ALK Expression Defines a Distinct Group of T/Null Lymphomas (“ALK Lymphomas”) with a Wide Morphological Spectrum
1998; Elsevier BV; Volume: 153; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)65629-5
ISSN1525-2191
AutoresBrunangelo Falini, Barbara Bigerna, Marco Fizzotti, Karen Pulford, Stefano A. Pileri, Georges Delsol, Antonino Carbone, Marco Paulli, Umberto Magrini, Fabio Menestrina, Roberto Giardini, Silvana Pilotti, Alessandra Mezzelani, Barbara Ugolini, Monia Billi, Alessandra Pucciarini, Roberta Pacini, Pier Giuseppe Pelicci, Leonardo Flenghi,
Tópico(s)Acute Myeloid Leukemia Research
ResumoThe t(2;5)(p23;q35) translocation associated with CD30-positive anaplastic large cell lymphoma results in the production of a NPM-ALK chimeric protein, consisting of the N-terminal portion of the NPM protein joined to the entire cytoplasmic domain of the neural receptor tyrosine kinase ALK. The ALK gene products were identified in paraffin sections by using a new anti-ALK (cytoplasmic portion) monoclonal antibody (ALKc) that tends to react more strongly than a previously described ALK1 antibody with the nuclei of ALK-expressing tumor cells after microwave heating in 1 mmol/L ethylenediaminetetraacetic acid buffer, pH 8.0. The ALKc monoclonal antibody reacted selectively with 60% of anaplastic large cell lymphoma cases (60 of 100), which occurred mainly in the first three decades of life and consistently displayed a T/null phenotype. This group of ALK-positive tumors showed a wide morphological spectrum including cases with features of anaplastic large cell lymphoma "common" type (75%), "lymphohistiocytic" (10%), "small cell" (8.3%), "giant cell" (3.3%), and "Hodgkin's like" (3.3%). CD30-positive large anaplastic cells expressing the ALK protein both in the cytoplasm and nucleus represented the dominant tumor population in the common, Hodgkin's-like and giant cell types, but they were present at a smaller percentage (often with a perivascular distribution) also in cases with lymphohistiocytic and small cell features. In this study, the ALKc antibody also allowed us to identify small neoplastic cells (usually CD30 negative) with nucleus-restricted ALK positivity that were, by definition, more evident in the small cell variant but were also found in cases with lymphohistiocytic, common, and "Hodgkin's-like" features. These findings, which have not been previously emphasized, strongly suggest that the neoplastic lesion (the NPM-ALK gene) must be present both in the large anaplastic and small tumor cells, and that ALK-positive lymphomas lie on a spectrum, their position being defined by the ratio of small to large neoplastic cells. Notably, about 15% of all ALK-positive lymphomas (usually of the common or giant cell variant) showed a cytoplasm-restricted ALK positivity, which suggests that the ALK gene may have fused with a partner(s) other than NPM. From a diagnostic point of view, detection of the ALK protein was useful in distinguishing anaplastic large cell lymphoma cases of lymphohistiocytic and small cell variants from reactive conditions and other peripheral T-cell lymphoma subtypes, as well as for detecting a small number of tumor cells in lymphohemopoietic tissues. In conclusion, ALK positivity appears to define a clinicopathological entity with a T/null phenotype ("ALK lymphomas"), but one that shows a wider spectrum of morphological patterns than has been appreciated in the past. The t(2;5)(p23;q35) translocation associated with CD30-positive anaplastic large cell lymphoma results in the production of a NPM-ALK chimeric protein, consisting of the N-terminal portion of the NPM protein joined to the entire cytoplasmic domain of the neural receptor tyrosine kinase ALK. The ALK gene products were identified in