The NPM-ALK and the ATIC-ALK Fusion Genes Can Be Detected in Non-Neoplastic Cells
2001; Elsevier BV; Volume: 158; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)64690-1
ISSN1525-2191
AutoresBrigitte Maes, Vera Vanhentenrijk, Iwona Włodarska, Jan Cools, Benjamin Peeters, Peter Marynen, Christiane De Wolf‐Peeters,
Tópico(s)Genetic factors in colorectal cancer
ResumoAnaplastic large cell lymphoma (ALCL) is frequently associated with the t(2;5)(p23;q35) translocation. It creates a NPM-ALK fusion gene, fusing the anaplastic lymphoma kinase (ALK) gene (2p23) and the nucleophosmin (NPM) gene (5q35). Other rearrangements involving the ALK gene have recently been shown to be associated with ALCL, among which the ATIC-ALK rearrangement resulting from the inv(2)(p23q35) translocation is probably the most recurrent. The aims of the present study were to investigate the presence of NPM-ALK and ATIC-ALK fusion genes in ALCL, using a real-time 5′ exonuclease-based reverse-transcription polymerase chain reaction (RT-PCR). This sensitive technique was also applied to investigate whether both fusion genes might be detected in Hodgkin’s disease cases and in reactive lymphoid tissue. Results of the RT-PCR were compared to ALK immunostaining, cytogenetics, and fluorescence in situ hybridization (FISH) results. RT-PCR detected the NPM-ALK and ATIC-ALK fusions at high levels in 8 and 3 of a total of 13 ALK-positive ALCL cases. One ALK-positive ALCL case was negative for both fusion genes analyzed but revealed a new ALK-related translocation t(2;17)(p23;q25) by cytogenetic and FISH analysis. In addition, of the eight ALK-positive ALCL cases that were strongly positive for the NPM-ALK fusion, three cases also showed the presence of the ATIC-ALK fusion, although at much lower levels. Similarly, out of the three strongly positive ATIC-ALK cases, one case was positive for the NPM-ALK fusion, at low levels. Finally, the NPM-ALK and the ATIC-ALK fusions were detected, at equally low levels, respectively in 13 and 5 ALK-negative ALCL cases, in 11 and 5 Hodgkin’s disease cases and in 20 and 1 non-neoplastic lymphoid tissues. The distinction between the high- and low-level detection was confirmed by relative quantitative RT-PCR for a representative number of cases. Of interest is the fact that the high-level detection coincided with the presence of ALK gene rearrangement detected by cytogenetics and FISH and may reflect a central role of the transcript in the oncogenic mechanism of ALK-positive ALCL. Low-level detection is not supported by cytogenetics and FISH, presumably due to the presence of the transcripts in only a small minority of normal cells not detectable by these techniques. Our findings demonstrate that NPM-ALK and ATIC-ALK fusion transcripts may be detected in conditions other than ALK-positive ALCL including reactive lymphoid tissues, although at low levels, suggesting the presence of the transcripts in normal (bystander) cells. Moreover, they suggest that the ALK gene rearrangement by itself might be insufficient to induce tumor formation. They further question the validity of quantitative real-time RT-PCR for monitoring minimal residual disease in ALCL. Finally, the newly identified translocation t(2;17)(p23;q25) can be added to the list of ALK gene rearrangements occurring in ALK-positive ALCL. Anaplastic large cell lymphoma (ALCL) is frequently associated with the t(2;5)(p23;q35) translocation. It creates a NPM-ALK fusion gene, fusing the anaplastic lymphoma kinase (ALK) gene (2p23) and the nucleophosmin (NPM) gene (5q35). Other rearrangements involving the ALK gene have recently been shown