Download Chromosomal Aberration of the 11q23 Locus in Acute Leukemia

Hematopathology / 11Q23 ABERRATIONS AND MLL GENE STATUS IN ACUTE LEUKEMIA
Chromosomal Aberration of the 11q23 Locus in Acute
Leukemia and Frequency of MLL Gene Translocation
Results in 378 Adult Patients
M. Christina Cox, MD, PhD,1 Paola Panetta,1 Francesco Lo-Coco, MD,1 Giovanni Del Poeta, MD,1
Adriano Venditti, MD, PhD,1 Luca Maurillo, MD, PhD,1 M. Ilaria Del Principe, MD, PhD,1
Alessandro Mauriello, MD,2 Lucia Anemona, MD, PhD,2 Antonio Bruno,1 Carla Mazzone, MD,1
Paolo Palombo, MD,3 and Sergio Amadori, MD, PhD1
Key Words: MLL; 11q23; AML; Acute myeloblastic leukemia; ALL; Acute lymphoblastic leukemia; AL; Acute leukemia;
Chromosomal aberrations; FISH; Fluorescence in situ hybridization; 11q22~25
DOI: 10.1309/RX27R8GJQM330C22
Abstract
Structural abnormality of the 11q23 band (11q23+)
bearing the MLL gene translocation (MLL+) is a
recurrent chromosome change observed in 3% to 7% of
acute lymphoblastic leukemias and in 3% to 4% of
acute myeloblastic leukemias. The resolution of
conventional cytogenetics (CC) in detecting 11q23
rearrangement is limited when the translocative partner
has a telomeric location; furthermore, CC can barely
discriminate between true 11q23+/MLL+ and
rearrangements clustering within the 11q22~25 region
without MLL involvement (MLL–). We characterized a
series of 378 consecutive patients with adult acute
leukemia by using CC, fluorescence in situ
hybridization (FISH), and multiplex karyotyping (MFISH) analysis. Our aim was to define the frequency of
cryptic MLL+ cases and the frequency of MLL+ within
11q22~25+ cases. As expected, FISH was more
sensitive than CC in detecting MLL+ cases, but rather
unexpectedly, 9 (45%) of 20 patients with 11q22~25+
were MLL–. A better characterization of
11q22~25+/MLL– leukemias is relevant for the
identification of new, recurrent translocations.
Moreover, these cases should be readily distinguishable
from 11q23+/MLL+ cases. We recommend that
karyotypic analysis always be complemented by
molecular or FISH methods to unravel MLL
rearrangements.
298
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DOI: 10.1309/RX27R8GJQM330C22
Structural abnormality of the 11q23 band (11q23+)
bearing the MLL gene translocation (MLL+) is a recurrent chromosome change in leukemia described in acute myeloblastic
leukemia (AML) and in acute lymphoblastic leukemia (ALL),
with a peak incidence in infant leukemia.1,2 A proposal by the
World Health Organization specifies a separate category for
AML with 11q23+/MLL+.3 This notion has been supported
recently by biologic studies: microarray analyses have shown
that MLL+ acute leukemias (ALs) have a peculiar geneprofiling pattern that distinguishes them from all other ALs and
that MLL+ leukemic blasts resemble very immature progenitor
cells.4 Furthermore, these studies showed that MLL+ leukemias
are a separate entity when compared with AML with MLL
partial tandem duplication (MLL-PTD), a recently identified
genetic aberration observed in a sizable proportion of AMLs.5
Extensive cytogenetic and molecular studies have shown
that 11q23/MLL is a highly promiscuous locus: more than 50
chromosomal loci have been described as 11q23 chromosome partners, whereas more than 30 MLL partner genes
have been characterized.6 It also should be mentioned that
t(11q23) might involve genes other than MLL7 and that
conventional cytogenetics can barely discriminate between
true 11q23+/MLL+ and rearrangements clustering within the
11q22~25 region without MLL involvement.8
Because t(9;11)(p21;q23) bearing the MLL/AF9 gene
fusion in AML and t(4;11) with MLL/AF4 gene fusion in
infant leukemia are the most common types,6 these translocations often are referred to as classic translocation, whereas
all other variants are reported as v11q23.
11q23+/MLL+ is described in 3% to 4% of AML cases
and is more frequent in younger subjects with de novo (5%-7%)
AML or with t-AML (10%-15%) evolving after chemotherapy.
