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Blood, 1 December 2000, Vol. 96, No. 12, pp. 3681-3695

REVIEW ARTICLE

CD30+ anaplastic large cell lymphoma: a review of its histopathologic, genetic, and clinical features

Harald Stein, Hans-Dieter Foss, Horst Dürkop, Theresa Marafioti, Georges Delsol, Karen Pulford, Stefano Pileri, and Brunangelo Falini

From the Institute of Pathology and Consultation and Reference Center for Lymph Node Pathology and Haematopathology, University Hospital Benjamin Franklin, Free University, Berlin, Germany; Laboratoire d' Anatomie et Cytologie Pathologiques, Hôpital Purpan, Toulouse, France; Nuffield Department of Clinical Laboratory Sciences, LRF Immunodiagnostics Unit, John Radcliffe Hospital, University of Oxford, Oxford, England; Servizio di Anatomia Patologica---Sezione di Emolinfopatologia, Università degli Studi di Bologna, Italy; and Dipartimento di Medicina Clinica e Sperimentale, Sezione di Ematologia e Immunologia Clinica, Policlinico Monteluce, Università degli Studi di Perugia, Italy.


    Abstract
Top
Abstract
Introduction
How ALCL was recognized
Morphologic features and...
ALCL may overlap with...
Immunophenotype
Immunoglobulin (Ig) and TCR...
Putative normal counterparts of...
Structure and pathogenic role...
Epstein-Barr virus
Clinical features and subforms
Soluble CD30 in ALCL...
Reproducibility of the...
Summary and conclusions
References

Anaplastic large cell lymphoma (ALCL) represents a generally recognized group of large cell lymphomas. Defining features consist of a proliferation of predominantly large lymphoid cells with strong expression of the cytokine receptor CD30 and a characteristic growth pattern. With the use of molecular and clinical criteria, 3 entities of ALCL have been identified: primary systemic anaplastic lymphoma kinase (ALK)+ ALCL, primary systemic ALK- ALCL, and primary cutaneous ALCL. ALK expression is caused by chromosomal translocations, most commonly t(2;5). ALK+ ALCL predominantly affects young male patients and, if treated with chemotherapy, has a favorable prognosis. It shows a broad morphologic spectrum, with the "common type," the small cell variant, and the lymphohistiocytic variant being most commonly observed. The knowledge of the existence of these variants is essential in establishing a correct diagnosis. ALK- ALCL occurs in older patients, affecting both genders equally and having an unfavorable prognosis. The morphology and the immunophenotype of primary cutaneous ALCL show an overlap with that of lymphomatoid papulosis. Both diseases have an excellent prognosis, and secondary systemic dissemination is only rarely observed. The described ALCL entities usually derive from cytotoxic T cells. In contrast, large B-cell lymphomas with anaplastic morphology are believed to represent not a separate entity but a morphologic variant of diffuse large B-cell lymphoma. Malignant lymphomas with morphologic features of both Hodgkin disease and ALCL have formerly been classified as Hodgkin-like ALCL . Recent immunohistologic studies, however, suggest that ALCLs Hodgkin-like represent either cases of tumor cell-rich classic Hodgkin disease or (less commonly) ALK+ ALCL or ALK- ALCL. (Blood. 2000;96:3681-3695)

© 2000 by The American Society of Hematology.

    Introduction
Top
Abstract
Introduction
How ALCL was recognized
Morphologic features and...
ALCL may overlap with...
Immunophenotype
Immunoglobulin (Ig) and TCR...
Putative normal counterparts of...
Structure and pathogenic role...
Epstein-Barr virus
Clinical features and subforms
Soluble CD30 in ALCL...
Reproducibility of the...
Summary and conclusions
References

In 1985, a number of the authors1 described---under the term anaplastic large cell lymphoma (ALCL)---a novel lymphoma category defined by a frequently cohesive proliferation of large pleomorphic blasts and a constant expression of the CD30 molecule on all neoplastic cells. Despite these common features, heterogeneity in the cytology and in the antigen profile of the tumor cells, as well as in the clinical features of patients affected by this condition, was noticed in the original description.1 This led to the distinction of several morphologic, immunophenotypic, and clinical subforms of ALCL.1-17 A morphologic and immunophenotypic overlap with classic Hodgkin disease was also recognized.1,10,13,18,19 In the late 1980s, a translocation between chromosomes 2 and 5 was assigned to a proportion of ALCL cases.20-23 All of these findings raised the questions of whether the morphologic, immunophenotypic, clinical, or genetic forms represent variants of the same disease or different diseases and what their relation is to Hodgkin disease. In the last 5 years, there has been great progress in the molecular characterization of ALCL and Hodgkin disease, the result being that most of the questions raised can now be answered.


