residual disease monitoring.
• A new BCR-ABL fusion gene transcript (e18a2) has been described (Van der Velden et al, Leukemia 2007). A reference material for BCR-ABL mRNA quantitation by RQ-PCR has been characterized (Saldanha et al, Leukemia 2007).
• Flowcytometric analysis of minimal residual disease in Dutch and British children with acute myeloid leukemia shows that the MRD level after the first treatment course is a strong and independent prognostic factor.
• Several parameters that affect the efficacy of gemtuzumab ozogamicin (an CD33-immunotoxin) have been identified. In collaboration with the Fred Hutchinson Cancer Research Center (Seattle), it was shown that CD33 expression and P-glycoprotein- mediated drug efflux inversely correlate and predict clinical outcome in patients with acute myeloid leukemia treated with gemtuzumab ozogamicin monotherapy (Walter et al, Blood 2007). In addition, in collaboration with the Institute for Medical BioMathematics (Israel), a mechanism-based pharmacokinetic model has been designed. This mathematical model can serve as a tool for optimizing treatment strategies and for prediction of individual responses (manuscript submitted).
• In collaboration with VUMC, Amsterdam, it was demonstrated that immunophenotyping of MDS patients allows identification of aberrancies in the myelomonocytic lineage not otherwise determined by cytomorphology. In addition, flow cytometry identified patients at risk for transfusion dependency and/or progressive disease independent of known risk groups, which might have impact on treatment decisions and the prognostic scoring system in the near future (Van de Loosdrecht et al, Blood 2008). Within the European LeukemiaNet, standardization of flowcytometric analysis of MDS patients has been initiated (Van de Loosdrecht et al, Haematologica 2009).
• In pair-wise diagnosis-relapse comparisons for ALL it was found that about half of relapses are due to 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. 

• Studies into the occurrence and mechanisms of illegitimate (oncogenic) recombinations plus their impact on leukemogenesis / lymphomagenesis will be further evaluated in the human immunodeficiency syndrome model (Nijmegen breakage syndrome, NBS) that shows an increased frequency of trans-rearrangements as well as an increased risk of lymphoma formation.
• TCR gene translocations are key aberrations in human T-cell acute lymphoblastic leukemia (T-ALL). Translocations will be studied in detail with respect to the presence of RSS-like elements around the breakpoints and/or other fragile sites in flanking sequences. Furthermore, using thymocyte (subsets) as a model the role of nuclear proximity and synchronous accessibility of TCR loci and oncogenes will be studied in much detail. Collectively, this will eventually shed light on the mechanism of translocation-mediated leukemogenesis in T-ALL. 
• Studies into the pathogenesis of mature, chronic B- and T-cell leukemias will be extended with a special focus on Ig / TCR-mediated antigenic stimulation including the involved antigens. Furthermore, since only limited information is available about oncogenic events in mature T-cell leukemias, such as T-cell large granular lymphocyte (T-LGL) leukemia, the transition of smoldering T-LGL proliferations into clinically malignant T-LGL leukemias plus the involved secondary genetic events will be another important research topic in our group.
• A protocol for PCR based MRD detection will be developed and applied in the context of a clinical trial on B-cell chronic lymphocytic leukemia (B-CLL) aiming to evaluate effectiveness of immunotherapy using a next generation CD20 antibody (start: end of 2009).
• Development of fully integrated 8-color antibody panels for advanced diagnosis of hematological malignancies (EuroFlow protocol).
• Design of immunobeads for detection of various types of fusion proteins in acute leukemias in a single-tube assay.
• MRD studies can identify specific risk group with high relapse rates, but several types of relapses remain difficult to predict: extramedullary relapses, relapses with lineage switch, and late T-ALL relapses. These three categories of “special” relapses will be studied in detail by analysis of the precise clonal relationship between diagnosis and relapse and by gene expression profiling for recognition of new diagnostic markers that can contribute to prediction of relapse (see also theme 3.6).
• We will investigate immature acute leukemia (undifferentiated leukemia, bi-phenotypic leukemia, bi-clonal leukemia, lineage-switch leukemia) in detail to identify factors involved in the processes of lineage commitment and lineage specification. Such analysis will not only provide better insight in normal hematopoiesis but will also improve the diagnostics of immature acute leukemia.
• MRD diagnostics (via RQ-PCR analysis of Ig/TCR gene rearrangements) will be implemented within the HOVON100 protocol for adult patients with ALL
• Recently a new immunotoxin (calicheamicin-conjugated CD22, CMC-544) has becoma available. In collaboration with Wyeth and the Department of pediatrics we will analyze the efficacy of this drug in children with ALL. In addition, we will try to identify parameters that can be modulated in order to increase the efficacy of CMA-544 and to develop diagnostic tools that may indicate which patients will benefit form CMC-544 therapy and what dosing schedule will be most optimal.
• Together with the Department of Hematology, we will evaluate how the diagnostics of patients with B-PLL can be improved To this end, cells from B-PLL patients of comparable malignancies (MCL, CLL) will be characterized in detail with respect to immunophenotype, gene expression profile, Ig/TCR gene rearrangement pattern, morphology, genetic abnormalities and clinical features. In addition, gene expression profiles of these patients will be compated with gene expression profiles of normal B-cell subsets to obtain more insight into the the differentiation stage of malignant transformation.
