Diagnosis of t(2;5)(p23;q35)-associated Ki-1 lymphoma with immunohistochemistry

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1994 84: 3648-3652
Diagnosis of t(2;5)(p23;q35)-associated Ki-1 lymphoma with
M Shiota, J Fujimoto, M Takenaga, H Satoh, R Ichinohasama, M Abe, M Nakano, T Yamamoto
and S Mori
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Diagnosis of t(2; 5)(p23; q35)-Associated Ki-l Lymphoma
With Immunohistochemistry
By Mami Shiota, Jiro Fujimoto, Masato Takenaga, Hitoshi Satoh, Ryo Ichinohasama, Masafumi Abe,
Masaru Nakano, Tadashi Yamamoto, and Shigeo Mori
Some Ki-l lymphomas carry a specific chromosomal translo- ent on biopsied lymphomas, reverse transcriptass-polymercation, t(2;51(p23;q35). W e have recently found a novel hyas8 chain reaction (RT-PCR) covering the fusion junction of
perphosphorylated 80-kD protein tyrosine kinase, p80, in a
p80 mRNA was performed. Among 10 Ki-l lymphomas and
human Ki-l lymphoma with this translocation. Subsequent
l 0 additional lymphomas other than the Ki-l lymphomas,
cDNA cloning showed that p80 is a fusion protein of two
expression of p80 mRNA was detectedin three cases excludifferent genes on chromosome 2p23 and 5q35, the novel
sively. When these 20 cases and additional 30 lymphomas
tyrosine kinase gene and nucleophosmin gene, respectively.were immunostained with anti-p80, positive staining was
In this study, we intended to detect p80 on lymphoma tisnoted exclusively in the three cases found by PCR to have
sues with immunologic methods. Thus,we developed rabbit
harbored the p80 mRNA. Thus,the present immunostaining,
as well as PCR, wasshown to beefficientfordetecting
polyclonal antibody using a synthetic peptide corresponding
to a part of its kinase domain. The antibody (anti-p80) immu-lymphomas producing this chimeric protein/mRNA.
0 1994 by The American Societyof Hematology.
AMS3. Then, to examine whether t{2;5)(p23;q35)was pres-
1-1 LYMPHOMA, also called anaplastic large cell
lymphoma or large cell anaplastic lymphoma, is a
subtype of human non-Hodgkin's lymphoma (NHL) characterized by expression of CD30 (Ki-l antigen) and its peculiar
large neoplastic cells, which mimick HodgkidReed-Sternberg cells.'.* Recent studies have shown that Ki-l lymphomas comprise a heterogeneous group, differing in their clinicalcourse,histology,immunohistology,and
Around one third of all Ki-l lymphomas carry a chromosomal translocation, t(2;5)(~23;q35),"~and these cases are
suggested to constitute a unique subgroup sharing an identical genetic background. However, the clinical and biological
specificity of cases with t(2; 5 ) have not yet been elucidated.
We recently established a Ki-l lymphoma cell line with
t(2;5) inmicewith
severe combined immunodeficiency
(SCID)." Subsequent analysis of signal transduction in this
cell line showed hyperphosphorylation of a unique, phosphotyrosine-containing protein of Mr 80,000, designated p80.
Amino acid sequence analysis of tryptic digests showed that
p80 is a novel protein tyrosine kinase similar to LTK."
Subsequent cloning of p80 cDNA showed that the p80 gene
is a fusion gene made up partly of a novel protein tyrosine
kinase and partly of nucleophosmin (unpublished observations, October 1994). As characterization of this cDNA was
being completed, the same cDNA was identified by posiFrom The Departments of Pathology and Oncology, The Institute
of Medical Science, The University of Tokyo; and Nichirei, CO,
Tokyo, Japan.
Submitted August 19, 1994; accepted September 26, 1994.
Supported in part by the Ministry of Education, Science, and
Culrure, Japan (grant no. 04557019).
Address reprint requests to Mami Shiota, MD, Department of
Pathology, TheInstitute
of Medical Science, The University of
Tokyo, Tokyo 108, Japan.
