Alk<sup>+</sup> CD30<sup>+</sup> lymphomas: a distinct molecular genetic subtype of non‐hodgkin's lymphoma (2001)

Conventional methods of non-Hodgkin's lymphoma (NHL) diagnosis and classification are based principally on morphology and immunophenotype and have significant shortcomings in the diagnosis of large-cell lymphoma. Rather than being discriminatory, morphological and immunophenotypic parameters have revealed a significant heterogeneity in the large-cell lymphomas and for the most part have failed to detect distinct clinically and biologically meaningful subsets. More specific pathogenic molecular markers are clearly needed to define further and to separate specific clinicopathological entities within large-cell lymphoma for targeted therapy. As detailed in this review, the discovery of anaplastic lymphoma kinase (ALK) has permitted the definition of a distinct molecular genetic subtype of NHL within the clinically and pathologically heterogeneous group of CD30+ lymphomas. In 1985, Stein et al (1985) described anaplastic large-cell lymphoma (ALCL) as a pleomorphic large-cell lymphoma characterized by anaplastic morphology, frequent infiltration of lymph node sinuses and strong expression of the activation antigen Ki-1 (CD30). Because of its unusual growth pattern, ALCL had previously been misdiagnosed frequently as metastatic carcinoma, melanoma or malignant histiocytosis. Several problems were quickly recognized in the diagnosis of ALCL. CD30 expression was not definitive and could be present in reactive and neoplastic conditions, including other NHLs, Hodgkin's disease and embryonal carcinoma (for a review, see Chiarle et al, 1999). In addition, the histological appearance varied and numerous ALCL subtypes were described, including pleomorphic, monomorphic, small-cell, Hodgkin's disease-related and lymphohistiocytic variants, and rare forms having sarcomatoid, giant cell-rich, signet ring-cell and neutrophil-rich histologies (for reviews, see Kadin, 1997; Chan, 1998; Kinney & Kadin, 1999; Skinnider et al, 1999). The phenotype could be T-, B- or null cell, with variable expression of epithelial membrane antigen (EMA) (50–80% of cases) and CD15 (approximately 15–20%) (Stein et al, 1985; Agnarsson & Kadin, 1988; Delsol et al, 1988; Kinney et al, 1990, 1991; Shulman et al, 1993). Clinical manifestations of ALCL were also heterogeneous and included a systemic nodal or extranodal presentation, primary cutaneous disease, ALCL arising in HIV+ patients and secondary ALCL occurring after other lymphoproliferative disorders such as lymphomatoid papulosis, mycosis fungoides or Hodgkin's disease. This heterogeneity in ALCL initially led to considerable controversy regarding its acceptance as a specific clinicopathological entity (Penny et al, 1991). Shortly after the initial descriptions of ALCL, a recurrent cytogenetic abnormality, t(2;5)(p23;q35), was recognized in approximately 45% of the small number of ALCLs karyotyped (Kaneko et al, 1989; Le Beau et al, 1989; Morgan et al, 1989; Rimokh et al, 1989; Bitter et al, 1990; Ebrahim et al, 1990). In 1994, the t(2;5) was cloned and the NPM–ALK fusion gene generated by the rearrangement was identified (Morris et al, 1994; Shiota et al, 1994a). This discovery enabled many cases of ALCL to be studied for the t(2;5) or evidence of ALK gene dysregulation and led to the description of the specific entity ALK+ lymphoma within the heterogeneous lymphomas possessing ALCL morphologies or CD30 expression. The positional cloning of t(2;5) identified fusion of the chromosome 5 gene encoding nucleophosmin (NPM), a nucleolar phosphoprotein, to the chromosome 2 gene encoding a novel receptor tyrosine kinase (RTK), anaplastic lymphoma kinase (ALK) (Morris et al, 1994). The chimaeric NPM–ALK mRNA is transcribed from the der(5) chromosome and encodes a protein composed of the N-terminal segment of NPM linked to the kinase domain of ALK (Fig 1). The reciprocal ALK–NPM fusion gene on the der(2) is not transcribed or translated into protein at significant levels and does not appear to contribute to lymphomagenesis (Morris et al, 1994; Delsol et al, 1997; Beylot-Barry et al, 1998; Cordell