Case 6 -

Anaplastic Large Cell Lymphoma of T/null Cell Phenotype, Systemic Type (ALK Positive), Lymphohistiocytic Variant D. Ashley Hill, M.D. Mihaela Onciu M.D.

Case History: A 10-year-old boy presented with left cervical lymphadenopathy. An excisional lymph node biopsy was performed. The excised cervical lymph node measured 2.3 cm in greatest dimension. The staging work-up revealed additional lymphadenopathy in the submandibular, paratracheal, axillary and inguinal areas. Microscopic Findings: Within the enlarged lymph node, the architecture was entirely effaced by a tumor with partially diffuse and partially nodular growth patterns, and associated fibrosis and vascular proliferation. The tumor involved the lymph node capsule focally. The tumor consisted of a heterogeneous mixture of inflammatory cells including histiocytes, plasma cells and small lymphocytes. Within this background there were large immunoblastic cells occurring singly and in variably-sized clusters. These cells had dark amphophilic cytoplasm and round nuclei with coarsely clumped chromatin. Only rare large "hallmark" cells with embryoid nuclei and anaplastic morphology were present. Differential Diagnosis: Peripheral T-cell lymphoma, unspecified Anaplastic large cell lymphoma, lymphohistiocytic variant Angioimmunoblastic T-cell lymphoma (T-cell lymphoma, AIL-like). Hodgkin lymphoma, classical type Reactive lymphadenopathy (infectious process). Immunophenotypic Findings (Immunohistochemistry): The large cells were positive for CD30, ALK (cytoplasmic expression), CD43, CD45 and CD45RO, a subset of cells expressed CD3; tumor cells were negative for CD15, and CD20. Molecular/Cytogenetic Analysis: Not performed. Of note, the pattern of ALK expression (i.e. limited to the cytoplasm) suggests that the tumor might contain a variant translocation involving ALK, other then the t(2;5) leading to the NPM-ALK fusion. Diagnosis: Anaplastic large cell lymphoma of T/null cell phenotype, systemic type (ALK positive), lymphohistiocytic variant. Anaplastic Large Cell Lymphoma (ALCL) Definition: ALCL is classified by the WHO as a lymphoma of T-lineage with characteristic morphology (large cells with abundant cytoplasm and pleomorphic nuclei) and consistent CD30 positivity. ALCL encompasses a biologically heterogeneous group of neoplasms that include: Primary systemic ALCL ALK-positive ALK-negative ('anaplastic variant of peripheral T-cell lymphoma, unspecified'). Primary cutaneous ALCL (ALK-negative). The distinction between ALK-negative systemic ALCL and primary cutaneous ALCL with systemic dissemination often requires knowledge of the history regarding the initial (primary) lesion, because when involving lymph nodes these tumors are essentially similar in morphology and immunophenotype. Epidemiology: ALCL accounts for 20-30% of childhood lymphomas in large series (St. Jude experience: ~50%), in contrast to ~3% of adult lymphomas. In children and young adults it presents almost exclusively (>90%) as ALK-positive ALCL (even in rare 'primary cutaneous' tumors), with a striking male predominance (male/female ratio 6.5). Clinical Features: ALK-positive ALCL commonly presents with involvement of extranodal sites. These include skin, bone (solitary or multiple polyostotic lesions), soft tissue, lung (discrete lesions or diffuse interstitial involvement), and liver. Other less common primary sites include paranasal sinuses and brain. Like other T-cell lymphomas, patients with ALCL often have pronounced systemic symptoms, which include fever, malaise and respiratory distress that may require respiratory support (the latter finding is commonly seen in ALCL with leukemic peripheral blood involvement). This clinical presentation often raises the differential diagnosis with an inflammatory/infectious process. Molecular Biology and Cytogenetics: All ALK-positive ALCLs show aberrant expression of the anaplastic lymphoma kinase (ALK) a membrane-bound receptor tyrosine kinase which is not expressed by any normal lymphoid elements. Across normal tissues, ALK expression can only be found within the brain, in scattered neurons. The aberrant ALK expression seen in ALCL is the result of various chromosomal translocations (See Table 6.1) that juxtapose the ALK locus (chromosome 2p23) to other partner genes, which in turn lead to the activation and determine the subcellular localization of the ALK (as seen by immunohistochemistry). For instance, the most common translocation present in up to 75% of ALCL is t(2;5)(p23;q35). The result of this translocation is a fusion protein (p80) that includes the portion