to be associated with ALCL, among which the ATIC-ALK rearrangement resulting from the inv(2)(p23q35) translocation is probably the most recurrent. The aims of the present study were to investigate the presence of NPM-ALK and ATIC-ALK fusion genes in ALCL, using a real-time 5′ exonuclease-based reverse-transcription polymerase chain reaction (RT-PCR). This sensitive technique was also applied to investigate whether both fusion genes might be detected in Hodgkin’s disease cases and in reactive lymphoid tissue. Results of the RT-PCR were compared to ALK immunostaining, cytogenetics, and fluorescence in situ hybridization (FISH) results. RT-PCR detected the NPM-ALK and ATIC-ALK fusions at high levels in 8 and 3 of a total of 13 ALK-positive ALCL cases. One ALK-positive ALCL case was negative for both fusion genes analyzed but revealed a new ALK-related translocation t(2;17)(p23;q25) by cytogenetic and FISH analysis. In addition, of the eight ALK-positive ALCL cases that were strongly positive for the NPM-ALK fusion, three cases also showed the presence of the ATIC-ALK fusion, although at much lower levels. Similarly, out of the three strongly positive ATIC-ALK cases, one case was positive for the NPM-ALK fusion, at low levels. Finally, the NPM-ALK and the ATIC-ALK fusions were detected, at equally low levels, respectively in 13 and 5 ALK-negative ALCL cases, in 11 and 5 Hodgkin’s disease cases and in 20 and 1 non-neoplastic lymphoid tissues. The distinction between the high- and low-level detection was confirmed by relative quantitative RT-PCR for a representative number of cases. Of interest is the fact that the high-level detection coincided with the presence of ALK gene rearrangement detected by cytogenetics and FISH and may reflect a central role of the transcript in the oncogenic mechanism of ALK-positive ALCL. Low-level detection is not supported by cytogenetics and FISH, presumably due to the presence of the transcripts in only a small minority of normal cells not detectable by these techniques. Our findings demonstrate that NPM-ALK and ATIC-ALK fusion transcripts may be detected in conditions other than ALK-positive ALCL including reactive lymphoid tissues, although at low levels, suggesting the presence of the transcripts in normal (bystander) cells. Moreover, they suggest that the ALK gene rearrangement by itself might be insufficient to induce tumor formation. They further question the validity of quantitative real-time RT-PCR for monitoring minimal residual disease in ALCL. Finally, the newly identified translocation t(2;17)(p23;q25) can be added to the list of ALK gene rearrangements occurring in ALK-positive ALCL. Anaplastic large cell lymphoma (ALCL) was identified in 19851Stein H Mason DY Gerdes J O'Connor N Wainscoat J Pallesen G Gatter K Falini B Delsol G Lemke H The expression of the Hodgkin's disease associated antigen Ki-1 in reactive and neoplastic lymphoid tissue: evidence that Reed-Sternberg cells and histiocytic malignancies are derived from activated lymphoid cells.Blood. 1985; 66: 848-858Crossref PubMed Google Scholar as a lymphoid neoplasm showing anaplastic large cells, preferentially infiltrating the paracortical and intrasinusoidal lymph node regions and expressing the Ki-1 antigen (later CD30). This characteristic morphology led to the inclusion of ALCL (more specifically T/Null ALCL) in the revised European-American lymphoma (REAL) classification2Harris NL Jaffe ES Stein H Banks PM Chan JK Cleary ML Delsol G De Wolf-Peeters C Falini B Gatter KC A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group.Blood. 