© American Society for Clinical Pathology
Hematopathology / ORIGINAL ARTICLE
In older patients with AML (60 years or older), it is observed
rarely.9 The majority of 11q23+/MLL+ AML cases have monocytoid differentiation features and are classified in the M4 and
M5 leukemia French-American-British (FAB) subtypes.10 In
adult ALL, the overall incidence of 11q23+/MLL+ is reported
to be around 3% to 7%,11 but in pro-B-cell ALL, it accounts for
more than 30% of chromosomal aberrations.12,13
While t(4;11) ALL has an established dismal prognosis,
the clinical outcome of 11q23+/MLL+ AML is more heterogeneous. 14-16 The Medical Research Council 14 and the
Southwest Oncology Group15 classify the risk for patients
with AML with t(9;11) as intermediate and poor, respectively. Even more disagreement surrounds the prognostic
relevance of classic t(9;11) vs v11q23: some clinical trials
reported that patients with t(9;11) fared better than patients
with v11q23,16,17 whereas other studies failed to identify
differences.9,14 These discrepancies probably reflect the
marked biologic heterogeneity of 11q23 aberrations. Furthermore, because many v11q23 translocations are rare translocations, the clinical impact of specific single variants is difficult to extrapolate, even from large studies on 11q23 AL.14-16
Recently, the combined use of conventional cytogenetics, reverse transcriptase–polymerase chain reaction (RTPCR), Southern blot analysis, and fluorescence in situ
hybridization (FISH) in limited AL series has revealed that
discrepant results with MLL involvement might become
evident,18 and a high incidence of patients with cryptic
MLL+ leukemia were observed in 2 pediatric series.19,20
Because the majority of clinical trials include only karyotype
data,14-16 it is reasonable to speculate that beyond biologic
heterogeneity, these discrepancies also are due partly to the
low accuracy of conventional cytogenetics.
We describe our findings in a series of 378 consecutive
cases of adult AL, studied with conventional cytogenetics,
FISH, and multiplex karyotyping (M-FISH) analysis. The
aim of the study was to define the incidence of cryptic MLL
gene translocation and the incidence of MLL gene rearrangement within 11q22~25+ cases.
Materials and Methods
Cases
Between November 1992 and September 2003, 478 AL
samples from newly diagnosed adult patients (older than 14
years) were sent to our laboratory for conventional cytogenetic analysis. Conventional cytogenetics was done
following standard methods, and residual pellets were stored
at –20°C in Carnoy solution. From January 2000, FISH
analysis with the MLL gene probe (Vysis, Downers Grove,
IL) was combined routinely with conventional cytogenetics
in newly diagnosed patients with AML and ALL (n = 170).
Furthermore, all residual archival AL samples (n = 208) also
were analyzed by FISH for MLL rearrangement. Overall,
378 samples from consecutive patients with newly diagnosed
AL were the basis of this study without further selection.
Leukemia was classified according to FAB criteria21,22 and
immunophenotyping of leukemic cells.23
Of the 378 cases, 327 (86.5%) were classified as AML,
47 (12.4%) as ALL, and 4 (1.1%) as biphenotypic leukemia.
Leukemia subtypes are summarized in ❚Table 1❚.
In 56 (17.1%) of 327 patients, AML had developed after
a primary malignancy; all ALL and B-cell AL cases were de
novo. The mean age was 58 years in patients with AML
(range, 14-81 years), 33 years in ALL (range, 14-74 years),
and 26 years in B-cell AL (range, 19-32 years).
All patients with AML who were eligible for intensive
chemotherapy were enrolled in consecutive trials of the
GIMEMA (Gruppo Italiano Malattie ematologiche
dell’adulto)–European Organization for Research and Treatment of Cancer cooperative group (AML8, AML10, AML12,
AML11, AML13, AML15). Patients younger than 60 years
who had an HLA-identical sibling donor underwent allogeneic bone marrow or peripheral blood transplantation. ALL
patients eligible for intensive treatment were enrolled in the
conventional induction regimen ALL 0288 (GIMEMA)24;
some patients with standard-risk and most with high-risk
disease were given intensive induction chemotherapy based
on high-dose cytarabine and mitoxantrone plus prednisone,
which, after a similar consolidation cycle, was followed by
autologous or allogeneic transplantation.25
❚Table 1❚
Distribution of Patients With AL in Subcategories and
Incidence of MLL+ and 11q22~25+/MLL– Cases Within
Different ALs*
Subcategory
No. of Patients
Acute myeloblastic leukemia
M0
27
M1
69
M2
82
M3
25
M4
47
M5a
30
M5b
30
M6
13
M7
4
Acute lymphoblastic leukemia
Pro-B cell
4
Pre-B cell, common
25
Burkitt
7
Pre-T cell
4
T cell
7
B-cell AL
4
Total
378
MLL+
11q22~25/MLL–
2 (7)
1 (1)
1 (1)
0 (0)
2 (4)
6 (20)
3 (10)
0 (0)
0 (0)
2 (7)
1 (1)
0 (0)
0 (0)
1 (2)
1 (3)
0 (0)
2 (15)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
15 (4.0)
0 (0)
1 (4)
0 (0)
0 (0)
0 (0)
1 (25)
9 (2.4)
AL, acute leukemia.