    How ALCL was recognized
Top
Abstract
Introduction
How ALCL was recognized
Morphologic features and...
ALCL may overlap with...
Immunophenotype
Immunoglobulin (Ig) and TCR...
Putative normal counterparts of...
Structure and pathogenic role...
Epstein-Barr virus
Clinical features and subforms
Soluble CD30 in ALCL...
Reproducibility of the...
Summary and conclusions
References

In 1982, Stein's group24,25 discovered a new molecule that was initially termed Ki-1 and subsequently designated CD30.26-28 CD30 is strongly expressed on Hodgkin and Reed-Sternberg cells of classic Hodgkin disease, but is absent from the cells of all normal tissues except for scattered activated large lymphoid blasts preferentially located around B-cell follicles. Biochemical studies29 and molecular cloning30 have revealed that CD30 is a 120-kd transmembrane cytokine receptor of the tumor necrosis factor receptor family, for which the ligand (CD30L) was identified.31 A soluble 85-kd form of CD3032 was found to be released from the membrane-bound molecule by proteolytic cleavage33 and can be detected in the sera of patients with CD30+ reactive and neoplastic lesions. Immunohistochemical analysis of a large range of human tumors has shown that CD30 is constantly expressed not only by Hodgkin and Reed-Sternberg cells, but also by a subset of diffuse large cell neoplasms, most of which had originally been diagnosed as malignant histiocytosis, regressing atypical histiocytosis, anaplastic (metastatic) carcinoma, malignant melanoma, seminoma, or even as malignant fibrous histiocytoma.1,3,10,28,34 The frequent presence of lymphoid markers and the consistent absence of molecules associated with histiocytic or other cell lineages indicated a lymphocytic origin for this new tumor category.1 The lymphoid nature was further confirmed by the demonstration of clonal rearrangements in antigen receptor genes.1,35,36 Because of the constant Ki-1/CD30 expression and the frequent anaplastic features, this tumor form was initially called anaplastic large cell lymphoma (ALCL)1 and then, variably, lymphoma large cell anaplastic CD30+ (Kiel classification37), Ki-1 lymphoma,2 or Ki-1+ large cell lymphoma.3 However, the 2 latter terms are inadequate because CD30 is also expressed in some unrelated neoplasms, such as Hodgkin disease1,25,28 or embryonal carcinoma.38 The term anaplastic large cell is also not adequate because the tumor cells in the small cell variant are not large and the monomorphic or immunoblastic variants are not anaplastic. Despite this and because of the absence of a better designation, the term ALCL has now been adopted by the Revised European American Lymphoma (REAL)13 and the new World Health Organization (WHO) classifications.39


    Morphologic features and subforms
Top
Abstract
Introduction
How ALCL was recognized
Morphologic features and...
ALCL may overlap with...
Immunophenotype
Immunoglobulin (Ig) and TCR...
Putative normal counterparts of...
Structure and pathogenic role...
Epstein-Barr virus
Clinical features and subforms
Soluble CD30 in ALCL...
Reproducibility of the...
Summary and conclusions
References

The histologic appearance of ALCL was originally described as a preferential paracortical involvement of lymph nodes with intrasinusoidal dissemination (Figure 1C); although this growth pattern is evident in partially involved lymph nodes, it remains otherwise diffuse.1 Because of the wide histologic spectrum of the tumor cell population and the admixture of reactive cells, several groups proposed the subclassification of ALCL into the subforms listed in Table 1.


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Figure 1. Morphology and immunophenotype of anaplastic large cell lymphoma, common type. (A) Cytologic features of ALCL, common type, in a paraffin section (hematoxylin and eosin). (B) Cytologic features of ALCL, common type, in a touch imprint (May-Grünwald-Giemsa). (C) Dissemination of ALCL cells in a marginal sinus. MS indicates marginal sinus; GC, reactive germinal center (hematoxylin and eosin). (D) CD30 immunoreactivity of ALCL, common type. All tumor cells are CD30+. Note the sinusoidal dissemination (arrow) and the perifollicular homing of the CD30+ tumor cells (antibody clone Ber-H2; alkaline phosphatase-antialkaline phosphatase [APAAP]). (E) Immunoreactivity of an ALCL of common type for the cytotoxic molecule perforin. The sinusoidal dissemination of the tumor cells (arrow) and a small reactive cytotoxic cell (arrowhead) are indicated (antibody clone P1-8; APAAP). (F) Immunoreactivity of an ALCL of common type for the anaplastic lymphoma kinase (ALK). There is strong labeling in the cytoplasm as well as in the nucleus (antibody clone ALKc; APAAP). (G) An ALCL of common type with labeling for ALK restricted to the cytoplasm (antibody clone ALKc; APAAP).


