1. No category

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1995 85: 3416-3422
Molecular detection of the (2;5) translocation of non-Hodgkin's
lymphoma by reverse transcriptase-polymerase chain reaction
JR Downing, SA Shurtleff, M Zielenska, AM Curcio-Brint, FG Behm, DR Head, JT Sandlund, DD
Weisenburger, AE Kossakowska and P Thorner
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RAPID COMMUNICATION
Molecular Detection of the (2;5) Translocation of Non-Hodgkin's Lymphoma
by Reverse Transcriptase-Polymerase Chain Reaction
By James R. Downing, Sheila A. Shurtleff, Maria Zielenska, Anita M. Curcio-Brint, Frederick G. Behm, David R. Head,
John T. Sandlund, Dennis D. Weisenburger, A.E. Kossakowska, Paul Thorner, Adonis Lorenzana, Marc Ladanyi,
and Stephan W. Morris
The t(2;5)(p23;q35) translocation was initially identified in
cases of anaplasticlarge-cell lymphoma (ALCL) that expressed the Ki-l (CD301 antigen. We have recently cloned
product
this translocation and shown it t o encode a chimeric
consisting of the N-terminal portion of a nonribosomal nucleolar phosphoprotein, nucleophosmin (NPM), from chromosome 5, fused t o the kinase domain of a novel transmembrane tyrosine-specificprotein kinase, anaplastic lymphoma
kinase (ALK), from chromosome 2. To better define the spectrum of lymphomasthat contain this translocation, we have
analyzed 70 cases of non-Hodgkin's lymphoma (NHL) for
expression of the t(2;5)-derived "/ALKchimeric message
by reverse transcriptase-polymerase chain reaction (RTPCR). Using a previously described set of oligonucleotide
primers, NPM/ALKchimeric transcripts were detected in 21
of 22 cases that contained the t(2;5) by cytogenetic analysis
and in l 0 of 48 cases that either lacked evidenceof the t(2;5)
In all but 1case, the NPM/
or had unsuccessful cytogenetics.
ALKPCR products were of identical size and sequence, suggesting that the genomic chromosome breaks are
clustered
in a single intron in both NPM and ALK. The NPM/ALK-expressing cases were not confined t o NHLs with anaplastic
6 immunoblastic
morphology and included 15
ALCLs,
lymphomas, and10 diffuse large-celllymphomas. Moreover,
only slightly greater than half of the cases with anaplastic
morphology and 59% of CD30-expressing caseswere NPM/
ALK positive. Thus, neither anaplastic morphology nor the
expressionof CD30 accurately predicted the presence ofthis
molecular geneticsubtype of lymphoma.
0 1995 by The American Societyof Hematology.
T
called Ki-l+ lymphomas (also called anaplastic large-cell
lymphoma [ALCL] or large-cell anaplastic lymphoma, and
in this report referred to as Ki-l+ lymphomas/ALCL), which
account for up to 30% to 40% of all pediatric large-cell
lymphomas.'-6 These cases are characterized by expression
of the CD30 antigen (recognized by the Ki-l and Ber-H2
monoclonal antibodies [MoAbs]), are primarily T cell in
lineage, and typically have anaplastic morphologic features."" Results of cytogenetic studies on Ki-l+lymphomas/
ALCL have shown the presence of a t(2;5)(p23;q35) translocation in a high proportion of these case^.^.^*"^
Clinically, Ki-l+ lymphomas/ALCL are aggressive, highgrade lymphomas that exhibit a bimodal age distribution
similar to that observed in Hodgkin's d i s e a ~ e . ~ . ~ . ' " ~P"a-~ ' ' ~ ' ~
tients present with peripheral lymphadenopathy and frequent
extranodal disease involving skin, bone, soft tissue, gastrointestinal tract, a n d o r l ~ n g . ~ , ~ , Several
~ , ' ~ . ' ' studies have suggested that Ki- I lymphomas/ALCL may have a better overall survival rate compared with other large-cell lymphoma
subtypes?,'6,'8-2n
Although Ki- l + lymphomas/ALCL have been suggested
to constitute a distinct clinicopathologic entity, the morphologic and immunophenotypic criteria required for this diagnosis have not been clearly defined. There is no uniformity
in cell lineage, expression of the CD30 antigen, presence of
the t(2;5), or morphologic appearance among cases diagnosed as Ki-l+ lymphomas/ALCL.'.2*6
Cases have been classified as ALCL that lack CD30 antigen expression andor
the t(2;5); conversely, cases that lacked anaplastic morphology have been shown to express Ki-l and/or contain the
t(2;5) translocation. In addition, morphologic variants of Kil + lymphomas have been recently described, including a
small-cell variant with exceedingly rare anaplastic cells.*'
Thus, the exact relationship between CD30 expression, the
presence of the t(2;5), and the histologic subtype of
lymphoma remains to beestablished. Because of the variable
criteria used to make a diagnosis of Ki-l' lymphomas/
HERAPY FORnon-Hodgkin's large-cell lymphoma has
greatly improved over the last 10 years, with diseasefree survival rates of greater than 50% being achieved with
current chemotherapy protocols. Unfortunately, a significant
number of patients continue to fail to respond to therapy.
