Clinical Features
ALK+ ALCL represents approximately 2-3% of non-Hodgkin lymphomas in adults and 20-30% of non-Hodgkin lymphomas in children.



These neoplasms can involve nodal or extranodal sites and are clinically aggressive [1, 3, 4, 5, 6] . Patients often have systemic symptoms (weight loss, fever, night sweats) and high International Prognostic Index score. Patients require combination chemotherapy and treatments such as bone marrow or stem cell transplantation have been used in some patients. Determination of ALK status in cases of ALCL has prognostic significance as most patients with ALK+ ALCL have a better prognosis than patients with ALK- ALCL as well as other peripheral T-cell lymphomas [7, 8] .

Histologic Findings
One of the confusing aspects of ALK+ ALCL is that its name suggests that all tumors have anaplastic cytologic features. This is not the case. A number of histologic variants have been described in ALCL (in both ALK+ and ALK- tumors).

Histologic Variants of ALCL	Frequency
Major	95%
Classical	65%
Monomorphous	10%
Lymphohistiocytic	10%
Small cell	10%
Minor	5% (or less)
Sarcomatoid	1%
Myxoid stroma (fasciitis-like)	1%
Giant cell-rich	1%
Neutrophil-rich	1%
Signet-ring	1%

In classical (or common) cases, as originally described by Stein and colleagues [1], ALCL cells grow cohesively and preferentially involve lymph nodes sinuses, particularly in lymph nodes not involved extensively. With greater involvement, ALCL replaces the paracortical regions or may diffusely replace lymph node



architecture. Cytologically, the neoplastic cells are large and bizarre, irregularly shaped, and often have polylobated nuclei. The nuclear chromatin is vesicular with prominent nucleoli. The tumor cell cytoplasm is abundant and usually basophilic. So-called hallmark cells are usually present. These cells have a horseshoe or kidney-shaped nucleus that partially or completely surrounds a clear, or more eosinophilic, paranuclear (Golgi) area. The classical type of ALK+ ALCL represents approximately 70% of all cases. Other histologic variants of ALK+ ALCL are more difficult to recognize without the knowledge that they express ALK. Their names are self-descriptive.

Immunophenotypic Features



Virtually all cases of ALK+ ALCL strongly express the activation antigen CD30, with a characteristic membranous and paranuclear (target-like) pattern. Approximately two thirds of cases are T-cell and the remainder are null cell. T-cell ALCLs express T-cell antigens, but an aberrant immunophenotype is common. Most cases are negative for CD3 and T-cell receptors, despite the presence of T-cell receptor gene rearrangements [9]. Null-cell ALCLs lack immunophenotypic (as well as molecular) evidence of lineage.

ALK+ ALCLs (both T- and null-cell) are commonly positive for markers expressed by cytotoxic T-cells, such as TIA-1, granzyme B and perforin [10]. Most tumors have high proliferation and apoptotic rates, and virtually all ALK+ ALCLs are negative for BCL-2 [11]. Some immunohistochemical markers, in addition to ALK, have been correlated with poorer prognosis, such as CD56, MUC1 (EMA), and survivin [12, 13, 14] .

Translocations Involving Chromosome 2p23



Chromosomal translocations involving the alk gene at chromosome 2p23 are the cause of ALK overexpression in ALCL [15, 16] . The most common abnormality is the t(2;5)(p23;q35) in 75% of cases. The variant abnormalities, in aggregate, occur in approximately 20% of cases.

Translocation	Fusion protein	Location of protein
t(2;5)(p23;q35)	NPM-ALK	Nucleus and cytoplasm
t(1;2)(p25;p23)	TPM3-ALK	Cytoplasm
t(2;3)(p23;q21)	TFG-ALK	Cytoplasm
inv(2)(p23q35)	ATIC-ALK	Cytoplasm
t(2;17)(p23;q23)	CLTC-ALK	Cytoplasm (granular)
t(2;X)(p23;q11-12)	MSN-ALK	Membrane
t(2;19)(p23;p13)	TPM4-ALK	Cytoplasm
t(2;17)(p23;q35)	ALO17-ALK	Cytoplasm
t(2;22)(p23;q11)	MYH9-ALK	Cytoplasm

