- Email: [email protected]
Backtracking of ALL to cord blood
Leukemia Research 33 (2009) e107–e108
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Letter to the Editor Backtracking of ALL to cord blood To the Editor, While data on prenatal origin of most cases of childhood B-cell precursor-acute lymphoblastic leukaemia (BCP-ALL) are conclusive [1], reports on natural history of T-lineage ALL are rather scarce—only few T-ALL cases were published with documented presence of a leukaemia-specific marker already at birth [2]. In most ALL patients the rearrangements of immunoglobulin (Ig) and T-cell receptor (TCR) genes were used as markers for the leukaemia backtracking, however, the in utero origin of ALL was confirmed also by positive backtracking of various leukaemia-specific fusion genes (e.g. MLL/AF4, TEL/AML1, AML1/ETO) [3–5] or mutations (NOTCH1) [6]. The data on the prenatal origin of childhood leukaemias are based primarily on the studies of concordant cases in twins and on the retrospective scrutiny of neonatal blood spots (Guthrie cards). A very limited amount of available material for detailed analysis of the preleukaemic clone is the main disadvantage of these approaches. To our knowledge, only one paper describes the availability of the whole cord blood sample for the backtracking study [7]. We had an opportunity to analyse banked cord blood samples from two patients with childhood ALL; the work has been approved by the Ethical committee of University Hospital Motol and subjects/guardians gave informed consent to the study. The first case was hyperdiploid BCP-ALL diagnosed at 3.7 years of age with pre-B immunophenotype (CD10+, CD19+, CD34+, CD38+, cytoplasmatic IgM+), DNA index 1.58 and gain of chromosomes 4, 7, 9, 12, 16, 17, 18, 20, 21 and X. The second child was diagnosed at the age of 4.9 with T-ALL presenting with only 30% of blasts in the bone marrow and suffered a very early relapse of the disease (90% of blasts) 11 months later with all the Ig/TCR markers and CDKN2A deletion (the only copy number change detected using comparative genomic hybridisation) maintained between the diagnosis and relapse. The patient was negative for all other T-ALL recurrent changes tested (TCR, HOX11, HOX11L2, LMO2, MLL, BCR/ABL, SIL/TAL, CALM/AF10 translocations, ABL1 amplification) with the exception of NOTCH1 mutation (deletion of 6 nucleotides in exon 26) present at the relapse of the disease. In both cases we performed an extensive search for all available clonotypic markers usable for the backtracking of the preleukaemic clone into the cord blood. In the hyperdiploid ALL we were finally able to use two immunoglobulin heavy chain gene (IGH) rearrangements (VH3-JH4 and VH1-JH4) and one rearrangement of TCR-delta (TCRD) with adequate sensitivity (1 leukaemic cell in 10,000–30,000 other cells). For the T-ALL case we optimised two TCR systems (TCRD and TCR-beta (TCRB). Moreover, the NOTCH1 mutation-specific PCR was established from the relapse sample. However, the diagnostic sample proved to be NOTCH1 mutationnegative (even after purification of the sample using cell sorting resulting in >99% purity of the leukaemic population). Similarly all follow-up specimens preceding relapse were negative for NOTCH1 0145-2126/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2009.01.013
mutation. Thus we did not use the NOTCH1 mutation for the cord blood analysis. In the hyperdiploid case we were able to confirm the presence of VH3-JH4 IGH rearrangement in the cord blood using quantitative PCR. The rearrangement was present at the sensitivity limit of the system (3 × 10−5 ). PCR for other rearrangements were negative, however, their sensitivity was slightly lower compared to VH3-JH4 system. In the T-ALL case, none of the two TCR rearrangements were detected in the cord blood. To further analyse the positive (hyperdiploid) case and to determine the immunophenotype of the preleukaemic clone we sorted the cord blood sample into different subpopulations—CD34+ stem cells (CD45+, CD34+, CD3−), more mature B-lineage committed cells (CD19+, CD45+, CD34−, CD3−) and (as a negative internal control) T-lineage subpopulation (CD19−, CD45+, CD34−, CD3+). We used FISH to examine the presence of trisomy of chromosome 7 (in this case the least typical extra chromosome for hyperdiploid ALL [8]) in these subpopulations. To determine the cut-off