* Data are given as number (percentage). AMLs are listed according to the FrenchAmerican-British Classification.
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Conventional Cytogenetics
Samples obtained before February 1997 were cultured
at 24 and 48 hours without synchronization. Starting from
1997, 3 short-term cell cultures were set up from each
harvest: 2 synchronized cultures at 24 and 48 hours and 1
overnight Colcemid-treated culture (0.0025 µg/mL) to obtain
a high rate of mitotic cells. The synchronization procedure
was carried out by incubating cells with methotrexate for 17
hours (final concentration, 10–7 mol/L), and then thymidine
solution (final concentration, 10–5 mol/L) was added for 5
additional hours. Cells were exposed to Colcemid (0.05
µg/mL) for the last 15 minutes before centrifugation and
standard processing.26 In the ALL samples, direct preparations were carried out after 1 hour of Colcemid exposure.
Karyotypes were set up on GTG-banded chromosomes
following the 1995 International System for Human Cytogenetic Nomenclature.27 To define a structural clonal aberration, at least 2 cells with the same chromosomal change were
to be found and at least 3 abnormal metaphases had to be
identified to define chromosomal aneuploidy. In 89% of
cases with a normal karyotype, 20 or more metaphases were
analyzed, while in the remaining cases, at least 10
metaphases were scored.
Fluorescence In Situ Hybridization
FISH was done using commercial double-colored probe
sets (Vysis). The SpectrumGreen-labeled probe covers a
350-kilobase portion centromeric to the MLL gene breakpoint, and the SpectrumOrange-labeled probe, a 190-kilobase portion largely telomeric of the breakpoint cluster
region.
Cytogenetic pellets from direct or short-term cultures
were used for hybridization: 20 µL of cytogenetic pellet
fixed in Carnoy solution was dropped with a micropipette on
a cleaned slide. The slides then were aged for 20 minutes at
80°C on a hot plate and dehydrated at room temperature in
70%, 80%, and 100% ethanol (2 minutes each). Geneframes (Abgene, Epsom, England) were applied to dried
slides to mark and separate the hybridization areas of single
probes. Slides were placed on a hot plate at 37°C, and 2.5 µL
of each probe-buffer solution was applied inside the area of
the slide delineated by the frame (probes were prepared
following the manufacturer’s instructions). The slide was
covered with a plastic coverslip (Abgene) and placed in the
PCR In Situ 1000 (Perkin-Elmer, Fremont, CA) device or
the Hybrite machine (Vysis). Codenaturation was carried out
at 72°C for 5 minutes and hybridization at 42°C for 90
minutes. If overnight hybridization was preferred, the codenaturation was carried out at 68°C for 4 minutes and
hybridization at 37°C. Slides then were removed and the
coverslip discarded. Posthybridization washing was done at
71°C in 0.4× saline sodium citrate (SSC) for 2 minutes and
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300
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at room temperature in 2× SSC for 1 minute. Slides then
were counterstained with 4'-6'-diamidino-8-phenylindole
(DAPI), 0.1 µg/mL (Vysis), and analyzed using an Olympus
BX2 microscope (Olympus, Tokyo, Japan) equipped with a
100-W lamp and a complete set of filters.
In the first 80 samples, 200 nuclei were analyzed per
patient, per probe; in the following 311 samples, only 100 cells
per patient were recorded for each probe tested. The slides
were analyzed blindly by 2 experienced operators (M.C.C. and
P.P.) who were unaware of each other’s results and of the
conventional cytogenetics results. The cutoff value of the MLL
probe (Vysis) was predetermined as 4.3% (mean plus 3 SD in
20,000 nuclei from 20 control bone marrow samples). Whenever abnormalities were found, a second test and incidental
cohybridization with a control probe were carried out. In the
presence of mitotic cells, metaphase FISH analysis also was
done. Additional FISH with centromeric, telomeric, or other
locus-specific probes was done when necessary to refine chromosomal breakpoints or identify aneuploidies.
Multiplex Karyotyping
We analyzed 11 AL samples by M-FISH. In 7 AML
cases, M-FISH was done to characterize a complex karyotype or ill-defined aberrations. In the other 4 AML cases,
with normal conventional cytogenetic results, M-FISH was
performed to check for cryptic translocations. None of these
cases had apparent 11q aberrations.
The slides, after aging overnight at room temperature,
were treated with pepsin (0.005%) for 2.5 minutes at 37°C
and then were fixed in a 10% formaldehyde solution for 2
minutes at room temperature. The slides were washed for 5
minutes in phosphate-buffered saline, dehydrated in an
alcohol series (70%, 85%, 100%) for 1 minute, and dried at
room temperature. The slides were codenatured with the
SpectraVysion assay probe (Vysis) at 68°C for 5 minutes
and hybridized overnight at 37°C in the PCR In Situ 1000.