paraffin sections by using a new anti-ALK (cytoplasmic portion) monoclonal antibody (ALKc) that tends to react more strongly than a previously described ALK1 antibody with the nuclei of ALK-expressing tumor cells after microwave heating in 1 mmol/L ethylenediaminetetraacetic acid buffer, pH 8.0. The ALKc monoclonal antibody reacted selectively with 60% of anaplastic large cell lymphoma cases (60 of 100), which occurred mainly in the first three decades of life and consistently displayed a T/null phenotype. This group of ALK-positive tumors showed a wide morphological spectrum including cases with features of anaplastic large cell lymphoma "common" type (75%), "lymphohistiocytic" (10%), "small cell" (8.3%), "giant cell" (3.3%), and "Hodgkin's like" (3.3%). CD30-positive large anaplastic cells expressing the ALK protein both in the cytoplasm and nucleus represented the dominant tumor population in the common, Hodgkin's-like and giant cell types, but they were present at a smaller percentage (often with a perivascular distribution) also in cases with lymphohistiocytic and small cell features. In this study, the ALKc antibody also allowed us to identify small neoplastic cells (usually CD30 negative) with nucleus-restricted ALK positivity that were, by definition, more evident in the small cell variant but were also found in cases with lymphohistiocytic, common, and "Hodgkin's-like" features. These findings, which have not been previously emphasized, strongly suggest that the neoplastic lesion (the NPM-ALK gene) must be present both in the large anaplastic and small tumor cells, and that ALK-positive lymphomas lie on a spectrum, their position being defined by the ratio of small to large neoplastic cells. Notably, about 15% of all ALK-positive lymphomas (usually of the common or giant cell variant) showed a cytoplasm-restricted ALK positivity, which suggests that the ALK gene may have fused with a partner(s) other than NPM. From a diagnostic point of view, detection of the ALK protein was useful in distinguishing anaplastic large cell lymphoma cases of lymphohistiocytic and small cell variants from reactive conditions and other peripheral T-cell lymphoma subtypes, as well as for detecting a small number of tumor cells in lymphohemopoietic tissues. In conclusion, ALK positivity appears to define a clinicopathological entity with a T/null phenotype ("ALK lymphomas"), but one that shows a wider spectrum of morphological patterns than has been appreciated in the past. Anaplastic large cell lymphoma (ALCL) was first described by Stein et al1Stein H Mason DY Gerdes J O'Connor N Wainscoat J Pallesen G Gatter K Falini B Delsol G Lemke H Schwarting R Lennert K The expression of Hodgkin's disease associated Ki-1 antigen in reactive and neoplastic lymphoid tissues: evidence that Sternberg-Reed cells and histiocytic malignancies are derived from activated lymphoid cells.Blood. 1985; 66: 848-858Crossref PubMed Google Scholar as a tumor with distinctive histological and immunohistological features, eg, preferential paracortical and intrasinusoidal lymph node involvement by sheets of large anaplastic cells. The tumor cells express the Ki-1 antigen,1Stein H Mason DY Gerdes J O'Connor N Wainscoat J Pallesen G Gatter K Falini B Delsol G Lemke H Schwarting R Lennert K The expression of Hodgkin's disease associated Ki-1 antigen in reactive and neoplastic lymphoid tissues: evidence that Sternberg-Reed cells and histiocytic malignancies are derived from activated lymphoid cells.Blood. 1985; 66: 848-858Crossref PubMed Google Scholar a molecule that was later renamed CD30 and shown to be a receptor for CD30L, a member of the tumor necrosis factor ligand family.2Falini B Pileri S Pizzolo G Durkop H Flenghi L Stirpe F Martelli MF Stein H CD30 (Ki-1) molecule: a new cytokine receptor of the tumor necrosis factor receptor superfamily as a tool for diagnosis and immunotherapy.Blood. 