Expression of Anaplastic Lymphoma Kinase in Non-Hodgkin's ...

Pathology Patterns Reviews

Expression of Anaplastic Lymphoma Kinase in Non-Hodgkin’s Lymphomas and Other Malignant Neoplasms Biological, Diagnostic, and Clinical Implications Mariusz A. Wasik, MD Key Words: Anaplastic lymphoma kinase; Non-Hodgkin’s lymphomas; Cell signal transduction therapy; Targeted lymphoma therapy DOI: 10.1309/3B7V25JMBXJJJ8X7

Abstract Accumulating evidence indicates that expression of anaplastic lymphoma kinase (ALK), typically due to t(2;5) translocation that creates an NPM-ALK fusion gene, defines a distinct type of T/null-cell lymphoma (TCL) within a vastly heterogeneous group of anaplastic large cell lymphomas. Through the translocation mechanism or as a full-length apparently intact protein, ALK also is expressed by a subset of inflammatory myofibroblastic tumors, glioblastomas, diffuse large B-cell lymphomas, and other malignant neoplasms. Owing to the recent progress in understanding its pathogenesis, ALK+ TCL has become a model malignant neoplasm in which morphology-based diagnosis and classification are gradually shifting toward biology-based diagnosis. Several lines of experimental evidence indicate that the ectopically expressed ALK is oncogenic in ALK+ TCL by being constitutively active owing to autophosphorylation and, consequently, by stimulating several critical signal transduction pathways involving phospholipase C-gamma, AKT, and STAT3 (signal transducer and activator of transcription 3). Targeting ALK and, perhaps, its downstream signaling effector proteins represents a promising novel therapeutic approach to ALK+ TCL. Diagnostic implications of the ALK expression in ALK+ TCL and other malignant neoplasms and the related current controversies are discussed.

© American Society for Clinical Pathology

CD30+ Anaplastic Large Cell Lymphomas Are Biologically Heterogeneous Although anaplastic large cell lymphomas (ALCLs) share a number of morphologic and immunophenotypic features, including expression of the CD30 antigen and epithelial membrane antigen (EMA),1-5 they are very heterogeneous. ALCL may arise de novo or represent a progression of underlying more indolent lymphomas and premalignant lymphoproliferations. Based on the cell type and organ of derivation, primary ALCLs can be divided into at least 3 broad categories with 1 or 2 subcategories ❚Table 1❚. The first category comprises B-cell lymphomas, those without the apparent predisposing factors and the AIDS-related lymphomas. The second is the group of primary cutaneous T-cell lymphoproliferative disorders that includes rare CD30– ALCL. The third comprises systemic T/null-cell ALCLs that can be further subdivided, based on the expression, or lack thereof, into anaplastic lymphoma kinase (ALK)+ and ALK–. The primary B-cell ALCLs usually are lymph node–based and are considered to represent a morphologic subtype of a diffuse large B-cell lymphoma.6 Their response to standard chemotherapy and survival rate seem similar to the other B-cell lymphomas in this broad category, 7,8 although 1 study9 concluded that they tend to have a better prognosis. The recent genome-scale gene expression– profiling studies that divided the diffuse large B-cell lymphoma into at least 2 prognostically separate groups10-12 raise the currently unanswered question: to which, if any, of these groups would the B-cell ALCLs belong? B-cell ALCLs can rarely be seen in the setting of an HIV infection. In contrast with the de novo B-cell ALCLs, these lymphomas usually are positive for the Epstein-Barr virus and share a Am J Clin Pathol 2002;118(Suppl 1):S81-S92 DOI: 10.1092/3B7V25JMBXJJJ8X7

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Wasik / EXPRESSION OF ANAPLASTIC LYMPHOMA KINASE

❚Table 1❚ General Types and Typical Features of Primary Anaplastic Large Cell Lymphomas Systemic B-Cell Feature Primary site Age group affected Prognosis

“True” De Novo

AIDS-Related

Lymph nodes Elderly people Moderate

Extranodal tissues Adults Poor

T/Null-Cell Primary Cutaneous T-Cell

ALK+

Skin Extranodal tissues Elderly people Children, young adults Good to excellent (less Very good favorable in CD30– cases)

ALK– Lymph nodes Elderly people Poor

ALK, anaplastic lymphoma kinase.