After 16 to 18 hours, slides were washed at 71°C in 0.4×
SSC/0.3% NP40 (Sigma-Aldrich, Milan, Italy) for 2
minutes, at room temperature in 2× SSC for 2 minutes, and
then dried and counterstained with DAPI III (Vysis). Fluorescent images were captured using an Olympus (Japan)
microscope equipped with a CCD camera. The images were
processed and analyzed using the QUIPS M-FISH program
(Applied Biosystems, Foster City, CA). A minimum of 5
metaphases were analyzed in each case and chromosomal
aberrations confirmed with additional hybridization using
specific whole-chromosome painting telomeric probes (QBiogene, Vysis) or region-specific probes (Q-Biogene).
Statistical Analysis
The χ2 test was used to determine differences between
variables in 2 × 2 tables. The Kaplan-Meier method was
© American Society for Clinical Pathology
Hematopathology / ORIGINAL ARTICLE
Results
Conventional Cytogenetics
Conventional cytogenetics showed clonal abnormalities
in 50.0% (189/378), a normal karyotype in 36.0% (136/378),
and failed in 14.0% (53/378) of the analyzed cases.
11q22~25 rearrangements were observed in 18 of 324 cases
(5.6%) with assessable conventional cytogenetics: 12 were
balanced translocations, and in 6 cases, there was no
evidence of a translocative partner chromosome ❚Table 2❚
and ❚Table 3❚.
Fluorescence In Situ Hybridization
FISH was carried out successfully in all samples
analyzed and revealed MLL+ in 15 (4.0%) of 378 cases. Of
the 18 cases showing rearrangement within the 11q22~25
bands by conventional cytogenetic analysis, only 11 had
MLL gene splitting (61%). Of 15 MLL+ samples, 4 (27%)
were missed by conventional cytogenetics. Two of these
were truly cryptic 11q23 rearrangements, add(16)(p13),
revised to t(16;11)(p13;q23) and t(10;11)(p13;q13). The
latter case had an unusual FISH pattern showing 2 yellow
spots and 1 green signal in interphase cells that was interpreted as MLL insertion.28 The 2 remaining MLL+ cases had
no metaphases suitable for evaluation. In 4 additional cases,
FISH detected abnormalities of the MLL gene pattern as
follows: 1 with MLL gene amplification; 1 with an extra copy
of the MLL gene owing to a cryptic unbalanced translocation
(Table 3) that was detected with M-FISH; 2 with an extra
copy of the MLL gene in which trisomy 11 was disclosed by
additional hybridization with a chromosome 11 centromeric
probe (no assessable conventional cytogenetics analysis).
Multiplex Karyotyping
M-FISH was carried out successfully in 11 cases and
identified 2 cases with an undetected 11q translocation,
respectively, t(9;11)(p21~22;q23~24), and an unbalanced
der(8)t(8;11)(p2?;q21~22). The latter case was mentioned in
the preceding section because FISH analysis showed an extra
copy of the MLL gene ❚Image 1❚.
Characteristics of Patients in MLL+ Cases
We did not find MLL+ cases in ALL (0/47) or B-cell AL
(0/4); all MLL+ cases were classified as AML. The overall
incidence of MLL+ in patients with AML was 4.6%
(15/327). All MLL+ cases involved younger patients
(younger than 60 years) with a mean age of 44 years (range,
24-57 years; mean age for all 327 patients with AML, 58
years; range, 14-81 years). Considering only the younger
patients, the incidence of MLL+ was 8.3% (13/157) in de
novo AML and 11% (2/19) in t-AML. Of the 2 patients with
t-AML, AML-M5a developed in one 18 months after
chemotherapy containing topoisomerase-II inhibitors for
testicular seminoma (unique patient number [UPN], 97111),
while the disease in the other evolved into AML-M0 after a
myelodysplastic phase diagnosed 8 months before (UPN,
03589).
The incidence of MLL+ cases was 7% in AML-M0 (n =
2), 6% in AML-M4 (n = 2), 20% in AML-M5a (n = 6), 10%
in AML-M5b (n = 3), 1% in AML-M1 and AML-M2 (n = 1
each), and 0% in other FAB subgroups (Table 1). In total,
73% of MLL+ cases (11/15) showed involvement of the
monocytic lineage. The partner chromosomes of the MLL
gene were as follows: 9p22 (2 [13%]); 10p12 (3 [20%]);
17q21-25 (2 [13%]); 19p13 (2 [13%]); 4q21, 6q22, 16p13,
22q13 (1 case each [7%]); and unknown (2 [13%]).
