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1995 86: 3517-3521
Transcripts of the npm-alk fusion gene in anaplastic large cell
lymphoma, Hodgkin's disease, and reactive lymphoid lesions
PG Elmberger, MD Lozano, DD Weisenburger, W Sanger and WC Chan
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Transcripts of the npm-alk Fusion Gene in Anaplastic Large Cell
Lymphoma, Hodgkin’s Disease, and Reactive Lymphoid Lesions
By P. Goran Elmberger, Maria D. Lozano, Dennis D. Weisenburger, Warren Sanger, and Wing C. Chan
tradictory information in the literature on the occurrence
Anaplastic large cell lymphoma (ALCL) and Hodgkin’s disease (HD) have some pathologic and immunohistochemical of the npm-a/& rearrangement in HD.We detected npm-elk
hybrid mRNA in 8 of 22 cases of ALCL (36%). but none of
similarities, and a histogenetic relationship between them
the 11 cases with reactivelesions
the 21 casesofHDor
has been suggested by some investigators.By cytogenetic
contained amplifiable template. All positive
ALCL had the T
study, the t(2;5)(p23;q35) translocation appears
to be unique
or indeterminate phenotype and occurred in young adults
of the t(2;5)(p23;q35) have reforALCL.Thebreakpoints
was very good correlation
between a cytocently been cloned and are reported
to involve a novel tyro- or children. There
RT-PCR.
genetically detectable t(2;5) and a positive signal
by
sine kinase gene, anaplastic lymphoma kinase (alk), on chroOur results indicate a selective but relatively infrequentasmosome
and
2
the nucleophosmin
gene
(npm) on
chromosome 5. Therefore,we studied the frequency of npm- sociation between the t(2;5) and ALCL ofT or indeterminate
elktranslocation in ALCL using a reverse transcriptase-poly- phenotype, not shared with HD or reactive hyperplasia.
0 1995 by The American Society of Hematology.
merase chain reaction (RT-PCR) assay. We also studied HD
there is conand a varietyof reactive lymphoid lesions since
A
NAPLASTIC LARGE-CELL lymphoma (ALCL) is a
recently recognized category of non-Hodgkin’s
lymphoma (NHL).14 Histologically, this lymphoma in its
classical form consists of large anaplastic cells that preferentially occupy the sinuses and T-cell areas of lymph nodes.
The tumor cells often exhibit a cohesive pattern of growth
and strongly express CD30 (Ki-l), which is an activationassociated marker belonging to the nerve growth factorhumor necrosis factor receptor family and expressed by subsets
of activated T and B cells, as well as by Reed-Stemberg
cells in Hodgkin’s disease (HD).
The histologic diagnosis of ALCL can be difficult and
many cases have previously been classified as lymphocyte
depleted HD, malignant histiocytosis, melanoma, or anaplastic carcinoma. Today, most misdiagnoses canbe avoided
with the application of modem analytical techniques including immunohistochemistry, electron microscopy, and molecular analysis of T- and B-cell antigen receptor gene rearrangements. However, ALCL can still be confused with
HD due to the presence of cells resembling Reed-Stemberg
cells and an overlapping immunohistochemical and genotypic pr~file.~”Although the reciprocal translocation
t(2;5)(p23;q35) has been associated with ALCL,8-I3 it has
not been described in HD. However, cytogenetic analysis is
hampered in HD by the generally small proportion of neoplastic cells that are not very active mitotically, resulting in
poor yieldof tumor cell metaphases in chromosome preparations.
Recently, Morris et al have shown that the t(2;5)(p23;q35)
results in the fusion of the nucleophosmin gene (nprn) on
chromosome 5 with the anaplastic lymphoma kinase gene
(alk) on chromosome 2.14 The alk gene shows sequence
similarity to the insulin receptor subfamily of tyrosine kinases. The abnormal fusion protein resulting from the translocation retains the tyrosine kinase domain and likely contributes to the malignant transformation of ALCL. Southern
blot analysis using probes close to the npm-alk breakpoints
or a reverse transcriptase-polymerase chain reaction (RTPCR) using nprn and alk primers have consistently detected
a fusion product in cases of ALCL or cell lines with a cytogenetically detected t(2;5).I5.l6The RT-PCRassay is an extremely sensitive technique that should be able to detect the
Blood, Vol 86, No 9 (November l), 1995 pp 3517-3521
presence of a small number of neoplastic cells with t(2;5),
independent of cell division. As such, RT-PCR appears ideally suited to investigate the possible relationship between
the t(2;5) and HD.