                              
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Table 1. Morphologic subforms of ALCL and their correlation with immunophenotype

The common type1,10,13,17 is characterized by sheets of large lymphoid cells with chromatin-poor horseshoe-shaped nuclei containing multiple nucleoli (Figure 1A). Cells with these cytologic features have been called hallmark cells40 because they are encountered in all ALCL variants, including the small cell and lymphohistiocytic variants. Multinucleated cells with Reed-Sternberg-like appearance may also occur. The tumor cells have an abundant cytoplasm which, in imprint preparations, frequently shows numerous vacuoles (Figure 1B). The monomorphic subform7 probably represents a variant of the common type. Because of the cytologic resemblance of the latter to immunoblastic lymphoma, it can easily be confused with nonanaplastic large cell lymphomas when immunohistochemistry is not applied. In the giant cell-rich type,3,10,13,17 a large number of the tumor cells contain more than one nucleus. The small cell variant11 is characterized by a mixture of small, medium-sized, and large lymphoid cells (Figure 2). The nuclei of the small and medium-sized cell population are often irregular. Large cells surrounding small vessels are a frequent and characteristic finding. This is particularly evident following immunostaining for CD30, which highlights the large anaplastic cells. In contrast, heterogeneity is seen in CD30 expression in the smaller cell population, with many small cells being CD30- (more commonly) or weakly positive (Figure 2B). In the past, the small cell variant was usually diagnosed as peripheral T-cell lymphoma, and some authors still prefer this interpretation (reported by D. Weisenburger at a workshop on the findings of the International Non-Hodgkin Lymphoma Classification Porject, organized by J. Armitage and D. Weisenburger, September 8-10, 1997, Omaha, NE). The small cell variant may, however, contain areas with the morphology of ALCL common type (ie, sheets of CD30+ blasts) and can transform into the common type and vice versa.11,40,41 These observations strongly suggest that the small cell variant is part of the histologic spectrum of ALCL. Cytogenetic studies11 showing the t(2;5) and more recently the nucleophosmin (NPM)-ALK fusion protein in the small and large cells of this subform have strongly supported this concept (discussed later in detail). The dominant feature of the lymphohistiocytic subform3,8,10,13 is the large number of histiocytes, which may mask the anaplastic tumor cell population (Figure 2). The latter can be highlighted by CD30 immunostaining. Occasionally, the histiocytes show signs of erythrophagocytosis and often display a monomorphic appearance with eccentric nuclei, a feature that in the past often led to the misdiagnosis of malignant histiocytosis.8 However, the histiocytes hardly proliferate, as revealed by their negativity for the nuclear proliferation marker Ki-67, and are therefore reactive. In contrast, the tumor cells, making up only a minor component of the entire infiltrate, are negative for histiocytic markers but strongly positive for CD30 and Ki-67.8 They are usually smaller than in the common ALCL type, and therefore this subtype may be related to the small cell variant. Because of the small-sized tumor cell component, the lymphohistiocytic subform was not regarded as a variant of ALCL in the Kiel classification, but was erroneously categorized as a peripheral T-cell lymphoma.37


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Figure 2. Morphology and immunophenotype of the small cell variant and the lymphohistiocytic variant of ALCL. (A) Small cell variant of ALCL. Most of the cells have a small irregular nucleus, the cytoplasm is light, and larger cells are located in close vicinity to small vessels (Giemsa). (B) Immunoreactivity of a small cell variant of ALCL for CD30. The larger tumor cells are strongly positive, whereas the small tumor cells are only weakly positive (alkaline phosphatase-antialkaline phosphatase [APAAP]). (C) Immunoreactivity of a small cell variant of ALCL for ALK. The larger cells (often located in the vicinity of small vessels) are positive in the cytoplasm and the nucleus, whereas in the smaller cells, the labeling is restricted mainly to the nucleus (APAAP). (D) Lymphohistiocytic variant of ALCL. Note the broad cytoplasm and the frequently slightly eccentrically located nuclei, which are revealed immunohistologically as belonging to reactive histiocytes (see panel E) (hematoxylin and eosin). (E) Immunoreactivity of the same case as in (D) with the CD68 antibody clone PG-M1. Nearly all of the larger cells are positive. These PG-M1-positive cells, representing nonneoplastic bystander macrophages, obscure the few CD30+ tumor cells (see Figure 2F) (APAAP). (F) Immunoreactivity of the same case as in (D) for CD30. Only a minority of the large cells are positive and represent the tumor cell population (APAAP). (G) Immunoreactivity of the same case as in (D) and (E) for ALK. The labeled cells vary greatly in size, with a predominance of smaller cells. The smaller the cells, the more the labeling is restricted to the nucleus. The negative cells are the bystander macrophages (antibody clone ALK1; APAAP). (H) Immunoreactivity of the same case as in (D), (E), and (G) for perforin. The tumor cells are strongly positive, whereas the bystander macrophages are negative (antibody clone P1-8;APAAP).

