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Regular Articles |
From the Institutes of Hematology*
and Internal
Medicine,
University of Perugia, Perugia, Italy;
University Department of Cellular Science,
John
Radcliffe Hospital, Oxford, United Kingdom; Institute of
Pathology,§
University of Bologna, Bologna, Italy;
Institute of Pathology,¶
Purpan Hospital, Toulouse,
France; Department of Pathology,||
Centro Oncologico
Aviano, Aviano, Italy; Institute of Pathology,**
University of Pavia,
Pavia, Italy; Institute of Pathology,
University of
Verona, Verona, Italy; Istituto Nazionale Tumori,
Milan, Italy; and Istituto Oncologico Europeo,§§
Milan, Italy.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ALCL is associated with a t(2;5) chromosomal translocation5 that fuses the ALK (anaplastic lymphoma kinase) and the NPM (nucleophosmin) genes,6,7 leading to the formation of a chimeric NPM-ALK protein (p80)6-8 consisting of the N-terminal portion of NPM9 linked to the cytoplasmic domain of the neural receptor tyrosine kinase ALK.10,11 The chimeric NPM-ALK protein is thought to play a key role in lymphomagenesis by aberrant phosphorylation of intracellular substrates.12,13
These discoveries allowed the development of reverse transcription (RT)-polymerase chain reaction (PCR) assays for the detection of NPM-ALK transcripts14 and the generation of polyclonal15-17 and monoclonal antibodies (mAbs)18 directed against the cytoplasmic portion of the ALK molecule. Extensive studies have demonstrated the presence of NPM-ALK gene and/or its protein product in cases of ALCL. The percentage of NPM-ALK-positive cases in different studies has varied between 30% and 60%, but a picture has emerged of a tumor that consistently presents with primary, systemic disease, shows a T/null phenotype, and usually occurs in the first three decades of life.16,19-28
A poorly investigated issue in this field concerns the correlation between histological features of ALCL and NPM-ALK protein expression. After the first description of ALCL by Stein et al in 1985,1 it became evident that neither anaplastic morphology nor CD30 expression could be regarded as absolute defining criteria for ALCL. Several pathologists reported morphological variants (eg, "common type," "lymphohistiocytic," "small cell," "neutrophil-rich," "sarcomatoid," or "Hodgkin's like"),29-36 which shared the same basic architectural features of ALCL, but which differed in terms of tumor cell cytology and the admixture of inflammatory cells. This raised the question whether the heterogeneous morphological features of the ALCL represent different clinicopathological entities or are just variants of a single disease.
In this paper, we have addressed this point by immunohistological labeling of a series of 100 cases of ALCL for ALK protein expression using a new mAb (ALKc) and also a previously reported anti-ALK antibody ALK1.18 We also investigated the nature of the small atypical cells that represent the predominant neoplastic population in the so-called lymphohistiocytic31 and small-cell variants32 of ALCL, an issue that has not been addressed in previous studies.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An NPM-ALK cDNA fragment corresponding to the whole open reading frame of the NPM-ALK protein was generated by PCR using oligonucleotide primers spanning the NPM ATG and the ALK TGA triplets. The PCR product was cloned into the pCRII vector of the TA cloning system (Invitrogen, San Diego, CA), checked by sequencing, and subcloned in PGEX-4T-1 (Pharmacia Biotech, Piscataway, NJ) to produce a glutathione S-transferase NPM-ALK full-length fusion protein. The protein was expressed in the HB101 Escherichia coli strain and purified by affinity chromatography following the manufacturer's instructions.
Production of the ALKc mAb
A fusion between the spleen cells of BALB/c mice previously immunized intraperitoneally with 150-µg aliquots of recombinant protein and the NS-1 myeloma cell line was carried out, as described previously.37 Hybridoma supernatants were screened by the immunoalkaline phosphatase (alkaline phosphatase-antialkaline phosphatase, APAAP) technique38 in cytocentrifuge preparations of a human cell line (Karpas 299) that carries the t(2;5).39 Selected hybridomas were cloned by limiting dilution. Five hybridoma supernatants out of approximately 1000 tested showed strong immunocytochemical staining of the Karpas 299 cell line but were unreactive on cryostat sections of normal human tonsil. Further testing on paraffin sections of ALCL bearing the (2;5) translocation showed that one of the supernatants reacted strongly with tumor cells but did not stain normal cells. The hybridoma was cloned to produce the ALKc clone used in subsequent studies.