1995; 85: 1-14Crossref PubMed Google Scholar The tumor, which represents about 5 to 10% of non-Hodgkin lymphomas in adults and 30 to 40% of large-cell lymphomas in children, is a highly aggressive lymphoma that usually presents as stage III to IV disease frequently associated with systemic symptoms and extranodal involvement, especially skin and bone.3Kadin ME Primary Ki-1 positive anaplastic large cell lymphoma: a distinct clinicopathologic entity.Ann Oncol. 1994; 5: 25-30Crossref PubMed Google Scholar However, despite its aggressive features, the disease can be cured in a high percentage of cases.3Kadin ME Primary Ki-1 positive anaplastic large cell lymphoma: a distinct clinicopathologic entity.Ann Oncol. 1994; 5: 25-30Crossref PubMed Google Scholar, 4Zinzani PL Bendandi M Martelli M Falini B Sabattini E Amadori S Gherlinzoni F Martelli MF Mandelli F Tura S Pileri S Anaplastic large cell lymphoma: clinical and prognostic evaluation of 90 adult patients.J Clin Oncol. 1996; 14: 955-962Crossref PubMed Scopus (87) Google Scholar ALCL is associated with a t(2;5) chromosomal translocation5Mason DY Bastard C Rimokh R Dastugue N Huret J-L Kristoffersson U Magaud J-P Nezelof C Tilly H Vannier J-P Hemet J Warnke R CD30-positive large cell lymphomas (Ki-1 lymphoma) are associated with a chromosomal translocation involving 5q35.Br J Haematol. 1990; 74: 161-168Crossref PubMed Scopus (285) Google Scholar that fuses the ALK (anaplastic lymphoma kinase) and theNPM (nucleophosmin) genes,6Morris SW Kirstein MN Valentine MB Dittmer KG Shapiro DN Saltman DL Look AT Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma.Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1980) Google Scholar, 7Ladany M The NPM/ALK gene fusion in the pathogenesis of anaplastic large cell lymphoma.Cancer Surv. 1997; 30: 59-75PubMed Google Scholar leading to the formation of a chimeric NPM-ALK protein (p80)6Morris SW Kirstein MN Valentine MB Dittmer KG Shapiro DN Saltman DL Look AT Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma.Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1980) Google Scholar, 7Ladany M The NPM/ALK gene fusion in the pathogenesis of anaplastic large cell lymphoma.Cancer Surv. 1997; 30: 59-75PubMed Google Scholar, 8Fujimoto J Shiota M Iwahara T Seki N Satoh H Mori S Yamamoto T Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5).Proc Natl Acad Sci USA. 1996; 93: 4181-4186Crossref PubMed Scopus (249) Google Scholar consisting of the N-terminal portion of NPM9Chan PK Chan FY Morris SW Liu QR Isolation and characterization of the human nucleophosmin/B23 (NPM) gene: identification of the YY1 binding site at the 5′ enhancer region.Nucleic Acids Res. 1996; 25: 1225-1232Crossref Scopus (46) Google Scholar linked to the cytoplasmic domain of the neural receptor tyrosine kinase ALK.10Morris SW Naeve C Mathew P James PL Kirstein MN Cui X Witte DP ALK, the chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin's lymphoma, encodes a novel neural tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK).Oncogene. 1997; 14: 2175-2188Crossref PubMed Scopus (422) Google Scholar, 11Iwahara T Fujimoto J Wen D Cupples R Bucay N Arakawa T Mori S Ratzkin B Yamamoto T Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system.Oncogene. 1997; 14: 439-449Crossref PubMed Scopus (564) Google Scholar The chimeric NPM-ALK protein is thought to play a key role in lymphomagenesis by aberrant phosphorylation of intracellular substrates.12Bischof D Pulford K Mason DY Morris SW Role of nucleophosmin (NPM) portion of the non-Hodgkin's lymphoma-associated NPM-anaplastic kinase fusion protein in oncogenesis.Mol Cell Biol. 1997; 17: 2312-2325Crossref PubMed Scopus (322) Google Scholar, 13Kuefer MU Look AT Pulford K Behm FG Pattengale PK Mason DY Morris SW Retrovirus-mediated gene transfer of NPM-ALK causes lymphoid malignancy in mice.Blood. 