1994; 84: 1361-1392PubMed Google Scholar as a distinct clinicopathological entity, showing a bimodal age distribution and presenting as a clinically aggressive disease, but curable in a high percentage of cases. Despite these characteristic features, morphological (eg, lymphohistiocytic and small cell)3Kadin ME Anaplastic large cell lymphoma and its morphological variants.Cancer Surv. 1997; 30: 77-86PubMed Google Scholar as well as clinical variants (systemic and cutaneous)4Paulli M Berti E Rosso R Boveri E Kindl S Klersy C Lazzarino M Borroni G Menestrina F Santucci M Gambini C Vassallo G Magrini U Sterry W Burg G Geerts ML Meijer CJLM Willemze R Feller AC Müller-Hermelink HK Kadin ME CD30/Ki-1-positive lymphoproliferative disorders of the skin —clinicopathologic correlation and statistical analysis of 86 cases: a multicentric study from the European Organization for Research and Treatment of Cancer Cutaneous Lymphoma Project Group.J Clin Oncol. 1995; 13: 1343-1354Crossref PubMed Scopus (223) Google Scholar, 5Vergier B Beylot-Barry M Pulford K Michel P Bosq J de Muret A Beylot C Delaunay MM Avril MF Dalac S Bodemer C Joly P Groppi A de Mascarel A Bagot M Mason DY Wechsler J Merlio JP Statistical evaluation of diagnostic and prognostic features of CD30+ cutaneous lymphoproliferative disorders: a clinicopathologic study of 65 cases.Am J Surg Pathol. 1998; 22: 1192-1202Crossref PubMed Scopus (96) Google Scholar have been described. In 1990,6Mason DY Bastard C Rimokh R Dastugue N Huret JL Kristoffersson U Magaud JP Nezelof C Tilly H Vannier JP Hemet J Warnke R CD30-positive large cell lymphomas ( a “Ki-1 lymphoma”) are associated with a chromosomal translocation involving 5q35.Br J Haematol. 1990; 74: 161-168Crossref PubMed Scopus (280) Google Scholar the association between ALCL and t(2;5)(p23;q35) was established and confirmed in a number of studies, with frequencies ranging from 15 to 75%.7Chan WC The t(2;5) or NPM-ALK translocation in lymphomas: diagnostic considerations.Adv Anat Pathol. 1996; 3: 396-399Crossref Scopus (7) Google Scholar The breakpoint involved in this translocation was cloned by Morris and colleagues,8Morris 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 receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase.Oncogene. 1997; 14: 2175-2188Crossref PubMed Scopus (407) Google Scholar demonstrating that the nucleophosmin (NPM) gene on chromosome 5q35 is fused to the previously unidentified anaplastic lymphoma kinase (ALK) gene on chromosome 2p23. The resulting chimeric NPM-ALK protein is thought to play a key role in the pathogenesis of t(2;5)-positive ALCL. Subsequently, antibodies (Abs) reacting with the ALK kinase became available, which allowed the immunohistochemical identification of ALCL cases with a 2p23/ALK- rearrangement, as the ALK protein is absent in normal lymphoid cells.9Pulford K Lamant L Morris SW Butler LH Wood KM Stroud D Delsol G Mason DY Detection of anaplastic lymphoma kinase (ALK) and nucleolar protein nucleophosmin (NPM)-ALK proteins in normal and neoplastic cells with the monoclonal antibody ALK1.Blood. 1997; 89: 1394-1404Crossref PubMed Google Scholar Importantly, expression studies with ALK Abs resulted in the description of a distinct ALK-positive ALCL subgroup, the so-called ALKomas. This particular subgroup of ALCL was shown to be associated with the presence of characteristic hallmark cells, and to have a much more favorable prognosis as compared to the ALK-negative group.10Falini B Bigerna B Fizzotti M Pulford K Pileri SA Delsol G Carbone A Paulli M Magrini U Menestrina F Giardini R Pilotti S Mezzelani A Ugolini B Billi M Pucciarini A Pacini R Pelicci PG Flenghi L ALK expression defines a distinct group of T/null lymphomas (“ALK lymphomas”) with a wide morphological spectrum.Am J Pathol. 