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Anaplastic lymphoma kinase-positive large B-cell lymphoma (ALK+ LBCL) represents a distinct subtype of mature B-cell neoplasms in the most recent WHO classification of hematolymphoid neoplasms. It has a characteristic immunoblastic/plasmablastic morphology, a distinct immunophenotypic profile and recurrent cytogenetic/molecular genetic abnormalities, and has been reported in both the adult and pediatric populations. With the advent of new ALK inhibitors for possible targeted therapy clinical trials, it is important to recognize this new entity, particularly in the pediatric population because the prognosis is worse than the more common ALK+ anaplastic large cell lymphoma. Though rare, awareness of its existence will avoid potential misdiagnosis and facilitate appropriate management.
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase of the insulin receptor superfamily. ALK was first discovered as part of the nucleophosmin (NPM)-ALK fusion protein as a result of the t(2;5)(p23;q35) chromosomal translocation frequently seen in anaplastic large cell lymphoma, a subtype of mature T-cell neoplasms [1, 2]. The native ALK is mainly expressed in the developing central and peripheral nervous system, and is normally not expressed in hematopoietic cells [3-5]. Besides ALK-positive anaplastic large cell lymphoma, various solid tumors, including inflammatory myofibroblastic tumor and other soft tissue tumors [6-10], lung cancer  and brain tumors [12-16] were found to aberrantly express ALK. The most common mechanism of ALK overexpression is through formation of a fusion protein with a partner due to chromosomal translocations. However, activation through point mutation and gene amplification has also been demonstrated.
ALK was initially believed to be expressed only in anaplastic large cell lymphoma. In 1997, Delsol et al reported a small series of diffuse large B-cell lymphoma with expression of ALK (ALK+ LBCL) . To date, approximately 40 cases of ALK+ LBCL have been described in the English literature and those cases share similar morphologic, immunophenotypic and molecular genetic characteristics. In fact, ALK+ LBCL is now considered to be a distinct entity of mature B-cell neoplasms in the new WHO classification of hematolymphoid neoplasm . Most patients with ALK+ LBCL presented with stage III/IV disease and were clinically worse than the more common ALK+ anaplastic large cell lymphoma, particularly in the pediatric population. Therefore, recognition of this rare entity will further our understanding of its pathobiology and development of more efficient treatment including targeted therapy.
Since the initial description of ALK+ LBCL by Delsol et al in 1997 , about 40 cases have been described. Their clinical features are summarized in Table 1. The youngest patient affected was 9 years old and the oldest one was 71 years old, with a mean age of 44.5 years. Approximately 27% of the cases occurred in the pediatric population (younger than 18 years). There is a male predominance with a male to female ratio of about 3.6:1 (32 vs 9). 23 patients presented with higher stage disease (III and IV) while 15 with lower stage disease (I and II or IIE). Interestingly, in patients younger than 18 years old, more patients presented with lower stage than higher stage diseases, a fact that may be attributed to early diagnosis in the pediatric population.
The most common anatomic site of involvement is cervical lymph node. However, any lymph node can be involved and systemic lymphadenopathy and extranodal presentation is not uncommon. Despite aggressive treatment, approximately half of the patients died of disease 4-26 months after therapy, a prognosis similar to other diffuse large B-cell lymphomas, but worse than the more common ALK-positive anaplastic large cells lymphoma [35, 36]. The outcome is not much different in patients younger than 18 with relatively early stage disease at diagnosis (Table 1).
The lymph node architecture in almost all cases is partially or completely effaced by a diffuse proliferation of large neoplastic lymphoid cells (Figure 1). Focal sinusoidal infiltration, coagulative necrosis and starry-sky pattern may be present [17, 31]. Cases with a prominent intravascular component has also been described .