1997; 90: 2901-2910Crossref PubMed Google Scholar These discoveries allowed the development of reverse transcription (RT)-polymerase chain reaction (PCR) assays for the detection ofNPM-ALK transcripts14Downing J Shurtleff S Zielenska M Curcio-Brint AM Behm FG Head DR Sandlund JT Weinburger D Kossakowska AE Thorner P Lorenzana A Ladanyi M Morris S Molecular detection of the t(2;5) translocation of non-Hodgkin's lymphoma by reverse transcriptase-polymerase chain reaction.Blood. 1995; 85: 3416-3422PubMed Google Scholar and the generation of polyclonal15Shiota M Fujimoto J Takenaga M Satoh H Ichinohasama R Abe M Nakano M Yamamoto T Mori S Diagnosis of t(2;5)(p23;q35)-associated Ki-1 lymphoma with immunohistochemistry.Blood. 1994; 84: 3648-3652PubMed Google Scholar, 16Shiota M Nakamura S Ichinohasama R Abe M Akagi T Takeshita M Mori N Fujimoto J Miyauchi J Mikata A Nanba K Takami T Yamabe H Takano Y Izumo T Nagatani T Mohri N Nasu K Satoh H Katano H Fujimoto J Yamamoto T Mori S Anaplastic large cell lymphomas expressing the novel chimeric protein p80NPM/ALK: a distinct clinicopathologic entity.Blood. 1995; 86: 1954-1960Crossref PubMed Google Scholar, 17Hutchinson RE Banki K Shuster JJ Barrett D Dieck C Berard CW Murphy SB Link MP Pick TE Laver J Schwenn M Mathew P Morris SW Use of an anti-ALK antibody in the characterization of anaplastic large-cell lymphoma of childhood.Ann Oncol. 1997; 8: 37-42Google Scholar and monoclonal antibodies (mAbs)18Pulford K Lamant L Morris S Butler LH Wood KM Stroud D Delsol G Mason DY Detection of ALK and NPM-ALK protein in normal and neoplastic cells with the monoclonal antibody ALK1.Blood. 1997; 89: 1394-1404Crossref PubMed Google Scholar directed against the cytoplasmic portion of the ALK molecule. Extensive studies have demonstrated the presence ofNPM-ALK gene and/or its protein product in cases of ALCL. The percentage of NPM-ALK-positive cases in different studies has varied between 30% and 60%, but a picture has emerged of a tumor that consistently presents with primary, systemic disease, shows a T/null phenotype, and usually occurs in the first three decades of life.16Shiota M Nakamura S Ichinohasama R Abe M Akagi T Takeshita M Mori N Fujimoto J Miyauchi J Mikata A Nanba K Takami T Yamabe H Takano Y Izumo T Nagatani T Mohri N Nasu K Satoh H Katano H Fujimoto J Yamamoto T Mori S Anaplastic large cell lymphomas expressing the novel chimeric protein p80NPM/ALK: a distinct clinicopathologic entity.Blood. 1995; 86: 1954-1960Crossref PubMed Google Scholar, 19Herbst H Anagnostopoulos J Heinze B Durkop H Hummel M Stein H ALK gene products in anaplastic large cell lymphomas and Hodgkin's disease.Blood. 1995; 86: 1694-1700PubMed Google Scholar, 20Elmberger PG Lozano MD Weisenburger DD Sanger W Chan WC Transcripts of the npm-alk fusion gene in anaplastic large cell lymphoma, Hodgkin's disease, and reactive lymphoid lesions.Blood. 1995; 86: 3517-3521PubMed Google Scholar, 21Wellmann A Otsuki T Vogelbruch M Clark HM Jaffe ES Raffeld M Analysis of the t(2;5)(p23;q35) translocation by reverse transcription polymerase chain reaction in CD30+ anaplastic large cell lymphomas, in other non-Hodgkin's lymphomas of T-cell phenotype, and in Hodgkin's disease.Blood. 1995; 86: 2321-2328PubMed Google Scholar, 22Lopategui JR Sun LH Chan JK Gaffey MJ Frierson Jr, HF Glackin C Weiss LM Low frequency association of the t(2;5)(p23;q35) chromosomal translocation with CD30+ lymphomas from American and Asian patients: a reverse transcriptase-polymerase chain reaction study.Am J Pathol. 