et al, 1999). Independent of the positional cloning efforts of Morris et al (1994), Shiota et al (1994a) simultaneously identified an abnormal 80-kilodalton (kDa) activated protein tyrosine kinase in the t(2;5)-positive lymphoma cell line AMS3; subsequent cloning of the cDNA encoding this kinase, which was designated p80, established its identity with NPM–ALK (Fujimoto et al, 1996). Schematic of the normal ALK receptor tyrosine kinase, the NPM–ALK fusion and the variant ALK fusion proteins identified to date. The number of amino acids from each ALK fusion partner present in the respective chimaeric proteins is indicated, as well as the total number of amino acids in each chimaera. The identical portion of ALK is present in all known fusions. At the time of preparation of this review, NPM–ALK, ATIC–ALK, TFG–ALKLand TFG–ALKS have been identified only in NHL; TPM3–ALK and CLTC–ALK are known to occur in both NHL and inflammatory myofibroblastic tumours (IMTs), whereas RanBP2–ALK and TPM4–ALK have been described only in IMTs (see text). The chromosomal aberration that generates each fusion is indicated; because the chromosomal abnormality that produces RanBP2–ALK has not yet been studied, it is not known whether a rearrangement between the two chromosomes 2 or an inversion of one chromosome 2 is associated with this ALK fusion. NPM is a highly evolutionarily conserved 38-kDa non-ribosomal RNA-binding protein that shuttles ribosomal ribonucleoproteins between the nucleus and the cytoplasm and is involved in the late stages of preribosomal particle assembly (Borer et al, 1989; Chan, W.Y. et al, 1989). NPM is one of the most abundant nucleolar proteins and is present within the cell normally as a homohexamer (Chan & Chan, 1995). Elegant heterokaryon experiments have demonstrated bidirectional shuttling of NPM between the nucleus and cytoplasm, a property thought to be important for its role in ribosomal assembly and function (Borer et al, 1989). Although NPM is ubiquitously expressed, its level varies with the cell cycle, increasing with mitogenic stimulation and being responsive in different settings to oestrogen and transforming growth factor β (TGF-β) stimulation (Feuerstein et al, 1988a, b; Chakrabarty et al, 1990; Peter et al, 1990; Feuerstein & Randazzo, 1991; Derenzini et al, 1995; Koike et al, 1996). Thus, NPM biosynthesis appears to be related to cell proliferation and mitogenesis, and a variety of functions for the protein other than its role in ribosomal biogenesis have been put forth, including involvement in the processes of DNA replication and transcriptional regulation of gene expression and also binding and inhibition of interferon regulatory factor 1 (IRF-1), a transcription activator that possesses tumour-suppressor activities (Peter et al, 1990; Feuerstein & Randazzo, 1991; Inouye & Seto, 1994; Takemura et al, 1994; Wang et al, 1994; Kondo et al, 1997). Interestingly, NPM overexpression in NIH-3T3 fibroblasts induces anchorage-independent soft agar growth and tumour formation in nude mice but no evident cell morphology changes – a phenotype very similar to that seen in NIH-3T3 that overexpresses IRF-2, the antagonistic repressor of IRF-1 that binds the same cis elements (Kondo et al, 1997). As described below, only a single definite function (i.e. homodimerization) has been ascribed to the portion of NPM present in NPM–ALK, and it is not clear that alteration of the various putative normal functions of NPM contributes significantly to lymphomagenesis mediated by NPM–ALK (Bischof et al, 1997; Mason et al, 1998). The human NPM gene spans more than 25 kilobases (kb) and contains 12 exons that parallel the exon/intron structure of the rat gene homologue; characterization of the human NPM promoter region has revealed a TATA box and a single GC box and also a binding site for the transcription factor YY1 (Chang & Olson, 1990; Chan et al, 1997). A large number of human processed NPM pseudogenes exist (Liu & Chan, 1993), making the use of NPM cDNA probes in Southern blot hybridization to detect gene rearrangements problematic (Bullrich et al, 1994). Interest in the normal, and possible oncogenic, functions