Clinical follow-up data were available for all 15 MLL+
cases. Complete remission (CR) was achieved in 9 patients
(60%); 5 (33%) had resistant disease, and 1 patient died
during induction chemotherapy. After a mean follow-up of
14.6 months, 9 patients died (60%). Of the remaining 6
patients, follow-up was very short for 2 (Table 2), and the
other 4 were in prolonged, continuous CR after having
undergone autotransplantation (n = 2) or allotransplantation
(n = 2) during the first (n = 3) or second (n = 1) CR. Of
these 4 patients (Table 3), 1 had a classic t(9;11), and the
100
MLL+
Unfavorable
Intermediate
Favorable
90
80
70
Percent
used to calculate the survival curves and the log-rank test to
determine significance ❚Figure 1❚.
60
50
40
30
20
10
0
0
20
40
60
80
100
Time (mo)
120
140
160
❚Figure 1❚ Cumulative proportion of surviving patients
(Kaplan-Meier survival curves). Patients were grouped
according to cytogenetic or fluorescence in situ hybridization
data in 4 classes (favorable, intermediate, unfavorable, and
MLL+). The log-rank test was used to determine
significance. Patients older than 60 years (MLL+, 0) or
diagnosed with t-AML (MLL+, 2) were excluded from the
computation. circles, complete data; +, censored.
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❚Table 2❚
Clinical and Cytogenetic Features of 15 Patients With MLL+ Acute Leukemia
UPN/Sex/Age (y) AML Type*
Secondary
98257/F/41
97111/M/32
M0
M5a
No
Yes (CHT)
97210/M/50
98079/F/44
97198/F/24
01424/M/44
96259/F/57
97212/M/24
M5a
M4
M5a
M1
M5b
M2
No
No
No
No
No
No
03535/M/51
03589/F/51
96334/F/38
97222/F/41
95078/M/28
98124/M/51
97025/F/56
M5b
M0
M5a
M5a
M5a
M5b
M4
No
Yes (MDS)
No
No
No
No
No
Karyotype
MLL Localization
CR
OS (mo)
4q21;11q23
11q23; 9p2?
Yes
Yes
14
10
9p22; 11q23
10p12;11q23
NE
11q23; 17q2?
11q23; 17q25
11q23; 19p13
Yes
No
No
Yes
Yes
Yes
+57
8
20
+24
+77
+56
11q23; 22q13
11q23; 16p13
6q22; 11q23
10p11-12; 11q23
11q23;19p13
NE
NE
Yes
Yes
No
Yes
No
NE
No
+1
+4
7
12
1
1
1
47,XX,t(4;11)(q21;q23),+m[6]
47,XY,t(9;11)(p21;q23),+der(8)t(8;22)
(q24;q12)c[25]
46,XY,t(9;11)(p22;q23)[12]
46,XX,t(10;11)(p12;q23)[25]
46,XX,t(10;11)(p13;q13)[8]/46,XX[2]
46,XY,t(11;17)(q23;q2?)[15]/[20]
46,XX,t(11;17)(q23;q24)[20]/46,XX[5]
46,XY,t(11;19)(q23;p13)/47,XY,t(11;19)
(q23;p13),+19/46,XY
46,XYt(11;22)(q23;q13)[20]
46,XX,add(16)(p13)[6]
46,XX,add(11)(q23)[3]/46,XX[4]
46,XX,add(11)(q23)[12]
46,XY,del(11)(q23)[11]
—
—
AML, acute myeloblastic leukemia; CHT, chemotherapy; CR, complete remission; MDS, myelodysplastic syndrome; NE, not evaluable; OS, overall survival; UPN, unique
patient number.
* Diagnoses are listed according to the French-American-British Classification.
❚Table 3❚
Clinical and Cytogenetic Features of 9 Patients With 11q22~25+/MLL– Acute Leukemia
UPN/Sex/Age(y)
Diagnosis
Secondary
95056/M/45
98230/F/24
99042/F/62
98207/F/14
00383/M/33
02058/F/58
AML-M6
B-cell AL
AML-M0
AML-M1
AML-M0
ALL, pre-B cell
Yes (MDS)
No
No
Yes (CHT)
No
No
95035/M/59
AML-M6
No
02418/M/40
AML-M4
Yes (MDS)
02061/F/46
AML-M5a
No
Karyotype
Breakpoint
46,XY,t(2;11)(p21;q23)[30]
46,XX,t(11;16)(q23;p13)[6]/46,XX[4]
46,XX,t(11;12)(q23;q24)[20]/46,XX[4]
46,XX,t(11;15)(q23;q12)[15]
46,XY,add(1)(q42),del(11)(q13q23),del(12)(p13)
46,XX,t(2;3)(q2?;q3?),add(11)(q23)ish.dup(11)
(q23q24)(MLLx1,Tel11q+)[8]/46,XX[3]
46-48,XY,–11,–17,–18,del(4)(q21),inv(6)
(p21.3q13),der(12)t(11;12)(q13;p13),
+(3-5)markers.ish der(11)amp(11)(q23)
(WCP11+,MLL>30 )/idem,–Y,–5
45,XY,–7,dic(12;22)(p13;q10),der(13;13)
(q10;q10),+13
M-FISH: der(8)t(8;11)(p22;q21-22)[20]
46,XX,der(7)add(7)(p10)
M-FISH: 46,XX,ish.der(7)t(7;19)(p10;p10)
(WCP7+,WCP19+),t(9;11)(p21-22;q23-24)
(WCP9+,WCP11+)[15]
CR
OS (mo)
Telomeric to MLL
NE
Telomeric to MLL
Telomeric to MLL
Centromeric to MLL
NE
Yes
Yes
Yes
Yes
1
23
12
24
20
Telomeric to MLL
MLL amplification
(>30 spots)
Yes
No
1
1
Centromeric to MLL
NE
NE
NE
No
1
AL, acute leukemia; ALL, acute lymphoblastic leukemia; AML, acute myeloblastic leukemia; CHT, chemotherapy; CR, complete remission; MDS, myelodysplastic syndrome;
NE, not evaluable; OS, overall survival; UPN, unique patient number; WCP, whole chromosome painting.