level for false negativity we screened 9000 cells from the same subpopulations in three cord bloods of healthy newborns. While the level of cells with trisomy of chromosome 7 in healthy cord bloods and also in the T-lineage subpopulation of the patient’s cord blood was 15 positive cells in 12,000 tested (control cord bloods: CD34+ fractions 4/3000, CD19+ fractions 3/4000, CD3+ fractions 6/4000; patient’s cord blood: CD3+ fraction 2/1000), we found 20/2000 cells in the CD34+ cord blood subpopulation of the hyperdiploid ALL patient (>3 standard deviations above the control). Importantly, we did not find any positive cell in 1000 of the pre-B (CD19+, CD34−) cells of the patient’s cord blood. Our results confirmed the prenatal origin of hyperdiploid ALL and we show that the preleukaemic cells reside in the early CD34+ subpopulation. While the presence of leukaemia-specific Ig/TCR rearrangements at birth was demonstrated repeatedly [7,9–11], the more detailed specification of the positive cells was shown only in the study by Maia et al. [7] proving unequivocally B-lineage commitment of the preleukaemic clone. We used different strategy for the analysis and we confirmed the immature phenotype (CD34+) of the preleukaemic cells. Our data strongly support the hypothesis that the preleukaemic hyperdiploid cells reside in the stem cell population and their maturation into more committed B-cell progenitors is very limited. Despite the availability of the cord blood material and very good sensitivity of PCR systems we were not able to prove a prenatal origin of T-ALL case. There are three possible explanations: (i) the leukaemia is not initiated during in utero development, (ii) the markers we have chosen for analysis are not specific for the preleukaemic cells, or (iii) the size of the preleukaemic clone at birth is too small to detect it with the PCR sensitivity. However, our detailed analysis of the case revealed unusual combination of biological and clinical features of this ALL. The patient presented with an “oligoblastic” ALL which became resistant to the standard chemotherapy with high MRD levels detected in all samples tested during the follow-up. The only additional change we
e108
Letter to the Editor / Leukemia Research 33 (2009) e107–e108
detected between diagnosis and very early relapse of the disease was the mutation of NOTCH1. Thus, we can speculate that this secondary change led to the aggressive course of this leukaemia. Conflict of interest statement None. Acknowledgements We would like to thank to Eva Fronkova (CLIP) and Dario Papi (TIB MOLBIOL) for their help with optimisation of Ig/TCR and NOTCH1 mutation-specific PCRs, respectively. This work was supported by grants from Charles University (GAUK 65/2006) and Czech Ministry of Education (MSM0021620813). Contributions: J. Zuna led the study and wrote the manuscript. Z. Prouzova was responsible for the molecular backtracking. T. Kalina carried out cell sorting of cord bloods. L. Lizcova and Z. Zemanova performed cytogenetics and FISH experiments. K. Muzikova analysed the Ig/TCR rearrangements. S. Rahmatova processed the cord bloods. J.P.P. Meijerink performed molecular analysis of the T-ALL case and J. Trka designed the study and participated in writing the manuscript. References [1] Greaves M. In utero origins of childhood leukaemia. Early Hum Dev 2005;81(January (1)):123–9. [2] Fischer S, Mann G, Konrad M, Metzler M, Ebetsberger G, Jones N, et al. Screening for leukemia- and clone-specific markers at birth in children with T-cell precursor ALL suggests a predominantly postnatal origin. Blood 2007;110(October (8)):3036–8. [3] Gale KB, Ford AM, Repp R, Borkhardt A, Keller C, Eden OB, et al. Backtracking leukemia to birth: identification of clonotypic gene fusion sequences in neonatal blood spots. Proc Natl Acad Sci USA 1997;94(December (25)):13950– 4. [4] Wiemels JL, Cazzaniga G, Daniotti M, Eden OB, Addison GM, Masera G, et al. Prenatal origin of acute lymphoblastic leukaemia in children. Lancet 1999;354(October (9189)):1499–503. [5] Wiemels JL, Xiao Z, Buffler PA, Maia AT, Ma X, Dicks BM, et al. In utero origin of t(8;21) AML1-ETO translocations in childhood acute myeloid leukemia. Blood 2002;99(May (10)):3801–5. [6] Eguchi-Ishimae M, Eguchi M, Kempski H, Greaves M. NOTCH1 mutation can be an early, prenatal genetic event in T-ALL. Blood 2008;111(January (1)):376–8.
