Anaplastic Large Cell Lymphoma, ALK Positive and ALK Negative

Epidemiology

ALCL accounts for 5% of all non-Hodgkin's lymphomas and 10% to 30% of childhood lymphomas. ALK-positive ALCL is most frequent in the first 3 decades of life and shows a slight male predominance.

Etiology

No pathogenic factor has been demonstrated. However, in rare cases, an association with recent insect bites has been observed. Occasional cases occur in human immunodeficiency virus (HIV)-positive patients or after solid organ transplantation. It is unlikely that these conditions play a primary etiologic role, but emergence of the disease may be facilitated by abnormal cytokine production.

Clinical Features

The majority of patients (70%) with systemic ALCL present with advanced stage III to IV disease with peripheral or abdominal lymphadenopathy, often associated with extranodal infiltrates and involvement of the bone marrow. Patients often show B symptoms (75%), especially high fever. Several cases with a leukemic presentation have been reported.

Primary systemic ALCL positive for the ALK protein frequently involves both lymph nodes and extranodal sites. Extranodal sites commonly include skin (26%), bone (14%), soft tissues (15%), lung (11%), and liver (8%). Retinal infiltration responsible for blindness and placental involvement have also been reported. Involvement of the gut and central nervous system is rare. However, occasional cases of primary ALCL in the stomach, bladder, or central nervous system have been observed (authors' unpublished observations and reference ). Mediastinal disease is less frequent than in Hodgkin's lymphoma. The incidence of bone marrow involvement is approximately 10% when analyzed with hematoxylin-eosin but increases significantly (30%) when immunohistochemical stains for CD30, EMA, or ALK are used ( Fig. 37-1 ). This is due to the fact that bone marrow involvement is often subtle, with only scattered malignant cells that are difficult to detect by routine examination. Most patients have circulating antibodies against nucleophosmin (NPM)-ALK protein, and these antibodies may persist in patients who are apparently in complete remission.

Morphology

The morphologic features of ALCL are wider than was initially described, ranging from small-cell neoplasms, which many pathologists might mistake for peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS), to tumors in which very large cells predominate.

ALCLs positive for the ALK protein exhibit a broad morphologic spectrum. However, all cases contain a variable proportion of large cells with eccentric horseshoe- or kidney-shaped nuclei, often with an eosinophilic region near the nucleus. These cells have been referred to as hallmark cells ( Fig. 37-2, A ) because they are present in all morphologic patterns. Although the hallmark cells are typically large, smaller cells with similar cytologic features may be seen and can greatly aid in making the diagnosis. Depending on the plane of the section, some cells may appear to contain cytoplasmic inclusions. These are not true inclusions, however, but invaginations of the nuclear membrane. Cells with these features have been referred to as donut cells (see Fig. 37-2, A ). In some cases, the nuclei are round to oval, and the proliferation appears quite monomorphic (see Fig. 37-7, A ).

The tumor cells have more abundant cytoplasm than most other lymphomas. The cytoplasm may appear clear, basophilic, or eosinophilic. On lymph node imprints, these cells show vacuolated cytoplasm (see Fig. 37-2, B ). Multiple nuclei may occur in a wreathlike pattern, giving rise to cells resembling Reed-Sternberg cells. The nuclear chromatin is usually finely clumped or dispersed, with multiple small basophilic nucleoli. Prominent inclusion-like nucleoli are relatively uncommon, aiding in the differential diagnosis with Hodgkin's lymphoma.

ALCLs exhibit a very broad range of cytologic appearances. Five morphologic patterns were recognized in the fourth edition of the WHO classification.

Anaplastic Large Cell Lymphoma, Common Pattern

ALCL, common pattern (70%) is composed predominantly of pleomorphic large cells with the hallmark features described earlier. Tumor cells with more monomorphic, rounded nuclei also occur, either as the predominant population or mixed with the more pleomorphic cells. Rarely, erythrophagocytosis by malignant cells may be seen. When the lymph node architecture is only partially effaced, the tumor characteristically grows within the sinuses and thus may resemble a metastatic tumor ( Fig. 37-3 ). Tumor cells may also colonize the paracortex and often grow in a cohesive manner ( Fig. 37-4 ).

Anaplastic Large Cell Lymphoma, Lymphohistiocytic Pattern

ALCL, lymphohistiocytic pattern (10%) is characterized by tumor cells admixed with a large number of histiocytes ( Fig. 37-5, A to C ). The histiocytes may mask the malignant cells, which are often smaller than in the common pattern (see Fig. 37-5, D ). The neoplastic cells often cluster around blood vessels and can be highlighted by immunostaining with antibodies to CD30 (see Fig. 37-5, E and F ), ALK, or cytotoxic molecules. Occasionally the histiocytes show signs of erythrophagocytosis. The histiocytes typically have finely granular eosinophilic cytoplasm and small, round, uniform nuclei. Well-formed granulomas are absent, and clusters of epithelioid cells (as may be seen in the lymphoepitheloid cell variant of PTCL, NOS) are not seen.