The identification of biologic subtypes of large-cell lymphomas that have either an inherently higher risk of failing
therapy or that comprise a disease subgroup amenable to a
more directed form of therapy remains a necessary goal to
further improve survival rates for this disease. A recently
described subgroup of large-cell lymphomas are the so-
From the Departments of Pathology, Tumor Cell Biology, Experimental Oncology, and Hematology/Oncology, St Jude Children's
Research Hospital, Memphis, TN;the Departments of Pathology
and Pediatrics, University of Tennessee College of Medicine, Memphis, TN; the Department of Pathology and Microbiology, University
of Nebraska Medical Center, Omaha, NE; the Department of Pathology, Foothill Hospital, Calgary, Alberta, Canada; the Departments
of Pathology and Hematology/Oncology, Hospital for Sick Children,
Toronto, Ontario, Canada; and the Departments of Pathology and
Human Genetics, Memorial Sloan-Kettering Cancer Center, New
York, NY.
Submitted March 9, 1995; accepted March 27, 1995.
Supported in pari by National Institutes of Health Grants No. CA01429 (J.R.D.) and CA-01702 (S.W.M.), by Cancer Center Core
Grant No. CA-21 765, by
American Cancer Society (ACS) Grant No.
BE-218 (J.R.D.),by ACS Clinical Oncology Career Development
Award 93-38 (M.L.),and by the American Lebanese Syrian Associated Charities of St Jude Children's Research Hospital.
Address reprint requests to James R. Downing, MD, Department
of Pathology, StJude Children's Research Hospital, 575 StJude
Place, Memphis, TN 38105.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
woS-4971/95/8512-0043$3.00/0
3416
Blood, Vol 85, No 12 (June 15), 1995: pp 3416-3422
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3417
DETECTION OF T(2;5) BY RT-PCR
In addition, all difficult cases were centrally reviewed and a consenALCL, ithas become difficult in practice to accurately define
sus diagnosis was reached. Cytogenetic analysis was performed sucthis entity. The presence of the t(2;5) translocation in a sigcessfully on 70% of cases using standard techniques at the participatnificant proportion of these cases suggests that it might serve
ing institution. Control cell lines included the leukemic cell lines
as a useful genetic abnormality to define a distinct lymphoma
H L 6 0 M * 3 ’ and K56232and the t(2;5)-containing lymphoma cell lines
subtype. Once defined, the clinical relevance of this subSU-DHL-l and SUP-MZ.~~
group could be determined through prospective treatment
Immunophenotyping. Immunophenotypic analysis was
perprotocols.