1995; 146: 323-328PubMed Google Scholar, 23Weisenburger DD Gordon BG Vose JM Bast MA Chan WC Greiner TC Anderson JR Sanger WG Occurrence of the t(2;5)(p23;q35) in non-Hodgkin's lymphoma.Blood. 1996; 87: 3860-3868PubMed Google Scholar, 24Lamant L Meggetto F Al Saati T Bruggieres L Bressac de Paillerets B Dastague N Bernheim A Rubie H Terrier-Lacombe MJ Robert A Brousset P Rigal F Schlaifer D Shiota M Mori S Delsol G High incidence of the t(2;5)(p23;q35) translocation in anaplastic large cell lymphoma and its lack of detection in Hodgkin's disease: comparison of cytogenetic analysis, reverse transcriptase-polymerase chain reaction, and P-80 immunostaining.Blood. 1996; 87: 284-291PubMed Google Scholar, 25Sarris AH Rajyalakshmi L Papadimitracopoulou V Waasdorp M Dimopoulos M McBride JA Cabanillas F Duvic M Deisseroth A Morris S Pugh WC Amplification of genomic DNA demonstrates the presence of the t(2;5)(p23;q35) in anaplastic large cell lymphoma, but not in other non-Hodgkin's lymphomas, Hodgkin's disease, or lymphomatoid papulosis.Blood. 1996; 88: 1771-1779PubMed Google Scholar, 26DeCoteau JF Butmarc JR Kinney MC Kadin ME The t(2;5) chromosomal translocation is not a common feature of primary cutaneous CD30+ lymphoproliferative disorders: comparison with anaplastic large-cell lymphoma of nodal origin.Blood. 1996; 87: 3437-3441PubMed Google Scholar, 27Shiota M Mori S Anaplastic large cell lymphomas expressing the novel chimeric protein p80NPM/ALK: a distinct clinicopathologic entity.Leukemia. 1997; 11: 538-540PubMed Google Scholar, 28Nakamura S Shiota M Nakagawa A Yatabe Y Kojima M Motoori T Suzuki R Kagami Y Ogura M Morishima Y Mizoguchi Y Okamoto M Seto M Koshikawa T Mori S Suchi T Anaplastic large cell lymphoma: a distinct molecular pathologic entity: a reappraisal with special reference to p80 (NPM/ALK) expression.Am J Surg Pathol. 1997; 21: 1420-1432Crossref PubMed Scopus (142) Google Scholar A poorly investigated issue in this field concerns the correlation between histological features of ALCL and NPM-ALK protein expression. After the first description of ALCL by Stein et al in 1985,1Stein H Mason DY Gerdes J O'Connor N Wainscoat J Pallesen G Gatter K Falini B Delsol G Lemke H Schwarting R Lennert K The expression of Hodgkin's disease associated Ki-1 antigen in reactive and neoplastic lymphoid tissues: evidence that Sternberg-Reed cells and histiocytic malignancies are derived from activated lymphoid cells.Blood. 1985; 66: 848-858Crossref PubMed Google Scholar it became evident that neither anaplastic morphology nor CD30 expression could be regarded as absolute defining criteria for ALCL. Several pathologists reported morphological variants (eg, "common type," "lymphohistiocytic," "small cell," "neutrophil-rich," "sarcomatoid," or "Hodgkin's like"),29Stein H Ki-1 anaplastic large cell lymphoma: is it a discrete entity?.Leuk Lymphoma. 1993; 10: 81-84Crossref PubMed Scopus (44) Google Scholar, 30Chan JKC Ng CS Hui PK Leungs TW Lau WH McGuire LJ Anaplastic large cell Ki-1 lymphoma: delineation of two morphological types.Histopathology. 1989; 15: 11-34Crossref PubMed Scopus (185) Google Scholar, 31Pileri S Falini B Delsol G Stein H Baglioni P Poggi S Martelli MF Rivano MT Mason DY Stansfeld AG Lymphohistiocytic T-cell lymphoma (anaplastic large cell lymphoma CD30+/Ki-1+ with a high content of reactive histiocytes).Histopathology. 1990; 16: 383-391Crossref PubMed Scopus (183) Google Scholar, 32Kinney MC Collins RD Greer JP Whitlock JA Sioutos N Kadin ME A small cell-predominant variant of primary Ki-1 (CD30)+ T-cell lymphoma.Am J Surg Pathol. 1993; 17: 859-868Crossref PubMed Scopus (238) Google Scholar, 33Mann KP Hall B Kamino J Borowitz MJ Ratech H Neutrophil-rich, Ki-1-positive anaplastic large cell malignant lymphoma.Am J Surg Pathol. 