number of clinical and prognostic features with other AIDSrelated lymphomas.13 Primary cutaneous CD30+ T-cell lymphoproliferative disorders represent a spectrum of conditions ranging from an apparently premalignant lymphomatoid papulosis to ALCL.14 These lymphoproliferations, in general, have an excellent prognosis. Some of the histologically overt ALCLs show signs of spontaneous remission that, however, frequently is followed by recurrence. Primary cutaneous ALCLs that fail to express CD30 seem to have a more aggressive clinical course. The systemic ALK+ T/null-cell ALCLs usually express T-cell markers and contain cytotoxic granules; the “null” cell designation is reserved for the cases in which T-cell derivation cannot be documented by immunophenotyping or T-cell receptor gene rearrangement studies. 1-6 Rarely, ALK+ ALCLs coexpress myeloid lineage–associated markers such as CD13,15 most likely a sign of malignancy-related genomic instability. An NK-cell derivation was proposed in a single case of ALK+ ALCL.16 However, owing to the common origin and related similarities, including coexpression of the T- and NK-cell (KIR) receptors by some normal lymphocytes, further improvements leading to the reliable distinction between T- and NK-cell lineages in malignant cells are required before the “true” NK-cell derivation of some ALK+ T/null-cell ALCLs can be broadly accepted. The ALK+ T/null-cell ALCLs tend to manifest as a disseminated nodal disease with frequent involvement of extranodal sites such as skin, bone, soft tissues, lung, and liver.17-19 The cutaneous involvement may lead to a diagnostic dilemma of distinguishing this kind of systemic lymphoma from the primary cutaneous CD30+ T-cell lymphoproliferative disorder. Lack of ALK expression by the primary cutaneous lesions facilitates the distinction in most cases. The frequency of systemic ALK+ compared with the ALK– T/null-cell ALCL is inversely related to the patient’s age, with ALK+ T/null-cell ALCL comprising the vast majority of ALCLs occurring in children. Most studies indicate that expression of ALK represents an independent, favorable prognostic factor in all patient age groups.17,20,21 However, other variables such as high stage of the S82 S82

Am J Clin Pathol 2002;118(Suppl 1):S81-S92 DOI: 10.1092/3B7V25JMBXJJJ8X7

lymphoma20; expression of the CD56 adhesion molecule22; the presence of reactive, activated cytotoxic T lymphocytes23; and origination from the bone24 negatively affect the treatment outcome. These modifying factors may explain why several retrospective studies, frequently performed on mostly adult patient populations, did not reveal the prognostic advantage of ALK expression in ALCL.8,25-28 ALK– T/null-cell lymphomas (TCLs) typically occur in older patients. Comparative studies to explain their relatively poor response to chemotherapy17-19 revealed several biologic features distinguishing them from ALK+ T/null-cell ALCLs. ALK– TCLs tend to have lower mitotic and proliferative rates and lower degree of expression of cell cycle–associated molecules.29 These lymphomas also tend to have a lower apoptotic rate30 and concentration of the apoptosis-mediating active caspase 3.31 They preferentially express another antiapoptotic protein, granzyme B–specific protease inhibitor (PI9).31 Roughly half of them express an antiapoptotic BCL2 protein, in variance with the ALK+ T/null-cell ALCLs that are consistently negative for BCL-2.30,31 ALK– T/null-cell ALCLs also preferentially express BCL-XL (40% vs 5%), but not proteins with known proapoptotic function, BAX and BCL-XS (approximately 70% vs 40% for both).31 Finally, their expression of chemokine receptors is variable, in contrast with ALK+ T/null-cell ALCLs, which frequently express CCR4 and lack CD134/OX40 and CXCR3.32

ALK Expression in Normal and Malignant Hematopoietic and Nonhematopoietic Cells Identification of ALK33-35 and development of ALKspecific antibodies36,37 has permitted characterization of the protein and determination of the ALK expression patterns in normal tissues and malignant tumors. They revealed that ALK is a growth factor receptor selectively expressed within the nervous system. ALK is expressed by a narrow yet diverse group of malignant neoplasms and displays the apparent cell-transforming properties in some of them. © American Society for Clinical Pathology