formed at the participating institutions and the reported results are
We have recently cloned the t(2;5) and have shown it to
those obtained by chart review. A variety of different standard tecbinvolve the genes encoding nucleophosmin (NPM; also
niques were used including immunoperoxidase on flash-frozen tissue
or formalin-fixed, paraffin-embedded sections and immunofluoresknown as B23 or numatrin), a nonribosomal nucleolar phoscence microscopy or flow cytometry. The CD30 antigen was detected
phoprotein on chromosome 5 , and the novel transmembrane
with either the Ber-H2 MoAb (Dako, Santa Barbara, CA) or the Kityrosine-specific protein kinase, anaplastic lymphoma kinase
1 MoAb (Dako). Samples were determined to be positive if the
(ALK) on chromosome 2.” The translocation results in the
majority of the neoplastic cells in adequately fixed portions of the
fusion of these genes on the der(5) chromosome, producing a
specimen displayed a membrane and/or Golgi pattern of staining
chimeric NPM/ALK gene and message. The resulting fusion
or were positive by immunofluorescent analysis. Cell lineage was
product consists of the N-terminal end of NPM fused indetermined by assessing the reactivity of the neoplastic cells with
frame to the intracellular kinase domain of ALK.22*23 NPM antibodies specific for various T- and B-cell-associated antigens
is an ubiquitously expressed 38-kD phosphoprotein that is
including CD45 (leukocyte common antigen; Dako), CD3 (Dako),
involved in the late stages of preribosomal particle assembly
CD43 (MTl; Biotest, Danville, NJ), CD45RO (UCHL-l; Dako),
CD20 (L26; Dako), MB-2 (Biotest), and CD45RA (4KB5; Dako).
and plays a role in shuttling ribosomal ribonucleoproteins
A case was defined as T lineage if it reacted with one or more of the
between the nucleolus and the
Currently, little
antibodies directed against the T-cell-associated antigens (UCHL-l,
is known about the normal function of the ALK-encoded
CD3, and MT-l) and lacked reactivity with antibodies to the Btyrosine-specific protein kinase receptor or the nature of the
cell-associated antigens (L26,4KB5, and MB-2). A B-cell lineage
ALK ligand(s). ALK is a member of the insulin receptor
was assigned if the opposite pattern of reactivity was observed.
subfamily, with greatest homology to the leukocyte tyrosine
RT-PCR. Total RNA was extracted and cDNA was synthesized
kinase (LTK), and is expressed in the small intestine, brain,
as previously de~cribed.’~
Amplification of the NPM/ALK chimeric
prostate, and testis.” However, no expression of ALK has
message was performed using Taq polymerase (Perkin-Elmer-Cetus,
been detected within the hematopoietic system. The (2;5)
Norwalk, CT) and the oligonucleotide primers 5’(A) and 3’(A) (Fig
translocation results in transcription of the ALK tyrosine
lB), which amplify a 177-bp product.’’ To increase the sensitivity
of detection, nested primer PCR analysis was performed using 5%
kinase domain driven off the strong NPM gene promoter
of the original PCR products and the oligonucleotide primers, 5’(B)
and leads to its inappropriate expression in lymphoid cells.
and 3’(B) (Fig lB), which amplify a 123-bp product. The presence
Using primers derived from NPM and ALK sequences
of amplifiable RNA was confirmed by RT-PCR amplification of
bracketing the breakpoint, we have developed a nested reor by amplificaeither &actin transcripts, as previously des~ribed?~
verse transcriptase-polymerase chain reaction (RT-PCR)
tion of the ubiquitously expressed normal NPM mRNA using the 5’
assay that allows the rapid, accurate, and sensitive detection
oligonucleotide 5’(A) (Fig 1B) and a 3’ oligonucleotide (3‘NPM)
of the fusion mRNA resulting from this t r a n s 1 o ~ a t i o n . ~ ~ ~having
~ ” ~ ~ the sequence 5‘-GCTACCACCTCCAGGGGCAGA-3’ that
In the present study, we have used this assay to analyze 70
is homologous to normal NPM coding sequences located 3’ to the
pediatric and adult cases of non-Hodgkin’s lymphoma
point at which the NPM/ALK fusion occurs. To prevent sample cross(NHL) for the presence of the t(2;5)-derived NPM/ALK
contamination, extreme care was taken in handling samples and
chimeric message. Our data show that neither anaplastic
negative controls were included inboth the RNA extraction and
amplification reacti0ns.3~After amplification, PCR products were
morphology nor the expression of CD30 accurately predict
size fractionated by electrophoresis through a 1.2% agarose gel in
the presence of this molecular genetic subtype of lymphoma.
MATERIALS AND METHODS
Patientsamples.