1998; 153: 875-886Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 11Benharroch D Meguerian-Bedoyan Z Lamant L Amin C Brugieres L Terrier-Lacombe MJ Haralambieva E Pulford K Pileri S Morris SW Mason DY Delsol G ALK-positive lymphoma: a single disease with a broad spectrum of morphology.Blood. 1998; 91: 2076-2084PubMed Google Scholar, 12Pittaluga S Wlodarska I Pulford K Campo E Morris SW Van den Berghe H De Wolf-Peeters C The monoclonal antibody ALK1 identifies a distinct morphological subtype of anaplastic large cell lymphoma associated with 2p23/ALK rearrangements.Am J Pathol. 1997; 151: 343-351PubMed Google Scholar, 13Shiota 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 Recently, it has been demonstrated that ALK protein expression in ALCL may occasionally be the result of ALK rearrangements other than NPM-ALK. An indirect estimate of the frequency of these other rearrangements can be made based on the number of reported ALKoma cases without t(2;5) or on the number of cases without nuclear staining. The cases without nuclear staining are unlikely to be associated with the NPM-ALK rearrangement, as it results in both cytoplasmic and nuclear staining of the neoplastic cells attributable to dimerization of NPM-ALK fusion protein with wild-type NPM, which carries nuclear localization motifs.14Mason DY Pulford KA Bischof D Kuefer MU Butler LH Lamant L Delsol G Morris SW Nucleolar localization of the nucleophosmin-anaplastic lymphoma kinase is not required for malignant transformation.Cancer Res. 1998; 58: 1057-1062PubMed Google Scholar Ten to twenty percent of ALKomas may thus carry ALK rearrangements other than NPM-ALK.15Pulford K Falini B Cordell J Rosenwald A Ott G Muller-Hermelink HK MacLennan KA Lamant L Carbone A Campo E Mason DY Biochemical detection of novel anaplastic lymphoma kinase proteins in tissue sections of anaplastic large cell lymphoma.Am J Pathol. 1999; 154: 1657-1663Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar Examples already described are t(1;2)(q25;p23) resulting in a newly identified TPM3-ALK fusion gene,16Lamant L Dastugue N Pulford K Delsol G Mariame B A new fusion gene TPM3-ALK in anaplastic large cell lymphoma created by a (1;2)(q25;p23) translocation.Blood. 1999; 93: 3088-3095Crossref PubMed Google Scholar t(2;3)(p23;q21) fusing the TFG and ALK genes,17Hernandez L Pinyol M Hernandez S Bea S Pulford K Rosenwald A Lamant L Falini B Ott G Mason DY Delsol G Campo E TRK-fused gene (TFG) is a new partner of ALK in anaplastic large cell lymphoma producing two structurally different TFG-ALK translocations.Blood. 1999; 94: 3265-3268PubMed Google Scholar inv(2)(p23q35) creating the ATIC-ALK fusion gene,18Trinei M Lanfrancone L Campo E Pulford K Mason DY Pelicci PG Falini B A new variant anaplastic lymphoma kinase (ALK)-fusion protein (ATIC-ALK) in a case of ALK-positive anaplastic large cell lymphoma.Cancer Res. 2000; 60: 793-798PubMed Google Scholar, 19Colleoni GW Bridge JA Garicochea B Liu J Filippa DA Ladanyi M ATIC-ALK: A novel variant ALK gene fusion in anaplastic large cell lymphoma resulting from the recurrent cryptic chromosomal inversion, inv(2)(p23q35).Am J Pathol. 2000; 156: 781-789Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 20Ma Z Cools J Marynen P Cui X Siebert R Gesk S Schlegelberger B Peeters B De Wolf-Peeters C Wlodarska I Morris SW Inv(2)(p23q35) in anaplastic large-cell lymphoma induces constitutive anaplastic lymphoma kinase (ALK) tyrosine kinase activation by fusion to ATIC, an enzyme involved in purine nucleotide biosynthesis.Blood. 