of the ALK protein with associated tyrosine kinase activity and the oligomerization domain of nucleophosmin (NPM) encoded in 5q35. Normal NPM molecules dimerize and shuttle between the cytoplasm and the nucleus (nucleolus). This is why, in most ALCL that contain the NPM-ALK fusion ALK expression can be detected in the nucleus and the cytoplasm. Several other translocations have been described in ALK-positive ALCL (frequency 2-5%). (See Table 6.1) In all these cases the fusion partners for ALK are proteins that dimerize (aspect that appears to be essential for activating the kinase function of ALK) but typically localize to the cytoplasm. Therefore, in ALCL containing these translocations ALK expression can be detected only in the cytoplasm. The presence of NPM-ALK can be explored for diagnostic purposes using PCR, RT-PCR, or FISH. Conventional cytogenetic analysis will detect the presence of the characteristic translocation. Aberrant ALK expression appears to play an essential role in tumorigenesis in ALCL. ALK-inhibitors are currently being studied as potential therapeutic agents for this type of lymphoma. Histologic Features: ALCL was initially described by Stein et al in 1985 as a lymphoma composed of large anaplastic cells with pleomophic nuclei and sinusoidal lymph node involvement (hence the designation), that expressed CD30 (Ki-1). This histologic subtype of ALCL is now designated as the common (classic) subtype. The availability of an immunohistochemical assay for ALK expression has allowed the recognition of lymphomas with very heterogeneous morphology as ALCL (See Table 6.3). Consequently, the term 'ALK lymphoma' or 'ALKoma' was proposed for this category of tumors, to better accommodate morphologic variants that lack obvious anaplastic morphology. ALCL typically involves the lymph node sinuses and interfollicular area, with subtotal effacement of the lymph node architecture. Regardless of their morphologic subtype, ALK-positive ALCL's consist of a mixture in variable proportions of large anaplastic "hallmark" cells with kidney- or horseshoe-shaped nuclei, and small atypical lymphoid cells with irregular nuclear outlines, as well as inflammatory cells that may include histiocytes and plasma cells. In the common (classical) type the hallmark cells predominate, sometimes forming cohesive clusters and sheets, while in the small cell type, these cells are sparse and the neoplastic infiltrate consists predominantly of small lymphoid cells. In the lymphohistiocytic type the neoplastic cells may be difficult to identify within an exuberant polymorphous inflammatory background that includes histiocytes and plasma cells, is usually poor in neutrophils and eosinophils, and sometimes shows associated fibroblastic proliferation, vascular proliferation and fibrosis. Occasionally, the fibrotic component may impart a partial nodularity to the tumor. Within this infiltrate, immunohistochemical staining for CD30 or ALK will highlight large tumor cells that typically aggregate around blood vessels. In the monomorphic type the hallmark cells may be difficult to find, with the tumor consisting predominantly of a monotonous, highly mitotic population of large immunoblastic cells. This latter variant may be associated with a "starry-sky" appearance due to frequent tingible-body macrophages. Rarely, ALCL may present with prominent (leukemic) peripheral blood involvement (at diagnosis or at relapse). In such cases, the lymphoma cells present on the peripheral blood smear include a mixture in variable proportions of small lymphoid cells with marked nuclear membrane irregularity ("cerebriform") and large immunoblastic cells with deeply basophilic and occasionally vacuolated cytoplasm, and coarsely clumped nuclear chromatin. In most cases, there is a predominance of small cells, very similar to the cellular composition of the small cell variant. Notably, in such cases a significant proportion of the peripheral blood leukocytosis usually consists of granulocytes that may show prominent left shift and toxic changes. This findings combined with the clinical picture of fever, malaise and respiratory distress may distract from the atypical lymphoid population present. Thus, many of the leukemic ALCL cases are correctly diagnosed only after an extensive infectious disease work-up and broad spectrum antibiotic coverage have been unsuccessful. In the sarcomatoid type the tumor cells are sparse and present within an edematous stroma that contains atypical proliferating fibroblasts. In these cases the tumor cells may have the classical morphology or a spindle-cell