haematologica online 2006
haematologica/the hematology journal | 2006; 91(1) | 29 |
Two consecutive immunophenotypic switches in a
child with MLL-rearranged acute lymphoblastic
An 18-month-old girl was diagnosed with pre-
pre-B ALL/t(4;11) leukemia, which during the
treatment and after matched bone marrow trans-
plantation (BMT), underwent two consecutive
switches from lymphoid to myeloid lineage and
vice versa. The high expression of HOXA9 and
FLT3 genes remaining genotypically stable in a
leukemia throughout phenotypic switches, sug-
gests that this leukemia may have originated as a
common B/myeloid progenitors.
Haematologica 2006; 91:(2)e29-e31
C Ca as se e r re ep po or rt t
An 18-month-old girl was admitted to the San
Giovanni Rotondo Casa Sollievo della Sofferenza IRCCS
Hospital presenting with a WBC of 201,000/mm3(80%
blasts), hemoglobin of 7.1 g/dL, and platelet count of
42,000/mm3. After obtaining informed consent from the
patient’s parents, a bone marrow (BM) aspirate was per-
formed. The sample was submitted to the University of
Padua’s Laboratory of Pediatric Onco-Hematology labo-
ratory, as National reference laboratory for all pediatric
leukemias enrolled in the AIEOP protocols, for morpho-
logic, cytochemical, cytogenetic, molecular, and flow
cytometric evaluations. The BM aspirate contained 95%
of tumor cells having the morphology of lymphoblasts
(French-American-British [FAB] L1) (Figure 1A). Flow
cytometric immunophenotypic analysis of blast cells
showed: CD19+, CD34+, CD45+, CD133+, CD135+,
NG2+, TdT+, HLA-DR (bright) (Table 1). Conventional
cytogenetic analysis revealed 46, XX, t(4;11)(q21;q23) in
all 20 metaphases analyzed. RT-PCR analysis showed the
presence 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 relevant
cytoreduction (4,000/mm3on day 5), the girl was
assigned to the high-risk group in accordance with the
AIEOP-BFM-ALL-2000 protocol. Thirteen days later her
WBC count increased a second time to 96,600/mm3, the
hemoglobin level was 9.8 g/dL, and platelets of
56,000/mm3. Peripheral blood and BM examination
revealed, respectively, 98% and 85% blasts characteristic
of monoblastic features and consistent with M5 mor-
phology. Cytochemistry was strongly positive for
myeloperoxidase (Figure 1B,C). Flow cytometric analysis
of BM showed: CD15 (bright), CD33 (bright), CD45+,
CD64+, CD135+, HLA-DR (dim) but CD19-, CD34-,
TdT- (Table 1). The results were consistent with a typical
AML (FAB M5) diagnosis. The translocation 46, XX,
t(4;11)(q21;q23) was still present (20 of 20 analyzed
metaphases). Consequently, her treatment was changed
to the AIEOP-AML-2002 protocol. Five months later,
after complete remission was achieved, she received allo-
geneic BMT from an HLA-matched normal donor. Two
months after the BMT, the patient relapsed again. The
leukemic cells displayed morphology (Figure 1D) and
immunophenotype (Table 1) of the initial diagnosis of
ALL. Cytogenetic analysis again revealed a 46, XX,
t(4;11)(q21;q23) in all 20 metaphases analyzed. She died
a few weeks later without responding to any further
DNA and RNA from BM samples were collected
throughout the disease course (diagnosis, myeloid phase
and relapse) of this patient. Samples were studied for
Ig/TCR clonality evaluation by PCR heteroduplex analy-
sis and for gene expression profiling. A heteroduplex and
homoduplex bands corresponding respectively to VH-JH
and VH-JH gene rearrangements were identified from the
diagnosis, 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.1Additional RT-PCR analyses showed
Figure 1. Morphologic analysis of
the leukemia cells in bone mar-
row smears. (A) Blast cells at
diagnosis. (B) Blast cells at 13-
day when ALL switched to AML
and (C) cytochemical myeloper-
oxidase staining. (D) Blast cells
at relapse (Wright-Giemsa ×
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 relapse
RNA 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 analysis
of 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 a
12% non-denaturing polyacrylamide minigel. Mononuclear cells were separated by Ficoll-Paque centrifugation and DNA was extracted
and 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 RNA
Transcript 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 cloud
around the diagonal stronger the correlation among samples. The correlation between ALL-1 and ALL-2 is stronger than between ALL
and AML samples.
the presence of two MLL-AF4 amplified products also Download full-text
during the myeloid phase and relapse (Figure 2A). The
sequencing of these PCR products showed two alterna-
tively spliced MLL-AF4 transcripts joining MLL exon 10
to either AF4 exon 4 or exon 5. Gene expression analysis
confirmed the MLL signature for each disease phase ana-
lyzed (ALL and AML). Furthermore, a closer examination
of these genes showed a significant over-expression of
HOXA9 and FLT3 gene targets during all phases of the
acute leukemia (data not shown).