ALK-1 Immunostaining Pattern Correlates with alk Gene Abnormalities



The immunostaining pattern with anti-ALK antibodies predicts, in part, the type of abnormality involving the alk gene in ALK-positive ALCLs [2, 15] . The npm gene involved in the t(2;5) encodes for nucleophosmin (NPM), a protein that normally shuttles proteins from the cytoplasm to the nucleus. As a result of the t(2;5), the amino-terminal of NPM is fused to the carboxy-terminal portion of ALK. Although NPM portion of the fusion protein does not have its normal nuclear localization signal (NLS), via interaction with normal cytoplasmic NPM (with its NLS), the NPM-ALK fusion protein is transported from the cytoplasm into the nucleus. Thus, in a t(2;5)-positive case of ALCL, ALK immunostaining is both cytoplasmic and nuclear. In most other translocations involving the alk gene, the pattern of ALK-1 immunostaining is restricted to the cytoplasm. Two other rare translocations result in unique staining patterns for ALK. In the t(2;17) involving the clathrin heavy chain gene (cltc), the CLTC protein is a component of cytoplasmic vesicles. Thus, ALK staining results in a granular cytoplasmic pattern. In the t(2;X) involving the moesin gene (msn), MSN is membrane associated and, thus, the ALK staining pattern is membranous.

npm-alk Can Be Detected in Healthy Individuals
Studies of peripheral blood and tissue specimens obtained from patients without evidence of ALCL have shown the presence of npm-alk. For example, Trumper and colleagues [17] used reverse transcription (RT)-PCR to detect npm-alk cDNA in 14 of 29 (48%) patients. Similarly, Maes and colleagues [18] detected npm-alk cDNA by real-time RT-PCR in 8 of 13 (62%) reactive lymph node biopsy specimens, respectively. The npm-alk is present at very low levels and ALK protein was not expressed.

As an aside, npm-alk DNA fusion sequences also have been detected in cases of cutaneous ALCL by long-range PCR [19].

ALK Expression Is Not Specific For ALCL
In normal tissues, ALK is only expressed in the central and peripheral nervous systems. Thus, expression of ALK at other sites is abnormal [20]. However, ALK expression is not specific for ALCL. Other lesions that overexpress ALK include subsets of rhabdomyosarcoma and neuroblastoma, a rare subtype of diffuse large B-cell lymphoma (DLBCL), and a subset of inflammatory myofibroblastic tumors [15].

Rare cases of DLBCL are ALK+. These tumors often exhibit plasmacytoid differentiation, are IgA positive, and are CD30 negative. These cases can be divided into two groups. In the first group, the DLBCLs carry the t(2;5)(p23;q35) [21]. In the second group, the DLBCLs carry the t(2;17) involving the alk and clathrin genes [22]. These cases exhibit a distinctive pattern of ALK staining that is granular and cytoplasm. There also may be cases of DLBCL that express full length ALK [23], however, some of these cases reported previously have since been shown to carry the t(2;17) [22].

In inflammatory myofibroblastic tumors, ALK-1 immunostaining is cytoplasmic. A number of fusion genes have been identified in these lesions [24, 25, 26] .

Translocation	Fusion protein	Location of protein
t(1;2)(p25;p23)	TPM3-ALK	Cytoplasm
t(2;17)(p23;q23)	CLTC-ALK	Cytoplasm (granular)
t(2;19)(p23;p13)	TPM4-ALK	Cytoplasm
t(2;11;2)(p23;p15;q31)	CARS-ALK	Cytoplasm
t(2;2)(p23;q13) or inv(2)(p23q11-13)	RANB2-ALK	Nuclear membrane

Role of NPM-ALK Fusion Protein in Lymphomagenesis
A number of mouse models have shown that NPM-ALK is important in the pathogenesis of ALCL. Both aggressive B-cell and T-cell lymphomas and plasma cell tumors have developed in mice, either transplanted with bone marrow cells infected with retroviral constructs containing npm-alk, or in transgenic animals [27, 28, 29] . The lineage of these tumors is dependent, in part, on the promoter used and on the degree of forced expression of NPM-ALK.

Not surprisingly, the pathways downstream of NPM-ALK have been a hot area of research, and a number of signaling pathways are now implicated. In addition, there are other pathways that are being identified. One recent and study of interest in this area by Crockett and colleagues [30] who used lysates of an ALK+ ALCL cell line (Karpas 299), performed co-immunoprecipitation with either anti-ALK or anti-NPM antibodies, and then used mass spectrophotometry to identify proteins bound to either ALK or NPM. They identified 46 proteins unique to the ALK complex and 11 proteins unique to the NPM complex. Focusing only on the ALK complex here, they identified many molecules already identified by others as being involved in signaling in ALCL, such as PLC-γ, JAK2, JAK3, STAT3, GRB2, IRS-1, and PI3-kinase/AKT. However, they also identified a number of other proteins as being involved that they grouped into four general families: adaptor proteins (e.g. SOCS), kinases (e.g. components of RAS/MAPK), phosphatases (e.g. meprin) and heat shock proteins (e.g. HSP60). These pathways involve many cell functions, such as mitogenic response, apoptosis, cell cycle control, and proliferation. The authors cautiously point out that not all of these proteins may interact in a direct manner, as some may be substrates of ALK or regulatory proteins that modulate ALK. The authors also point out that binding of a protein in the ALK complex, although suggestive, is not proof of in vivo importance.

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