Representative portions of diagnostic tissue
from patients with NHL were obtained with informed consent. Samples consisted of either dispersed mononuclear cells frozen as viable
cell suspensions or tissue blocks frozen in OCT embedding media
(Miles Inc, Elkhart, IN). Samples were obtained from St Jude Children’s Research Hospital (SJCRH; 19 cases); Memorial Sloan-Kettering Cancer Center (15 cases); The Hospital for Sick Children,
Toronto (14 cases); The Foothill Hospital, Calgary (12 cases); and
The University of Nebraska Medical Center (10 cases). Cases were
selected based on a diagnosis of NHL and the presence of cryopreserved material but were selectively biased toward cases containing
a (25) translocation and/or expression of CD30, and thus do not
represent a random sampling of this malignancy. The morphology
of each case was reviewed by an experienced hematopathologist at
the participating institution and 64% of the cases were also centrally
reviewed by three hematopathologists, (J.R.D., D.R.H., and F.G.B.).
the presence of ethidium bromide and either directly visualized under
UV light or transferred to nylon membranes and hybridized with
specific probes. The NPM/ALK chimeric message was detected using
a ”P-end-labeled junction-specific probe (Fig lB), and negative
cases were reprobed with the 3’(B) nested oligonucleotide radiolabeled with ”P to identify products thathad variant junction sequences. The normal amplified NPM product was detected in Southem blots with an NPM specific probe having the sequence
5’-GTGCTGTCCACTAATATGCAC-3‘.26
RESULTS
The (2;5) translocation leads to the fusion of the ALK
receptor tyrosine kinase gene on chromosome 2 to the NPM
gene on chromosome 5 and results in the generation of a
der(5)-encoded NPMIALK chimeric mRNA and protein (Fig
IA). The RT-PCR assay used in this study for the detection
of the NPMIALK chimeric mRNA has been previously describedz3and results in the specific amplification of a 177-
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DOWNING ET AL
3418
A
"1" 2
Chromosome 5
COOH
294
4
4
NPM
MB
AC
N
AC
N
4
Chromaome 2
NH 2
AM
1
COOH
Tyrosine KJnase Domain
.c
"1" 2
der(5)
I
NPMIALK
SA
4
Tyrosine Kinase Domain
3.A
U
n
5'B
-
3'B
PROBE
5'(A): 5'-CC CT T GGG GGC T T T G M ATA ACA CC-3'
3'(A): 5'-GAG GTG CGG AGC T TGCTC AGC-3'
5'(B): 5'-CCA GTGGTC T TA AGG T TG-3'
3'(B): 5"TACTCAGGGCTCTGCAGC-3'
Probe: SAG CAC T TA GTAGTGTAC CGC CGG A-3'
bp NPM/ALK chimeric product from cases containing the
(2;5) translocation (Fig IB). Mixing experiments between
the leukemic cell line K562, which lacks this translocation,
andthe t(2;S)-containing lymphoma cell line SU-DHL-l
demonstrated an ability to detect the presence of the NPM/
ALK chimeric transcript at a sensitivity of 1 in 1 X IO5 cells
(data not shown). This level of sensitivity was increased a
further IO-fold by use of the described nested PCR approach.
In this study, we have applied this assay to a group of
70 cases of NHL collected from both pediatric and general
hospitals in the United States and Canada. This group of
patients washighly selected with two goals in mind: ( l )
to first determine whether this assay detects the NPMIALK
chimeric transcript in all cases with cytogenetic evidence of
the t(2;S) or whether variant mRNAs exist that are unamplifiable with these oligonucleotide primers; and (2) to assess
the correlation of anaplastic morphology andKi-l expression
with the presence of the NPM/ALK chimeric message. The
patients analyzed ranged in age from 2 to 86 years, with a
meanof25.6
years. The morphologic spectrum of cases
included 28 ALCL (15 T-cell, 7 B-cell, 5 non-Thon-B, and
1 lineage undefined), 21 immunoblastic lymphomas (7 Tcell, 6 B-cell, I non-Thon-B, and 7 lineage undefined), 19
diffuse mixed or diffuse large-cell lymphomas (9 T-cell, 6
B-cell,and
4 non-Thon-B), and 2 cases of Burkitt's
lymphoma that were initially diagnosed as diffuse largecelllymphomabuton
re-review were classified as small,
~ 0
679
Fig 1. Schematic of the NPM
and ALK proteins and the fusion
product derived from the der(5)
chromosome. IAI
is
an
. NPM
.
ubiquitously
expressed
nucleolar phosphoproteinthat shuttles
ribosomalcomponents between
the nucleolus
and
cytoplasm.