[7] Maia AT, Tussiwand R, Cazzaniga G, Rebulla P, Colman S, Biondi A, et al. Identification of preleukemic precursors of hyperdiploid acute lymphoblastic leukemia in cord blood. Genes Chrom Cancer 2004 May;40(1):38–43. [8] Moorman AV, Richards SM, Martineau M, Cheung KL, Robinson HM, Jalali GR, et al. Outcome heterogeneity in childhood high-hyperdiploid acute lymphoblastic leukemia. Blood 2003;102(October (8)):2756–62. [9] Gruhn B, Taub JW, Ge Y, Beck JF, Zell R, Hafer R, et al. Prenatal origin of childhood acute lymphoblastic leukemia, association with birth weight and hyperdiploidy. Leukemia 2008;22(September (9)):1692–7. [10] Maia AT, van der Velden VH, Harrison CJ, Szczepanski T, Williams MD, Griffiths MJ, et al. Prenatal origin of hyperdiploid acute lymphoblastic leukemia in identical twins. Leukemia 2003;17(November (11)):2202–6. [11] Panzer-Grumayer ER, Fasching K, Panzer S, Hettinger K, Schmitt K, StocklerIpsiroglu S, et al. Nondisjunction of chromosomes leading to hyperdiploid childhood B-cell precursor acute lymphoblastic leukemia is an early event during leukemogenesis. Blood 2002;100(July (1)):347–9.
Jan Zuna a,∗ Zuzana Prouzova a Tomas Kalina a Libuse Lizcova b Zuzana Zemanova b Katerina Muzikova a Sarka Rahmatova c Jules P.P. Meijerink d Jan Trka a a CLIP (Childhood Leukaemia Investigation Prague), Department of Paediatric Haematology and Oncology, Charles University Prague, 2nd Faculty of Medicine, V Uvalu 84, 150 06 - Prague 5, Czech Republic b Center of Oncocytogenetics, Institute of Clinical Biochemistry and Laboratory Diagnostics, General Faculty Hospital and Charles University Prague, 1st Faculty of Medicine, Prague, Czech Republic c Cord Blood Bank, Institute of Haematology and Blood Transfusion, Prague, Czech Republic d Department of Paediatric Oncology/Haematology, Erasmus MC/Sophia Children’s Hospital, Rotterdam, The Netherlands ∗ Corresponding
author. Tel.: +420 224436580; fax: +420 224436521. E-mail address: [email protected] (J. Zuna) 19 December 2008 Available online 8 February 2009
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Letter to the Editor Backtracking of ALL to cord blood To the Editor, While data on prenatal origin of most cases of childhood B-cell precursor-acute lymphoblastic leukaemia (BCP-ALL) are conclusive [1], reports on natural history of T-lineage ALL are rather scarce—only few T-ALL cases were published with documented presence of a leukaemia-specific marker already at birth [2]. In most ALL patients the rearrangements of immunoglobulin (Ig) and T-cell receptor (TCR) genes were used as markers for the leukaemia backtracking, however, the in utero origin of ALL was confirmed also by positive backtracking of various leukaemia-specific fusion genes (e.g. MLL/AF4, TEL/AML1, AML1/ETO) [3–5] or mutations (NOTCH1) [6]. The data on the prenatal origin of childhood leukaemias are based primarily on the studies of concordant cases in twins and on the retrospective scrutiny of neonatal blood spots (Guthrie cards). A very limited amount of available material for detailed analysis of the preleukaemic clone is the main disadvantage of these approaches. To our knowledge, only one paper describes the availability of the whole cord blood sample for the backtracking study [7]. We had an opportunity to analyse banked cord blood samples from two patients with childhood ALL; the work has been approved by the Ethical committee of University Hospital Motol and subjects/guardians gave informed consent to the study. The first case was hyperdiploid BCP-ALL diagnosed at 3.7 years of age with pre-B immunophenotype (CD10+, CD19+, CD34+, CD38+, cytoplasmatic IgM+), DNA index 1.58 and gain of chromosomes 4, 7, 9, 12, 16, 17, 18, 20, 21 and X. The second child was diagnosed at the age of 4.9 with T-ALL presenting with only 30% of blasts in the bone marrow and suffered a very early relapse of the disease (90% of blasts) 11 months later with all the Ig/TCR markers and CDKN2A deletion (the only copy number change detected using comparative genomic hybridisation) maintained between the diagnosis and relapse. The patient was negative for all other T-ALL recurrent changes tested (TCR, HOX11, HOX11L2, LMO2, MLL, BCR/ABL, SIL/TAL, CALM/AF10 translocations, ABL1 