other 3 had t(11;17)(q23;q21), t(11;17)(q23;q25), and
t(11;19)(q23;p13), respectively.
We compared overall survival in patients with MLL+
AML and 122 patients with MLL– AML. The latter group of
patients was divided by karyotype into 3 categories: (1)
favorable (n = 41), ie, t15;17; t8;21, inv16, or t16;16; (2)
intermediate (n = 61), ie, AML with a normal karyotype or
with abnormalities not defined as favorable or unfavorable;
and (3) unfavorable (n = 20), ie, 5q–/–5, 7q–/–7, inv3/t3;3;
t(6;9); t(9;22), 12p–; 9q–; 17p and 21q abnormalities; or
20q–, complex aberrant karyotype with 3 or more aberrations. The median overall survival was 8.2 months in the
MLL+ group compared with 35 months, 11.5 months, and
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2.4 months in the favorable, intermediate, and unfavorable
groups, respectively (P < .001; Figure 1). Patients older than
60 years (MLL+, 0) or diagnosed with t-AML (MLL+, 2)
were excluded from the computation.
Characteristics of Patients With 11q22~25+/MLL–
Leukemia
Of 327 cases analyzed by cytogenetics, 9 showed
rearrangements clustering within the 11q22~25 region
without MLL gene splitting (2.8%). In 8 of these cases, the
involved region was within the 11q23~25 bands, and in 1
case, it was within the 11q13~22 bands. Seven rearrangements were detected by conventional cytogenetics and 2 only
© American Society for Clinical Pathology
Hematopathology / ORIGINAL ARTICLE
B
A
MLL probe
MLL probe
t (2;11)(p21;q23)
t (11;15)(q23;q12)
der(11)
11
11
der(11)
D
C
E
WCP11 probe; WCP19 probe
MLL probe
11
der(7)
11
der(8)
der(11)
t (11;12)(q23~24;q24)
19
19
der(9)
11
der(7)t(7;19)(p10;p10); t(9;11)(p21;q23)
❚Image 1❚ A, Partial karyotype showing t(2;11)(p21;q23) (left); fluorescence in situ hybridization (FISH) analysis with the MLL doublecolor break-apart probe (Vysis, Downers Grove, IL) (right). The probe localized on normal chromosome 11 and on der(11) (unique
patient number [UPN], 95056). The red and the green spots colocalized in a fusion red-green signal; the MLL gene is not
rearranged. B, Partial karyotype showing t(11;15)(q23;q12) (left); FISH with the MLL probe, which localized on chromosome 11 and
on der(11) (right). The MLL probe appears as a fusion red-green signal; MLL is not rearranged (UPN, 98207). C, FISH analysis with
the MLL probe that localized on normal chromosome 11 (2×) and on der(8). The probe appears as a fusion red-green signal (MLL
not rearranged) (UPN, 02418). D, Partial karyotype showing t(11;12)(q23~24;q24). E, FISH analysis with whole chromosome
painting 11(red) and whole chromosome painting 19 (green) probes, disclosing der(7)t(7;19)(p10;p10); t(9;11)(p21;q23) (UPN, 02061).
by M-FISH. In 4 cases, the rearranged locus was telomeric
to MLL, in 2 it was centromeric to MLL, in 1 case the MLL
gene was amplified, and in the remaining 2 cases, no
metaphase was available to assess MLL status. In 3 cases, the
11q rearrangement was associated with a complex karyotype
(3 or more aberrations), in 4 it was the sole chromosomal
change, and in 2 it was associated with another structural
abnormality (Table 3).