• Within the new HOVON protocol for patients with MDS, flow cytometric immunophenotyping will be performed both at intake as well as during and/or after therapy in order to evaluate the clinical significance of this assay in more detail.

1. Dik WA, Pike-Overzet K, Weerkamp F, De Ridder D, De Haas EF, Baert MRM, Van der Spek P, Koster EEL, Reinders MJT, Van Dongen JJM, Langerak AW, Staal FJT. New insights on human T-cell development by quantitative T-cell receptor gene rearrangement studies and gene expression profiling. J Exp Med 2005;201:1715-1723. IF 15.2 - 5%
2. Sandberg Y, Almeida J, Gonzalez M, Lima M, Barcena P, Szczepański T, Van Gastel-Mol EJ, Wind HK, Balanzategui A, Van Dongen JJM, San Miguel JF, Orfao A, Langerak AW. TCRgd⁺ large granular lymphocyte leukemias reflect the spectrum of antigen-selected normal TCRgd⁺ T-cells in peripheral blood. Leukemia 2006;20:503-513. IF 8.6 - 10%
3. Dik WA, Nadel B, Przybylski GK, Asnafi V, Grabarczyk P, Navarro JM, Verhaaf B, Schmidt CA, Macintyre EA, Van Dongen JJM, Langerak AW. Different chromosomal breakpoints impact LMO2 expression levels in T-ALL. Blood 2007;110:388-392. IF 10.4 - 5%
4. Rodriguez-Caballero A, Garcia-Montero AC, Barcena P, Almeida J, Ruiz-Cabello F, Taberno MD, Garrido P, Munoz-Criado S, Sandberg Y, Langerak AW, Balanzategui A, Orfao A. Expanded cells in monoclonal TCRab+/CD4+/Nka+/CD8-/+dim T-LGL lymphocytosis recognize hCMV antigens. Blood 2008;112:4609-4616. IF 10.4 - 5%
5. Walter RB, Gooley TA, van der Velden VHJ, Loken MR, van Dongen JJM, Flowers DA, Bernstein ID, Appelbaum FR. CD33 expression and P-glycoprotein-mediated drug efflux inversely correlate and predict clinical outcome in patients with acute myeloid leukemia treated with gemtuzumab ozogamicin monotherapy. Blood 2007;109:4168-4170. IF 10.4 - 5%
6. van der Velden VHJ, Cazzaniga G, Schrauder A, Hancock J, Bader P, Panzer-Grumayer ER, Flohr T, Sutton R, Cave H, Madsen HO, Cayuela JM, Trka J, Eckert C, Foroni L, Zur Stadt U, Beldjord K, Raff T, van der Schoot CE, van Dongen JJM; European Study Group on MRD detection in ALL (ESG-MRD-ALL). Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data. Leukemia. 2007;21:604-611. IF 8.6 - 10%
7. van der Velden VHJ, Corral L, Valsecchi MG, Jansen MWJC, de Lorenzo P, Cazzaniga G, Panzer-Grümayer ER, Schrappe M, Schrauder A, Meyer C, Marschalek R, lo Nigro L, Metzler M, Basso G, Mann G, den Boer ML, Biondi A, Pieters R, van Dongen JJM. Prognostic significance of minimal residual disease in infants with acute lymphoblastic leukemia treated within the Interfant-99 protocol. Leukemia 2009;23:1073-1079. IF 8.6 - 10%
8. Howe SJ, Mansour MR, Schwarzwaelder K, Bartholomae C, Hubank M, Kempski H, Brugman MH, Pike-Overzet K, Chatters SJ, de Ridder D, Gilmour KC, Adams S, Thornhill SI, Parsley KL, Staal FJT, Gale RE, Linch DC, Bayford J, Brown L, Quaye M, Kinnon C, Ancliff P, Webb DK, Schmidt M, von Kalle C, Gaspar HB, Thrasher AJ. Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J Clin Invest 2008;118:3143-3150. IF 16.6 - 5%
9. Pike-Overzet K, de Ridder D, Weerkamp F, Baert MR, Verstegen MM, Brugman MH, Howe SJ, Reinders MJ, Thrasher AJ, Wagemaker G, van Dongen JJM, Staal FJT. Ectopic retroviral expression of LMO2, but not IL2Rgamma, blocks human T-cell development from CD34+ cells: implications for leukemogenesis in gene therapy. Leukemia 2007;21:754-763. IF 8.6 - 10%
10. Weerkamp F, Dekking E, Ng YY, van der Velden VHJ, Wai H, Böttcher S, Brüggemann M, van der Sluijs AJ, Koning A, Boeckx N, Van Poecke N, Lucio P, Mendonça A, Sedek L, Szczepański T, Kalina T, Kovac M, Hoogeveen PG, Flores-Montero J, Orfao A, Macintyre E, Lhermitte L, Chen R, Brouwer-De Cock KA, van der Linden A, Noordijk AL, Comans-Bitter WM, Staal FJT, van Dongen JJM; EuroFlow Consortium. Flow cytometric immunobead assay for the detection of BCR-ABL fusion proteins in leukemia patients. Leukemia 2009;23:1106-1117. IF 8.6 - 10%