Although genomic structure of ALK remains largely undefined, it is known that the ALK gene encodes a 1,620–amino acid protein that undergoes cotranslational Nlinked glycosylation to a fully mature form weighing 200 kd.33,38 ALK belongs to the insulin receptor family of cell membrane–spanning receptors that possess intrinsic tyrosine-kinase activity. ALK is structurally the most closely related to leukocyte tyrosine kinase and shares 57% of its amino acid sequence.38 Physiologic ALK expression is restricted to the nervous system, with expression seen primarily in various nerve ganglia and thalamic and hypothalamic nuclei.19,37,38 Other, nonneural tissues, including hematopoietic organs, show no detectable expression of ALK protein. Recently, the growth factor pleiotrophin has been identified as a ligand for ALK.39 Its expression parallels the expression of ALK; however, further studies are needed to establish its exact role in the ALK-mediated signaling and development and function of the nervous system.39 ALK protein is expressed in malignant tumors as either a full-length or a chimeric protein encoded by 2 partner genes fused together as a result of chromosomal translocation ❚Table 2❚ . Translocations involving the ALK gene located on the short arm of chromosome 2 and at least 9 different partner genes33,40-54 are responsible for the ectopic ALK expression in at least 2 types of malignant neoplasms: ALK+ T/null-cell ALCLs and inflammatory myofibroblastic tumors (IMTs); the latter represent a rare entity occurring in soft tissues and the gastrointestinal tract of children and young adults. The best characterized t(2;5) occurring in approximately 80% of ALK+ T/null-cell ALCLs fuses the ALK gene with a gene encoding nucleophosmin (NPM) that is a ubiquitously expressed protein involved in shuttling of ribosomal

components between the cytoplasm and the nucleus.55,56 The resulting 80-kd NPM-ALK chimeric protein contains the oligomerization motif of NPM fused to the cytoplasmic portion of ALK that includes an intact kinase catalytic domain.33,34,38 Of interest, NPM is involved via chromosomal translocations in formation of 2 other oncoproteins by gene fusion with myelodysplasia/myeloid leukemia factor 1 (MLF1)57 and retinoic acid receptor alpha58 seen in subsets of myelodysplastic syndrome/acute myelogenous leukemia and acute promyelocytic leukemia, respectively. With exception of 1 translocation involving the moesin (MSN) gene,52 the other known ALK translocations33,42-51,53-54 occur within the same intron of the ALK gene. As a result, these chimeric ALK proteins contain either the whole intracytoplasmic portion of ALK or, in the case of MSN/ALK, a slightly shorter ALK fragment that contains, nevertheless, the entire kinase domain. Whereas some of these ALK gene translocation variants have been identified in both ALK+ T/null-cell ALCL and IMT, the others seem to occur selectively in either one of these entities. Similar to NPM, the other ALK gene fusion partners are expressed by a broad range of tissues. Although they are functionally quite diverse (Table 2), the kind of fusion partner does not seem to have a noticeable effect on lymphoma pathogenesis; ALK+ T/nullcell ALCLs seem to be similar regardless of whether they express NPM-ALK or one of the ALK gene fusion variants. These gene fusion partners, however, determine the cellular localization of the chimeric ALK protein. Since the native NPM shuttles between the nucleus and cytoplasm,55,56 NPMALK can be found in both. Most of the other chimeric ALK proteins display a diffuse cytoplasmic staining pattern; the only exceptions are the ones containing clathrin50 that shows

❚Table 2❚ Full-Length and Chimeric ALK Proteins: Characterization of Gene Fusion Partners, Expression Pattern in Malignant Cells, and Cellular Localization Gene Fusion Partner

Translocation

None

None

Nucleophosmin (NPM) 5-Aminoimidazole-4-carboxamide ribonucleotide formyltransferase/ IMP cyclohydrolase (ATIC) Clathrin heavy chain (CLTC)

Tumor Type Expression

Protein Molecular Weight

Protein Cellular Localization

t(2;5)(p23;q35) inv(2)(p23;q35)

Glioblastoma, B-cell ALK (200 kd) lymphoma, neuroblastoma, other (eg, breast) T/null-cell ALCL NPM-ALK (80) T/null-cell ALCL ATIC-ALK (95)

Membranous Nuclear and cytoplasmic Cytoplasmic

Moesin (MSN) TRK-fused gene (TFG)

t(2;17)(p23;q23) t(2;2)(p23;q13) t(X;2)(q11-12;p23) t(2;3)(p23;q21)