.
.
The metal binding (MW, acidic
amino acid clusters (ACI, and nu0
clear~localization signals (NI of
NPM are
indicated.
ALK is a
transmembrane receptor tyrosine-specific protein kinase. The
membrane-spanning domain is
indicated by the solidbox and
the intracytoplasmic kinase domain is shown. The positions of
the breakpoints in the t12;5)
translocation
by
are indicated
arrows. The dw(5)-encoded
NPM/
ALKchimeric product consists of
the N-terminal portion of NPM
fused inframe to the cytoplasmic
domain ofALK. The position of
the oligonucleotide primers and
probe used for RT-PCR analysis
are shown. (8) Sequence
of
the amplification oligonucleotide primers for standard I5'(AI
and 3'(A)1 and nested [5'(81and
3'(8)]RT-PCRanalysisand the
detection probe.
noncleaved cell lymphomas. Cytogenetic analysis was successfully performed on 49 of these cases andshowedthe
presence of the t(2;5) in 22 patients.
Total RNA was isolated fromdiagnostic lymph node biopsies from these 70 patients and analyzed by RT-PCR for both
the der(5)-encoded NPM/ALK message and the ubiquitously
expressed NPM transcript as a control for the presence of
amplifiable RNA. Figure 2 shows representative results of
the analysis of 7 patients with cytogenetically proven t(2;5)containing lymphomas, including examples of anaplastic and
nonanaplastic cases, and 2 patients whose tumors lacked
cytogenetic evidence of this translocation. As shown, a standard RT-PCRreactionwas
able to detect the NPM/ALK
fusion messages in the majority of patients; however, in rare
cases, a nested RT-PCR approach was required for detection
of the chimeric product (Fig 2, lane 10).
Samples from 22 patients were found to contain a (23)
translocation by cytogenetic analysis. RT-PCR analysis of
these cases resulted in the amplification of a specific NPM/
ALK fusion transcript in allbut l case. The single t(2;5)containing case that lacked a detectable NPM/ALK chimeric
transcript was from a 63-year-old patient with a B-lineage,
Ki-l+ ALCL. In addition, 10 of 48 cases that either had no
evidence of the t(2;5) by cytogenetics (27) or were not analyzed by cytogenetics (21) were found to express the NPM/
ALK fusion transcript. The identification of the NPM/ALK
chimeric mRNA in cases analyzed by cytogenetics but lack-
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DETECTION OF T(2;5)BYRT-PCR
3419
Fig 2. Representative RT-PCR analysis forthe
NPM/ALKfusion transcript. Total
RNA was extracted
from the t(2;5)-containing cell line SU-DHL-1 and 9
diagnostic tissue samples from patients with largecell NHL. Seven of these cases had cytogenetically
proven t(2;5), whereas 2 cases lacked this translocation. The extracted RNA was reverse transcribed and
then amplified for theNPM/ALK chimeric transcript
in standard (Std) and nestedPCR assays. PCR products were separated by electrophoresis, transferred
t o a nylon membrane, and hybridizedwith either an
NPM/ALK junction-specific probe (Std and Nested)
oran NPM-specific probeINPM-control)and
detectedby autoradiography. RT-PCR amplification
was performed for the ubiquitouslyexpressed NPM
message as a control for thepresence of amplifiable
RNA.
Std.
Nested
NPM- control
1 2 3 4 5 6 7 8 91011
ing evidence of the t(2;5) translocation (6 of 27 cases) indicates that occasional cases exist that either have cryptic
translocations or contain a malignant clone refractory to cytogenetic analysis.
In all but 1 case, the NPM/ALK PCR products were of
identical size and sequence, suggesting that the genomic
breakpoints are clustered in a single intron in both the NPM
and ALK genes. The single exception was a 33-year-old
patient with a T-lineage, Ki-l+ ALCL that expressed a
smaller in-frame NPM/ALK chimeric product that resulted
from an ALK fusion point 30 bp 3' to the usual ALK fusion
point joined to the same portion of NPM (M. Ladanyi, unpublished data). This product was amplifiable with the
primer set used and, although it was undetectable with the
junction-specific probe, it hybridized with an oligonucleotide
probe homologous to ALK.