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 1994 by The American Society of Hematology.
tional cloning of this chromosomal breakpoint by another
group." Thus, it is now evident that p80 is a chimeric protein
that characterizes Ki- 1 lymphomas bearing t(2; 5)(p23;q35).
These results promoted us to study Ki- l lymphomas using
reverse transcriptase-polymerase chain reaction (RT-PCR)
and immunohistology, anticipating that the results would be
of value for diagnosis of this specific subtype, as well as the
study of p80-mediated intracellular events.
Cells and tissues. A human Ki-l lymphoma cell line AMS3,
bearing the reciprocal chromosomal translocation t(2;5)(p23;q35),
was maintained in SCID mice, The details of AMS3 have been
described previously." SurgicalIy excised and fresh-frozen human
tissues, comprising three nontumorous tonsils and 50 malignant
lymphomas, were selected from our tissue library. The lymphomas
comprised 10 Ki-l lymphomas, 6 HLs, and 24 NHLs. The 24 NHLs
consisted of 6 T-cell NHLs including 3 diffuse large noncleaved
cell type, 2 immunoblastic type, and l lymphoblastic type; 16 Bcell NHLs including 2 follicular lymphoma, 3 diffuse small lymphocytic type, 9 diffuse large noncleaved type, and 2 immunoblastic
type; and 2 non T-non B cell NHL with the histology of immunoblastic type. Three of them were known to ear t(2;5)(p23;q35), whereas
cytogenetic data were not available for most of the other cases. The
specimens had been embedded in OCT compound (Miles Laboratories, Kankakee, IL), snap-frozen in n-hexane precooled with dry
ice-acetone and stored at -70°C until use. All of these cases were
used for immunohistologic study, and 20 rather well-presereved
cases were also used for RT-PCR assay. The paraffin-embedded
sections of some of these cases were also used for immunohistology.
In vitro-maintained human T-cell leukemia cell lines, Jurkat and
MT- 1, were used for Western blotting. None bore the t(2;5) chromosomal translocation. The cells were cultured in RPM1 1640 (Nissui
Pharmaceutical, Tokyo, Japan) containing 10% (voVvol)fetal bovine
serum (ICN Biochemical, Tokyo, Japan) at 37°C and 5% COZ.Peripheral blood mononuclear cells (PBMC) isolated by centrifugation
with Ficoll-Hypaque (Pharmacia LKB Biotechnology, Uppsala,
Sweden) from healthy donors were also used as another control.
RT-PCR. RT-PCR was performed to clarify the presence of p80
fusion mRNAs in stained lymphomas. RNAs were isolated from 20
lymphomas, comprising 10 Ki-l lymphomas, 6 T-cell lymphomas,
4 B-cell lymphomas, and also I nontumorous tonsil, using the guanidinium-acidphenol extraction method." To make cDNA pools,
Blood, Vol 84, No 11 (December I). 1994: pp 3648-3652
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total RNAs (10 pg) were reverse-transcribed with 100 U of modified
RT from Moloney Murine Leukemia Virus (Superscript; BXL) in a
total volume of 25 pL according tothe manufacturer’s protocol
Each cDNA pool (0.5 pL) was PCR-amplified in 50 pL of solution
containing 10 mmol/L Tris (pH 8.3), 50 mmoVL KC1, 1.5 mmoVL
MgCl,, 200 p m o K d N T P s , 30 pm01 of primers, and 2.5 U of Taq
polymerase (Toyobo, Japan). Three primers were prepared to detect
two cDNAs, those of nucleophosmin for nonfusion gene and p80
for fusion gene. Thus, the forward primer for both reactions was
the reverse primer for
nucleophosmin cDNA (M23613), 5’-TGGAACC”TGCTACCACCTC-3‘, and for p80, 5’-GAGCTTGCTCAGCTGTACTC-3’.