The sarcomatoid form of ALCL42 mimics soft-tissue tumors, especially of malignant fibrous histiocyte type. The neoplastic cells of this rare variant are large, bizarre, and often spindle-shaped, and express CD30. Multinucleated forms are present in varying numbers. The distinction from malignant fibrous histiocytoma is easily accomplished by immunohistology because these and other soft-tissue tumors consistently lack CD30 and other lymphoid markers. Other rare subforms of ALCL are characterized by an abundant admixture of eosinophils or neutrophils.13,43-45 Such cases may easily be mistaken as Hodgkin disease, true histiocytic malignancies, or even as an acute inflammatory process.44,45 This is especially valid for the neutrophil-rich subform because it may mimic an acute inflammation and, in the skin, a pustular lesion.44 An ALCL subform with signet-ring appearance has also been described.15,46


    ALCL may overlap with Hodgkin disease
Top
Abstract
Introduction
How ALCL was recognized
Morphologic features and...
ALCL may overlap with...
Immunophenotype
Immunoglobulin (Ig) and TCR...
Putative normal counterparts of...
Structure and pathogenic role...
Epstein-Barr virus
Clinical features and subforms
Soluble CD30 in ALCL...
Reproducibility of the...
Summary and conclusions
References

Although there is accumulating evidence that ALCL and Hodgkin disease are biologically distinct, the morphologic and immunophenotypic border between these disease categories is not sharp in all instances.10,18,19,47 This applies especially to Hodgkin disease cases rich in tumor cells, with lymphocyte depletion, nodular sclerosis grade 2, or syncytial growth pattern. To keep the entities of Hodgkin disease and ALCL distinct, investigators in the late 1980s created a category (basket) under the term ALCL-HD-related. The borderline cases, or gray-zone cases, could then be collected in this basket for further studies.3,10,48 Under the designation Hodgkin-like ALCL, this type was adopted by the REAL classification as a preliminary category.13 The tumors falling into this category show features of both ALCL and Hodgkin disease. These ambiguous cases contain relatively dense nodules or sheets of tumor cells with features of classic Hodgkin and Reed-Sternberg cells (Figure 3). Tumor cells are usually present within sinuses and, because of capsule thickening and nodular or diffuse fibrosis, the sinusoidal dissemination is on occasion recognizable only by immunolabeling for CD30. The proportion of admixed reactive inflammatory cells is lower than that found in typical cases of Hodgkin disease. The change from the term ALCL-HD-related to ALCL-HD-like reflects the tendency in the early 1990s to believe that most of these gray-zone lymphomas represent ALCL mimicking Hodgkin disease. However, the frequent expression of the B-cell-specific activation protein BSAP (PAX5) in the absence of the protein ALK (discussed later) favors the opinion that most cases of ALCL-HD-like represent a tumor cell-rich variant of classic Hodgkin disease and not a true ALCL because the mentioned expression pattern is characteristic for Hodgkin disease.49 Accordingly, the new WHO classification has abandoned this subform and subsumes these cases under classic Hodgkin disease.39,50,51


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Figure 3. Hodgkin-like ALCL. (A-D) Hodgkin-like ALCL, ALK+. (A) Morphology at low magnification (hematoxylin and eosin). Note the nodular sclerosis. (B) Same case as in (A) at higher magnification. The large tumor cells resemble Hodgkin- and Reed-Sternberg cells. (C) Same case as in (A) immunostained for EMA (antibody clone E29). All tumor cells are labeled. (D) Same case as in (A) immunostained for ALK (antibody clone ALK1). The tumor cells show a strong positivity of their cytoplasm. (E,F) Hodgkin-like ALCL, ALK-. (E) Morphology by Giemsa stain. (F) Immunoreactivity for CD30 (antibody clone Ber-H2; alkaline phosphatase-antialkaline phosphatase). All tumor cells are strongly CD30-positive, but ALK-negative (not shown).