amplification) with the exception of NOTCH1 mutation (deletion of 6 nucleotides in exon 26) present at the relapse of the disease. In both cases we performed an extensive search for all available clonotypic markers usable for the backtracking of the preleukaemic clone into the cord blood. In the hyperdiploid ALL we were finally able to use two immunoglobulin heavy chain gene (IGH) rearrangements (VH3-JH4 and VH1-JH4) and one rearrangement of TCR-delta (TCRD) with adequate sensitivity (1 leukaemic cell in 10,000–30,000 other cells). For the T-ALL case we optimised two TCR systems (TCRD and TCR-beta (TCRB). Moreover, the NOTCH1 mutation-specific PCR was established from the relapse sample. However, the diagnostic sample proved to be NOTCH1 mutationnegative (even after purification of the sample using cell sorting resulting in >99% purity of the leukaemic population). Similarly all follow-up specimens preceding relapse were negative for NOTCH1 0145-2126/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2009.01.013
mutation. Thus we did not use the NOTCH1 mutation for the cord blood analysis. In the hyperdiploid case we were able to confirm the presence of VH3-JH4 IGH rearrangement in the cord blood using quantitative PCR. The rearrangement was present at the sensitivity limit of the system (3 × 10−5 ). PCR for other rearrangements were negative, however, their sensitivity was slightly lower compared to VH3-JH4 system. In the T-ALL case, none of the two TCR rearrangements were detected in the cord blood. To further analyse the positive (hyperdiploid) case and to determine the immunophenotype of the preleukaemic clone we sorted the cord blood sample into different subpopulations—CD34+ stem cells (CD45+, CD34+, CD3−), more mature B-lineage committed cells (CD19+, CD45+, CD34−, CD3−) and (as a negative internal control) T-lineage subpopulation (CD19−, CD45+, CD34−, CD3+). We used FISH to examine the presence of trisomy of chromosome 7 (in this case the least typical extra chromosome for hyperdiploid ALL [8]) in these subpopulations. To determine the cut-off level for false negativity we screened 9000 cells from the same subpopulations in three cord bloods of healthy newborns. While the level of cells with trisomy of chromosome 7 in healthy cord bloods and also in the T-lineage subpopulation of the patient’s cord blood was 15 positive cells in 12,000 tested (control cord bloods: CD34+ fractions 4/3000, CD19+ fractions 3/4000, CD3+ fractions 6/4000; patient’s cord blood: CD3+ fraction 2/1000), we found 20/2000 cells in the CD34+ cord blood subpopulation of the hyperdiploid ALL patient (>3 standard deviations above the control). Importantly, we did not find any positive cell in 1000 of the pre-B (CD19+, CD34−) cells of the patient’s cord blood. Our results confirmed the prenatal origin of hyperdiploid ALL and we show that the preleukaemic cells reside in the early CD34+ subpopulation. While the presence of leukaemia-specific Ig/TCR rearrangements at birth was demonstrated repeatedly [7,9–11], the more detailed specification of the positive cells was shown only in the study by Maia et al. [7] proving unequivocally B-lineage commitment of the preleukaemic clone. We used different strategy for the analysis and we confirmed the immature phenotype (CD34+) of the preleukaemic cells. Our data strongly support the hypothesis that the preleukaemic hyperdiploid cells reside in the stem cell population and their maturation into more committed B-cell progenitors is very limited. Despite the availability of the cord blood material and very good sensitivity of PCR systems we were not able to prove a prenatal origin of T-ALL case. There are three possible explanations: (i) the leukaemia is not initiated during in utero development, (ii) the markers we have chosen for analysis are not specific for the preleukaemic cells, or (iii) the size of the preleukaemic clone at birth is too small to detect it with the PCR sensitivity. However, our detailed analysis of the case revealed unusual combination of biological and clinical features of this ALL. The patient presented with an “oligoblastic” ALL which became resistant to the standard chemotherapy with high MRD levels detected in all samples tested during the follow-up. The only additional change we
