An 18-month-old girl was diagnosed with prepre-B ALL/t(4;11) leukemia, which during thetreatment and after matched bone marrow transplantation(BMT), underwent two consecutiveswitches from lymphoid to myeloid lineage andvice versa. The high expression of HOXA9 andFLT3 genes remaining genotypically stable in aleukemia throughout phenotypic switches, suggeststhat this leukemia may have originated as acommon B/myeloid progenitors

A., Zangrando

G., Basso

G., Germano

L., Del Giudice

M., Campo Dellorto

M., Pigazzi

M., Spinelli

Paolucci, Paolo

S., Ladogana

English

G. GERMANO

M. PIGAZZI

L. DEL GIUDICE

M. CAMPO DELLORTO

M. SPINELLI

A. ZANGRANDO

PAOLUCCI, Paolo

S. LADOGANA

G. BASSO

Archivio istituzionale della ricerca - Università di Modena e Reggio Emilia

haematologica online 2006 haematologica/the hematology journal | 2006; 91(1) | 29 |Two consecutive immunophenotypic switches in achild with MLL-rearranged acute lymphoblasticleukemiaAn 18-month-old girl was diagnosed with pre-pre-B ALL/t(4;11) leukemia, which during thetreatment and after matched bone marrow trans-plantation (BMT), underwent two consecutiveswitches from lymphoid to myeloid lineage andvice versa. The high expression of HOXA9 andFLT3 genes remaining genotypically stable in aleukemia throughout phenotypic switches, sug-gests that this leukemia may have originated as acommon B/myeloid progenitors.Haematologica 2006; 91:(2)e29-e31Case reportAn 18-month-old girl was admitted to the SanGiovanni Rotondo Casa Sollievo della Sofferenza IRCCSHospital presenting with a WBC of 201,000/mm3 (80%blasts), hemoglobin of 7.1 g/dL, and platelet count of42,000/mm3. After obtaining informed consent from thepatient’s parents, a bone marrow (BM) aspirate was per-formed. The sample was submitted to the University ofPadua’s Laboratory of Pediatric Onco-Hematology labo-ratory, as National reference laboratory for all pediatricleukemias enrolled in the AIEOP protocols, for morpho-logic, cytochemical, cytogenetic, molecular, and flowcytometric evaluations. The BM aspirate contained 95%of tumor cells having the morphology of lymphoblasts(French-American-British [FAB] L1) (Figure 1A). Flowcytometric immunophenotypic analysis of blast cellsshowed: CD19+, CD34+, CD45+, CD133+, CD135+,NG2+, TdT+, HLA-DR (bright) (Table 1). Conventionalcytogenetic analysis revealed 46, XX, t(4;11)(q21;q23) inall 20 metaphases analyzed. RT-PCR analysis showed thepresence of two amplified MLL-AF4 fusion transcripts(Figure 2A). The diagnosis of pre-pre-B ALL was made.Considering the insufficient clearance of circulating blasts(36,000/mm3) on day 8 after a steroid prophase (pred-nisone poor response; PPR) and despite an initial relevantcytoreduction (4,000/mm3 on day 5), the girl wasassigned to the high-risk group in accordance with theAIEOP-BFM-ALL-2000 protocol. Thirteen days later herWBC count increased a second time to 96,600/mm3, thehemoglobin level was 9.8 g/dL, and platelets of56,000/mm3. Peripheral blood and BM examinationrevealed, respectively, 98% and 85% blasts characteristicof monoblastic features and consistent with M5 mor-phology. Cytochemistry was strongly positive formyeloperoxidase (Figure 1B,C). Flow cytometric analysisof BM showed: CD15 (bright), CD33 (bright), CD45+,CD64+, CD135+, HLA-DR (dim) but CD19-, CD34-,TdT- (Table 1). The results were consistent with a typicalAML (FAB M5) diagnosis. The translocation 46, XX,t(4;11)(q21;q23) was still present (20 of 20 analyzedmetaphases). Consequently, her treatment was changedto the AIEOP-AML-2002 protocol. Five months later,after complete remission was achieved, she received allo-geneic BMT from an HLA-matched normal donor. Twomonths after the BMT, the patient relapsed again. Theleukemic cells displayed morphology (Figure 1D) andimmunophenotype (Table 1) of the initial diagnosis ofALL. Cytogenetic analysis again revealed a 46, XX,t(4;11)(q21;q23) in all 20 metaphases analyzed. She dieda few weeks later without responding to any furthertreatment forms. DNA and RNA from BM samples were collectedthroughout the disease course (diagnosis, myeloid phaseand relapse) of this patient. Samples were studied forIg/TCR clonality evaluation by PCR heteroduplex analy-sis and for gene expression profiling. A heteroduplex andhomoduplex bands corresponding respectively to VH-JHand VH-JH gene rearrangements were identified from thediagnosis, myeloid phase and relapse samples (Figure 2B).The sequence analysis revealed an identical VH4-JH6, VH4-JH4 and Vγ11-Jγ1.3/2.3 junctions region between the spec-imens analyzed.1 Additional RT-PCR analyses showedFigure 1. Morphologic analysis ofthe leukemia cells in bone mar-row smears. (A) Blast cells atdiagnosis. (B) Blast cells at 13-day when ALL switched to AMLand (C) cytochemical myeloper-oxidase staining. (D) Blast cellsat relapse (Wright-Giemsa ×1000). G. Germano et al.