    Immunophenotype
Top
Abstract
Introduction
How ALCL was recognized
Morphologic features and...
ALCL may overlap with...
Immunophenotype
Immunoglobulin (Ig) and TCR...
Putative normal counterparts of...
Structure and pathogenic role...
Epstein-Barr virus
Clinical features and subforms
Soluble CD30 in ALCL...
Reproducibility of the...
Summary and conclusions
References

Immunohistochemical screening of a large number of undifferentiated large cell malignancies has revealed that the tumor cells of all ALCL cases show a strong expression of CD30 on the cell membrane and in the Golgi region (Figure 1D; diffuse cytoplasmic CD30 positivity is of dubious significance), so that the membrane-associated expression of CD30 was included in the definition of ALCL.1,3,10,13,28 The analysis of conventional T- and B-cell markers revealed 3 immunophenotypes (Table 2), with the T-cell type being the most frequent.1,6,52 The epsilon -chain of the T-cell receptor CD3 complex (TCR) is one of the most constantly expressed T-cell antigens; a minority of cases express CD4 or CD8, with a predominance of CD4.1,10,53 The frequency of the null cell type depends on the number of T-cell antigens investigated. Most, if not all, of the null cell cases belong to the T-cell type. This becomes evident when a large number of T-cell antigens, or the configuration of the TCR genes (see below), is investigated and the studies are extended to cytotoxic molecules. The vast majority of the T/null ALCLs proved to harbor clonally rearranged TCR gamma  and beta  genes and expressed the cytotoxic molecules perforin, granzyme B, and T-cell-restricted intracellular antigen-1 (TIA-1) (Figures 1E, 2H, 12D), regardless of the expression of CD4 or CD8.36,53,54 Because cytotoxic molecules are expressed not only by cytotoxic T cells but also by natural killer (NK) cells,55,56 and activated NK cells may express CD30,57 there is the possibility that a minority of the T/null ALCLs are derived from NK cells rather than cytotoxic T cells. In support of this possibility is the expression of the NK cell-associated marker CD56 in some cases of ALCL54 and the finding that approximately 10% of ALCLs lack detectable TCR gene rearrangements.36 Further studies are required to clarify unequivocally the relation of some ALCL cases to NK cells.

                              
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Table 2. CD30+ large cell lymphomas with anaplastic morphology: immunophenotype, antigen-receptor genotype, and EBV infection

Large cell lymphomas with anaplastic morphology that express B-cell antigens are relatively rare1,6,58 (Table 2). They have been incorporated into the Kiel classification as a separate entity. However, according to the REAL and the new WHO classifications, they are not accepted as a distinct entity but are regarded as a morphologic and immunophenotypic variant of diffuse large B-cell lymphoma.13 Recent studies further support this view.59 Therefore, in the current article, these tumors are referred to as anaplastic large B-cell lymphomas. ALCLs that express both B-cell and T-cell antigens have been detected so far only by immunolabeling of frozen sections,1 possibly because of the higher sensitivity of this approach. The meaning of the double expression of T- and B-cell markers is obscure.

Antigenic markers can be useful in the distinction of the different clinical subforms of ALCL (further discussed later). Unlike the systemic form, the primary cutaneous ALCL is usually negative for epithelial membrane antigen (EMA)4,12 and for the ALK protein60-63 (Table 3). Moreover, nearly half of the cases arising in the skin are positive for the cutaneous lymphocyte antigen recognized by monoclonal antibody HECA 452.12

                              
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Table 3. Correlation between clinical features, morphology, and immunophenotype in ALCL

Immunophenotypic differential diagnosis of ALCL versus Hodgkin disease

As mentioned earlier, ALCL and Hodgkin disease share several morphologic and immunophenotypic features and, in some cases, assignment to one of these entities is not possible.3,10,13,64,65 This situation is particularly true for cases that lack expression of T-cell and B-cell antigens. The overlap between these conditions has vanished for the cases expressing ALK fusion proteins (chimeric ALK) because this protein is consistently absent from the tumor cells of all cases of Hodgkin disease.66-69 However, there is still an overlap (Figure 4) between ALK- ALCL and Hodgkin disease. In 1994, at the European Association of Hematopathology Workshop in Toledo, Spain, it became evident that markers such as CD15, BNH.9, and EMA---despite initial optimism---do not help in this distinction.19 This proved to be valid also for cytotoxic molecules because these may also be expressed by Reed-Sternberg cells of Hodgkin disease36,53,70 as well as by the tumor cells in ALCL. Recently, BSAP71 has been found to be expressed by Reed-Sternberg cells but not by cells of T-cell or null-cell-type ALCL.49,72 A preliminary study has demonstrated the usefulness of this antigen in the differential diagnosis of ALCL and Hodgkin disease.49