1995; 19: 407-416Crossref PubMed Scopus (119) Google Scholar, 34Chan JKC Buchanan R Fletcher CDM Sarcomatoid variant of anaplastic large-cell lymphoma.Am J Surg Pathol. 1990; 14: 983-988Crossref PubMed Scopus (159) Google Scholar, 35Falini B Liso A Pasqualucci L Flenghi L Ascani S Pileri S Bucciarelli E CD30+ anaplastic large cell lymphoma, null type, with signet-ring appearance.Histopathology. 1997; 30: 90-92Crossref PubMed Scopus (31) Google Scholar, 36Kadin ME Anaplastic large cell lymphoma and its morphological variants.Cancer Surv. 1997; 30: 77-86PubMed Google Scholar which shared the same basic architectural features of ALCL, but which differed in terms of tumor cell cytology and the admixture of inflammatory cells. This raised the question whether the heterogeneous morphological features of the ALCL represent different clinicopathological entities or are just variants of a single disease. In this paper, we have addressed this point by immunohistological labeling of a series of 100 cases of ALCL for ALK protein expression using a new mAb (ALKc) and also a previously reported anti-ALK antibody ALK1.18Pulford K Lamant L Morris S Butler LH Wood KM Stroud D Delsol G Mason DY Detection of ALK and NPM-ALK protein in normal and neoplastic cells with the monoclonal antibody ALK1.Blood. 1997; 89: 1394-1404Crossref PubMed Google Scholar We also investigated the nature of the small atypical cells that represent the predominant neoplastic population in the so-called lymphohistiocytic31Pileri S Falini B Delsol G Stein H Baglioni P Poggi S Martelli MF Rivano MT Mason DY Stansfeld AG Lymphohistiocytic T-cell lymphoma (anaplastic large cell lymphoma CD30+/Ki-1+ with a high content of reactive histiocytes).Histopathology. 1990; 16: 383-391Crossref PubMed Scopus (183) Google Scholar and small-cell variants32Kinney MC Collins RD Greer JP Whitlock JA Sioutos N Kadin ME A small cell-predominant variant of primary Ki-1 (CD30)+ T-cell lymphoma.Am J Surg Pathol. 1993; 17: 859-868Crossref PubMed Scopus (238) Google Scholar of ALCL, an issue that has not been addressed in previous studies. An NPM-ALK cDNA fragment corresponding to the whole open reading frame of the NPM-ALK protein was generated by PCR using oligonucleotide primers spanning the NPM ATG and theALK TGA triplets. The PCR product was cloned into the pCRII vector of the TA cloning system (Invitrogen, San Diego, CA), checked by sequencing, and subcloned in PGEX-4T-1 (Pharmacia Biotech, Piscataway, NJ) to produce a glutathione S-transferase NPM-ALK full-length fusion protein. The protein was expressed in the HB101Escherichia coli strain and purified by affinity chromatography following the manufacturer's instructions. A fusion between the spleen cells of BALB/c mice previously immunized intraperitoneally with 150-μg aliquots of recombinant protein and the NS-1 myeloma cell line was carried out, as described previously.37Flenghi L Bigerna B Fizzotti M Venturi S Pasqualucci L Pileri S Ye BH Gambacorta M Pacini R Baroni C Pescarmona E Anagnostopoulos I Stein H Asdrubali G Martelli MF Pelicci PG Dalla Favera R Falini B Monoclonal antibodies PG-B6a and PG-B6p recognize, respectively, a highly conserved and a formol-resistant epitope on the human BCL-6 protein amino-terminal region.Am J Pathol. 1996; 148: 1543-1555PubMed Google Scholar Hybridoma supernatants were screened by the immunoalkaline phosphatase (alkaline phosphatase-antialkaline phosphatase, APAAP) technique38Cordell JL Falini B Erber WN Ghosh AK Abdulaziz Z MacDonald S Pulford KAF Stein H Mason DY Immunoenzymatic labelling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes).J Histochem Cytochem. 