2000; 95: 2144-2149Crossref PubMed Google Scholar, 21Wlodarska I De Wolf-Peeters C Falini B Verhoef G Morris SW Hagemeijer A Van den Berghe H The cryptic inv(2)(p23q35) defines a new molecular genetic subtype of ALK-positive anaplastic large-cell lymphoma.Blood. 1998; 92: 2688-2695PubMed Google Scholar the CLTCL-ALK fusion gene22Touriol C Greenland C Lamant L Pulford K Bernard F Rousset T Mason DY Delsol G Further demonstration of the diversity of chromosomal changes involving 2p23 in ALK-positive lymphoma: 2 cases expressing ALK kinase fused to CLTCL (clathrin chain polypeptide-like).Blood. 2000; 95: 3204-3207Crossref PubMed Google Scholar and the not further characterized t(1;2)(q21;p23).23Rosenwald A Ott G Pulford K Katzenberger T Kuhl J Kalla J Ott MM Mason DY Muller-Hermelink HK t(1;2)(q21;p23) and t(2;3)(p23;q21): two novel variant translocations of the t(2;5)(p23;q35) in anaplastic large cell lymphoma.Blood. 1999; 94: 362-364Crossref PubMed Google Scholar Interestingly, NPM-ALK transcripts have been detected in Hodgkin’s disease (HD) cases24Orscheschek K Merz H Hell J Binder T Bartels H Feller AC Large-cell anaplastic lymphoma-specific translocation (t[2;5] [p23;q35]) in Hodgkin's disease: indication of a common pathogenesis?.Lancet. 1995; 345: 87-90Abstract PubMed Google Scholar, 25Trumper L Daus H Merz H von Bonin F Loftin U Cochlovius C Moller P Feller AC Pfreundschuh M NPM/ALK fusion mRNA expression in Hodgkin and Reed-Sternberg cells is rare but does occur: results from single-cell cDNA analysis.Ann Oncol. 1997; 8-: 83-87Crossref PubMed Scopus (17) Google Scholar, 26Yee HT Ponzoni M Merson A Goldstein M Scarpa A Chilosi M Menestrina F Pittaluga S De Wolf-Peeters C Shiota M Mori S Frizzera G Inghirami G Molecular characterization of the t(2;5) (p23; q35) translocation in anaplastic large cell lymphoma (Ki-1) and Hodgkin's disease.Blood. 1996; 87: 1081-1088PubMed Google Scholar suggesting it to be not as specific as previously assumed. Alternatively, these findings may indicate a common pathogenesis of ALCL and HD.24Orscheschek K Merz H Hell J Binder T Bartels H Feller AC Large-cell anaplastic lymphoma-specific translocation (t[2;5] [p23;q35]) in Hodgkin's disease: indication of a common pathogenesis?.Lancet. 1995; 345: 87-90Abstract PubMed Google Scholar However, other investigators failed to detect t(2;5) in HD and considered the studies demonstrating t(2;5) in HD as controversial.24Orscheschek K Merz H Hell J Binder T Bartels H Feller AC Large-cell anaplastic lymphoma-specific translocation (t[2;5] [p23;q35]) in Hodgkin's disease: indication of a common pathogenesis?.Lancet. 1995; 345: 87-90Abstract PubMed Google Scholar, 27Sarris AH Luthra R Papadimitracopoulou V Waasdorp M Dimopoulos MA McBride JA Cabanillas F Duvic M Deisseroth A Morris SW 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, 28Lamant L Meggetto F al Saati T Brugieres L de Paillerets BB Dastugue N Bernheim A Rubie H Terrier-Lacombe MJ Robert A Rigal F Schlaifer D Shiuta 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, 29Wellmann 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 Finally, the detection of t(2;5) in peripheral blood of healthy individuals has also been reported.30Trumper L Pfreundschuh M Bonin FV Daus H Detection of the t(2;5)-associated NPM/ALK fusion cDNA in peripheral blood cells of healthy individuals.Br J Haematol. 