Other Antibodies
The mAb ALK1 raised against a fragment (amino acids 419 to 520) of the cytoplasmic portion of ALK protein has been described previously.18 Immunophenotyping of ALCL in paraffin sections was performed with antibodies directed against the following antigens: CD45, CD45RO, CD3, and CD20 (all obtained from DAKO A/S, Glostrup, Denmark); CD30/Ber-H2 (kindly provided by Prof. H Stein, Free University of Berlin, Berlin, Germany); and CD45RA, CD68, CD79a, and PML proteins (generated in the investigators' laboratories).
Expression of the NPM-ALK Protein in HeLa Cells
NPM-ALK cDNA corresponding to the whole open reading frame of the protein was subcloned in the pcDNA3 expression vector (Invitrogen) and used for transient transfection of HeLa cells by the calcium chloride-HEPES-buffered-saline (HBS) method.40 As negative control, HeLa cells were transfected in parallel with the plasmid vector containing no insert.
Western Blotting
Western blotting of cell lysates of the human cell lines U937, Karpas 299, Daudi, and Rh30 rhabdomyosarcoma6 was performed as previously described18 using mAbs ALKc and ALK1 (diluted 1:5).
Enzyme-Linked Immunosorbent Assay
The reactivity of the antibodies ALKc and ALK1 or the negative control reagent MR12 (mouse anti-rabbit MR12, prepared in the laboratory of Mason et al)18 was tested against DHFR-ALK (a recombinant protein containing amino acids 1359 to 1460 of the full-length ALK receptor protein used to raise the antibody ALK1), by using a previously described enzyme-linked immunosorbent assay technique.18
Cell Lines
A variety of human cell lines of different origin, MOLT-4, Jurkat, and Peer (T cell); Daudi, Nalm 12, and Cess (B cell); L-428 and L540 (Hodgkin's); Karpas 299, JB6, Su-DHL1 (ALCL bearing t(2;5)); K-562 (erythroid); U937, HL60, KG1, and NB4 (myeloid); and HeLa (carcinoma), were maintained in culture in RPMI 1640 containing 10% fetal calf serum (Life Technologies, Inc., Grand Island, NY). Cytospins were prepared from exponentially growing cells, fixed in acetone for 10 minutes at room temperature, and then used for immunocytochemical studies.
Tissue Processing for Immunohistochemistry
Paraffin-embedded tissue samples had been fixed either in 10% buffered formalin for 24 hours to 1 week (most cases) or in Brasil-Dubosq or B5 for 2 hours (a minority of cases). Paraffin sections on silane-coated slides were rehydrated and subjected to microwaving (750 W for three cycles of 5 minutes each) using either 0.01 mol/L citrate buffer, pH 6.0,41 or 1-mmol/L ethylenediaminetetraacetic acid buffer, pH 8.0,42 as antigen retrieval solution. After microwave heating, sections were allowed to cool at room temperature for approximately 20 minutes, washed with Tris-buffered saline, and immunostained.
Frozen sections from snap frozen samples (when available) were air dried overnight and fixed in acetone for 10 minutes.
Normal Human Tissues
Normal lymphohemopoietic tissues comprised tonsil (n = 10), spleen (n = 5), bone marrow (n = 5), and thymus (n = 3). Samples representative of all extrahemopoietic tissues were also investigated. All tissues were diagnostic biopsies or were obtained at the time of autopsy.
Reactive and Neoplastic Lymphoid Samples
The following nonneoplastic conditions were studied: follicular hyperplasia (n = 10), toxoplasmic lymphadenitis (n = 5), tubercular lymphadenitis (n = 2), Kikuchi's lymphadenitis (n = 5), sarcoidosis (n = 3), and reactive T-immunoblastic proliferations (n = 2).
A total of 510 cases of lymphoid neoplasms that included 100 cases of ALCL and 40 cases of acute and chronic myeloid disorders were retrieved from the authors' institutions. Lymphomas and leukemias were categorized according to the REAL43 and FAB44 classifications. In all cases, diagnosis was based on morphological examination of conventionally stained tissue sections supplemented by immunophenotyping. Diagnostic immunomorphological criteria for ALCL were those originally established by Stein et al,1 and an attempt was made to assign each case to one of the following morphological subtypes of ALCL: common, lymphohistiocytic, small cell, giant cell, or Hodgkin's-like, according to previously defined criteria.29,31,32,36,43 The assessment was made independently by two investigators (BF and SAP). All cases were reviewed for the second time after immunostaining for the ALK protein, and controversial cases were discussed to reach a consensus.