Therefore, we have undertaken a study of the utility of a
RT-PCR assay in the detection of the t(2;5) in ALCL, and
compared the results with available cytogenetic data. A series of classical HD was also studied to determine whether
the t(2;5) with the same breakpoints was detectable in this
condition. Tissues showing reactive lymphoid hyperplasia
were also examined for the presence of rare cells with the
translocation.
MATERIALS AND METHODS
Patient samples. Cases of primary ALCL were identified in the
Nebraska Lymphoma Study Group (NLSG) Registry. All the cases
were reviewed and those showing the typical histopathologic features
of ALCL, ie, large anaplastic cells with strong expression of CD30
on tumor cells, along with snap-frozen tissue were selected for further study. None of the morphologic variants of ALCL was included
in this study. Three of the tumors occurred in extranodal sites including two skin lesions and a thigh mass, but one of the skin lesions
represented a recurrent tumor after a nodal primary and the other
skin tumor was associated with axillary lymphadenopathy. Human
immunodeficiency virus infection was not diagnosed in any of the
patients studied. Two cases without anaplastic morphology were
From the Division of Clinical Cytology, the Department of Pathology and Cytology, Karolinska Institute and Hospital, Stockholm,
Sweden; the Department of Pathology and Microbiology, and the
Department of Pediatrics, University of Nebraska Medical Center,
Omaha.
Submitted May 8, 1995; accepted June 26. 1995.
Supported in part by Grant No. USPHS CA36727 from the National Institutes of Health.
Address reprint requests to Wing C. Chan, MD, Department of
Pathology and Microbiology, University of Nebraska Medical Center, 600 42nd St, Omaha, NE 68198-3135.
The publication costs of this article were defrayedin part by page
charge payment. This article must therefore be herebymarked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
ooaS-4971/95/8609-01$3.00/0
3517
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ELMBERGER ET AL
3518
identified from our cytogenetic dat;hse because of the presence of
the 1(2;5)(~23:q35).
These two cases were analyzed for the correlation of cytogenetic findings with the RT-PCR assay. The most recent
21 cases of HD. including 15 cases of the nodular sclerosis and six
cases of the mixed cellularity subtypes. with classical features and
snap-frozen tissue were also identified in the NLSG database. In
addition. I I cases of various types of reactive lymphoid hyperplasia
were included in the study.
Cofrrrol rissrrc. Frozen placental tissue and K562 cells, an erythroleukemia cell line, were used as negative controls for the npm-crlk
rearrangement assay. The JB6 cell line (kindly provided by Dr M.
Kadin. Beth Israel Hospital. Harvard Medical School. Boston. MA)
derived from a case of ALCL and carrying a t(2:S) was used as a
positive control.
RNA isokrrion m t d cDNA prqwrorion. Tissue samples thathad
been either directly snap-frozen or embedded in OCT medium and
snap-frozen were used. Intact tissue (3 mm’) was minced with a
fresh razor blade. transferred to a prelabeled Eppendorf tube with I
mL TRlzol (GIBCO BRL, Gaithersburg. MD) containing 5 pg glycogen carrier (BoehringerMannheim. Indianapolis. I N ) and immediately vortexed. The OCT-embedded tissue was sectioned on a cryostat with disposable knife blades, and three 20-pm sections with
approximately I cm‘ area were collected in I mL TRIzol. Every
step was taken to avoid the possibility of cross contamination. Tissue
was minced in ;I laminar flow hood with new towels over the cutting
area and U V irradiation between each sample. An extensive cleaning
procedure was applied between the cutting of each sample in the
cryostat, including dismantling of the cutting knife holder and five
cleansing cycles with ethanol and 0. I mollL HCI. A new blade was
used for each sample. Gloves were changed between each step in the
procedure. To monitor the efficacy of the contamination prevention
procedures. negative control placental tissue was introduced as every
fifth sample.