Anaplastic Large Cell Lymphoma, Small-Cell Pattern

ALCL, small-cell pattern (10%) shows a predominant population of small to medium-sized neoplastic cells with irregular nuclei ( Fig. 37-6, A to C ). However, morphologic features vary from case to case, and cells with round nuclei and clear cytoplasm (“fried egg” cells) may predominate. Hallmark cells are always present and are often concentrated around blood vessels (see Fig. 37-6, D ). Usually there is massive infiltration of the perinodal connective tissue. This morphologic variant of ALCL is often misdiagnosed as PTCL, NOS by conventional examination. When the blood is involved, atypical cells reminiscent of flowerlike cells may be observed in smear preparations. It is likely that the small-cell and lymphohistiocytic patterns are closely related.

Anaplastic Large Cell Lymphoma, Hodgkin-Like Pattern

ALCL, Hodgkin-like pattern (1% to 3%) is characterized by morphologic features mimicking nodular sclerosis classical Hodgkin's lymphoma. These cases show a vaguely nodular fibrosis associated with capsular thickening and a significant number of tumor cells resembling classic Reed-Sternberg cells associated with hallmark cells ( Fig. 37-7, E ). In the past, many tumors with similar features were referred to as Hodgkin-like ALCL . However, most cases designated as such were ALK negative and were more likely variants of classical Hodgkin's lymphoma rich in Hodgkin cells or lymphomas with features intermediate between Hodgkin's lymphoma and diffuse large B-cell lymphoma—so-called gray-zone lymphomas. It must be stressed that CD30-positive lymphomas, with or without a sinusoidal growth pattern, should not be diagnosed as ALCL, Hodgkin-like unless they are positive for ALK. In cases negative for ALK protein, additional immunophenotypical and molecular studies usually permit their classification as aggressive B-cell or T-cell lymphomas, including the new WHO category of B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin's lymphoma.

Immunophenotype

By definition, all ALCLs are positive for CD30. In most cases, virtually all neoplastic cells show strong CD30 staining on the cell membrane and in the Golgi region ( Fig. 37-8, A ). In the small-cell variant, the strongest immunostaining is seen in the large cells; smaller tumor cells may be only weakly positive or even negative for CD30. In the lymphohistiocytic and small-cell patterns, the strongest CD30 expression is also present in the larger tumor cells, which often cluster around blood vessels (see Figs. 37-5, F and 37-6, D ). The majority of ALCLs are positive for EMA. The staining pattern for EMA is usually similar to that seen with CD30, although in some cases only a proportion of malignant cells is positive (see Fig. 37-8, B ).

The great majority of ALCLs express one or more T-cell or natural killer (NK)-cell antigens. However, owing to the loss of several pan–T-cell antigens, some cases may have an apparent null-cell phenotype. Because no other distinctions can be found in cases with a T-cell versus null-cell phenotype, T/null ALCL is considered a single entity. CD3, the most widely used pan–T-cell marker, is negative in more than 75% of cases. This tendency for loss of CD3 is also seen in ALK-negative ALCL. CD5 and CD7 are often negative as well. CD2 and CD4 are more useful and are positive in a significant proportion of cases. CD43 is expressed in more than two thirds of cases, but this antigen lacks lineage specificity (see Fig. 37-8, C ). Furthermore, most cases exhibit positivity for the cytotoxic-associated antigens TIA-1, granzyme B, and perforin (see Fig. 37-8, D and E ). CD8 is usually negative, but rare CD8-positive cases exist. Occasional cases are positive for CD68/KP1 but not CD68/PGM1.

Tumor cells are variably positive for CD45 and CD45RO but strongly positive for CD25. Blood group antigens H and Y (detected with antibody BNH.9) have been reported in more than 50% of cases (see Fig. 37-8, F ). CD15 expression is rarely observed, and, when present, only a small proportion of neoplastic cells is stained. ALCLs are consistently negative for Epstein-Barr virus (EBV) (i.e., EBV-encoded small RNA [EBER] and latent membrane protein-1 [LMP-1]). A study with array technology to detect new genes expressed in ALCL found that clusterin is aberrantly expressed in all cases of systemic ALCL but not in primary cutaneous ALCL. Most ALK-positive ALCLs are negative for BCL2 (see Fig. 37-8, G ). A number of other antigens are expressed in ALCL, but they are not of diagnostic value. They include CD56 ; SHP1 phosphatase ; BCL6, C/EBPβ, and serpinA1 ; myeloid-associated antigens CD13 and CD33 ; and p63.

The ALK staining may be cytoplasmic, nuclear, and nucleolar, or it may be restricted to either the cytoplasm or, more rarely, the cell membrane ( Fig. 37-9 ). In the group of hematopoietic neoplasms, ALK expression is virtually specific for ALCL because it is absent from all normal postnatal human tissues except for rare cells in the brain and absent from hematopoietic neoplasms other than ALCL, with the exception of ALK-positive large B-cell lymphomas (see Fig. 37-11 ) and a novel form of ALK-positive histiocytosis seen in infancy. It is important to note that in the small-cell pattern and, to a lesser extent, in the lymphohistiocytic pattern, ALK staining may be restricted to scattered large cells. However, ALK staining performed without a nuclear counterstain reveals a large population of small cells showing restricted nuclear staining.