Cytologically, the lymphoma cells in all reported cases exhibit either an immunoblastic or plasmablastic morphology with a round, centrally or eccentrally located nucleus, a prominent central nucleolus and a moderate amount of eosinophilic or amphophilic cytoplasm (Figure 1). Occasional binucleated or multinucleated cells mimicking Reed-Stern berg cells may also be seen . In extranodal sites, the lymphoma cells in ALK+ LBCL cells may form cohesive sheets resembling nonhematolymphoid neoplasms [21, 24].
Extensive immunophenotypic profiling of ALK+ LBCL by flow cytometry has been unsuccessful due to the cohesiveness and immunoblastic/plasmablastic morphology of the lymphoma cells. Among the reported cases, only one had a flow cytometric immunophenotyping analysis performed successfully . The lymphoma cells in this case demonstrated increased side angle light scatter properties, and expressed CD45, CD4, HLA-DR and moderate density CD38. As expected, they were negative for mature B cells markers including CD19, CD20, CD23, FMC7 and surface immunoglobulin light chains. They also failed to express T cell (CD2, CD3, CD5, CD7 and CD8), myelomonocytic (CD14 and CD33) lineage markers as well as CD10.
Immunohistochemical staining with a panel of antibodies has been the mainstay to characterize the neoplastic cells of ALK+ LBCL (Figures 2 and and3).3). As summarized in Table 2, the lymphoma cells are uniformly positive for CD138/VS38c, MUM1 and epithelial membrane antigen (EMA). Most of them demonstrate intracellular immunoglobulin light chain restriction and show a preferential usage of IgA over IgG (18 vs 3). They have lost the B cell lineage-specific markers CD19, CD20, CD22 and CD79a with only focal and weak expression of these markers in few cases, suggesting terminal plasma cell differentiation. Interestingly, CD4 expression was observed in three quarter and CD57 in about one third of the cases examined. The expression of other T cell, natural killer cell and myelomonocytic cell lineage markers, including CD56 and CD68, was absent. There was no evidence of EBV infection.
In contrast to ALK-positive anaplastic large cell lymphoma, CD30 expression is essentially absent. Though a few cases were reportedly positive for CD30, the expression was either focal or weak. Expression of CD45 is absent in about a quarter of the cases reported. In light of its cytomorphology, uniform expression of EMA and, in rare cases, focal expression of cytokeratins, ALK+ LBCL may occasionally be confused with metastatic carcinoma.
As the name implies, the neoplastic cells in all cases of ALK+ LBCL strongly express ALK (Figure 2C). The majority of them have a granular cytoplasmic staining pattern with rare exceptions, which demonstrate cytoplasmic and nuclear staining [19, 22, 33].
Initial studies by Delsol et al failed to demonstrate the presence of NPM-ALK by RT-PCR in 3 of 7 ALK+ LBCL cases studied . Subsequent reports have shown that the majority of ALK+ LBCL cases contain CLTC-ALK fusion gene secondary to t(2;17) transiocation as demonstrated by either conventional karyotyping, FISH or RT-PCR (Table 3). It is possible that the cases originally reported by Delsol et al may instead harbor the t(2;17) and RT-PCR designed specifically for NPM-ALK fusion gene may have failed to detect CLTC-ALK. A small subset of ALK-positive ana plastic large cell lymphoma and inflammatory myofibroblastic tumor also harbor the t(2;17) transiocation and aberrantly overexpress ALK [38-41], suggesting a broader role of CLTC-ALK fusion protein in tumorigenesis.
The ALK gene is located on chromosome 2p23. It is normally expressed in the developing central nervous system, but not in hematopoietic cells [3-5]. Aberrant expression of ALK has been demonstrated in ALK-positive anaplastic large cell lymphoma via chromosomal translocations, causing fusion of ALK with a variety of partners . The most common transiocation is t(2;5)(p23;q25), resulting in a 80 kd fusion NPM-ALK . It occurs in about 75% of anaplastic large cell lymphoma. Other translocations include t(1;2)(q25;q23)[TPM3-ALK], t(2;3)(p23;q21) [TGF-ALK], inv2(p23;q35)[ATIC-ALK], t(2;X)(p23;q11-12) [MSN-ALK], t(2;19)(p23;q13) [TPM4-ALK], t(2;17)(p23;q25)[AL017-ALK] and t(2;22)(p23;q11.2)[MYH9-ALK].