of NPM has intensified recently with the realization that the protein is involved not only with NPM–ALK but also with the NPM–MLF1 (myelodysplasia/myeloid leukaemia factor 1) fusion produced by the t(3;5) found in myelodysplastic syndromes and acute non-lymphoblastic leukaemia (ANLL), as well as the NPM–RARα (retinoic acid receptor alpha) fusion formed by the t(5;17) in occasional cases of acute promyelocytic leukaemia (Redner et al, 1996; Yoneda-Kato et al, 1996). The genomic chromosome break occurs within intron 4 of NPM in the t(2;5) and t(5;17) and within intron 6 in the t(3;5), incorporating the N-terminal 117 residues of NPM in NPM–ALK and NPM–RARα and the N-terminal 175 residues in NPM–MLF1. ALK is a member of the insulin receptor subfamily of RTKs with greatest homology to leucocyte tyrosine kinase (LTK), the two proteins exhibiting 57% amino acid identity and 71% similarity in their region of overlap (Iwahara et al, 1997; Morris et al, 1997). Anaplastic lymphoma kinase should not be confused with the unrelated activin-like kinase genes that are designated ALK-1 to ALK-7, which encode cell-surface receptors with serine/threonine kinase activity (ten Dijke et al, 1993). Other than the intron in which the breakpoint of the t(2;5) almost invariably occurs, the genomic structure of ALK has not been reported (Ladanyi & Cavalchire, 1996a; Luthra et al, 1998). The 6226-bp ALK cDNA contains a single open reading frame (ORF) that encodes a 1620-amino-acid protein with a predicted mass of approximately 177 kDa (Fig 1), which undergoes co-translational N-linked glycosylation, emerging in its mature form as a 200-kDa single-chain membrane-spanning receptor. In situ hybridization and immunostaining studies of embryonic and adult mice have revealed Alk expression to begin at approximately embryonic day 11 and to persist into the neonate and adult, although the abundance of Alk decreases in the postnatal period (Iwahara et al, 1997; Morris et al, 1997). Alk expression is confined to the nervous system, being found in several thalamic and hypothalamic nuclei; the trigeminal, facial and acoustic cranial ganglia; the olfactory bulb; the dorsal root ganglia; the anterior horns of the spinal cord in the region of the developing motor neurones; the sympathetic chain; and the ganglion cells of the gut. Notably, haematopoietic tissues show no detectable expression of Alk. Studies examining ALK expression in human tissues have corroborated this restricted expression pattern (Shiota et al, 1994b; Pulford et al, 1997). The neural-restricted expression of ALK suggests that it serves as a receptor for a neurotrophic factor(s) that probably plays a role in the normal development and function of the nervous system, analogous to the TRK receptors. Indeed, ALK shares a partially overlapping expression with the genes encoding the TRK family of neurotrophin receptors, the TRK-C expression pattern being most similar with expression in the spinal cord and enteric nervous system like ALK, but nonetheless with substantial differences both temporally and spatially (Barbacid, 1995). Intensive efforts to identify the ALK ligand(s) have been unsuccessful, and the knockout of Alk in the mouse does not reveal an essential function in neural development (unpublished observations), however. Thus, the normal function of ALK remains an enigma at this time. Because of t(2;5), the strong NPM promoter drives transcription of the portion of ALK encoding its kinase domain (the entire intracellular segment of the normal receptor), leading to its inappropriate expression in lymphoid cells. The NPM–ALK transcript contains an ORF of 2040 bp, encoding a 680-amino-acid protein that includes the first 117 amino acids of NPM and the final 563 amino acids of ALK (Fig 1). The portion of ALK in NPM–ALK (and all other known ALK fusions) corresponds to residues 1058–1620 of the normal receptor (Iwahara et al, 1997; Morris et al, 1997). As mentioned, the genomic breaks in NPM–ALK occur in NPM intron 4, which contains two Alu repetitive sequences and measures 911 bp, and in a 1935-bp intron that separates exon sequences encoding the transmembrane and