Anaplastic Large Cell Lymphoma: A Study Of Clinicopathologic, Immunohistochemical And Molecular Biological Features With Emphasizing The ALK Fusion Gene And Its Expression

[Background and Objective] Anaplastic large cell lymphoma (ALCL) is a rare non-Hodgkin's lymphoma. In 60%-85% of ALCL cases the expression of anaplastic large cell lymphoma kinase (ALK) fusion protein could be detected. The expression of the chimeric protein is caused by the translocation between chromosome 2 and 5, or t(l;2), t(2;3) et al. All these translocations involve the ALK gene located in chromosome 2 and create a new fusion ALK gene. ALK (+) ALCL appears to benefit from chemotherapy more than ALK (-) forms of systemic ALCL. So it is important to detect the ALCL with ALK fusion gene or not. In this study 30 cases of ALCL were analyzed in order to observe their clinicopathologic, immunophenotypic and molecular biological features, especially the ALK fusion gene and its expression in mRNA and protein levelsby using FISH, RT-PCR and immunohistochemistry. Otherwise the relationship between Epstein-Barr virus infection and ALCL was also observed. [Materials and Methods] 30 cases of ALCL, including 28 cases of systemic ALCL and 2 cases of primary cutaneous ALCL were from the Department of Pathology, West China Hospital. The histopathological classification used in this study was new WHO classification (2001). Paraffin blocks of these cases were cut?> for immunostaining, EBER in situ hybridization and DNA/RNA extraction. SP method was used for immunostaining and the first antibodies were ALK-1, CD30, CD45RO, CD3, CD20, CD79a, EMA, granzyme B and TIA-1. In situ hybridization was used for detecting EBER1/2. For detecting ALK fusion gene FISH and RT-PCR were applied[Results] Histopathologically, there were 17 cases of common variant (60.7%), 8 of small cell variant (28.6%), 2 of lymphohistocytic variant (7.1%) and one of giant cell rich variant (3.6%) in 28 systemic cases. Immunohistochemically, all cases were CD30 strong positive in which 19 cases (63.3%) with CD3 and/or CD45RO positivity, whereas 11 cases (36.7%) without CD3, CD45RO, CD20 and CD79a positivities. Cytotixic markers granzyme B and TIA-1 expressed in 5 of 10 performed cases and 7 of 12 performed cases, respectively. EMA expressed in 5 of 8 performed cases. There were 10 cases (34.5%) with clonal TCR-y chain gene rearrangement in 29 performed cases. EB virus infection was found in one of 30 cases (3.3%) by EBER probe and in situ hybridization.ALK fusion protein was detected in 19 of 28 systemic ALCLs (67.9%) in which 17 with nuclear and cytoplasmic positive products, whereas 1 with cytoplasmic positivity and 1 with cytoplasmic and membranous positivities. In the two cases of primary cutaneous ALCL, there was a negative reaction for ALK fusion protein. By RT-PCR technique there were 19 of 27 performed systemiccases with ALK fusion gene transcription in which 16 with expected NPM-ALK transcript, 1 with a long NPM-ALK transcript (had an additional 129 bp fragment from ALK gene introns by DNA sequencing), 1 with TPM3-ALK transcript, and 1 with TFG-ALKs transcript. ALK gene translocation was found in 12 of 20 performed cases by FISH. Comparing with the three methods to detect ALK fusion gene or its product, the results by FISH and RT-PCR matched 100% case by case with ALK imrnunostaining.Clinically, in this group there were 20 males and 10 females. The male to female ratio was 2:1. The age range was from 4 to 74 years and averagely 27.63 + 17.57 years. The patients with ALK gene aberrations were much younger than those without this abnormalities (20.84± 10.65 vs 39.36+21.27 years, p=0.003). The two years survival rate of patients with ALK gene aberrations was 82.4%, whereas 27.8% of the patients without ALK gene abnormalities. Survival curves of these two groups showed a statistic significant distinguishability (p=0.0217). [ Conclusions ] 1. It is necessary to distinguish the ALCL with ALK fusion gene from ALCLwithout ALK fusion gene because of clinical and molecular genetic differences.2.The three methods to detecting ALK fusion gene or its product show the samespecificity and sensitivity. Immunostaining of ALK fusion protein is the firstchoice in routine surgical pathological works.3. As we know this is the first discovery of a splicing variant of NPM-ALK fusiongene with an additional 129 bp fragment from introns of ALK gene.4.ALCLs in this group does not show a close relationship with EB virus infection(1/30 EBER+).