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REVIEW ARTICLE
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 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 In 1985, a number of the authors1
described 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
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.
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
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
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
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 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
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 optimismdo 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
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
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
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
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-
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.66,69,106ALK 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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Abstract
Introduction
-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.
and 
genes
and expressed the cytotoxic molecules perforin, granzyme B, and
T-cell-restricted intracellular antigen-1 (TIA-1) (Figures 