T/null-cell ALCL, IMT T/null-cell ALCL, IMT T/null-cell ALCL T/null-cell ALCL

Cytoplasmic (granular) Cytoplasmic Membranous Cytoplasmic

Tropomyosin 3 (TPM3) Tropomyosin 4 (TPM4) ALO17 Cysteinyl-tRNA synthetase (CARS)

t(1;2)(q25;p23) t(2;19)(p23;p13.1) t(2;17)(p23;q25) t(2;11;2)(p23;p15;q31)

T/null-cell ALCL, IMT IMT T/null-cell ALCL IMT

CLTC-AKL (248) RanBP2-ALK (80 kd) MSN-ALK (125) TFG-ALK (85, short; 97, long; 113, extralong) TPM3-ALK (104) TPM4-ALK (100) ALO17-ALK (unknown) CARS-ALK (unknown)

Cytoplasmic Cytoplasmic Unknown Unknown

ALCL, anaplastic large cell lymphoma; ALK, anaplastic lymphoma kinase; IMT, inflammatory myofibroblastic tumor.



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granular cytoplasmic staining, probably due to binding of the fusion protein to clathrin-coated vesicles, and a moesin52 membrane-bound protein cross-linker that shows membranous staining. Cellular localization and the molecular weight of the 2 recently identified chimeric ALK proteins containing ALO17 and CARS (cysteinyl-tRNA synthetase) remain to be determined.53 There currently is much less information about expression in malignant cells of ALK as a full-length protein. Such a form of ALK has been found, so far only in tumor-derived cultured cell lines, in a variety of tumors, including a subset of neuroblastomas, glioblastomas, breast carcinomas, melanomas, and diffuse large B-cell lymphomas.33,37,59-61 It seems that the ALK expressed in such malignant cells is structurally intact and functional. ALK expression by neuroblastomas and, perhaps, gliomas may simply reflect their derivation from the central nervous system in which ALK is physiologically, although selectively, expressed. 19,37,38 However, its expression in the other malignant neoplasms is clearly ectopic and ought to result from an aberrant activation of ALK gene transcription. In addition to the cell of origin, B-cell lymphomas that express full-length ALK display several other important differences compared with the ALK+ T/null-cell ALCLs.61 These rare lymphomas are composed of large rather than anaplastic cells and lack expression of CD30 but not of EMA. Interestingly, they express non–B-cell lineage markers CD4 and CD57 and usually contain cytoplasmic IgA. Most affected patients have advanced stage disease at diagnosis and are almost exclusively adults. They have a rather poor prognosis,61 which suggests that expression of a full-length ALK may not be prognostically beneficial, in contrast with the expression of the chimeric ALK protein by ALK+ T/null-cell ALCLs.17,20,21

Mechanisms of the ALK-Induced Oncogenesis Several lines of evidence indicate that ectopic expression of the chimeric ALK protein has a critical role in malignant cell transformation35,62,63 by the mechanisms similar to other chimeric tyrosine kinases such as BCR/ABL.64 In contrast with the native ALK, wild-type NPM and the other fusion partner genes are, under physiologic conditions, widely expressed including in lymphoid and soft tissues. Furthermore, these proteins, with the apparent exception of one of them, MSN,57 occur naturally as homo-oligomers owing to the interactions of their proximal ends. Therefore, fusion of their gene promoter and the proximal coding regions with the distal portion of the ALK gene containing the entire kinase domain leads to the abundant and constitutive expression of S84 S84