Total R N A extraction. precipitation. and washings were performed according to the TRlzol protocol. Total RNA was quantitated
using spectrophotometric absorbance at 260/280 nm. diluted to 0.2
pglpL, and stored in 10 pL fractions at -70°C for further analysis.
Contaminating DNA was removed by DNAase treatment (GlBCO
BRL). cDNA was prepared using random hexamer primers and reverse transcriptase (GIBCO BRL) according to the manufacturer‘s
instruction.
RT-PCR. The presence of amplifiable cDNA was confirmed by
performing PCR for glyceraldehyde 3 phosphate dehydrogenase
(G3PDH) mRNA with a PCR product of 497 bp.” The S’ r r p n and
3’ d k primers and the junction oligonucleotide used for probing
have been previously described.”’ and their sequences are S’-CCCTTCCGGGCTTTGAAATAACACC-3‘. 5”GAGGTGCGGAGCTTGCTCAGC-3’. and 5’-AGCACITAGTAGTGTACCGCCGGA3’. respectively. Amplification was performed with addition of 10
pmol of each primer, I U of Taq polymerase, 125 nmol MgCI?. 10
nmol deoxynucleotide triphosphates in I X PCR buffer (50 mmollL
KCI. 10 mmollL Tris HCI at pH 8.3. 0.1 m g h L gelatin. 0.45%
Nonidet-P40. and 0.45% Tween 20) in a total volume of 45 pL. Hot
start technique was used and the reaction started with the addition
of 5 pL of cDNA when the reaction mixture reached 94°C. The
cycling parameters were 40 cycles at 94°C for 30 seconds. 62°C for
I minute, and 72°C for 30 seconds, followed by a final extension at
72°C for I O minutes. The RT-PCR products were electrophoresed
on 1.5% NuSieve agarose gels and visualized with ethidium bromide
staining. The PCR products were then blotted by capillary transfer
onto nylon membranes, which were hybridized with end-labeled
/rprn-olk junctional oligonucleotide. washed at the appropriate stringency, and then autoradiograph was performed.
/nttnrrtto/o,~ic
srrrdies. lmmunophenotyping was performed on
Fig1. Sensitivity of the RT-PCR assay.Serial dilutions ofcDNA
from JB6 cell line were amplified and the products electrophoresed
as follows: lanes 1 through 6 at 10.’. l 0 10 ‘,
and lo”
dilutions, respectively. Lane 7. reaction performed with no template
DNA. M, molecular-weight markers at 100-bp interval. A signal is
detectable down to 10 dilution.
’,
paraffin and/or frozen sections using a panelof monoclonal and
polyclonal antibodies against leukocyte-associated antigens. The
cases were classified as being of T-cell typc when theneoplastic cells
in frozen sections were positive with one or more of the antibodies to
CD2. CD3. or CD5 and negative for CDIY, CD20. and CD22 and/
or paraffin sections were positive withatleast
two antibodies to
CD45RO. CD43. or CD3 in the absence of CD20 positivity. The
cases were classified :IS being of B-cell type when frozen sections
were positive with one or more antibodies to CD19. CD20, or CD22
in the absence of CD2. CD3. and CDS. and/or when paraffin sections
showed CD20 positivity with no more than one of either CD43 or
above
CD45RO being positive. When a case did not tit any of the
criteria. it was considered to have an indeterminate phenotype.
RESULTS
Perfonnonce of RT-PCR for npm-alk. Using the control
cell line JB6, the sensitivity of the optimized ntpm-dk RTPCR assay was determined. cDNA was prepared from I pg
of RNA (equivalent to approximately IO5 cells) andserial
IO-fold dilutions of the RT-reaction product from JB6 were
made in the RT-reaction product from 1 pg of K562 RNA
to simulate a progressively decreasing amount of tumor cells
with npnt-alk transcripts in irrelevant cells. Theexpected
175-bp rtpnt-alk product was clearly visible on agarose gels
when a dilution of IO-‘ JB6 cDNAwasused as template
(Fig l ) .