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Figure 4. Current morphologic and immunophenotypic overlaps of true ALCLs and anaplastic large B-cell lymphomas with classic Hodgkin disease. The REAL classification refers to the overlapping neoplasms as ALCL-HD-like because they cannot, because of missing morphologic and immunophenotypic criteria, be allocated to one of the established lymphoma entities. The overlap between ALK+ ALCL and Hodgkin disease has disappeared with the availability of monoclonal antibodies to ALK protein, which strongly stain the tumor cells of all cases of ALCL with a rearranged ALK gene but consistently fail to stain Hodgkin and Reed-Sternberg cells of Hodgkin disease.


    Immunoglobulin (Ig) and TCR gene rearrangements
Top
Abstract
Introduction
How ALCL was recognized
Morphologic features and...
ALCL may overlap with...
Immunophenotype
Immunoglobulin (Ig) and TCR...
Putative normal counterparts of...
Structure and pathogenic role...
Epstein-Barr virus
Clinical features and subforms
Soluble CD30 in ALCL...
Reproducibility of the...
Summary and conclusions
References

Initial studies73,74 on the configuration of the antigen receptor genes in ALCL, which were performed using the Southern blot technique, demonstrated a surprising divergence between immunophenotype and Ig and TCR gene rearrangements. More recent investigations using the PCR in conjunction with family-specific primers have, however, demonstrated an almost complete concordance between the T-cell and B-cell antigen profile and the presence of clonally rearranged TCR genes (Table 2).36 The demonstration of clonally rearranged TCR beta  and gamma  genes in 90% of cases of T- and null-type ALCL36 indicates that most, if not all, of the null ALCLs are immunophenotypic variants of T-type ALCL and that ALCLs originating from NK cells seem to be rare, despite the recently reported (although somewhat controversial) expression of CD56 in approximately one third of ALCLs.54


    Putative normal counterparts of ALCL
Top
Abstract
Introduction
How ALCL was recognized
Morphologic features and...
ALCL may overlap with...
Immunophenotype
Immunoglobulin (Ig) and TCR...
Putative normal counterparts of...
Structure and pathogenic role...
Epstein-Barr virus
Clinical features and subforms
Soluble CD30 in ALCL...
Reproducibility of the...
Summary and conclusions
References

Normal lymphoid tissue contains a small population of large lymphoid blasts that express CD30.1,24,25 These nonneoplastic CD30+ blasts resemble the neoplastic cells of systemic ALCL1,25 in their cytologic features and tissue distribution (preferentially perifollicular and occasionally intrasinusoidal localization). It is therefore tempting to assume that they represent the normal precursor cells of systemic ALCL. The question to be answered now is whether these normal CD30+ blasts have the same cytotoxic T-cell phenotype and genotype as systemic ALCLs. Such studies are in progress in the authors' laboratory.

So far, no likely candidate for the normal precursor cell for the primary cutaneous ALCL can be identified. The anaplastic large B-cell lymphoma might be derived from CD30+ germinal center B cells (as are most diffuse large B-cell lymphomas) because they carry---like reactive germinal center B cells and the nonanaplastic CD30- diffuse large B-cell lymphoma---somatic mutations in their rearranged variable Ig genes.75 This supports the close relation between diffuse large B-cell lymphomas with and without anaplastic morphology and is consistent with the concept of the REAL classification that anaplastic large B-cell lymphoma is a variant of diffuse large B-cell lymphoma.


    Structure and pathogenic role of the NPM-ALK gene
Top
Abstract
Introduction
How ALCL was recognized
Morphologic features and...
ALCL may overlap with...
Immunophenotype
Immunoglobulin (Ig) and TCR...
Putative normal counterparts of...
Structure and pathogenic role...
Epstein-Barr virus
Clinical features and subforms
Soluble CD30 in ALCL...
Reproducibility of the...
Summary and conclusions
References