Results from all patients are tabulated according to their
morphologic diagnosis in Table 1. As indicated, only slightly
greater than half of the ALCLs expressed NPM/ALK (pa-
Table l. Histology of70 Cases of NHL Analyzed by RT-PCR for
NPMIALK Expression
~~
Results of RT-PCR Analysis
Histology
ALCL
13
lmmunoblastic
Diffuse mixed and diffuse large-cell
Burkitt's lymphoma
NPMIALK
NPMIALK
Positive
15
6
10
0
*This number includes 4 follicular large-cell lymphomas.
Negative
15
9"
2
tients no. 1 through 3 in Fig2,and
see Fig 3A). Clear
examples were observed of immunoblastic and diffuse mixed
or large-cell lymphomas that lacked anaplastic features but
expressed the NPM/ALK chimeric message (Fig I , patients
no. 4 through 7, and see Fig 3B). Similarly, cases were
observed thathad classic anaplastic large-cell morphology
butlacked molecular evidence of the t(2;5) translocation
(patient no. 9 in Fig 2. and see Fig 3C).
The results of immunophenotypic analysis of these cases
are presented in Table 2. With the exception of I B-lineage
case, all of the NPM/ALK-expressing cases analyzed were
eitherT cell or non-Thon-B cell in lineage. The immunophenotype of the B-lineage tumor was performed by both Row
cytometry and immunohistochemistry of tissue sections with
complete concordance between results (data not shown). Expression of the Ki-l antigen was detected in all but 2 of the
analyzed cases expressing the NPM/ALK transcript. However, expression of Ki-l was also detected in 49% of cases
that lacked NPM/ALK expression (Table 2). Taken together,
these results indicate that neither anaplastic morphology nor
the expression of the Ki-l antigen predicted the presence
of the NPM/ALK fusion transcript with absolute accuracy.
However, NPM/ALK expression was most commonly seen
in a subset of Ki-l expressing T-cell lymphomas.
DISCUSSION
The diagnosis of Ki-l + IymphomdALCL appears to identify a somewhat heterogenous group of NHLs with variable
clinical, morphologic, and immunophenotypic features. Cytogenetic analysis suggests that a proportion of these cases
contain a (2;5) translocation. To explore the possibility that
From www.bloodjournal.org by guest on November 18, 2014. For personal use only.
3420
DOWNING ET AL
Fig 3. Photomicrographs of representative cases.
Sections were stained with hematoxylin and eosin.
(A) A classicKi-l+ALCL that had the
t(2;5) by cytogenetics and expressed the NPM/ALK chimeric transcript (patient no. 1 in Fig 2). (B) An immunoblastic
lymphoma that contained the t(2;5) and expressed
the NPM/ALKchimeric transcript (patientno. 4 in Fig
21. (C) An example of a classicKi-l+ ALCL that lacked
cytogenetic evidence of t(2;5) and did not express
the NPM/ALK chimeric transcript (patient no. 9 in
Fig 2). Magnification was x 630 for (A) and (B) and
x1.575 for (C).
detection of this genetic lesion might identlfy a more uniform
that the RT-PCR assay used in this study was able to detect
clinicopathologic subgroupof lymphomas, we have used an
the NPWALK chimericmessage in 21 of22 t(2;5)-conRT-FCR assay for the t(2;5)-derived N P W M K chimeric
taining cases and in 10 of 48 cases that either lacked this
message to examine 70 cases of N H L . Our results showed
cytogenetic lesion or had unsuccessful cytogenetics. In all
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3421
DETECTION OF ~ ( 2 ; 5 )BY RT-PCR
Table 2. Imrnunophanotypeof Cases Analyzed by RT-PCR for
NPMIALK Expression
Results of
RT-PCR for
NPWALK
Ki-l*
+
27
+NPMIALK
-NPMIALK 11 19
-
2
Lineage
ND
T
B
Non-TlNon-B
ND
2
9
21
10
1
20
6
3
5
4
Abbreviation: ND, not determined.