Nonhemopoietic Tumors
The following nonhemopoietic tumors were investigated: 50 carcinomas from various sites, 10 melanomas, 50 soft tissue tumors of different types (including 35 cases of rhabdomyosarcomas), and 25 tumors of the central nervous system.
Immunoenzymatic Labeling
All sections were stained by the immunoalkaline phosphatase (APAAP) technique, as previously described.38 Briefly, paraffin sections were incubated with the primary mAbs, followed by rabbit anti-mouse immunoglobulin (Dako, Glostrup, Denmark) and APAAP complexes. To maximize the sensitivity of the method, steps 2 and 3 were repeated once each. All antibody steps were for 30 minutes with intervening 5-minute washes in 0.05 mol/L Tris-buffered saline, pH 7.6. Endogenous alkaline phosphatase was blocked with 1 mmol/L levamisole.45 Slides were then counterstained for 5 minutes in Gill's hematoxylin and mounted in Kaiser's glycerol gelatin (Merck, Darmstadt, Germany).
RT-PCR Analysis
RT-PCR studies were performed as previously described.24
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Western blotting of the t(2;5)-positive Karpas 299 cell line using
antibody ALKc revealed an 80-kd immunoreactive polypeptide that
corresponded in size to the NPM-ALK fusion protein (Figure 1)
. ALKc also detected the full-length
200 kd ALK protein present in the rhabdomyosarcoma cell line Rh30
(Figure 1)
. Identical bands, although at lower intensity, were detected
by the ALK1 antibody (not shown). No bands were detected by the ALKc
mAb in lysates of the U937 and Daudi cell lines that served as negative
controls (Figure 1)
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in ALCL cell lines bearing t(2;5) (Karpas 299, JB6 and SuDHL1). No
other human cell lines were labeled by the ALKc mAb. As expected (due
to expression of full-length ALK), ALKc gave strong diffuse cytoplasmic
positivity in the absence of nuclear labeling (Figure 2C)
in a
rhabdomyosarcoma sample that had been previously shown to react with
the ALK1 antibody. Staining of the same biopsy with the ALK1 and ALKc
antibodies at high dilution showed expression of the ALK protein even
at the level of cell membrane (Figure 2D)
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ALK protein was not detected with antibody ALKc in any of the normal and reactive lymphohemopoietic tissues tested on cryostat and paraffin sections. The only reactivity in normal tissues was observed in the brain (weak positivity of a few neural cells).
ALK Protein Expression in Human Lymphomas
Paraffin sections from 510 cases of lymphomas representative of
the different categories in the REAL classification,43
were
analyzed for expression of ALK protein(s) (Table 1)
. Reactivity was restricted to
approximately 60% of ALCL (all of T or null phenotype) (Table 1)
.
These ALK-positive lymphoma cases usually presented in the first three
decades of life, whereas ALK-negative ALCL cases were most frequent in
older patients with a plateau in the sixth decade (Figure 3)
. The lower incidence of ALK-positive
cases in the first decade as compared to the second and third ones
(Figure 3)
is a bias due to the limited number of pediatric patients
(less than 14 years old) who are usually referred to the institutions
participating to this study.
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About 75% of the ALK-positive cases showed the morphology of the
classic or common type (Table 2)
characterized by a proliferation of large, bizarre cells (Figure 4A)
, which tended to invade lymph node
sinuses and infiltrate the paracortex, sometimes in a pseudocohesive
pattern. In most cases, the large cells expressed the ALK protein
strongly both in the cytoplasm and in the nucleus (Figure 4B)
, a small
proportion of large cells showing nucleus-restricted ALK positivity
being rarely observed (Figure 4C)
. In about one-third of these cases, a
percentage of small cells (range, 5 to 30%) that showed
nucleus-restricted reactivity for the ALK protein (Figure 4D)
was also
present. This population of cells was usually not recognized on
conventionally stained paraffin sections. About 15% of all
ALK-positive cases showed restricted expression of the ALK protein in
the cytoplasm of large neoplastic cells (Figure 4E)
. With the exception
of one case of giant cell type (see below), all samples showed a
"common" type morphology and were devoid of ALK-positive small
cells.