1998; 103: 1138-1144Crossref PubMed Scopus (73) Google Scholar The present study was aimed at investigating the presence of ALK gene rearrangements in various lymphoid tissues including ALCL cases, HD cases and reactive lymphoid tissue. Results obtained by a real-time 5′ exonuclease-based reverse transcription-polymerase chain reaction (RT-PCR) to detect NPM-ALK and ATIC-ALK fusion genes were compared to those obtained by ALK protein immunostaining, cytogenetics and fluorescence in situ hybridization (FISH). Thirty-three ALCL cases (cases 1 to 33), 22 HD cases (cases 34 to 55) (Table 1 ), and 31 cases of reactive lymphoid tissue without evidence of lymphoma (cases 56 to 86, not listed in Table 1), were selected from the database of the Department of Pathology of the University Hospitals of the Catholic University of Leuven, Belgium. All cases were documented by a freshly frozen tissue block. The reactive lymphoid tissue samples included lymph nodes (n = 29) and spleens (n = 2).Table 1Summary of the Results of the Anaplastic Large Cell Lymphoma and Hodgkin's Disease CasesRT-PCR*Positive results are expressed by CT values.KaryotypeCaseDiagnosisALK IHCGAPDHNPM-ALKATIC-ALKFISHStatusChromosome 2 abnormalities1ALCLpos211737NDA-Mt(2;5)(p23;q35)2†Cases included in previous studies.12,21ALCLpos1519negNDA-Ct(2;5)(p23;q35)3†Cases included in previous studies.12,21ALCLpos2119negNPM-ALKND4ALCLpos1819negNPM-ALKND5†Cases included in previous studies.12,21ALCLpos1920negNPM-ALKA-Ct(2;5)(p23;q35)6†Cases included in previous studies.12,21ALCLpos1720negNPM-ALKND7†Cases included in previous studies.12,21ALCLpos2421negNPM-ALKND8ALCLpos1923negNPM-ALKND9ALCLpos2237negNPM-ALKA-Mt(2;5)(p23;q35)10†Cases included in previous studies.12,21ALCLpos153321ALK-RA-Minv(2)(p23q35)11†Cases included in previous studies.12,21ALCLpos243625ALK-RA-Cinv(2)(p23q35)12†Cases included in previous studies.12,21ALCLpos213722ALK-RA-Cinv(2)(p23q35)13ALCLpos15negnegALK-RA-Mt(2;17)(p23;q25)14ALCLneg1637negNDA-CN15ALCLneg1730negNo ALK-RA-C+2;+216ALCLneg173230NDA-CN17ALCLneg173233NDND18ALCLneg2033negNDND19ALCLneg173436NDND20ALCLneg2135negNDND21ALCLneg1635negNDNN22ALCLneg233536NDND23ALCLneg153537NDNDN24ALCLneg1736negNo ALK-RA-CN25ALCLneg1637negNDND26ALCLneg1638negNDND27ALCLneg19negnegNDND28ALCLneg23negnegNDNDN29ALCLneg20negnegNDND30ALCLneg22negnegNDND31ALCLneg18negnegNDA-Cadd(2)(q37)32ALCLneg16negnegNo ALK-RND33ALCLneg15negnegNDA-CN34HDneg183334NDA-SN35HDneg193334NDA-CN36HDneg1834negNDND37HDneg203636NDND38HDneg1836negNDA-MN39HDneg1736negNDA-CN40HDneg1636negNDND41HDneg2037negNDND42HDneg193738NDN43HDneg1638negNDN44HDneg1739negNDN45HDneg16negnegNDA-CN46HDneg16negnegNDND47HDneg20neg38NDA-CN48HDneg18negnegNDND49HDneg21negnegNDND50HDneg17negnegNDND51HDneg17negnegNDA-CN52HDneg21negnegNDND53HDneg23negnegNDND54HDneg15negnegNDND55HDneg21negnegNDNDIHC, immunohistochemistry; neg, negative; ND, not done; N, normal; A, abnormal; -S, simple (1 chromosomal aberration); -M, moderate (1 to 4 chromosomal aberrations); -C, complex (>4 chromobsomal aberrations);ALK-R; ALK rearrangement.* Positive results are expressed by CT values.† Cases included in previous studies.12Pittaluga S Wlodarska I Pulford K Campo E Morris SW Van den Berghe H De Wolf-Peeters C The monoclonal antibody ALK1 identifies a distinct morphological subtype of anaplastic large cell lymphoma associated with 2p23/ALK rearrangements.Am J Pathol. 1997; 151: 343-351PubMed Google Scholar, 21Wlodarska I De Wolf-Peeters C Falini B Verhoef G Morris SW Hagemeijer A Van den Berghe H The cryptic inv(2)(p23q35) defines a new molecular genetic subtype of ALK-positive anaplastic large-cell lymphoma.Blood. 