The methods used for detection of Ki-l antigen expression and
lineage assignment are described in the Materials and Methods.
but 1 NPWALK-expressing case, identically sized RT-PCR
products were observed, suggesting that breakpoints were
clustered in a single intron in both genes. The 1 exception
expressed a variant product that resulted from a more 3’
fusion point in ALK. In addition, 1 case was identified that
had cytogenetic evidence of the t(2;5) but lacked RT-PCR
evidence of NPM/ALK expression. The absence of amplifiable NPM/ALK in this case most likely reflects additional
breakpoint heterogeneity, resulting in the inability to amplify
the chimeric transcript with the oligonucleotide primers
used. Despite these 2 exceptions, the RT-PCR assay described here should allow the amplification of the NPMIALK
chimeric product in the vast majority of cases containing
this translocation and thus appears to be a highly effective
method for detecting this genetic lesion.
Analysis of the clinicopathologic characteristics of the
NPM/ALK-expressing cases showed that this genetically defined subgroup of lymphomas only partially overlapped with
classically defined Ki-l+1ymphomdALCL. All but 2 of the
NPM/ALK expressing cases for which immunophenotypic
data were available expressed the Ki-l antigen and, with 1
exception, were either T cell or non-Thon-B cell in lineage.
However, the morphologic spectrum of these cases was quite
variable and included 15 ALCLs, 10 diffuse mixed or diffuse
large-cell lymphomas, and 6 immunoblastic lymphomas.
Moreover, only slightly greater than half of the lymphomas
with anaplastic morphology were found to express NPM/
ALK, and only 59% of %-l+cases expressed this chimeric
message. In addition, within the ALCL histologic subgroup
the mean age of the NPM/ALK-expressing cases was 18.4
years, whereas in the NPM/ALK-negative cases it was 46
years. These data suggest that classically defined Ki-l+
ALCL in young patients is more frequently associated with
molecular evidence of t(2;5) than in the adult patient population. However, this latter result should be interpreted with
caution because this patient cohort was highly selected and,
although nearly half of the patients were more than 21 years
of age, the analyzed cases do not represent a random sampling of NHL.
In addition to aiding in the identification of this molecular
genetic subtype of NHL, the described RT-PCR assay should
also help in differentiating this neoplasm from several morphologically related malignancies. The pleomorphic nature
of the malignant cells in ALCL has frequently led to the
misdiagnosis of these cases as metastatic carcinoma, melanoma, malignant fibrous histiocytoma, or malignant histiocytosis. In fact, nearly one third of the Ki-l+ALCLs reported
in a recent clinical study were initially misdiagnosed.’” Because the (2;5) translocation has not been identified by cytogenetics in any nonhematopoietic malignancy, the identification of this genetic lesion should be
of
value in
differentiating NPM/ALK-expressing lymphomas from these
other tumors. In addition, differentiation of Hodgkin’s disease and Ki-l+ lymphomas/ALCL can occasionally be difficult; however, we and others have failed to detect expression
of the t(2;5)-derived NPM/ALK chimeric transcript in more
than 100 cases of Hodgkin’s di~ease.**,~~.~’
Thus, this RTPCR assay should also be of aid in differentiating NPM/
ALK-expressing cases from Hodgkin’s disease. Because of
the high level of sensitivity and specificity of the RT-PCR
assay for NPM/ALK, as few as 200 cells obtained from a
fine needle aspirate are sufficient to perform this analysis.
Thus, application of this assayin certain clinical settings
may eliminate the need for an open biopsy. Moreover, this
assay has been used successfully on formalin-fixed and paraffin-embedded material; therefore, this analysis is possible
in cases in which frozen tissue is ~navailable.~’
In summary, t(2;5)-containing large-cell lymphomas appear to comprise a distinct genetic subgroup of NHLs that
can have variable histologic and immunophenotypic characteristics. Whether this genetic subgroup identifies a clinically
relevant subset of NHLs remains to be defined. However,
classification of this group of lymphomas based ona putative
etiologic mechanism holds promise for the ultimate development and use of specifically targeted therapy.
ACKNOWLEDGMENT
The authors thank John F. Zacher for photographic work, S. Patricia Odum and Margaret Griffith for technical assistance in the immunophenotypic analysis of SJCRH patients, and Drs Daniel Filippa
and R.S.K. Chaganti for providing pathologic and cytogenetic data,
respectively, on the MSKCC patients.
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