In the late 1980s, it was found that a proportion of ALCLs were associated with a 2;5 chromosomal translocation.20-23 As demonstrated by Morris et al76 in 1994, the 2;5 translocation causes the NPM gene located at 5q35 to fuse with a gene at 2p23 encoding the receptor tyrosine kinase anaplastic lymphoma kinase (ALK). The properties of wild-type NPM and ALK as well as their chimerized genes and proteins are summarized in Table 4 and in Figure 5. Wild-type NPM (also known as B23) was first identified in the late 1970s and early 1980s78,79 as a ubiquitous acidic 37-kd phosphoprotein associated with nucleoli. NPM shuttles continuously between the cytoplasm and the nucleolus and thus functions as a carrier of newly synthesized proteins into the nucleolus.80 The NPM molecule exercises this function through an oligomerization motif81 at the N-terminal region and 2 nuclear localizing signals at the C-terminal domain82 (Figure 5). The wild-type ALK protein is a 200-kd transmembrane receptor that is most closely related to leukocyte tyrosine kinase (LTK)76,83-85 and whose postnatal expression is restricted physiologically to a few scattered cells in the nervous system (some glial cells, a few endothelial cells, and some pericytes).66 The intracellular tail of the ALK molecule carries the tyrosine kinase catalytic domain (Figure 5), which becomes physiologically activated as a result of homodimerization following ligand binding.86

                              
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Table 4. Properties of NPM, ALK, and NPM-ALK



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Figure 5. Molecular structure of nucleophosmin (NPM), anaplastic lymphoma kinase (ALK), and ALK fusion proteins. The NPM molecule consists of an oligomerization domain (residues 1-83), a metal-binding domain (MB; residues 104-115), 2 acidic amino acid clusters (AC; residues 120-132 and 161-188) that function as acceptor regions for nucleolar targeting signals,82 and 2 nuclear localization signals (NLS). The oligomerization domain was defined by means of deletion mutants.81 The ALK protein is a transmembrane tyrosine kinase receptor containing a transmembrane domain (TM) and a tyrosine kinase domain (TKD) in the N-terminal part of the intracytoplasmic tail. In the NPM-ALK fusion protein, the extracellular and transmembrane domains of ALK are replaced by the oligomerization domain of NPM (in approximately 75%) or of other proteins (X), schematically represented in Figure 7. The fused part of NPM contains, in addition to the oligomerization domain, the metal-binding region. The fusion point is at codon 117. N indicates amino terminal; C, carboxy terminal.

The 2;5 translocation juxtaposes the portion of the NPM gene encoding the N-terminal domain of NPM (amino acids 1-117) (Figure 5) to the part of the ALK gene that codes for the entire cytoplasmic region of the ALK protein.76,87 As a consequence, the ALK gene comes under the control of the NPM promoter, which induces a permanent and ubiqitous transcription of the NPM-ALK hybrid gene, resulting in the production of an 80-kd chimeric protein termed NPM-ALK76 or p80.88 This NPM-ALK protein contains the NPM oligomerization domain and the intracytoplasmic region of ALK. The C-terminal NPM domain carrying the nuclear localization signals and the extracellular and transmembrane region of the ALK are absent.76,87 The NPM-ALK protein can form homodimers (by cross-linking with other NPM-ALK molecules) or heterodimers (by cross-linking with wild-type NPM) (Figure 6). The formation of homodimers results in the constitutive activation of the catalytic ALK domain contained in the NPM-ALK fusion protein.86,87 The activated ALK domain has been shown to bind GRB288,89 and the SH2 domains of phospholipase C-gamma ,89 interactions that have been demonstrated to induce mitogenic activity and are likely to be involved in neoplastic transformation.86,89 Transfection of murine hematopoietic cells with the NPM-ALK fusion gene induces transplantable lymphoid tumors.90 NPM-ALK was also found to transform rat fibroblasts in vitro.91 Both of these latter findings further support the oncogenic property of this fusion protein.


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Figure 6. Patterns of oligomerization and cellular distribution of NPM-ALK fusion and variant ALK-fusion proteins in the various subforms of ALK+ ALCL.

Methods for the demonstration of NPM-ALK and the subcellular distribution of this fusion protein