the chimeric ALK oligomers. The homo-oligomerization leads to the ligand-independent constitutive activation of the ALK domains by transphosphorylation and autophosphorylation. As a result, chimeric ALK continuously activates multiple intracellular signaling pathways that contribute to malignant transformation of the affected cells.62,63,65 Interestingly, duplication of the ALK gene on the other, nontranslocated chromosome also has been noted in the NPMALK–expressing cells, suggesting that structural alteration of the nonfused gene also might, perhaps, have a role in the pathogenesis of ALK+ T/null-cell ALCLs.66 Oncogenic properties of NPM-ALK and the other ALK chimeric proteins have been documented in both in vitro35,62,67 and in vivo63,68 experimental systems. Accordingly, expression of NPM-ALK confers a malignant phenotype and renders cytokine-independent cultured fibroblastic and hematopoietic cell lines.35,62,63,68 NPM and kinase-deficient variants of the ALK construct genes failed to transform the target cells, stressing the importance of both the NPMmediated protein homo-oligomerization and the ALK-mediated tyrosine phosphorylation and the resulting activation of the cell-signaling pathways for the oncogenic properties of NPM-ALK. Injection into mice of the murine bone marrow cells engineered to express the NPM-ALK gene has resulted in generalized ALK+ lymphomas that display anaplastic large cell morphologic features.68 As expected, recent data indicate that the other ALK gene fusions formed with the other partners display similar cell-transforming and signal transduction–inducing properties.56 Similar to BCR/ABL and other oncogenes, low concentrations of NPM-ALK and ATIC-ALK transcripts were found in normal, nonneoplastic lymphoid cells when ultrasensitive reverse transcriptase–polymerase chain reaction was applied.54,69 This raises the important question of whether expression of the chimeric ALK is sufficient for malignant cell transformation or whether additional, ALK-independent genetic and/or environmental changes must occur for the effective oncogenesis of the affected cells. Notably, in contrast with the transcripts, ALK protein has not been detected so far in nonmalignant cells, despite the fact that it can be detected easily on a single-cell level in malignant ALK+ T/null-cell ALCL.37 This suggests that the chimeric ALK protein is synthesized by normal cells in a small amount that, perhaps, may be insufficient for an effective cell transformation or is not synthesized at all. Solving this puzzle would provide a better understanding of ALK-mediated oncogenesis. The role of the full-length ALK in oncogenesis is far less certain. Indeed, there is no evidence that the expression of full-length ALK in neuroblastoma and several other types of malignant cells, such as breast carcinoma and melanoma, has any role in their pathogenesis.59,60 Similarly, there is no evidence that ALK is activated in the diffuse large B-cell © American Society for Clinical Pathology


lymphomas expressing the full-length protein.61 However, a recent report70 indicates that ALK-mediated signaling may have a role in glioblastomas by the classic receptor-ligand interaction. Glioblastomas were found to overexpress fulllength, membrane-bound ALK and use it for autocrine signaling by pleiotrophin, the newly identified ligand of the ALK receptor. 39 Inhibition of ALK expression in the glioblastoma cells reduced pleiotrophin-induced phosphorylation of the antiapoptotic protein AKT. Furthermore, this depletion of ALK reduced tumor growth of xenografts in athymic nude mice and prolonged survival of the animals because of increased apoptosis in the tumors. Additional studies focused on the primary tumor tissues are needed to further elucidate the role of the pleiotrophin–ALK receptor axis in glioblastomas and other malignant neoplasms expressing full-length ALK. Substantial progress has been made in elucidating signal transduction pathways activated by the NMP/ALK protein. Early studies have shown a direct interaction of NPM-ALK with Shc and IRS-1,35,62 raising the possibility that these adapter proteins may have a role in ALK-mediated lymphomagenesis. However, interaction of both Shc and IRS-1 may not be essential for NPM-ALK–mediated cell transformation.35,71 The potential role of another adapter molecule, GRB2, in such process also is controversial.35,71 More recently, phospholipase C-gamma has been identified as a major downstream target of NPM-ALK.72 Activation of phospholipase C-gamma, which in normal cells leads to the generation of diacylglycerol and inositol triphosphate, activation of protein kinase C, and calcium mobilization, may have a role in the NPM-ALK–mediated oncogenesis by transducing mitogenic signals.72 Recently, NPM-ALK has been found to activate also the PI3-kinase (PI3k)/AKT signaling pathway.73,74 Recruitment of the p85 regulatory subunit of PI3k, which is then phosphorylated, leads to the constitutive activation of the known proto-oncogene, serine/threonine kinase AKT. Signaling via this pathway has been implicated in protecting lymphoma cells from apoptosis by phosphorylating and inhibiting function of BAD and caspase 9 and expression of the FAS ligand. The PI3k/AKT pathway seems critical for the ALK-mediated oncogenicity, because inhibitors of PI3k impair growth and induce massive apoptosis of NPM-ALK–carrying cells.73,74 NPM-ALK was found to phosphorylate and activate STAT3 (signal transducer and activator of transcription 3) in both transfected cells and primary lymphoma tissues75,76 ❚Image 1❚. STATs are members of the ubiquitously expressed family of transcription factors activated in response to growth factors and cytokines. Tyrosine-phosphorylated STATs form dimers that translocate into the nucleus and initiate gene transcription. STATs have a critical role in promoting cell proliferation and protecting cells from apoptosis, both © American Society for Clinical Pathology