Abnormal expression of ALK has also been observed in solid tumors [6-16]. Soda et al first demonstrated inv(2)(p21;p23) in a subset of non-small cell carcinoma of lung, resulting in formation EML4-ALK fusion protein [11, 43]. More recently, several groups have identified ALK gene mutation and amplification in familiar as well as sporadic neuroblastomas [12, 13, 15, 16]. NPM-ALK transgenic mice developed both B and T cell lymphomas [44-48]. These findings suggest an important role of ALK activation in the pathogenesis of malignant lymphoma and solid tumors.
The physiological target(s) or substrate(s) of ALK remains elusive. Much of the studies have been focusing on the fusion protein NPM-ALK with constitutive tyrosine kinase activity [42, 49]. NPM-ALK has been shown to interact with numerous intracellular targets involved in signal transduction pathways important for cell proliferation, cell cycle progression and apoptosis [42, 44, 50]. These include the JAK/STAT pathway, m-TOR pathway as well as the SHIP2 tyrosine phosphatase negative regulatory loop. More recently, NPM-ALK has been shown to upregulate the expression of an immunosuppressive molecule on the cell surface, CD274 , suggesting a role in tumor evasion of the human immune surveillance. Little is known about the targets of CLTC-ALK fusion protein. Momose et al  demonstrated hyperactivation of STAT3 in ALK+ LBCL compared to ALK- LBCL, suggesting that the CLTC-ALK fusion protein may also act through the JAK/STAT pathway to induce malignant transformation.
The characteristic morphologic and immunophenotypic profiles should allow for distinction of ALK+ LBCL from other entities including anaplastic large cell lymphoma, plasmablastic myeloma, metastatic carcinoma and other morphologic variants of diffuse large B-cell lymphoma (immunoblastic, plasmablastic and anaplastic). Anaplastic large cell lymphoma is usually strongly positive for CD30 with a T-cell phenotype, negative for plasma cell markers CD138, MUM1 and intracellular monoclonal immunoglobulin light or heavy chain proteins, and frequently demonstrates molecular evidence of clonal T-cell receptor gene rearrangement. Plasmablastic myeloma has not been reported to express ALK, and would be associated with other myeloma features such as lytic bone lesions and serum or urine paraproteins. Plasmablastic lymphoma has an immunophenotype similar to ALK+ LBCL, but they tend to occur in the oral cavity of patient with HIV infection. They are usually EBV-positive and always ALK-negative. Anaplastaic variant of diffuse large B-cell lymphoma can be easily distinguished from ALK+ LBCL because B-cell lineage specific markers such as CD20 and CD79 are strongly positive, and ALK is always negative. Occasionally, metastatinc carcinoma may enter the differential diagnosis because focal cytokeratin staining has been seen in rare ALK+ LBCL cases. However, evidence of plasma cell differentiation with light chain or heavy chain restriction distinguishes ALK+ LBCL from metastatic carcinoma.
In conclusion, ALK+ LBLC is a rare subtype of diffuse large B-cell lymphoma with a characteristic histomorphology, immunophenotypic profile, recurrent cytogenetic abnormality and dismal prognosis. It should be distinguished from other subtypes of diffuse large B-cell lymphoma, ALK-positive anaplastic large cell lymphoma, plasmablastic myeloma, and nonhematolymphoid neoplasms using a panel of antibodies and molecular techniques if needed. Recent in vitro and animal studies have shown promise of immunotherapy using ALK as a vaccine or targeted therapy with small molecule inhibitors of ALK [52-55], providing potential new treatment modalities for ALK+ LBCL.