With the use of these primers, a 252-bp product was expected for
nucleophosmin cDNA, and a 225-bp product for p80 cDNA. The
reaction was performed for 2 minutes at 94°C for 1 cycle, 30 seconds
at 9 4 T , 30 seconds at W C , and 2 minutes at 72°C for 40 cycles.
Antibody againsrp80 (mti-p80). The antibody against p80 (antip80) was raised by immunizing rabbits with a synthetic peptide
SNQEVLEFVTSGGR, the sequence obtained in our previous
study,’’ corresponding to the putative kinase domain. The specific
antibody was purified by affinity chromatography on columns to
which the synthetic peptide had been linked covalently.
Western bloning. AMs3 cells were lysed with RIPA buffer (1%
vol/vol, Triton X-100) and the cleared lysates were immunoprecipitated with anti-p80. The whole lysates and the immunoprecipitates
were then subjected to sodium dodecyl sulfate (SDS)B% polyacrylamide gel electrophoresis (PAGE) under reducing conditions and
then transferred to polyvinylidene difluoride membrane (Bio-Rad,
CA), which was subsequently blocked by incubation with bovine
serum albumin (50 mg/mL). The blot was probed in turn with the
anti-p80 antibody (1 mg/mL), diluted 1:1,O00, and the preimmune
serum and the antibody preabsorbed with excess synthetic peptide
used as the antigen.
Irnrnunohisrochernical study. The labeled avidin-biotin (LAB)
method was used to demonstrate p80 and other antigens on tissue
section^.'^ To detect p80 and some other lymphocyte markers, antip80 and several CD antibodies described in the Results section were
used as the first immunohistology reagents. The preimmune serum,
previously taken from the rabbit that harbored anti-p80 after immunization, was also used as a control serum. For the second- and thirdphase reagents, biotinylated antirabbit Ig (E353; DAKOPA’ITS,
Denmark) and streptavidin (E364; DAKOPAITS) were used. The
staining procedures were as follows: Frozen sections (5-pm thick)
were cut with a cryostat and fixed with periodate-lysine paraformaldehyde (PLP) solution for 15 minutes. They were then washed with
phosphate-buffered saline (PBS), incubated with appropriately diluted antibody for 1 hour, washed with PBS, incubated with the
second- and third-phase reagents for 30 minutes, and washed. They
were then stained with 0.6 mg/mL 3.3’-diaminobenzidine tetrahydrochloride (Sigma Laboratories CO, St Louis, MO) in PBS containing
0.01% HZOZ
for 5 minutes. The immunostaining of paraffin sections
was done by the method of Shi et al.’5.’6
RT-PCR analysis was performed to show the expression
of the fusion gene. Although 252-bp products corresponding
to the control nucleophosmin cDNA were found in all 21
samples including 20 NHLs and 1 normal tonsil, the 225bp products corresponding to the p80 cDNA were found
exclusively in 3 cases (Fig 1). These 3 cases were found to
be the same ones found in the previous analysis to bear the
chromosomal translocation t(2; 5)(p23;q35), suggesting that
this RT-PCR detected this chromosomal translocation spe-
cifically. All 3 of these cases were Ki-l lymphomas and not
other subtypes.
On Western blotting, anti-p80 demonstrated an 80-kD
band on AMs3 but not on the other cell lines, Jurkat and
MT-1, or on PBMC from a healthy donor. No extrabands
were observed in any of these cells. The 80-kD band was
eliminated by preabsorbing the 1:1,000-diluted antibody
with excess synthetic peptide that was used as the immunogen (Fig 2).
On immunostaining with anti-p80, most of the AMs3 cells
were heavily stained, the reaction being evident in the nuclei
and cytoplasm. This immune reaction was observed up to a
dilution of 1:13,500 (Fig 3). Meanwhile, in the control tonsil
tissues, a weak reaction was noted on follicular dendritic
cells and endothelial cells. However, this reaction was very
faint and easy to differentiate from the heavy reaction of
AMS3. In addition, such reactions were not observed in any
of the lymphoma tissues. AMs3 wasnot stained by the
1:1,000-diluted preimmune serum.