normal and malignant. Activated STAT3 has been shown to be directly oncogenic in vitro and in vivo in fibroblast transformation models,77,78 apparently by inducing resistance to apoptosis.79,80 NPM-ALK indeed causes enhanced transcription of the antiapoptotic factor BCL-XL, largely through activated STAT3, indicating that this mechanism may contribute to the successful outgrowth of ALK+ tumor cells.76 In addition, mechanisms that under physiologic conditions inactivate STAT3 seem to be dysregulated in the ALK+ cells, enhancing the cell-transforming effects of STAT3.75 Preliminary data indicate that NPM-ALK also activates STAT5.81 Recently, activation of another signaling pathway involving the Notch1 receptor has been identified in ALCL.82 Constitutive activation of this pathway is oncogenic, and its dysregulation by overexpression of the truncated, hence, constitutively activated Notch1 is present in a subset of T-cell acute lymphoblastic leukemia/lymphomas.83 Expression of the Notch1 ligand, Jagged1, also was found in ALCL tissues in malignant and adjacent normal cells, suggesting an autocrine and/or paracrine activation of the Notch1 pathway.82 In vitro interaction between intact Notch1 on tumor cells and Jagged1 leads to proliferation and inhibition of apoptosis. Activation of this pathway seems, however, to be ALK-independent because it also is present in Hodgkin lymphoma cells.82

ALK Expression and Signaling as Potential Therapeutic Targets Because, similar to other oncogenic kinases, NPM-ALK and the other variants of the chimeric ALK protein seem to have a fundamental role in malignant cell transformation, selective inhibition of their expression and/or function represents an attractive potential therapeutic approach. Lessons learned from the functional inhibition of the BCR/ABL kinase, a product of the t(9;22) translocation seen in chronic myelogenous leukemia and in a subset of acute myelogenous and lymphoblastic leukemias, indicate that such highly targeted treatment may be very effective and well tolerated.84 Similarly, encouraging therapeutic effects recently were obtained by inhibiting 2 other oncogenic kinases: the mutated, constitutively active c-kit occurring in gastrointestinal stromal tumors85 and the hybrid proteins involving the enzymatically active part of the beta chain of the receptor for platelet-derived growth factor86 seen in a unique type of chronic myeloproliferative disorders. Currently, these novel therapies use small organic compounds such as imatinib mesylate (STI571, Gleevec or Glivec) that are fairly specific for the targeted tyrosine kinase and act by blocking an ATPbinding site of the kinase.84 Am J Clin Pathol 2002;118(Suppl 1):S81-S92 DOI: 10.1092/3B7V25JMBXJJJ8X7

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A

B

E

F

“Proof of principle” experiments already have been performed in ALK+ T/null-cell ALCLs by treating lymphoma-derived cell lines with the broad-specificity tyrosine-kinase inhibitor Herbimycin A.87,88 Such treatment inhibited NPM-ALK kinase activity and autophosphorylation and phosphorylation of its signaling downstream effector AKT. These effects were associated with a timeand dose-dependent apoptosis accompanied by caspase 3 activation. However, a much more selective ALK inhibitor is needed for an effective, therapeutically valid inhibition of the kinase. Furthermore, combined therapy directly targeting not only the ALK protein but also its critical downstream effectors and/or their regulatory proteins may prove to be the most effective targeted therapy for ALK+ malignant neoplasms.75 The recent finding that NPM-ALK interacts with the heat shock protein (Hsp) 90, apparently through direct binding via the ALK portion of the chimeric protein, has S86 S86