The presence of the NPM-ALK translocation was initially demonstrated in tissue samples by Southern blot analysis,92 reverse transcriptase-polymerase chain reaction (RT-PCR),93-97 in situ hybridization,67 and, more recently, by 2-color fluorescence in situ hybridization (FISH).98 The application of these techniques has confirmed the association of ALCL with the 2;5 translocation. The Southern blot and RT-PCR techniques have, however, produced discrepant results over the frequency of the NPM-ALK fusion gene in ALCL and the occurrence of this anomaly in large B-cell lymphoma, Hodgkin disease, and even in normal cells.99-101 Because RT-PCR is prone to artifact102 and FISH is time-consuming and difficult to apply to paraffin sections, the production and use of polyclonal85,103,104 and monoclonal antibodies specific for fixative-resistant epitopes on the cytoplasmic tail of the ALK protein66,69 and also on the N-terminal domain of NPM105 represented a significant advance in the detection of the NPM-ALK anomaly. Because ALK protein is absent in all normal tissues, with the exception of scattered cells in the brain,66 a positive immunohistochemical staining in tissues (other than brain) indicates anomalous ALK expression, usually in the form of the t(2;5)-associated NPM-ALK fusion protein.66,69,103-105 Thanks to the generation of a monoclonal antibody against the N-terminus of NPM, the molecular association of the detected ALK with NPM can also be demonstrated immunohistologically because, in the presence of NPM-ALK, this antibody stains both the cytoplasm and the nucleus,105 whereas in tissues devoid of NPM-ALK, the labeling is restricted to the nucleus.105,106 The putative mechanism that might account for the different subcellular distribution of NPM-ALK is represented in Figure 6. The lack of nuclear localization signals in the chimeric NPM-ALK protein suggests that its transportation into the nuclei of tumor cells most likely occurs through the formation of heterodimers of NPM-ALK with wild-type NPM,107 which contains 2 nuclear localization signals.82 The availability of anti-ALK and anti-NPM antibodies applicable to archival paraffin-embedded tissues allowed the screening of large numbers of neoplasms, leading to a clear perception of the presence and frequency of the NPM-ALK fusion protein and the possibility of variant ALK proteins in lymphomas.66,69,106

ALK proteins other than NPM-ALK

In 3 large series of ALCL, 15% to 28% of chimeric ALK+ lymphomas were found to be negative for the t(2;5) translocation (as detected by immunohistochemistry), and it was suggested that they may represent cases in which the ALK gene fuses to a partner other than NPM to produce variant X-ALK protein(s).40,66,69,106 Such X-ALK+ lymphomas are characterized by a cytoplasm-restricted expression of the ALK protein (Figures 6 and 7) and a nucleus-restricted expression of wild-type NPM.106 Additional evidence to support the presence of chimeric ALK proteins other than NPM-ALK has been obtained from reports of genetic abnormalities affecting the ALK gene in ALK+ ALCL. These include the inversion (2)(p23;q35) and the translocations (1;2)(q21;p23) and (2;3)(p23;q21),108-110 suggesting the existence of genes other than NPM that can deregulate the ALK gene. The existence of variant ALK proteins has been confirmed by immunobiochemical studies using the monoclonal antibodies to ALK and NPM (N-terminal domain).111 Western blotting studies have demonstrated the presence of variant ALK proteins of 85, 97, 104, and 113 kd.111 These new ALK fusion partners have now been identified by 5' rapid amplification of cDNA ends (RACE) studies (Figure 7). Lamant et al112 described the 104-kd ALK protein as being TPM3 (nonmuscle tropomyosin)-ALK in a tumor exhibiting the (1;2)(q21;p23) translocation. The 85- and the 97-kd ALK proteins were found to be generated by a fusion of the ALK gene with the TFG (tropomyosin receptor kinase-fused gene).113 The larger TFG-ALK fusion protein (TFG-ALKlong) contains an additional 165-bp TFG sequence113 and is associated with the (2;3)(p23;q21) translocation.110 Both of the TPM3 and TFG genes have been found to be involved in the deregulation of the kinase domain of other oncogenic tyrosine kinases present in carcinomas.114,115 In common with NPM, both TFG and TPM3 proteins contain dimerization regions. The possibility therefore exists that the formation of homodimers of TPM3-ALK or TFG-ALK (to mimic ligand binding) results in the constitutive activation of the ALK kinase domain, conferring oncogenic activity on these variant ALK proteins. In support of this is the finding that both TFG-ALK and TPM3-ALK proteins are capable of auto-phosphorylation in vitro.112 The 2 other ALK fusion partners recently identified are ATIC (5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleotide transformylase/inosine monophosphate cyclohydrolase),116-118 caused by the inversion (2)(p23;q35), and CLTCL (clathrin heavy polypeptide-like gene),119 which occurs as a result of the (2;22)(p23;q11) translocation (Figure 7). Recent studies have also been able to document the frequency with which the newly found ALK fusion variants occur (Figure 7).112,113,116-119 It is of pathogenic significance that all chimeric ALK variants contain the same functional kinase domain of ALK as that present in the NPM-ALK protein (Figure 7). The lack of nuclear localization signals in the variant fusion proteins (other than NPM-ALK) accounts for the absence of these fusion proteins from the nucleus and their distribution only in the cytoplasm (Figure 6).


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