RT-PCR in ALCL. HI),crrtd reactive conditions. RNA
samples from 22 cases of CD30-positive ALCL. 21 cases of
classical HD, and 1 1 cases of reactive lymphoid hyperplasia
were suitable for analysis. Only cases with amplifiable products after the RT reaction using G3PDH primers were subjected to the rtprn-alk RT-PCR assay. All results were confirmedby probing themembranesforthedesired175-bp
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npm-alk FUSION GENE IN LYMPHOMA
3519
Table 1. npm-alk RT-PCR Analysis in CDBO-Positive ALCL,
Hodgkin's Disease, and Reactive Lymphoid Hyperplasia
the four cytogenetically negative ALCL cases showedthe
n p m - d k product with RT-PCR.
RT-PCR for t12;5)
DISCUSSION
Samples (n)
+
ALCL (22)
HD
Nodular sclerosis (15)
Mixed cellularity (6)
Reactive hyperplasia (11)
8
14
0
0
0
15
6
11
hybrid product using the breakpoint specific probe. Of the
22 ALCLcases. 8 (36%) were positive by the RT-PCR assay
(Fig 2). Noneofthe
21 cases ofHD or the I I cases of
reactive hyperplasia showedthe expected 175 bp product
(Table I ) . All HD and reactive cases remained negative following transfer and probing with the labeled npnt-dk junction oligonucleotide.
Noneof
IO placental negative control samples interspersed among thetest samples, the negative control cell
line RNA and cDNA, or the reaction mixtures without DNA
template added to the assay procedure was positive.
Clirticcrl jecrtlrres. The cases of ALCLwiththe
t(2;S)
differed fromthe negative cases withrespect to age and
tumor cell phenotype. The positive cases were younger and
were only of T-cell or indeterminant phenotype (Table 2).
Corre1crtiort.s with cytogenetic clatcr. Of the 22 cases of
ALCL, I O hadbeen previously analyzed cytogenetically.
Among these. the classical t(2;S)(p23,q3S) was found in six
cases, and in one additional case, the origin of a der 5q3S
was uncertain. The six cases with the classical t(2;S) were
all positive in the RT-PCR analysis.
Two additional cases ofNHLwiththe
t(2;S), butnot
included in our analysis of ALCL because of atypical morphologic features or the absence of CD30, were also studied.
These tissue samples weretestedwiththeRT-PCRassay
and a non-anaplastic, CD30'peripheral T-cell lymphoma
from a 7-year-old boy was found to be positive, whereas a
B-large-celllymphoma with monoclonalkappa expression
from a 73-year-old man was negative. Thus, of the eight
cases with a t(2;5)(p23,q3S) demonstrated cytogenetically,
seven (88%) were positive with the RT-PCR assay. None of
the positive control JB6. Lanes 2, 10, and 14 are
placental samples interspersedbetween test samples.Lanes 1, 4, and 15 through 18 are samples
with the characteristic band at 175 bp (upper
panel), which hybridizes strongly with the 32P-labeled junctional oligonucleotide (lower panel). M,
molecular-weight markers at 100-bp interval.
While the majority of cases of ALCL can be recognized
by a constellation of morphologic and immunohistochemical
features, there is no single characteristic feature that is pathognomonic of this lymphoma. The discovery of the
t(2;S)(p23;q35) in cases of ALCL was significant'"'
since
it wasthoughtto
be a specific marker for this form of
lymphoma, andtoprovide
a clue as toits pathogenesis.
However, the reported incidence of the t(2;S) is highly variable in ALCL,probably due tovariablemethods of case
selection in the various studies.