juxtamembrane segments of ALK (Ladanyi & Cavalchire, 1996a; Chan et al, 1997; Luthra et al, 1998). As the breaks in both genes occur within the same introns almost invariably — only a single lymphoma with a molecular variant has been described previously in which the genomic break was in NPM intron 4 but within the juxtamembrane ALK exon, creating an in frame fusion 30 bases distal to the usual ALK junction owing to use of a cryptic splice signal (Ladanyi & Cavalchire, 1996b) — essentially all t(2;5)-positive lymphomas can be diagnosed by either RNA-polymerase chain reaction (PCR) or genomic DNA-PCR using the same set of NPM and ALK primers. In addition to ectopic expression of the truncated cytoplasmic portion of ALK due to its fusion with NPM, the kinase domain of ALK is constitutively activated and relocalized from its normal position at the inner surface of the cell membrane in neural cells to within both the cytoplasm and nucleus/nucleoli in lymphoma cells (Bischof et al, 1997; Mason et al, 1998). Detailed studies of the portion of NPM present in NPM–ALK have revealed this segment to encode an oligomerization domain that can mediate self-association of the fusion protein, mimicking the aggregation and resultant activation of RTKs at cell membranes when they bind to their cognate ligand (Bischof et al, 1997). The constitutive self-association of NPM–ALK molecules produces conformational changes in the structure of the kinase domain of ALK that activate the auto- and trans-phosphorylation capabilities of the kinase, resulting in the phosphorylation and consequent activation of multiple mitogenic signalling substrates. Analysis of other truncated, fused tyrosine kinases has revealed that the only apparent common feature shared by the multiple identified fusion gene partners is their ability to mediate homo-oligomerization of the respective fusion protein, mimicking activation normally mediated by ligand binding to receptor (Rodrigues & Park, 1994). Consistent with reports that many different partners can fuse with and activate a given truncated kinase (Rodrigues & Park, 1994), an experimentally engineered mutant, TPR–ALK, in which the N-terminus of the nuclear pore protein TPR (Byrd et al, 1994) that contains two leucine zipper motifs known to mediate dimerization was substituted for the NPM segment of NPM–ALK, was shown to be a constitutively active protein with essentially the same transforming potency as wild-type NPM–ALK (Bischof et al, 1997; Mason et al, 1998). The exact nature of the NPM oligomerization motif remains to be determined; computer-assisted analysis of NPM failed to identify similarity to other oncogenic tyrosine kinase fusion partner proteins such as BCR, H4, RFP, TFG, TPM3 or TPR, which contain coiled-coil domains that mediate oligomerization (McWhirter et al, 1993; Rodrigues & Park, 1994; Greco et al, 1995), or to TEL, which contains a helix-loop-helix domain that serves this function (Golub et al, 1997). Subcellular localization studies of NPM–ALK-expressing cells revealed the fusion protein to be within both the cytoplasmic and nuclear compartments and to be abundant in both the nucleoplasm and nucleoli (Bischof et al, 1997; Mason et al, 1998). The localization of NPM–ALK within the nuclear compartment is due to heterodimerization of the fusion protein with normal NPM and subsequent shuttling of NPM–ALK into the nucleus and nucleoli as a result of the NPM transport activity (Borer et al, 1989; Bischof et al, 1997; Mason et al, 1998). Localization of NPM–ALK to the nuclear compartment is not functionally essential for its transforming activity, however, given that the engineered mutant TPR–ALK as well as naturally occurring variant ALK fusions (described below) are localized entirely within the cytoplasm and transform cells with high potency (Bischof et al, 1997; Mason et al, 1998). The mitogenicity of NPM–ALK has been demonstrated in vitro using immortalized rodent fibroblast lines such as NIH-3T3 and Fr3T3 and in vivo in mice; its mitogenicity is also evident from the ability of the fusion to render interleukin (IL)-3-dependent cells, such as the murine pro-B line Ba/F3, factor independent (Fujimoto et al, 1996; Bischof et al, 1997; Kuefer et al, 1997; Bai et al, 1998; Mason et al, 1998; Hubinger et al, 1999a). As expected, because of its ability to mediate self-association of the fusion, the NPM portion (or, alternatively, a heterologous dimerization motif such as TPR) is essential to these activities, expression of the truncated ALK kinase domain alone lacking function. Furthermore, mutations in the ALK portion of the fusion that render the kinase inactive abrogate the oncogenic activity of ALK fusions (Fujimoto et al, 1996; Bischof et al, 1997; Bai et al, 1998), indicating the necessity for ALK-mediated tyrosine phosphorylation in transformation. Tyrosine autophosphorylation sites on RTKs define binding motifs for SRC homology 2 (SH2) domain-containing signalling molecules such as SHC, GRB2 and phospholipase C-gamma (PLC-gamma), among many others (Pawson & Scott, 1997). Different signal transducing molecules bind via their SH2 domains to specific autophosphorylation sites in the cytoplasmic domain of RTKs such as ALK, undergoing phosphorylation and activation themselves. By recruiting a particular set of signal-transducing molecules, a given RTK is capable of inducing specific cellular responses. Several tyrosine kinase signalling substrates are known to interact with NPM–ALK and may be involved in transformation mediated by the protein. For example, the substrates SHC, insulin receptor substrate 1 (IRS-1) and GRB2 associate with NPM–ALK (Fujimoto et al, 1996; Bischof et al, 1997; Bai et al, 1998). However, NPM–ALK proteins containing mutations of tyrosine 156, to which IRS-1 binds, or tyrosine residue 567, to which SHC interacts either singly or doubly, still efficiently transform cells, indicating that neither of these substrates is essential for NPM–ALK-mediated oncogenesis (Fujimoto et al, 1996; Bischof et al, 1997; Bai et al, 1998). The necessity for GRB2 signalling in NPM–ALK-induced transformation has yet to be assessed. Additional tyrosine kinase substrates examined for their ability to associate with, and be activated by, NPM–ALK include PLC-gamma, the p85 subunit of PI′3-kinase, VAV, BTK, SHPTP2, NCK, SHB, SYK and SYP (Bai et al, 1998). Of these molecules, and p85 interact with The tyrosine phosphorylation and activation of by NPM–ALK, evident by of occurs because of of with NPM–ALK tyrosine a mutant, NPM–ALK no forms with or to its phosphorylation and activation (Bai et al, 1998). or fibroblasts that NPM–ALK to transformation. overexpression of partially the of to NPM–ALK that may be one of the most important of NPM–ALK that contribute to its Analysis of of all possible NPM–ALK phosphorylation sites revealed all that kinase activity, other than the mutant, are to render cells further the of activation (Bai et al, 1998). from these studies also that is more important for the mitogenic signal mediated by NPM–ALK, than the by the fusion, that signalling by NPM–ALK appears to occur in part by activation of the et al, 1999; Bai et al, is a of a subunit and an regulatory which is found in cellular with RTKs and tyrosine are mediated by the SH2 domains of the p85 and of p85 with activated tyrosine kinases is thought to a signal for activation of the which in in of that are to bind to homology domains of signalling proteins and to activate molecules, including the serine/threonine kinase et al, b; et al, 1999). In is capable of and the family member and cell & 1997). is also involved in activation of inhibition of and of transcription of all of which contribute to its function 1999). is also involved in signalling by such as leading to haematopoietic et al, 1997). The but not SH2 domain of the regulatory p85 subunit with NPM–ALK, activation of as well as phosphorylation of occurring as a result (Bai et al, NPM–ALK can the of partially cells from (Bai et al, Additional evidence of the of NPM–ALK includes of cells to by abundant expression of by these ALCL cells after This is with reports inhibition of by activation of the in and the ability of activation of and to et al, 1995; et al, 1998). and the growth of cells to a than in the of function in transformation mediated by the chimaeric