Of 9 patients, 7 had AML and were a median age of 42
years (range, 14-62 years). The FAB subtypes represented
were as follows: AML-M0, 2; AML-M1, 1; AML-M4, 1;
AML-M5a, 1; and AML-M6, 2 (Table 3). Three patients
(33%) had secondary AML: 2 were diagnosed with
myelodysplasia 12 (UPN, 02418) and 20 (UPN, 95056) months
before the leukemia outbreak; in 1 patient, AML recurred
after chemotherapy and allotransplantation for previous AML
that at disease onset showed a normal karyotype (UPN,
98207). Of 7 patients with AML, clinical outcome could be
evaluated for 6, and 1 was lost to follow-up. Two died during
the chemotherapy induction phase, 3 achieved CR, and the
remaining 1 had resistant disease (Table 3). All 6 patients died
(median survival, 10 months; range, 1-24 months).
The remaining 2 patients with 11q22~25+/MLL– were
characterized as having pre-B-cell ALL (UPN, 02058) and
B-cell AL with T-cell and myeloid markers (UPN, 98230);
both cases were de novo leukemias. Pre-B-cell ALL was
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diagnosed in a 58-year-old woman who died of disease recurrence after achieving CR that lasted 420 days. B-cell AL was
diagnosed in a 24-year-old woman who died of infection
during the course of aploidentical stem cell transplantation.
In 5 patients with 11q22~25+/MLL– leukemia, 5
different balanced translocations cytogenetically indistinguishable (Table 3) from typical 11q23+/MLL+ were
observed: (1) The t(2;11)(p21;q23) was found in a case of
AML-M6 evolved after myelodysplastic syndrome.
t(2;11)(p21;q23) has been described in t-AML and in
myelodysplastic syndrome.29 The MLL gene rearrangement
was shown at the molecular level in 2 cases and excluded in
1 patient. (2) The t(11;16)(q23;p13) was found in a young
patient with a B-cell AL with T and myeloid markers. To our
knowledge this is the only reported case of t(11;16)(q23;p13)
without MLL+ leukemia.30,31 (3) The t(11;12)(q23;q24) was
found. By studying printed research, we found only 1 other
case with a similar translocation, but it showed an MLL splitting pattern by FISH analysis.9 (4) The t(11;15)(q23;q12)
was observed in a patient with AML at the time of leukemia
recurrence. t(11;15)(q23;q12~15) is a rare 11q23v. In a few
cases studied at the molecular level, MLL rearrangement has
been ascertained and a partner gene named AF15 has been
cloned.32 (5) The t(9;11)(p21-22;q23-24) was identified incidentally by M-FISH and confirmed by whole chromosome
painting. This case was previously reported,30 and to our
knowledge, no other such case has been described.
The remaining 4 patients had various unbalanced
11q22~25 translocations always associated with other chromosomal changes: (1) The del(11)(q13q23) was associated
with 2 additional aberrations. (2) An unbalanced
t(8;11)(p21~22; q13~22) was identified through M-FISH in a
complex karyotype. (3) The add(11)(q23) was identified by
conventional cytogenetic analysis; it was revised after FISH
(using MLL and 11q telomeric probes) to dup(11)(q23~25).
(4) FISH analysis showed MLL amplification33 on the derivative markers of chromosome 11 within a complex karyotype
also bearing a t(11;12)(q13;p13) without MLL rearrangement.
The 9 11q22~25+/MLL– cases were clinically and cytogenetically heterogeneous, and statistical consideration regarding
survival is not feasible. Nevertheless, the overall outcome was
rather poor; no patients survived more than 24 months.
Discussion
In the forthcoming era of tailored, targeted therapy, the
identification of genetic aberration in leukemia will become
more and more important for assigning patients to more
specific or more intensive treatment. The combining of
conventional cytogenetics and molecular and FISH methods
greatly increases the accuracy of information; nevertheless,
304
304
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DOI: 10.1309/RX27R8GJQM330C22
the majority of multicenter clinical trials still base their data
only on karyotype results.14-17 In the present study, conventional cytogenetics was combined with FISH analysis19,28
using a commercial probe that should permit the identification of all MLL rearrangements (not MLL-PTD) and of the
translocated partner chromosome.34 The overall incidence of
MLL+ cases in patients with AML (4.6%) is comparable to
that recently reported by Schoch et al.9 MLL+ cases were not
detected in the 47 cases of ALL and 4 cases of B-cell AL
analyzed by FISH. We attribute this finding to the low
number of cases studied. Notably 2 (4%) of these 51 patients
had 11q23+ without MLL rearrangement.