The cloning of the breakpoint in the t(2;5)(p23;q3S) was
a significant step toward the understanding of the pathogenesis of ALCL, and it opened the way to the demonstration of
the t(2;5) by molecular techniques." The new assays have
allowed the study of a larger number of cases of ALCL since
tissues with adequately preserved DNA and/or RNA can
now be examined. Several such studies of ALCL have been
published15.1X'?"or presented at recent
the
and
results are summarized in Table 3. Bullrichet all5 studied
16 cases of ALCL by Southern blot analysis and found two
positive cases (12.5%). A similar percentage of positives
(16%) wasfound by Lopategui et allXwhen37paraffinembedded tissues werestudied by RT-PCR.while Orscheschek et all'' reported that four of five cases (80%) they
examined were positive.Variable percentages of ALCL have
been reported to be positive
( 13% to 43%) in abstracts by
other groups."'.'' Some of the differences in these reported
series may be due to case selection with regard toage, phenotype, cytogenetic findings, andthe diagnostic criteria for
inclusion in the ALCL category. Our finding of 36% is close
tothemedianofthereported
series. We havebeenvery
strict in the pathologic criteria for case selection, so our
results should be representative of classic cases of ALCL.
Morphologic variants, CD3O- cases, primary cutaneous
ALCL, and cases associated with the acquired immunodeficiency syndrome have notbeen
included. Our patients
spanned the entire age spectrum, with four of the patients
under I8 years of age. It is interesting that the t(2;S)-positive
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3520
ELMBERGER ET AL
Table 2. Age, Sex, and Tumor Phenotype of Cases of CD30+ ALCL
would give rise to some overlap in the cases assigned to the
two categories.
ALCL
Cases
Male/Female
T/B/I
However, this explanation cannot account forthevery
high percentage of positive cases reported by Orscheschek
All cases 1319 22
35 yrs
a1411o
et aI.l9 Theprimers used by these latter investigators encom(3-86)
RT-PCR(+)
a 18
414
yrs
41014
pass a larger DNA fragment than the primers usually used
(3-32)
by other investigators. However, because the products of
58
RT-PCR(-)
915
14
yrs
41416
all their positive cases hybridized with the same junctional
(9186)
oligonucleotide, it does not appear thattheir cases had
breakpoints that were undetectable by the primers used by
others. Although it is possible that rare Reed-Sternberg cells
in HD could harbor this translocation, the techniques used
cases appear more prevalent in the younger age group and all
by us and other investigators are highly sensitive and yet we
of the ALCLs were of the T-cell or indeterminate phenotype
were unable todetect any positive cases of HD in our series.
(Table 2). Whether the t(2;5) positive cases form a distinct
Therefore,
even if the t(2;5) is present in HD, it would have
clinicopathologic subset of ALCL deserves further investigato be present in a very small fraction of the Reed-Sternberg
tion.
cells. This is supported by our cytogenetic studies of 5 1 cases
There was a very good correlation between the cytogenetic
ofHDwith clonal or non-clonal karyotypic abnormalities,
findings and the RT-PCR results when cases with a non-Bwhereinnot a single metaphase showing t(2;5)(p23;q35)
cell phenotype were considered with concordance in all
was detected (W.S., unpublished observations, April 1995).
seven cases. However, two cytogenetically positive cases [ 1
Wedidnot detect the t(2;5) byRT-PCR in any of our
with t(2;5); 1 with a der 5q35J with a B-cell phenotype were
cases of reactive lymphoid hyperplasia. Similar results have
not positive by the RT-PCR assay. It is interesting to note
been obtained by ~ t h e r s . ’ ~Therefore,
,’~
it appears that the
that the one B-cell ALCL case examined by Southern blotRT-PCR assay should be a useful assay to detect minimal
ting by Bullrich et alls appeared to have a different
residual disease or tostudy
biopsies with questionable
breakpoint from the one commonly found in ALCL. In most
involvement in patients with tumors containing the translocacases of ALCL with t(2;S), the breakpoints seem to occur
tion.Whenusedasan
alternative to cytogenetic studies,
in a common intron on npm and alk, respectively. Whether
there
would
be
a
small
percentage
of cases that would be
B-cell ALCL is associated with breakpoints that are outside
missed, including t(2;5)(p23;q35) with breakpoints different
of these introns requires further investigation. The B-cell
fromthe classical one andvariant translocations such as
ALCL with der 5q35 in our series may indeed not have the
t(2;5)(p21;q35),t(3;5)(q12;q35), and t(5;6)(q35;p12).“’.”
classic t(2; S ) .