e108
Letter to the Editor / Leukemia Research 33 (2009) e107–e108
detected between diagnosis and very early relapse of the disease was the mutation of NOTCH1. Thus, we can speculate that this secondary change led to the aggressive course of this leukaemia. Conflict of interest statement None. Acknowledgements We would like to thank to Eva Fronkova (CLIP) and Dario Papi (TIB MOLBIOL) for their help with optimisation of Ig/TCR and NOTCH1 mutation-specific PCRs, respectively. This work was supported by grants from Charles University (GAUK 65/2006) and Czech Ministry of Education (MSM0021620813). Contributions: J. Zuna led the study and wrote the manuscript. Z. Prouzova was responsible for the molecular backtracking. T. Kalina carried out cell sorting of cord bloods. L. Lizcova and Z. Zemanova performed cytogenetics and FISH experiments. K. Muzikova analysed the Ig/TCR rearrangements. S. Rahmatova processed the cord bloods. J.P.P. Meijerink performed molecular analysis of the T-ALL case and J. Trka designed the study and participated in writing the manuscript. References [1] Greaves M. In utero origins of childhood leukaemia. Early Hum Dev 2005;81(January (1)):123–9. [2] Fischer S, Mann G, Konrad M, Metzler M, Ebetsberger G, Jones N, et al. Screening for leukemia- and clone-specific markers at birth in children with T-cell precursor ALL suggests a predominantly postnatal origin. Blood 2007;110(October (8)):3036–8. [3] Gale KB, Ford AM, Repp R, Borkhardt A, Keller C, Eden OB, et al. Backtracking leukemia to birth: identification of clonotypic gene fusion sequences in neonatal blood spots. Proc Natl Acad Sci USA 1997;94(December (25)):13950– 4. [4] Wiemels JL, Cazzaniga G, Daniotti M, Eden OB, Addison GM, Masera G, et al. Prenatal origin of acute lymphoblastic leukaemia in children. Lancet 1999;354(October (9189)):1499–503. [5] Wiemels JL, Xiao Z, Buffler PA, Maia AT, Ma X, Dicks BM, et al. In utero origin of t(8;21) AML1-ETO translocations in childhood acute myeloid leukemia. Blood 2002;99(May (10)):3801–5. [6] Eguchi-Ishimae M, Eguchi M, Kempski H, Greaves M. NOTCH1 mutation can be an early, prenatal genetic event in T-ALL. Blood 2008;111(January (1)):376–8.
[7] Maia AT, Tussiwand R, Cazzaniga G, Rebulla P, Colman S, Biondi A, et al. Identification of preleukemic precursors of hyperdiploid acute lymphoblastic leukemia in cord blood. Genes Chrom Cancer 2004 May;40(1):38–43. [8] Moorman AV, Richards SM, Martineau M, Cheung KL, Robinson HM, Jalali GR, et al. Outcome heterogeneity in childhood high-hyperdiploid acute lymphoblastic leukemia. Blood 2003;102(October (8)):2756–62. [9] Gruhn B, Taub JW, Ge Y, Beck JF, Zell R, Hafer R, et al. Prenatal origin of childhood acute lymphoblastic leukemia, association with birth weight and hyperdiploidy. Leukemia 2008;22(September (9)):1692–7. [10] Maia AT, van der Velden VH, Harrison CJ, Szczepanski T, Williams MD, Griffiths MJ, et al. Prenatal origin of hyperdiploid acute lymphoblastic leukemia in identical twins. Leukemia 2003;17(November (11)):2202–6. [11] Panzer-Grumayer ER, Fasching K, Panzer S, Hettinger K, Schmitt K, StocklerIpsiroglu S, et al. Nondisjunction of chromosomes leading to hyperdiploid childhood B-cell precursor acute lymphoblastic leukemia is an early event during leukemogenesis. Blood 2002;100(July (1)):347–9.
Jan Zuna a,∗ Zuzana Prouzova a Tomas Kalina a Libuse Lizcova b Zuzana Zemanova b Katerina Muzikova a Sarka Rahmatova c Jules P.P. Meijerink d Jan Trka a a CLIP (Childhood Leukaemia Investigation Prague), Department of Paediatric Haematology and Oncology, Charles University Prague, 2nd Faculty of Medicine, V Uvalu 84, 150 06 - Prague 5, Czech Republic b Center of Oncocytogenetics, Institute of Clinical Biochemistry and Laboratory Diagnostics, General Faculty Hospital and Charles University Prague, 1st Faculty of Medicine, Prague, Czech Republic c Cord Blood Bank, Institute of Haematology and Blood Transfusion, Prague, Czech Republic d Department of Paediatric Oncology/Haematology, Erasmus MC/Sophia Children’s Hospital, Rotterdam, The Netherlands ∗ Corresponding
author. Tel.: +420 224436580; fax: +420 224436521. E-mail address: [email protected] (J. Zuna) 19 December 2008 Available online 8 February 2009