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Six of the 60 ALK-positive cases displayed the histological features of
the so-called lymphohistiocytic variant of ALCL, as first described by
Pileri et al31
(Figure 5A)
.
In the majority of these cases, clustered or isolated CD30-positive
large anaplastic cells displayed both cytoplasmic and nuclear ALK
positivity and were accompanied by a variable percentage of small cells
showing nucleus-restricted ALK positivity (Figure 5B)
. These small
cells showed weak or absent CD30 staining (not shown). In some samples,
ALK-positive small cells were morphologically indistinguishable from
reactive lymphocytes, having scant cytoplasm and round nuclei (Figure 5, A and B)
, and ALK labeling was the only way they could be
recognized, especially when present at a low percentage in the
paracortex of the lymph node.
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were admixed with small
numbers of large anaplastic tumor cells, which tended to localize
around blood vessels (Figure 5E)
. The small neoplastic cells usually
showed a nucleus-restricted ALK positivity (Figure 5D)
, occasionally
associated with a faint cytoplasmic labeling (Figure 5F)
, which never
attained the intensity of the cytoplasmic positivity observed in the
scattered large cells located around the blood vessels (Figure 5F)
.
Two ALK-positive lymphomas showed Hodgkin's-like features, eg, bands
of sclerosis dividing the lymph node parenchyma into nodules that
contained tumor cells with a "lacuna-like" appearance. One of the
cases was characterized by common type cytology, whereas the other
showed the presence of two tumor cell populations segregated in
different areas of the lymph node (Figure 6, A and B)
, ie, large lacunar-like cells (ALK
positive both in the cytoplasm and nucleus) within the nodules and
small-sized elements with nucleus-restricted ALK positivity encased
within the fibrous bands surrounding the nodules, which were regarded
as reactive lymphocytes at conventional morphology. Minimal bone marrow
involvement in this case could be documented only by ALK staining
(Figure 6, C and D)
.
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ALK Protein Expression in Nonhemopoietic Tumors
One hundred biopsies representative of a large variety of
nonhemopoietic tumors were all ALK-negative, with the exception of a
single case of rhabdomyosarcoma (1 of 35 tested), which showed strong
cytoplasmic and cell membrane-associated labeling for the ALK protein
(Figure 2, C and D)
.
Comparison of ALKc and ALK1 Antibodies
Antibody ALK1 but not ALKc or the negative control antibody MR12 reacted with the recombinant protein ALK-DHFR in the enzyme-linked immunosorbent assay. No cross-blocking of ALK1 and ALKc antibodies was observed at Western blotting, where ALKc gave stronger bands than ALK1. At immunohistochemistry, the two antibodies gave essentially identical reactions in most cases, but ALKc reacted more strongly than ALK1 with the nuclei of tumor cells (especially those of the small size) in at least 15 to 20% of specimens. One of the 60 ALK-positive lymphomas reacted with ALKc but not with ALK1. Conversely, two cases (one fixed in formalin and the other in B5) were ALKc negative/ALK1 positive.
All together, these findings demonstrate that the mAb ALKc is directed against a different ALK epitope from that recognized by antibody ALK1 and suggest that the highest yield of information is obtained when both the antibodies are employed for the study of ALCL.
RT-PCR Studies
As shown in Table 3
, the finding of
ALK expression at the nuclear level correlated with a positive RT-PCR
test for NPM-ALK. However, in the two cases in which
labeling was restricted to the cytoplasm, RT-PCR was negative.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The production of polyclonal antibodies and mAbs to ALK protein15-18 made it possible to detect, by immunohistological techniques, the NPM-ALK fusion protein generated by the t(2;5) translocation. This has allowed the histological features of large numbers of ALK-positive lymphomas to be reviewed.16,18,28,47,48 In the present paper, we document by using a new anti-ALK mAb, ALKc, the range of morphological appearances found among these neoplasms.