| 30 | haematologica/the hematology journal | 2006; 91(1)Figure 2. (A) RT-PCR analysis of the MLL-AF4 fusion gene showing expression of two different products in diagnosis, 13-day and relapseRNA leukemic cells. Total RNA was extracted from BM cells of diagnosis, 13-day and relapse using TRIzol® (Life Technologies, NY, USA).Integrity of the RNA was assessed by capillary electrophoresis (Agilent Tecnologies, Palo Alto, CA, USA). (B) Heteroduplex PCR analysisof IGH and TCRG gene rearrangements. Blast cells at diagnosis as well as 13-day and relapse contained a biallelic heteroduplex (he)VH6-JH and a monoclonal homoduplex (ho) VÁIV-JÁ1.2/2.3 gene rearrangement. PCR products were detected by silver staining on a12% non-denaturing polyacrylamide minigel. Mononuclear cells were separated by Ficoll-Paque centrifugation and DNA was extractedand purified using Gentra kit (Gentra System, Minneapolis, MN, USA). Mw marker, molecular weight marker; Mock, negative control; nor-mal MNC, normal mononuclear cells. (C) Hierarchical clustering based on HG-U95Av2 expression data of diagnosis ALL (ALL-1), 13-day(AML) and relapse (ALL-2). The genes used in this analysis are the top 100 genes chosen by t-test statistic that are the most differenti-ated among the tumor samples. The normalized expression value for each gene is indicated by color, with red representing high expres-sion and blue representing low expression. cRNA was prepared according to the standard Affymetrix protocol (BioArray™ High Yield RNATranscript Labeling Kit; Enzo Diagnostics, Farmingdale, NY, USA). Expression values were determined using Affymetrix MAS 5.0 software.(D) Scatter plots indicating statistical correlations among the samples on the basis of their gene expression values. Tighter the dot cloudaround the diagonal stronger the correlation among samples. The correlation between ALL-1 and ALL-2 is stronger than between ALLand AML samples.the presence of two MLL-AF4 amplified products alsoduring the myeloid phase and relapse (Figure 2A). Thesequencing of these PCR products showed two alterna-tively spliced MLL-AF4 transcripts joining MLL exon 10to either AF4 exon 4 or exon 5. Gene expression analysisconfirmed the MLL signature for each disease phase ana-lyzed (ALL and AML). Furthermore, a closer examinationof these genes showed a significant over-expression ofHOXA9 and FLT3 gene targets during all phases of theacute leukemia (data not shown). Several hypotheses have been suggested to explain lin-eage conversion in acute leukemia, but its precise mech-anism remains unclear.2,3 We have described a case of anacute leukemia in which the blast cells rapidly changedlineage from pre-pre-B ALL to AML after 13 days of high-risk AIEOP-BFM chemotherapy. The patient achieved acomplete remission by conventional AML-type treat-ment which included BMT. Eight months later sherelapsed again and the blast cells showed a clear return tolymphoid B-cell phenotype. Analyzing their characteris-tics, as shown in Table 1, the Ig/TCR gene rearrange-ments and the cytogenetic t(4;11) abnormalities demon-strated that the leukemic cells switched throughout eachdisease phase while maintaining the same clonal relation-ship. Re-examining the blast cells (85%) at day 13showed that <1×10-3 were CD34+/CD19+, indicatingthat it was unlikely that this cells contaminated themyeloid phase. Therefore, the CD34+/CD19+ lymphoid-restricted cells at diagnosis were not fully B-lymphoidcommitted but also able to differentiate into monocytoidblasts retaining the Ig/TCR and MLL-AF4 leukemic-spe-cific rearrangements. In addition, the expression of theprogenitor/stem cell-related markers, such as CD133 andCD34, only during the lymphoid phase could indicatethat an immature lymphoid progenitor develops thepotential to address different lineages. Gene expressionstudies of MLL-rearranged ALL demonstrate that theseleukemias represent a unique disease when compared toother ALLs.4,5 Moreover, the differences in gene expres-sion support the hypothesis that the cell of origin of MLLis an early hematopoietic progenitor with both myeloidand B-lymphoid potential.4,6 In our patient this is furthersupported by the high level expression of HOXA9 andFLT3 genes in all phases of acute leukemia. HOXA9 andFLT3 are expressed in early hematopoietic progenitorsand both are necessary for the appropriate expansion ofthe hematopoietic stem cells.7-9 Considering this hypoth-esis together with our finding of related Ig/TCR andt(4;11)/MLL-AF4 gene rearrangements during the lineageswitches, indicates