Among the 50 lymphomas that were subjected to immunostaining, only three reacted with anti-p80. All of these three
cases were the Ki-l lymphomas that had been shown in the
preceding cytogenetic study to bear t(2;5)(p23;q35), and
also those that expressed p80 mRNA detected by RT-PCR.
The staining of these three specimens was clear and definite,
the reaction being found in most, if not all, of the neoplastic
large cells. However, the intracellular locations of the immune reaction differed among the cases. Thus, in case 1,
nuclei were most heavily stained, and definite nuclear staining was observed in about two thirds of the neoplastic cells
of this particular case. In cases 2 and 3, the cytoplasm was
most heavily stained, whereas nuclei were stained occasionally in about one third of the neoplastic cells. The cell membrane did not seem to be the main locus of immunostaining.
The paraffin sections of AMs3 also reacted with this antibody, clearly and at similar loci (Fig 4).
On hematoxylin-eosin staining, all of the 10 Ki-l lymphomas showed a similar anaplastic large cell morphology, and
no clear differences were noted between anti-p80-stained
and unstained cases. Also, on immunostaining with CD3
(Becton Dickinson [BD] CA), CD4 (BD), CD8 (BD), CD15
(BD), CD20 (Dakopatts, Denmark), CD25 (BD), CD71
(Nichirei, Tokyo, Japan) and antiepithelial membrane antigen (EMA, Dakopatts), no differences were noted between
p80-positive and -negative eases.
PCR and RT-PCR have been used to detect chromosomal
translocations in hematologic and other malignancies.” They
are especially useful for revealing the discrete loci of genomic DNA or cDNA containing the fusion junction. In the
present study, t(2;5)(p23;q35) was advantageous for RTPCR study because the cDNA sequences around the fusion
junction were identical in all five cases studied thus far,
including our AMS3 cells.11,12
The primer pairs used in the
present RT-PCR were prepared to show this fusion junction,
and 3 of 20 cases were found to harbor 225-bp products of
the expected size. As expected, these three cases were the
same Ki-l lymphomas that, on previous cytogenetic study,
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3 4
6 7 8 9 10 11
12 13 1 4 15 16 l7 18 19
Fig 1. Demonstration oft(2;51(p23;q35) by RT-PCR. The 225-bp product indicates thepresence of p80 chimeric mRNA from the fusiongene
junction, whereas the 252-bp product corresponds t o nucleophosmin mRNA from nontranslocated chromosome 5q35. The p80 cDNA was
demonstrated in cases 1 through 3 exclusively. (Lane 21,D-H,O as a negative control.)
had been shown to carry t(2;5)(p23;q35). The succeeding
immunohistologic study using anti-p80 also showed that
these three cases, and none of the other 47 cases, reacted
with this antibody. Thus, the results of RT-PCR and immunostaining corresponded perfectly.
The immune reaction on t(2;5)-carrying Ki-l lymphomas
was found in both cytoplasm and nuclei, although the ratio
of stained nuclei varied among the cases. Also, the intensity
of nuclear or cytoplasmic staining vaned among neoplastic
cells in the same tissue section. These variations in immunostaining may somehow be related to the molecular function
of p80 in these cells. Wang et allxreported that nucleophosmin was predominantly found in nucleoli, and partly in cytoplasm fractions, presumably associating with cytoskeletal
elements. Present results in terms of localization of p80 may
somehow be related to the intracellular localization of
In the meantime, it is necessary to be cautious about the
specificity of anti-p80. Anti-p80 was thought initially to recognize p80 specifically. Thus, an oligopeptide corresponding
to a unique part of the kinase domain was selected as the
immunogen. Later, upon cloning of p80 cDNA, as well as
upon recently published sequences of cDNA covering the
junction of t(2;5),'* this oligopeptide was confirmed to be
encoded by the kinase portion of unique tyrosine-kinase,
termed as ALKby Moms et al.'* Therefore, wehad to
consider the possibility that anti-p80 may also react with
kinase portion ALK. However, on immunostaining and
Western blotting, no lymphomas or peripheral blood lymphocytes not carrying t(2;5) gave positive signals, suggesting that anti-p80 can be used on human lymphomas and
non-neoplastic lymphocytes to detect p80 specifically.