C

D

❚Image 1❚ Expression of phosphorylated STAT3 (signal transducer and activator of transcription 3) in anaplastic lymphoma kinase (ALK)+ T/null-cell anaplastic large cell lymphoma. The images represent low-power (×100) and highpower (×600) magnification (A and B, H&E; C and D, anti-ALK antibody; E and F, anti–phospho-STAT3 antibody).75

created an option of indirectly inhibiting ALK expression and function.89 Hsp90 is a chaperone protein that has a critical role in the folding and maturation of several oncogenic protein kinases. Perturbation of Hsp90 structure by antibiotics from the benzoquinone ansamycin family affects the stability and degradation of its substrates. Treatment with one such compound decreased NPM-ALK expression and phosphorylation and, consequently, its association with several signaling effectors. 89 Furthermore, it increased binding of the chimeric protein to Hsp70, which is known to affect protein degradation.89 Further studies should determine the value of such Hsp targeting in the clinical setting. It seems that immunotherapy targeting ALK may represent another attractive therapeutic approach to ALK+ TCL, alternative or complementary to inhibition of ALK expression and function. Accordingly, it has been proposed that the host immune response may contribute to the overall good prognosis of ALK+ TCLs.90 A preliminary study not only © American Society for Clinical Pathology


found the presence of humoral immune response to the NPM-ALK protein but also suggested that a high titer of anti-ALK IgG antibodies may represent a favorable prognostic factor.90 Furthermore, it seems that ALK protein can also induce a cellular immune response91 that, in general, should be more effective than a humoral response in eliminating ALK+ cells. Accordingly, the immunogenic peptide sequences within the ALK protein capable of stimulating the production of ALK-specific, major histocompatibility complex–restricted, cytotoxic CD8+ T cells have been identified.91 Interestingly, cultured ALK+ TCL cells have been shown to be sensitive to infection with an adenovirus engineered to multiply in p53-defective cells both in vitro92 and in vivo in a mouse xenotransplanted lymphoma model.93 Although p53 has not been shown so far to be structurally and/or functionally defective in the primary, noncultured ALK+ TCL tissues, this novel therapeutic approach being tested in several types of p53-defective epithelial tumors also may prove beneficial for patients with ALK+ TCL. Finally, it can be argued that for malignant neoplasms expressing the full-length ALK, inhibition of its interaction with ligand also may be therapeutically beneficial. In principle, this can be accomplished by various mechanisms, including blocking of the ligand-binding site by antibodies or small molecules or by inhibiting function or expression of the ligand. This approach may prove particularly useful in glioblastomas in which ALK binding of its ligand pleiotrophin seems to have a pathogenic role.39,70

Diagnostic Implications of ALK Expression and Related Controversies Identification and characterization of ALK has broad implications for understanding biology, diagnosis, and, potentially, treatment of the ALK-expressing malignant neoplasms. Immunohistochemical analysis with ALK antibodies has stressed that even the apparently clinically and biologically rather homogeneous ALK+ T/null-cell ALCLs are morphologically very heterogeneous.17-19,94,95 The presence of medium to large cells with a reniform eccentric nucleus and perinuclear clearing in the Golgi region18,96 seems to be the common denominator seen in all morphologic variants. However, their frequency, as well as the morphologic features of other malignant cells, as well as the composition of the reactive component of ALK+ T/null-cell ALCL, is extremely variable. In some cases, most of the neoplastic cells may not appear anaplastic at all but may have a morphologic spectrum that includes monomorphic diffuse large cell and small cell–predominant and lymphohistiocytic types, among others.95 Therefore, the term ALK+ T/null-cell lymphoma seems more appropriate to include the © American Society for Clinical Pathology