1984; 32: 219-229Crossref PubMed Scopus (2927) Google Scholar in cytocentrifuge preparations of a human cell line (Karpas 299) that carries the t(2;5).39Fischer P Nacheva E Mason DY Sherrington PD Hoyle C Hayhoe FG Karpas A A Ki-1 (CD30)-positive human cell line (Karpas 299) established from a high-grade non-Hodgkin's lymphoma, showing a 2;5 translocation and rearrangement of the T-cell receptor β-chain gene.Blood. 1988; 72: 234-240PubMed Google Scholar Selected hybridomas were cloned by limiting dilution. Five hybridoma supernatants out of approximately 1000 tested showed strong immunocytochemical staining of the Karpas 299 cell line but were unreactive on cryostat sections of normal human tonsil. Further testing on paraffin sections of ALCL bearing the (2;5) translocation showed that one of the supernatants reacted strongly with tumor cells but did not stain normal cells. The hybridoma was cloned to produce the ALKc clone used in subsequent studies. The mAb ALK1 raised against a fragment (amino acids 419 to 520) of the cytoplasmic portion of ALK protein has been described previously.18Pulford K Lamant L Morris S Butler LH Wood KM Stroud D Delsol G Mason DY Detection of ALK and NPM-ALK protein in normal and neoplastic cells with the monoclonal antibody ALK1.Blood. 1997; 89: 1394-1404Crossref PubMed Google Scholar Immunophenotyping of ALCL in paraffin sections was performed with antibodies directed against the following antigens: CD45, CD45RO, CD3, and CD20 (all obtained from DAKO A/S, Glostrup, Denmark); CD30/Ber-H2 (kindly provided by Prof. H Stein, Free University of Berlin, Berlin, Germany); and CD45RA, CD68, CD79a, and PML proteins (generated in the investigators' laboratories). NPM-ALK cDNA corresponding to the whole open reading frame of the protein was subcloned in the pcDNA3 expression vector (Invitrogen) and used for transient transfection of HeLa cells by the calcium chloride-HEPES-buffered-saline (HBS) method.40Pear WS Nolan GP Scott ML Baltimore D Production of high-titer helper-free retrovirus by transient transfection.Mol Cell Biol. 1993; 90: 8392-8396Google Scholar As negative control, HeLa cells were transfected in parallel with the plasmid vector containing no insert. Western blotting of cell lysates of the human cell lines U937, Karpas 299, Daudi, and Rh30 rhabdomyosarcoma6Morris SW Kirstein MN Valentine MB Dittmer KG Shapiro DN Saltman DL Look AT Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma.Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1980) Google Scholar was performed as previously described18Pulford K Lamant L Morris S Butler LH Wood KM Stroud D Delsol G Mason DY Detection of ALK and NPM-ALK protein in normal and neoplastic cells with the monoclonal antibody ALK1.Blood. 1997; 89: 1394-1404Crossref PubMed Google Scholar using mAbs ALKc and ALK1 (diluted 1:5). The reactivity of the antibodies ALKc and ALK1 or the negative control reagent MR12 (mouse anti-rabbit MR12, prepared in the laboratory of Mason et al)18Pulford K Lamant L Morris S Butler LH Wood KM Stroud D Delsol G Mason DY Detection of ALK and NPM-ALK protein in normal and neoplastic cells with the monoclonal antibody ALK1.Blood. 1997; 89: 1394-1404Crossref PubMed Google Scholar was tested against DHFR-ALK (a recombinant protein containing amino acids 1359 to 1460 of the full-length ALK receptor protein used to raise the antibody ALK1), by using a previously described enzyme-linked immunosorbent assay technique.18Pulford K Lamant L Morris S Butler LH Wood KM Stroud D Delsol G Mason DY Detection of ALK and NPM-ALK protein in normal and neoplastic cells with the monoclonal antibody ALK1.Blood. 