that the leukemia population in ourpatient could have originated from a common B/myeloidprogenitors with the capacity to differentiate into com-mitted cells of either lymphoid or myeloid lineage. Incontrast, the translocation t(4;11) that leads to a MLL-AF4 fusion gene has been preferentially associated withB-cell phenotype lineage10, but in this case the leukemicclone retains the possibility to induce both myeloid orlymphoid gene expression, suggesting that an immatureprogenitor/stem cell may be the target of the chromoso-mal translocation. Therefore, an interpretation of thiscase is that the MLL-rearranged leukemic clone is able todifferentiate as lymphoid and myeloid under therapeuticeffects by amplifying or suppressing the normal differen-tiation programs for their survival/expansion.In conclusion, this report confirms that some forms ofacute leukemia may arise from very immature cellsbelonging to a common myeloid/lymphoid progenitor.Moreover, it provides further information into the mech-anism of leukemic lineage switches and underlines that itcould be useful to test new therapeutic protocols fit tothese particular severe leukemias.Giuseppe Germano,1,2 Martina Pigazzi,1 Laura del Giudice,1Marta Campo Dell’Orto1, Monica Spinelli,1 Andrea Zangrando1,3,Paolo Paolucci,4 Saverio Ladogana,4 Giuseppe Basso11Laboratory of Pediatric Onco-Hematology, Department ofPediatrics, University Hospital of Padua, Padua, Italy, 2Division ofOncology, Children's Hospital of Philadelphia, Philadelphia,Pennsylvania, PA 19104, USA, 3Department of Chemical ProcessEngineering, University of Padua, Padua, Italy, 4Department ofHematology, IRCCS Casa Sollievo della Sofferenza Hospital, SanGiovanni Rotondo, ItalyFunding: Research supported by Fondazione Città della Speranza,CNR, MURST ex 40% and 60%, and Associazione Italiana perla Ricerca sul Cancro (AIRC).Acknowledgments: The authors wish to thank Dr EmanuelaFrascella, Anna Leszl, Benedetta Accordi, Emanuela Giarin, SilviaDisarò and Katia Polato.Key word: Acute leukemia, switch, clonal Ig/TCR, MLL.Correspondence: Giuseppe Basso, Department of Pediatrics, University Hospital of Padua, viaGiustiniani 3, 35128 Padua, Italy. Tel: +39-049-8211454. Fax: international +39-049-8211462. E-mail: giuseppe.basso@unipd.itReferences1. Germano G, del Giudice L, Palatron S, Giarin E, Cazzaniga G,Biondi A, Basso G. Clonality profile in relapsed precursor-B-ALL children by GeneScan and sequencing analyses.Consequences on minimal residual disease monitoring.Leukemia 2003;17:1573-82.2. Ridge SA, Cabrera ME, Ford AM, Tapia S, Risueno C, Labra S,et al. Rapid intraclonal switch of lineage dominance in congen-ital leukaemia with a MLL gene rearrangement. Leukemia1995; 9: 2023-263. Bierings M, Szczepanski T, van Wering ER, Willemse MJ,Langerak AW, Revesz T, van Dongen JJ. Two consecutiveimmunophenotypic switches in a child with immunogenotyp-ically stable acute leukaemia. Br J Haematol. 2001;113:757-62.4. Armstrong SA, Staunton JE, Silverman LB, Pieters R, den BoerML, Minden MD, et al. MLL translocations specify a distinctgene expression profile that distinguishes a unique leukemia.Nat Genet. 2002;30:41-7. 5. Kohlmann A, Schoch C, Dugas M, Schnittger S, HiddemannW, Kern W, Haferlach T. New insights into MLL generearranged acute leukemias using gene expression profiling:shared pathways, lineage commitment, and partner genes.Leukemia. 2005;19:953-64. 6. Armstrong SA, Korsmeyer SJ. Comparison of humangenomics and genetic models of cancer to identify novel ther-apeutic targets.Cell Cycle. 2003;2:408-9.7. Lawrence HJ, Christensen J, Fong S, Hu YL, Weissman I,Sauvageau G et al. Loss of expression of the HOXA-9 home-obox gene impairs the proliferation and repopulating ability ofhematopoietic stem cells. Blood. 2005;106:3988-94.8. Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesisand leukemia. Blood. 2002 100:1532-42.9. Ayton PM, Cleary ML. Transformation of myeloid progenitorsby MLL oncoproteins is dependent on Hoxa7 and Hoxa9.Genes Dev. 2003;17:2298-307. 10. Johansson B, Moorman AV, Haas OA, Watmore AE, CheungKL, Swanton S, Secker-Walker LM. Hematologic malignancieswith t(4;11)(q21;q23)--a cytogenetic, morphologic,immunophenotypic and clinical study of 183 cases. European11q23 Workshop participants. Leukemia. 1998;12:779-87.haematologica online 2006 haematologica/the hematology journal | 2006; 91(1) | 31 |

Two consecutive immunophenotypic switches in a child with MLL-rearranged acute lymphoblastic leukemia.

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Two consecutive immunophenotypic switches in a child with MLL-rearranged acute lymphoblastic leukemia.

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