Several hypotheses have been suggested to explain lin-
eage conversion in acute leukemia, but its precise mech-
anism remains unclear.2,3We have described a case of an
acute leukemia in which the blast cells rapidly changed
lineage from pre-pre-B ALL to AML after 13 days of high-
risk AIEOP-BFM chemotherapy. The patient achieved a
complete remission by conventional AML-type treat-
ment which included BMT. Eight months later she
relapsed again and the blast cells showed a clear return to
lymphoid 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 each
disease phase while maintaining the same clonal relation-
ship. Re-examining the blast cells (85%) at day 13
showed that <1×10-3were CD34+/CD19+, indicating
that it was unlikely that this cells contaminated the
myeloid phase. Therefore, the CD34+/CD19+ lymphoid-
restricted cells at diagnosis were not fully B-lymphoid
committed but also able to differentiate into monocytoid
blasts retaining the Ig/TCR and MLL-AF4 leukemic-spe-
cific rearrangements. In addition, the expression of the
progenitor/stem cell-related markers, such as CD133 and
CD34, only during the lymphoid phase could indicate
that an immature lymphoid progenitor develops the
potential to address different lineages. Gene expression
studies of MLL-rearranged ALL demonstrate that these
leukemias represent a unique disease when compared to
other ALLs.4,5Moreover, the differences in gene expres-
sion support the hypothesis that the cell of origin of MLL
is an early hematopoietic progenitor with both myeloid
and B-lymphoid potential.4,6 In our patient this is further
supported by the high level expression of HOXA9 and
FLT3 genes in all phases of acute leukemia. HOXA9 and
FLT3 are expressed in early hematopoietic progenitors
and both are necessary for the appropriate expansion of
the hematopoietic stem cells.7-9Considering this hypoth-
esis together with our finding of related Ig/TCR and
t(4;11)/MLL-AF4 gene rearrangements during the lineage
switches, indicates that the leukemia population in our
patient could have originated from a common B/myeloid
progenitors with the capacity to differentiate into com-
mitted cells of either lymphoid or myeloid lineage. In
contrast, the translocation t(4;11) that leads to a MLL-
AF4 fusion gene has been preferentially associated with
B-cell phenotype lineage10, but in this case the leukemic
clone retains the possibility to induce both myeloid or
lymphoid gene expression, suggesting that an immature
progenitor/stem cell may be the target of the chromoso-
mal translocation. Therefore, an interpretation of this
case is that the MLL-rearranged leukemic clone is able to
differentiate as lymphoid and myeloid under therapeutic
effects by amplifying or suppressing the normal differen-
tiation programs for their survival/expansion.
In conclusion, this report confirms that some forms of
acute leukemia may arise from very immature cells
belonging to a common myeloid/lymphoid progenitor.
Moreover, it provides further information into the mech-
anism of leukemic lineage switches and underlines that it
could be useful to test new therapeutic protocols fit to
these particular severe leukemias.
Giuseppe Germano,1,2Martina Pigazzi,1Laura del Giudice,1
Marta Campo Dell’Orto1, Monica Spinelli,1Andrea Zangrando1,3,
Paolo Paolucci,4Saverio Ladogana,4Giuseppe Basso1
1Laboratory of Pediatric Onco-Hematology, Department of
Pediatrics, University Hospital of Padua, Padua, Italy, 2Division of
Oncology, Children's Hospital of Philadelphia, Philadelphia,
Pennsylvania, PA 19104, USA, 3Department of Chemical Process
Engineering, University of Padua, Padua, Italy, 4Department of
Hematology, IRCCS Casa Sollievo della Sofferenza Hospital, San
Giovanni Rotondo, Italy
Funding: Research supported by Fondazione Città della Speranza,
CNR, MURST ex 40% and 60%, and Associazione Italiana per
la Ricerca sul Cancro (AIRC).
Acknowledgments: The authors wish to thank Dr Emanuela
Frascella, Anna Leszl, Benedetta Accordi, Emanuela Giarin, Silvia
Disarò and Katia Polato.
Key word: Acute leukemia, switch, clonal Ig/TCR, MLL.
Correspondence: Giuseppe Basso,
Department of Pediatrics, University Hospital of Padua, via
Giustiniani 3, 35128 Padua, Italy.
Tel: +39-049-8211454. Fax: international +39-049-8211462.
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