1997; 89: 1394-1404Crossref PubMed Google Scholar A variety of human cell lines of different origin, MOLT-4, Jurkat, and Peer (T cell); Daudi, Nalm 12, and Cess (B cell); L-428 and L540 (Hodgkin's); Karpas 299, JB6, Su-DHL1 (ALCL bearing t(2;5)); K-562 (erythroid); U937, HL60, KG1, and NB4 (myeloid); and HeLa (carcinoma), were maintained in culture in RPMI 1640 containing 10% fetal calf serum (Life Technologies, Inc., Grand Island, NY). Cytospins were prepared from exponentially growing cells, fixed in acetone for 10 minutes at room temperature, and then used for immunocytochemical studies. Paraffin-embedded tissue samples had been fixed either in 10% buffered formalin for 24 hours to 1 week (most cases) or in Brasil-Dubosq or B5 for 2 hours (a minority of cases). Paraffin sections on silane-coated slides were rehydrated and subjected to microwaving (750 W for three cycles of 5 minutes each) using either 0.01 mol/L citrate buffer, pH 6.0,41Cattoretti G Suurmeijer AJH Antigen unmasking on formalin-fixed paraffin-embedded tissues using microwaves: a review.Adv Anat Pathol. 1995; 2: 2-9Crossref Google Scholar or 1-mmol/L ethylenediaminetetraacetic acid buffer, pH 8.0,42Pileri S Roncador G Ceccarelli C Piccioli M Briskomatis A Sabattini E Ascani S Santini D Piccaluga PP Leone O Damiani S Ercolessi C Sandri F Pieri F Leoncini L Falini B Antigen retrieval techniques in immunohistochemistry: comparison of different methods.J Pathol. 1997; 183: 116-123Crossref PubMed Scopus (288) Google Scholar as antigen retrieval solution. After microwave heating, sections were allowed to cool at room temperature for approximately 20 minutes, washed with Tris-buffered saline, and immunostained. Frozen sections from snap frozen samples (when available) were air dried overnight and fixed in acetone for 10 minutes. Normal lymphohemopoietic tissues comprised tonsil (n = 10), spleen (n = 5), bone marrow (n = 5), and thymus (n = 3). Samples representative of all extrahemopoietic tissues were also investigated. All tissues were diagnostic biopsies or were obtained at the time of autopsy. The following nonneoplastic conditions were studied: follicular hyperplasia (n = 10), toxoplasmic lymphadenitis (n = 5), tubercular lymphadenitis (n = 2), Kikuchi's lymphadenitis (n = 5), sarcoidosis (n = 3), and reactive T-immunoblastic proliferations (n = 2). A total of 510 cases of lymphoid neoplasms that included 100 cases of ALCL and 40 cases of acute and chronic myeloid disorders were retrieved from the authors' institutions. Lymphomas and leukemias were categorized according to the REAL43Harris NL Jaffe ES Stein H Banks P Chan JKC Cleary ML Delsol G De Wolf-Peeters C Falini B Gatter KC Grogan TM Isaacson PG Knowles DM Mason DY Muller-Hermelink HK Pileri SA Ralfkier E Warnke RA A Revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group.Blood. 1994; 84: 1361-1392PubMed Google Scholar and FAB44Bennet JM Catovsky D Daniel MT Flandrin G Galton DA Gralnick HR Sultan C Proposed revised criteria for the classification of acute myeloid leukemia: a report of the French-American-British Cooperative Group.Ann Int Med. 1985; 103: 620-625Crossref PubMed Scopus (2948) Google Scholar classifications. In all cases, diagnosis was based on morphological examination of conventionally stained tissue sections supplemented by immunophenotyping. Diagnostic immunomorphological criteria for ALCL were those originally established by Stein et al,1Stein H Mason DY Gerdes J O'Connor N Wainscoat J Pallesen G Gatter K Falini B Delsol G Lemke H Schwarting R Lennert K The expression of Hodgkin's disease associated Ki-1 antigen in reactive and neoplastic lymphoid tissues: evidence that Sternberg-Reed cells and histiocytic malignancies are derived from activated lymphoid cells.Blood. 1985; 66: 848-858Crossref PubMed Google Scholar and an attempt was made to assign each case to one of the following morphological subtypes of ALCL: co
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