inhibition with such as also induces significant in lymphoma cell lines such as et al, 1999; Bai et al, Studies of other apparent NPM–ALK substrates are in in several for example, analysis of the signal and activator of transcription proteins has revealed phosphorylation and activation by NPM–ALK and suggests that this activation may be for NPM–ALK-mediated (unpublished A final proteins a member of the tumour factor receptor et al, 1999). by Shiota et al (1994a) that with the that the two molecules functionally interact in a essential for Hubinger et al that NPM–ALK binds to the intracellular domain of the CD30 the ALK portion to mediate of the However, analysis of CD30 and NPM–ALK from lymphoma cells revealed that the is not by CD30 ligand and that CD30 activation to NPM–ALK activity as by either autophosphorylation of the or the phosphorylation of a NPM–ALK Thus, no between CD30 and NPM–ALK could be although with CD30 the localization of NPM–ALK in a that proteins to NPM–ALK-mediated these that no essential between and CD30 in In the the description of t(2;5), a number of variant rearrangements have been reported in Although of these cryptic or of t(2;5) that a NPM–ALK fusion in the cell (Bullrich et al, 1994), other such as and have not been further characterized et al, 1990; et al, 1996). In addition, a cell line designated from a lymphoma has been reported with a break in the of evident alteration et al, a number of rearrangements with other than have been including and et al, 1996; 1997; et al, 1997; et al, 1998; et al, 1998; et al, 1999). with the description of these variant several large studies examined the expression of ALK proteins in ALCL using (Shiota et al, 1995; et al, 1996; et al, 1997; et al, 1997; Pulford et al, 1999; et al, 1998; et al, b; et al, 1999). Interestingly, of lymphomas expression of ALK restricted to the cytoplasm of tumour cells, as to the cytoplasmic and localization of NPM–ALK, that these cases ALK fusions with partner proteins other than and in vitro kinase of such Pulford et al identified ALK proteins with molecular of kDa a containing kDa a with and of the of these proteins using an et al, that the portion of NPM in NPM–ALK also failed to evidence for NPM involvement in these variant proteins et al, 1999). A of and of lymphomas ALK fusions other than NPM–ALK these variant cases may not show the morphological found in lymphomas as no cases of variant were identified to be to this et al, The clinicopathological of these cases to be from given that all were or null in or with a and frequently with disease with systemic and extranodal also to the extranodal involvement included soft nervous system and gut. The of these variant lymphomas was and was not significantly different from that of t(2;5)-positive but clearly to that of ALCL and to the rare large lymphomas that ALK (see both of which to have et al, 1997; et al, Thus, for it appears that lymphomas variant ALK fusions can be with lymphomas as they a similar group of tumours to be from variant rearrangements that occur in lymphomas have been characterized — and — and a variant ALK fusion formed by a rearrangement not previously described has also been et al have shown to in which the TPM3 gene from encoding a is fused to the sequences of ALK encoding its cytoplasmic The TPM3 gene has previously been shown to be a fusion partner with the truncated RTK in carcinoma and et al, 1995). As in the TPM3 genomic breakpoint in TPM3–ALK is between exons and resulting in fusion of the N-terminal residues of TPM3 to ALK (Fig 1). In (and all variant ALK rearrangements characterized the ALK intron containing the genomic breakpoint is the same as that involved in the The portion of TPM3 into its fusion with ALK forms a coiled-coil structure that can mediate mimicking activation of normal ALK and analogous to self-association and activation of NPM–ALK by its NPM et al have shown to gene as the ALK fusion with two different and possible the breakpoint in (Fig 1). was initially identified as a partner for in carcinoma in the fusion gene et al, 1995). The chimaeric TFG–ALKS protein is of which are by TFG, by 563 residues of The protein