1998; 92: 2688-2695PubMed Google Scholar Open table in a new tab IHC, immunohistochemistry; neg, negative; ND, not done; N, normal; A, abnormal; -S, simple (1 chromosomal aberration); -M, moderate (1 to 4 chromosomal aberrations); -C, complex (>4 chromobsomal aberrations);ALK-R; ALK rearrangement. All cases were reviewed on hematoxylin and eosin-stained paraffin-embedded tissue sections. Immunophenotyping was performed on fixed paraffin-embedded sections or on frozen tissue sections. Monoclonal Abs reacting to CD30 (BerH2), CD15 (LeuM1), CD20 (L26) and CD3 (Leu4) were applied, using a streptavidin-biotin-peroxidase three-stage technique. The peroxidase reaction was developed using 3,3′-diaminobenzidine tetrahydrochloride (Dako, Glostrup, Denmark) and hydrogen peroxide, 0.01% v/v. Antibodies were purchased from Becton Dickinson (San Jose, CA) and from Dako. The tetrahydrochloride immunohistochemical labeling with the monoclonal ALK1 antibody was performed as previously described.12Pittaluga S Wlodarska I Pulford K Campo E Morris SW Van den Berghe H De Wolf-Peeters C The monoclonal antibody ALK1 identifies a distinct morphological subtype of anaplastic large cell lymphoma associated with 2p23/ALK rearrangements.Am J Pathol. 1997; 151: 343-351PubMed Google Scholar The diagnosis of ALCL and HD was made according to the REAL classification2Harris NL Jaffe ES Stein H Banks PM Chan JK Cleary ML Delsol G De Wolf-Peeters C Falini B Gatter KC A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group.Blood. 1994; 84: 1361-1392PubMed Google Scholar and according to the recommendations of the World Health Organization advisory committee.31Harris NL Jaffe ES Diebold J Flandrin G Muller-Hermelink HK Vardiman J Lister TA Bloomfield CD The World Health Organization classification of neoplastic diseases of the hematopoietic and lymphoid tissues. Report of the Clinical Advisory Committee meeting, Airlie House, Virginia, November.Ann Oncol. 1997; 10 (1999): 1419-1432Crossref Scopus (484) Google Scholar All cases were analyzed using a newly-developed real-time PCR assay. In short, total RNA was extracted from 5 to 8 sections of 25 μm thickness from freshly frozen tissue blocks using Trizol reagent (Life Technologies, Merelbeke, Belgium). One microgram of total RNA was converted into cDNA using Superscript reverse transcriptase according to the manufacturer’s recommendations (Life Technologies). Five microliters of the RT reaction (20 μl) were then used as template for a 50-μl reaction for the real-time detection of the NPM-ALK and ATIC-ALK fusion transcripts using the TaqMan Universal Master Mix and an ABI Prism 7700 Sequence Detection System (PE Corporation, Foster City, CA). Two sets of primers (15 pmol each) combined with a TaqMan probe (20 pmol) were used for the respective transcripts. The primer directed to the ALK portion of the transcripts was the same for both transcripts (primer ALKr: 5′-TGTACTCAGGGCTCTGCAGCT). Forward primers used were NPMf: 5′-GGGCCAGTGCATATTAGTGGA and ATICf: 5′-CTGTACACACTGCAGCCCAAG. The TaqMan probes were 6-carboxy-fluorescein (FAM)-labeled and bridged the breakpoints (N/A: 5′-AGCACTTAGT AGTGTACCGCCGGAAGCACC and A/A: 5′-CCATCACAGTGTACCGCCGGAAGC). The quality of the synthesized cDNA was verified during the same run by amplification of a 101-bp fragment of the GAPDH gene, which is constitutively expressed in all cells (primers 5′-AGCCTCAAGATCATCAGCAATG and 5′-ATGGACTGTGGTCATGAGTCCTT. TaqMan probe: 5′-JOE-CCAACTGCTTAGCACCCCTGGCC). Amplification conditions were 2 minutes at 50°C (allowing AmpEraseUNG treatment) and 10 minutes at 95°C, followed by 43 cycles of denaturation (95°C, 15 seconds), extension (60°C, 1 minute). Results of this real-time PCR method are expressed as the CTvalue, that represents the cycle at which fluorescence raises above a threshold value. CT values below 40 are