Some of the variant translocations may be three-way transloIn most of the reported series, HD has been found to be
cations that involve nprn and alk,I5but one cannot exclude
negative for the t(2;5) by molecular assays or only a low
the possibility of other partners for the nprn gene.
percentage of positive cases have been found.’5316,22.24.2s
A
In conclusion, the RT-PCR assay is a useful alternative
striking exception is the report by Orscheschek et al,”
to cytogenetic studies and has the advantages of high sensiwherein 11 of 13 cases of HD (85%) were found tobe
tivity, lesslabor intensiveness, and does not require cell
positive by RT-PCR. Because it may be difficult to differenculture. For CD30’ ALCL with classical morphology, about
tiate HD from ALCL in some cases, it is conceivable that
36%ofthe
cases had detectable t(2;5)(p23;q35) bythis
the different diagnostic criteria used by various investigators
No.
Median Age
(range)
Phenotype
Table 3. Review of the Literature: t(2;5) in ALCL, HD, and Benign Conditions
ALCL
No.
Studies
t
et
(14)
Current study
Bullrich et all5
Ladanyi et aIz5
Lopategui et all8
Orcheschek et all’
Downing
McBride et alz’
Ngan et alzz
Weilmann et aiz3
Yee et aiz4
Total 20
Method
(36)
RT-PCR
Southern
RT-PCR
RT-PCR
(80)
RT-PCR
RT-PCR
Southern
RT-PCR
RT-PCR
RT-PCR
148
8
blot
No.
No.
Studied
Positive
22
16
-
4
21
blot
8
37
5
49
(13)
15
24 15 (13)
22
17
(29) 198
2
Studied
113)
21
9
40
(16)
13
(43)
-
-
2
2
5
58
No.
No.
(Yo)
Studied
Positive
(0)
(22)
(0)
11
0
-
-
-
-
No.
(%)
6
Benign Conditions
HD
Positive
0
2
0
-
1 14
(36)
(30)
* Lymphomatoid papulosis in all six cases.
t Cases classified as ALCL and imrnunoblastic included for analysis.
-
-
41
2
-
-
-
0
(85)
5
-
(211
(5)
6*
0
-
-
-
-
20
0
0
(%l
(0)
(0)
(0)
(0)
(0)
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npm-a/&FUSION GENE IN LYMPHOMA
3521
12. Ebrahim SA, Ladanyi M, Desai SB, OffitK, Jhanwar SC,
Filippa DA, Liebeman PH, Chaganti RS: Immunohistochemical
molecular cytogenetic analysis of a consecutive series of 20 peripheral T-cell lymphomas of uncertain lineage including twelve Ki-1
positive lymphomas. Genes Chromosomes Cancer 2:27, 1990
13. Mason DY, Bastard C, Rimokh R, Dastugue N, Huret JL,
Kristoffersson U, Magaud JP, Nezelof C, Tilly H, Vannier JP, Hemet
J, Warnke R: CD30-positive large cell lymphomas (“Ki-l
lymphoma”) are associated with a chromosomal translocation inACKNOWLEDGMENT
volving 5q35. Br J Hematol 74:161, 1990
14. Moms SW, Kirstein MN, Valentine MB, Dittmer KG, ShaWe thank Robert Wickert for technical assistance, Martin Bast
piro DN, Saltman DL, Look A T Fusion of a kinase gene, ALK, to a
for data management, George Pallas for photography, andKaren
nucleolar protein gene, NPM, in non-Hodgkin’s lymphoma. Science
Hansen for preparation of the manuscript.
263:1281, 1994
15. Bullrich F, Moms SW, Hummel M, Pileri S, Stein H, Croce
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