LTK is another protein tyrosine kinase that may crossreact with anti-p80, because its catalytic domain is highly
homologous to p80. Thus, we immunostained a human erythroid leukemia cell line K562, which is known to express
Fig 2. Anti-p80 shows an 80-kD band corresponding t o p80 in the Ki-l lymphoma cell line AMS3, but not in control cells. Cell l y s a t e s
were immunoprecipitated with 25.2G4 (anti-phosphotyrosine antibody; Wako Biochemicals, Japan) and the immunoprecipitates were further
immunoblotted withantbp80 (lane 1).Whole lysates of AMs3 (lane 2). the controlcell lines MT1(lane 4) and Jurkat(lane 5).and mononuclear
cells from a healthy control (lane 6) were directly immunoblotted with anti-p80. Lane 3, AMs3 cell lysates immunoblotted with anti-p80
preabsorbed with an excess of the syntheticpeptide. Lane S, Rainbow proteinmolecular weight markers (Amersham, Japan). p80 is detected
only in lanes 1 and 2.
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Fig 3. lmmunostaining of four biopsied fresh-frozen tissues of Ki-l lymphomas. (a) lmmunostaining with CD30 (DAKO-BerH2, DAKO). (b)
lmmunostaining with anti-p80. (c) Hematoxylin-eosin staining. Cases 1,2, and 3, but notcase 4, are known to bear the chromosomaltranslocation t(2;51(p23;q35). Clear immunostaining isobserved in cases 1 through 3, but notin case 4, whereas all fourcases react strongly with CD30.
In case 1, nuclei are stained in about t w o thirds of the neoplastic cells, whereas in cases 2 and 3 the cytoplasm isstained.
Fig 4. lmmunostaining
paraffin section of case 1 with
anti-p80. Clear immune reaction,
equivalent t o Fig 3, is noted.
As the result, no staining was noted on K562, whereas
heavy staining was found on A M S 3 , the p80-carrying Ki-l
lymphoma cell line, apparently suggesting the absence of
cross-reaction between anti-p80 and LTK (data not shown).
Also, LTK is known to be expressed on normal immature
and mature B cells of miceZoas well as human (Hirai H.,
personal communication, October 1994). Our immunostaining of normal human tonsils or B-cell NHLs did not show
any positive reaction with non-neoplastic or neoplastic B
cells on these tissues, again suggesting the absence of cross-
reactivity of this antibody with LTK. Meanwhile, the reaction of follicular dendritic cells observed in non-neoplastic
tonsil must be studied further in detail to show their antigenic
specificities, even though these reactions were far weaker
and not evident in nuclei, and thus easy to differentiate from
reactions with t(2;5)-associated lymphomas.
The breakpoints of the chromosome translocation are often dispersed in certain restricted introns, leading to the production of, occasionally, a variety of different chimeric proteins." However, there remains a less likely possibility in
case of t(2;5)(p23;q35) that there exists another type of
translocation that does not produce p80 and is not detected
by the present RT-PCR. As yet, we cannot rule out this
possibility because a cytogenetic study was not done on a
large proportion of the present RT-PCR-negative or p80negative cases. However, this variant, if present, will remain
in a very minor group, because all the previously reported
or present cases with t(2;5)(p23;q35) studied so far showed
identical cDNA sequences around the fusion junction.
Ki-l lymphoma is known to constitute a heterogeneous
It is evident that cases with t(2;S) should be differentiated from other Ki- 1 lymphomas because they apparently
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differ in their genetic background. The demonstration of p80
will contributemuchtothisdifferentiation.Our
antibodyhas aspecificbenefitin
paraffin sections. The application of this antibody to a wide
range of paraffin-embedded Ki-l lymphomas will show the
identity of the t(2;5) subgroup in Ki- 1 lymphomas in terms
of their clinical course, histology, and other phenotypic characteristics.
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