Genetics and Molecular Findings

Approximately 90% of ALCLs show clonal rearrangement of the T-cell receptor genes, irrespective of whether they express T-cell antigens. The majority of ALCLs are associated with a reciprocal translocation, t(2;5)(p23;q35), which juxtaposes the gene at 5q35 encoding NPM, a nucleolar-associated phosphoprotein, with the gene at 2p23 coding for ALK, a receptor tyrosine kinase. Polyclonal and monoclonal antibodies recognizing the intracellular portion of ALK react with both NPM-ALK protein and the full-length ALK protein, but no normal lymphoid cells express full-length ALK; as a consequence, immunostaining with anti-ALK has been used to detect ALCL cases carrying the t(2;5) translocation. However, variant translocations involving ALK and other partner genes on chromosomes 1, 2, 3, 9, 17, 19, and 22 also occur ( Table 37-1 ). All result in the upregulation of ALK, but the distribution of the staining varies, depending on the translocation. The classic t(2;5) translocation leads to positive staining for ALK in the nucleolus, nucleus, and cytoplasm (see Fig. 37-9, A and B ). In the variant translocations, often only cytoplasmic staining is observed (see Fig. 37-9, C to E ). In the t(2;5)(p23;q35) translocation, the particular cytoplasmic, nuclear, and nucleolar staining can be explained by the formation of dimers between wild-type NPM and the fusion NPM-ALK protein. Wild-type NPM provides nuclear localization signals, whereby the NPM-ALK protein can enter the nucleus. The formation of NPM-ALK homodimers with dimerization sites at the N-terminus of NPM mimics ligand binding and is responsible for activation of the ALK catalytic domain (i.e., autophosphorylation of the tyrosine kinase domain of ALK), which is responsible for its oncogenic properties.

Besides the t(2;5) translocation, at least 11 variant translocations involving the ALK gene at p23 have been recognized. In all these translocations, the ALK gene is placed under the control of the promoter of genes that are constitutively expressed in lymphoid cells—hence the ALK gene expression. The most frequent variant translocation is t(1;2)(q25;p23), in which the TPM3 gene on chromosome 1 (which encodes a non-muscular tropomyosin protein) is fused to the ALK catalytic domain. However, in cases associated with the t(1;2) translocation, which express the TPM3-ALK protein (104 kDa), ALK staining is restricted to the cytoplasm of malignant cells, and in virtually all cases there is stronger staining on the cell membrane (see Fig. 37-9, C ). This staining pattern is found in 15% to 20% of ALK-positive ALCLs. Tropomyosins are known to form dimeric alpha-coiled structures that can induce dimerization of the chimeric TPM3-ALK protein and activation of the ALK catalytic domain (i.e., autophosphorylation of ALK protein). The genes fused with ALK in the t(2;3)(p23;q11) and inv(2)(p23q35) translocations have been identified (see Fig. 37-9, D ). Two different fusion proteins of 85 and 97 kDa (TFG-ALK _short and TFG-ALK _long ) are associated with the t(2;3)(p23;q11) translocation, which involves TFG (TRK-fused gene). The inv(2)(p23q35) translocation involves the ATIC gene (formerly known as pur-H ), which encodes 5-aminomidazole-4-carboxamide-ribonucleotide transformylase-IMP cyclohydrolase (ATIC), which plays a key role in the de novo purine biosynthesis pathways. In TFG-ALK–positive and ATIC-ALK–positive ALCLs, ALK staining is restricted to the cytoplasm in a diffuse pattern.

Rare cases of ALCL show a unique granular ALK cytoplasmic staining pattern (see Fig. 37-9, E ). In these cases, the ALK gene is fused to the CLTC gene, which encodes the clathrin heavy polypeptide (CLTC), which is the main structural protein of coated vesicles. The sequence of the fusion gene suggests that these tumors might have reciprocal translocations involving breakpoints at 17q11-qter and 2p23. In CLTC-ALK–positive ALCL, the implication of the CLTC in the hybrid protein accounts for the granular cytoplasmic staining pattern because the CLTC-ALK protein is involved in the formation of the clathrin coat on the surface of vesicles. Moreover, the process of clathrin coat formation mimics ligand binding; this allows the autophosphorylation of the carboxyterminal domain of the ALK protein, which is probably responsible for its oncogenic property. In a single report, the moesin (MSN) gene at chromosome Xq11-12 was identified as a new ALK fused gene (MSN-ALK fusion protein) in a case of ALCL with a distinct ALK membrane-restricted pattern. The particular membrane-staining pattern of ALK is probably due to the binding properties of the N-terminal domain of moesin to cell membrane–associated proteins. In this case, the ALK breakpoint was different from that described in all other translocations and occurred within the exonic sequence coding for the juxtamembrane portion of ALK. The recently described TRAF1-ALK fusion encodes part, but not all, of the C-terminal TRAF domain responsible for oligomerization of TRAF1. Thus, the potential dimerization and function of TRAF1-ALK requires further study.

In the recently reported translocation of dicentric (2;4)(p23;q33), the ALK partner was not identified.