considered positive. The lower the CT value, the higher the positivity of the sample, suggesting the higher concentration of the target sequence in the starting material. The sensitivity of the assay was determined by amplification of a NPM-ALK-positive and an ATIC- ALK-positive control sample, diluted 10 to 108 times in diethyl pyrocarbonate water, and was 10−7 for both. The sensitivity study is illustrated in Figure 1. A limited number (n = 3) (cases 59, 61, and 65) of NPM-ALK PCR products were sequenced to verify the specificity of the PCR. Cloning was performed using the pGEM-T easy vector system (Promega, Leiden, the Netherlands). In short, the PCR products were gel-purified and inserted in the pGEM-T vector. Blue-white screening with X-gal and IPTG (Life Technologies, Merelbeke, Belgium) was used to pick the appropriate colonies for sequencing. Six white colonies were checked for the presence of the insert and were sequenced over the full length in both directions. Quantification was performed in a representative part of the cases namely four ALK-positive ALCL cases (cases 1, 2, 3, and 6), four ALK negative ALCL cases (cases 17–19 and 21), eight HD cases (cases 34–37, 39, 40, 45, and 48) and eight reactive tissues (cases 56, 59–62, 66, 77, and 79). The NPM-ALK fusion gene in the samples was quantified by measuring CTand by using a standard curve to determine the starting target quantity. As the precise amount of total cDNA added to each reaction mix and its quality are both difficult to assess, the GAPDH transcripts were also quantified as an endogenous reference and each sample was normalized by dividing the NPM-ALK target amount by the GAPDH target amount. Each of the normalized target values was divided by a designated calibrator-normalized target value. Final results are expressed as N-fold differences in NPM-ALK level relative to the GAPDH transcript amount and the calibrator. The standard curves for both NPM-ALK and GAPDH were constructed with 10-fold serial dilutions in diethyl pyrocarbonate water of cDNA prepared from total RNA extracted from case 1. The series of diluted cDNA’s were aliquoted and stored at −20°C until use. Each PCR run was performed with triplicates, of which results were averaged, and included no template controls, 7 points of the standard curves and 8 unknowns. Amplification conditions were similar to those used for the qualitative RT-PCR (see above). Carryover of PCR product was avoided by using the real-time RT-PCR method as it is a closed tube assay that requires no post-PCR handling. Three tubes had to be opened for sequencing, but this was performed in a separate room, preventing contact of products with the PCR setup area. Moreover, the master mix that was used contains dUTP instead of dTTP, and the enzyme AmpEraseUNG (uracil-N-glycosylase). UNG treatment removes dUTP containing carryover PCR products32Longo MC Berninger MS Hartley JL Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions.Gene. 1990; 93: 125-128Crossref PubMed Scopus (727) Google Scholar and is followed by thermal inactivation of UNG before the actual PCR. In our laboratory neither NPM-ALK nor ATIC-ALK detection, was performed with a conventional RT-PCR lacking the dUTP-AmpEraseUNG system. Apart from these specific characteristics of the real-time RT-PCR, general recommendations were taken in account to prevent carryover of RNA/cDNA during handling before the real-time PCR, such as regularly changing gloves, maintaining separate areas, and using positive-displacement pipets and sterile filter tips. In addition, cutting an
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