appearance and may cluster around blood vessels. Immunophenotype: On immunohistochemical (IHC) staining ALCL cells are characteristically positive for CD30 and ALK (in the ALK-positive cases), and usually positive for CD45 (leukocyte common antigen). A variety of monoclonal and polyclonal antibodies are available for use in paraffin-embedded tissue (See Table 6.2). Of note, in the small cell variant (including the forms with leukemic presentation) the small cells may be CD30 negative and usually show a much lower level of expression of ALK than the large "hallmark cells" also present within the same tumors. The majority of ALK-positive ALCL are also EMA positive (and cytokeratin negative). Many tumors express CD43 (not T-lineage specific). While the majority of these tumors are of T-lineage when examined at the molecular level, many of them fail to express several pan T-cell antigens as detectable by IHC, hence the apparent "null-cell phenotype". More than 50% of these lymphomas fail to express CD3, CD5, CD7, and CD45RO. The majority of ALCL will express CD2 and CD4, while they are only exceptionally CD8 positive. Also, regardless of the CD4/CD8 status the tumor cells are very often positive for cytotoxic T-cell antigens (e.g. TIA-1, perforin, granzyme B). Flow cytometric analysis usually shows a similar immunophenotype. In addition, 40-50% of the analyzed cases have been found to express antigens typically associated with myeloid differentiation, such as CD11b, CD13, CD15, and CD33. Differential Diagnosis: The differential diagnosis of systemic ALCL is largely dependent on the histologic variant. (See Table 6.4) ALCL common variant: Non-hematopoietic tumors (metastatic carcinoma, melanoma) Hodgkin lymphoma, classical type (especially lymphocyte depleted and syncitial subtypes) Diffuse large B-cell lymphoma containing anaplastic large cells Diffuse large B-cell (plasmablastic) lymphoma expressing ALK Anaplastic large cell lymphoma, primary cutaneous (if involving skin) Reactive lymphadenopathy (infectious mononucleosis) ALCL small cell variant Peripheral T-cell lymphoma unspecified For leukemic cases: T-cell prolymphocytic leukemia (small cell variant), Sezary syndrome. ALCL lymphohistiocytic Peripheral T-cell lymphoma, unspecified Histiocytic sarcoma Reactive lymphadenopathy ALCL monomorphic Peripheral T-cell lymphoma, unspecified Burkitt lymphoma Diffuse large B-cell lymphoma ALCL sarcomatoid Inflammatory myofibroblastic tumor Sarcoma of any type Table 6.1: Chromosomal Translocations and Their Corresponding Gene Partners for the ALK Gene Encountered in ALCL Chromosomal translocation Fusion product Fusion partner t(2;5)(p23;q35) NPM-ALK Nucleophosmin t(1;2)(q21;p23) TPM3-ALK Tropomyosin 3 t(2;3)(p23;q21) TFG-ALK TFG (Tropomyosin receptor kinase-fused gene) t(2;22)(p23;q11) CLTCL-ALK Clathrin heavy chain inv(2)(p23;q35) ATIC-ALK ATIC (pur H gene) Table 6.2: Antibodies Specific for ALK Available for Immunohistochemical Staining Antibody Specificity Type p80 NPM-ALK (kinase domain) polyclonal (research) ALK-11 ALK, cytoplasmic portion polyclonal ALK-1 ALK, cytoplasmic portion monoclonal ALK-1c ALK, cytoplasmic portion monoclonal Table 6.3: Histologic Variants of ALCL and Their Relationship with ALK Expression Histologic variant Proportion of ALK+ cases Reference Common (classic) 60-90% Agnarsson et al, 1988. Small cell -leukemic presentation 100% Kinney et al, 1993 Onciu et al, 2003 Lymphohistiocytic 80-100% Pileri et al, 1990 Monomorphic Unknown Chott et al, 1990 Giant-cell rich 30-40% Agnarsson et al, 1988 Sarcomatoid Rare case reported, including ALK-positive and ALK-negative cases Chan et al, 1991 Neutrophil-rich 10 cases reported (2/2 cases examined for ALK were negative) McCluggage et al, 1998 Eosinophil-rich Rare cases, none assessed for ALK expression McCluggage et al, 1998 Signet-ring Rare cases, none assessed for ALK expression Fallini et al, 1997 Table 6.4: Immunohistochemistry in the Differential Diagnosis of ALCL Neoplasm ALK CD30 CD3 CD7 CD20 CD45 CD45RO EMA CK S100 ALCL + + -/+ -/+ - + -/+ + - - PTCL - -/+ + + - + + - - - Hodgkin lymphoma - + - - -/+ - - - - - DLBCL - -/+ - - + + - - - - DLBCL expressing ALK + - - - - + - + - - Burkitt lymphoma - - - - + + - - - - Histiocytic sarcoma - - - - - +/- - - - + IMT + - - - - - - - - - Sarcoma, NOS - - - - - - - -/+ - -/+ Carcinoma - - - - - - - +/- + -/+ Melanoma - - - - - - - - - + Reactive immunoblasts (infectious mononucleosis) - + -/+ -/+ + + -/+ - - - PTCL-peripheral T-cell lymphoma, unspecified; DLBCL – diffuse large B-cell lymphoma; IMT – inflammatory myofibroblastic tumor.