The ALK-positive lymphomas in this series included five cases that showed features of the small cell variant. These cases were of interest for several reasons. First, ALK reactivity was seen not only in the large neoplastic cells but also in the numerous smaller neoplastic cells. This implies that the genetic lesion (the NPM-ALK gene), which is presumed to play a direct causal role in the genesis of ALK-positive lymphomas,6,7 must be present in all of the neoplastic cells. The large cells cannot, therefore, represent a subclone that has arisen in a low-grade (small cell) lymphoma after the acquisition of the (2;5) anomaly. In this context, it may be noted that a recent study has addressed this possibility directly by studying a series of transformed T-cell lymphomas and has confirmed that histological progression is not accompanied by the appearance of the (2;5) translocation.49 Immunocytochemistry also identified a typical perivascular pattern of large cells. Similar findings have been recently reported by Benharroch et al.50
Small neoplastic cells expressing ALK protein were, by definition, most evident in the small cell variant but were also found in cases showing other histological patterns. In the lymphohistiocytic lymphomas, they were particularly obvious in immunostained sections, and it is possible that most cases of this neoplasm should be considered as examples of the small variant of ALCL in which large numbers of reactive histiocytes have accumulated. Approximately one-third of ALK-positive lymphomas cases showing the common histological pattern also contained a minor population of small cells showing nucleus-restricted ALK reactivity.
We suggest that many ALK-positive lymphomas lie on a spectrum, their
position being defined by the ratio of small to large neoplastic cells
(Figure 7)
. The tissue distribution of
tumor cells (eg, "perivascular pattern"), the occurrence of
sclerosis (occasionally imparting to the lesion a Hodgkin's-like
appearance), and the presence of reactive histiocytes or other cell
types, probably induced by the release of cytokines by tumor cells
(especially those of small size) may add to the heterogeneous
morphological pattern of ALK-positive lymphomas. The recognition of the
small neoplastic cells in this study was facilitated by the ALKc mAb,
which tends to label them more strongly than does ALK1,51
the first anti-ALK mAb to be described,18
presumably
because it detects a different epitope. Antigen retrieval in
ethylenediaminetetraacetic acid buffer, which acts by chelating calcium
ions (possibly responsible for masking of several nucleus-located
antigens),42
may have contributed further to these
findings.
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The ability to detect by immunocytochemistry a population of ALK-positive small cells also has important diagnostic implications in the following settings: 1) distinction of the lymphohistiocytic variant from benign reactive lymphadenopathies or infection-associated hemophagocytic syndromes,53 2) differential diagnosis between the small cell variant and peripheral T cell lymphomas or inflammatory infiltrates (especially in skin),36 and 3) detection of very small number of tumor cells in bone marrow and/or lymph nodes either at the time of initial diagnosis or after therapy.
A final observation of interest is that, in approximately 15% of the ALK-positive lymphomas in this study, labeling appeared to be confined to the cytoplasm. A similar observation has been recently reported in an independent study by Benharroch et al.50 Our hypothesis is that these cells may carry variant translocations, in which the ALK gene on chromosome 2 is linked to a gene other than NPM. We also assume that the resultant fusion protein(s) activates the ALK kinase by cross-linking (as occurs in the case of NPM-ALK), but that they do not contain any motifs that direct the protein to the nucleus. There is at least one precedent for this in a reported case of ALK-positive ALCL that carried the (1;2) translocation and in which immunolabeling was also confined to the cytoplasm.18,24 Furthermore, an engineered TPR-ALK construct has been reported that can transform cells but that remains confined to the cytoplasm.12 It is of interest that the cases in the present study that showed only cytoplasmic labeling for ALK seemed to lack the degree of variation in neoplastic cell size seen in the majority of ALK-positive lymphomas. This hints at the possibility that they may represent a subtype of ALK-positive lymphoma with distinctive histological and possibly clinical features. Further biochemical and molecular biological studies of neoplasms in which ALK positivity is confined to the cytoplasm are therefore clearly likely to be of interest.
In conclusion, this study emphasizes the importance of immunohistological labeling for ALK protein(s) in the evaluation of ALCL. All cases in this series were of T cell or null phenotype, and the age of these patients tended to be lower than that of ALK-negative ALCL (confirming other data).16 In consequence, ALK positivity appears to define a clinicopathological entity (ALK lymphomas), but one that shows a much wider spectrum of morphological patterns than has been appreciated in the past.
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Acknowledgements |
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Footnotes |
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Supported by A.I.R.C. (Associazione Italiana Ricerca Cancro).
Accepted for publication May 6, 1998.
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