small cell and other nonanaplastic variants. As a result, immunohistochemical staining for ALK using an appropriate antibody (eg, ALK1, DAKO, Carpinteria, CA, or anti-p80, Caltag Laboratories, Burlingame, CA) is imperative for making an accurate diagnosis in diagnostically difficult cases and is advisable in all cases of ALK+ TCL. The latter approach is supported by the prevailing data that expression of the chimeric ALK represents a favorable prognostic factor within the ALCL group.17,20,21 In addition, expression of the chimeric ALK protein distinguishes ALK+ TCL from other malignant entities such as classic Hodgkin lymphoma and primary cutaneous CD30+ T-cell ALCLs, both of which can display considerable morphologic overlap with ALK+ TCL. Indeed, deletion of the provisional category of the morphologically borderline cases of Hodgkin-like97 or Hodgkin-related98 ALCL99 was based to a large degree on their lack of expression of the chimeric ALK protein.18,19,37,100 Most of these controversial cases seem to represent Hodgkin lymphoma. The fact that Hodgkin lymphoma is essentially a malignancy of germinalcenter B lymphocytes 101 further stresses the need for discrimination between Hodgkin and T/null-cell ALCLs. Besides expression of the “conventional” markers such as CD15, EMA, and CD45, the expression of ALK and of the B cell vs T cell lineage-specific markers can facilitate making this distinction in many cases. Evaluation for expression of Epstein-Barr virus–related antigens may also be of some value, because Epstein-Barr virus–encoded RNA 1 was reported to be expressed only rarely and latent membrane protein 1 not expressed at all by ALK+ TCL.102 Considerable controversy existed in the past about ALK expression in Hodgkin lymphoma. However, the early studies performed using PCR-based technologies suggesting ALK expression in a subset of Hodgkin lymphoma103-106 were not confirmed by a large number of research groups, in particular when ALK expression was analyzed at the protein level by immunohistochemical analysis rather than on the nucleic acid level.37,100,105,107-116 The reasons for this discrepancy are not totally clear, but they may reflect the aforementioned detection of the ALK fusion gene DNA and RNA transcripts in even normal lymphocytes.54,69 Therefore, it is plausible that often the reactive and not the neoplastic cells were the major source of the ALK nucleic acids in the Hodgkin lymphoma cases deemed positive for ALK expression. One also cannot exclude that some diagnostically challenging T/null-cell ALCL cases were misclassified in the early studies, particularly when only a minor subset of Hodgkin lymphoma was reported to express ALK.104-106 Despite the earlier suggestions to the contrary, based again on the PCR-based data117 it is apparent that primary CD30+ cutaneous T-cell lymphoproliferative disorders are also ALK–.37,111-114,118 Therefore, evaluation for ALK expression Am J Clin Pathol 2002;118(Suppl 1):S81-S92 DOI: 10.1092/3B7V25JMBXJJJ8X7

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of cutaneous lesions with anaplastic or large cell morphologic features also is diagnostically useful. This applies particularly to younger patients based on the aforementioned inverse correlation between the frequency of ALK+ lymphomas and age17,20,21 and to patients in whom an extracutaneous disease is suspected. Although a few cases of ALK-positive ALCL apparently restricted to skin have been observed (Gary Wood, MD, oral communication, April 2001; Hans Konrad MullerHermelink, MD, oral communication, November 2001), they seem to belong to the same general biologic category as systemic ALK+ ALCLs, because they occurred in children in whom ALK+ ALCLs are frequent and the primary CD30+ cutaneous T-cell ALCLs are rare. Detection of ALK expression also is helpful in diagnosing IMT. It always should be done in conjunction with CD30 and other lymphoid markers to permit distinction of IMT from an extranodal ALK+ T/null-cell ALCL with atypical morphologic features. Whether expression of ALK has any prognostic value in this type of tumor is unclear. Detection of ALK in glioblastomas 70 and other malignant neoplasms that contain full-length ALK37,59,60,61 remains currently investigational. Identification of ALK and its detection in a subset of ALCLs has provided remarkable insight into the biology of this group of lymphomas. It also has provided evidence that biologically related groups of lymphomas may cover a broad morphologic spectrum. Accordingly, several groups of investigators have postulated that ALK expression defines a specific clinicopathologic type of lymphoma,17-19 analogous to the chronic myeloproliferative disorders expressing another oncogenic tyrosine kinase, BCR/ABL, as a result of the t(9;22) translocation.84 It may be argued that this concept has a major limitation of equating expression of a single gene with a specific disease entity, whereas the prevailing evidence indicates that malignant tumors are multigenetic.119 Numerous variables that modify prognosis in ALK+ T/nullcell ALCLs20,22-24 and, as some may argue, the detection of NPM-ALK and other ALK gene fusion transcripts in healthy individuals54,69 clearly indicate that this also is the case in this group of lymphomas. One can certainly agree, however, that expression of the constitutively active ALK fusion protein seems critical in the pathogenesis of T/null-cell ALCL. Furthermore, should an at least semispecific and clinically suitable ALK inhibitor be developed and prove as effective as imatinib mesylate in chronic myelogenous leukemia84 and certain other malignant neoplasms derived by constitutively active kinases,85,86 detection of the aberrantly expressed and activated ALK may well become a key diagnostic factor in ALK+ malignant neoplasms. From the Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia.

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Supported in part by the grant R01-CA96856 from the National Cancer Institute, Bethesda, MD. Address reprint requests to Dr Wasik: Dept of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, 7.103 Founders, Philadelphia, PA 19104.

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