Compared with FISH, the sensitivity of conventional
cytogenetics was 73%: FISH permitted the identification of 4
additional MLL+ samples (15/378 vs 11/378; Table 1). Of
the 15 cases, 2 (13%) were truly cryptic MLL+ cases, which
is similar to the rate reported in adult AML.9,10 In infant and
childhood AL,19,20 the incidence of cryptic MLL+ cases
might be higher. A recently published series showed that
25% of MLL+ cases were missed by conventional cytogenetic analysis,19 and several of these cases had insertion of
the MLL gene. We found only 1 case of cryptic MLL insertion in a patient with the t(10;11)(p12;q13) (UPN, 97198).
Extensive FISH studies have shown that t(10;11) is a
complex translocation that implies inversion of translocated
chromosomes with multiple breaks. 35 Furthermore, in
patients with the t(10;11), the possibility of AF10/CALM
gene fusion without MLL gene involvement should be ruled
out. AF10/CALM is a nonrandom translocation described in
AML, ALL, and non-Hodgkin lymphoma.36,37
Not infrequently, reciprocal MLL translocation appears
at the chromosomal level as an unbalanced rearrangement
and is referred to as add(11)(q23) or del(11)(q23).38 In the
present series, FISH permitted the revision of 3 (20%) of 15
cases reported as unbalanced 11q23+ aberrations. A
del(11)(q23) was revised to t(11;19)(q23;p13), and 2 cases
with add(11)(q23) were reclassified as t(6;11)(q22;q23)39
and t(10;11)(p12;q23), respectively.
Although the analysis of clinical outcome is beyond the
scope of this work, we briefly report that the overall survival
of patients with de novo MLL+ AML was comparable to that
of the intermediate-risk group (Figure 1). Notably 3 (75%) of
4 patients in prolonged, continuous CR had 11q23v (Table 3).
Several studies comparing FISH and conventional cytogenetics for the diagnosis of MLL+ have shown greater sensitivity of FISH. Conversely, except for sporadic articles,7,19
little emphasis has been given to the incidence of cases
displaying 11q22~25 aberrations without MLL rearrangement
in AL. Only recently, Tanaka et al8 reported a series of
11q+/MLL– cases in Japanese patients affected by various
hematologic malignant neoplasms. They identified several
restricted breakpoint sites involved in translocations, deletions,
© American Society for Clinical Pathology
Hematopathology / ORIGINAL ARTICLE
or both. The candidate targets of these rearrangements might
be a few genes known to map at the 11q23 locus that are
implicated in hematopoietic malignant neoplasms.40-46
Overall in our series, 9 (45%) of 20 11q22~25+ cases
showed no translocation of the MLL gene. Seven of these
were identified by conventional cytogenetic analysis, and 2
were observed incidentally in a group of 11 AML cases
analyzed by M-FISH.47,48 Cryptic rearrangement of 11q in
the form of balanced or unbalanced translocation has been
reported in published series of M-FISH and spectral karyotyping.47,48 Our findings further strengthen the notion that
wider use of these technologies could give a relevant hint
about the chromosomal changes in AL.
A better characterization of 11q22~25+/MLL–
leukemias is relevant for the identification of new recurrent
translocations, cloning of genes, and elucidation of the pathogenic mechanism involved in AL. Moreover, these cases
should be readily distinguished from 11q23+/MLL+ cases.
Beyond FISH, MLL rearrangement also can be detected
by Southern blot and RT-PCR. Southern blot is sensitive and
capable of identifying all MLL translocations and MLL-PTD
but is not used routinely because it is laborious and unable to
discriminate different MLL rearrangements. RT-PCR is the
most sensitive approach for detecting specific subtypes of MLL
rearrangements. The main drawback of this method is that the
partner gene needs to be known. To overcome this limitation, a
multiplex RT-PCR approach has been devised.49 This method
is useful because it identifies, in a single reaction, the most
common MLL translocations: (4;11)(q21;q23) (MLL/AF4);
t(6;11)(q27;q23) (MLL/AF6); t(9;11)(p21-22;q23) (MLL/AF9);
t(10;11)(p11-13;q23) (MLL/AF10); t(11;19)(q23;p13.1)
(MLL/ELL); and t(11;19)(q23;p13.3) (MLL/ENL).
Whatever the method for determining MLL gene status,
the diagnosis of 11q23+/MLL+ leukemia should not be done
without confirmation by molecular or FISH methods. In the
light of our increased knowledge of the complexity of genetic
aberrations in leukemias, this translocation is a good paradigm of the need for common criteria for genetic diagnosis.
From the Departments of 1Hematology and 2Anatomic Pathology,
S’Eugenio Hospital, University of Tor Vergata and 3Alte
Specialità, S’Eugenio, Hospital, Rome, Italy.
Ministero della Salute (Ricerca Finalizzata), MIUR (FIRB
Project) and AIRC.
Address reprint requests to Dr Cox: UOC Ematologia, P.le
Umanesimo 10, 00144 Rome, Italy.
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