API2-MALT1 Fusion Defines a Distinctive Clinicopathologic Subtype in Pulmonary Extranodal Marginal Zone B-Cell Lymphoma of Mucosa-Associated Lymphoid Tissue
2003; Elsevier BV; Volume: 162; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63908-9
ISSN1525-2191
AutoresMitsukuni Okabe, Hiroshi Inagaki, Koichi Ohshima, Tadashi Yoshino, Chunmei Li, Tadaaki Eimoto, Ryuzo Ueda, Shigeo Nakamura,
Tópico(s)Galectins and Cancer Biology
Resumot(11;18)(q21;q21) is associated with mucosa-associated lymphoid tissue (MALT)-type lymphoma and results in API2-MALT1 fusion. However, its clinicopathologic significance remains unclarified. API2-MALT1 fusion is detected most frequently in MALT lymphomas primarily involving the lung. We therefore screened 51 cases of pulmonary MALT lymphoma for API2-MALT1 fusion, and studied its relationship with clinicopathologic factors including the immunohistochemical expression of BCL10, another MALT lymphoma-associated molecule. The API2-MALT1 fusion transcript was detected in 21 of 51 (41%) cases, and was correlated with the absence of any underlying autoimmune disease, and with a normal serum lactate dehydrogenase, a "typical" histology without marked plasmacytic differentiation or an increased number of large cells, and aberrant nuclear BCL10 expression. However, its prognostic impact was not identified in the limited follow-up (6 to 187 months, median 27). These data suggest that the API2-MALT1 fusion may be a causative gene abnormality unrelated to autoimmune disease. In addition, this alteration may define a homogeneous MALT lymphoma subtype that is clinically more indolent and histologically more "typical." Aberrant nuclear BCL10 expression may have a possible role as a tool to screen for this API2-MALT1 fusion. A large-scale study with a long follow-up is necessary to establish the prognostic significance of API2-MALT1 fusion. t(11;18)(q21;q21) is associated with mucosa-associated lymphoid tissue (MALT)-type lymphoma and results in API2-MALT1 fusion. However, its clinicopathologic significance remains unclarified. API2-MALT1 fusion is detected most frequently in MALT lymphomas primarily involving the lung. We therefore screened 51 cases of pulmonary MALT lymphoma for API2-MALT1 fusion, and studied its relationship with clinicopathologic factors including the immunohistochemical expression of BCL10, another MALT lymphoma-associated molecule. The API2-MALT1 fusion transcript was detected in 21 of 51 (41%) cases, and was correlated with the absence of any underlying autoimmune disease, and with a normal serum lactate dehydrogenase, a "typical" histology without marked plasmacytic differentiation or an increased number of large cells, and aberrant nuclear BCL10 expression. However, its prognostic impact was not identified in the limited follow-up (6 to 187 months, median 27). These data suggest that the API2-MALT1 fusion may be a causative gene abnormality unrelated to autoimmune disease. In addition, this alteration may define a homogeneous MALT lymphoma subtype that is clinically more indolent and histologically more "typical." Aberrant nuclear BCL10 expression may have a possible role as a tool to screen for this API2-MALT1 fusion. A large-scale study with a long follow-up is necessary to establish the prognostic significance of API2-MALT1 fusion. Extranodal marginal zone B-cell lymphoma (MZBL) of mucosa-associated lymphoid tissue (MALT) is listed as a distinct clinicopathologic entity by the World Health Organization (WHO) Classification of Tumors of the Hematopoietic and Lymphoid Tissues.1Jaffe E Harris NL Stein H Vardiman JW Tumours of Haematopoietic and Lymphoid Tissues. World Health Organization Classification of Tumors, Pathology, and Genetics. IARC Press, Lyon2001Google Scholar Since first described, the clinical, histological, immunophenotypic, and genetic features of this lymphoma have been highlighted by many authors. In general, MALT lymphomas run an indolent clinical course, preferably involve extranodal sites, and exhibit varied histological spectra consisting of lymphoepithelial lesions, follicular colonization, and prototypically centrocyte-like (CCL) cells.1Jaffe E Harris NL Stein H Vardiman JW Tumours of Haematopoietic and Lymphoid Tissues. World Health Organization Classification of Tumors, Pathology, and Genetics. IARC Press, Lyon2001Google Scholar Interestingly, a pre-existing chronic inflammation, eg, Helicobacter pylori (H. pylori) gastritis, Hashimoto's thyroiditis, or Sjogren's syndrome, may make an important contribution to its development.2Wotherspoon AC Ortiz-Hidalgo C Falzon MR Isaacson PG Helicobacter pylori-associated gastritis and primary B-cell gastric lymphoma.Lancet. 1991; 338: 1175-1176Abstract PubMed Scopus (1760) Google Scholar, 3Royer B Cazals-Hatem D Sibilia J Agbalika F Cayuela JM Soussi T Maloisel F Clauvel JP Brouet JC Mariette X Lymphomas in patients with Sjogren's syndrome are marginal zone B-cell neoplasms, arise in diverse extranodal and nodal sites, and are not associated with viruses.Blood. 1997; 90: 766-775PubMed Google Scholar, 4Isaacson PG Mucosa-associated lymphoid tissue lymphoma.Semin Hematol. 1999; 36: 139-147PubMed Google Scholar However, MALT lymphomas are found in some patients with no evidence of such precursor lesions or conditions. The chromosome translocation t(11;18)(q21;q21) has been identified as a recurring cytogenetic abnormality of MALT lymphomas.5Ott G Katzenberger T Greiner A Kalla J Rosenwald A Heinrich U Ott MM Muller-Hermelink HK The t(11;18)(q21;q21) chromosome translocation is a frequent and specific aberration in low-grade but not high-grade malignant non-Hodgkin's lymphomas of the mucosa-associated lymphoid tissue (MALT-) type.Cancer Res. 1997; 57: 3944-3948PubMed Google Scholar Our group as well as others revealed that API2 at 11q21 and a novel gene, MALT1 at 18q21, are fused as a result of this translocation.6Dierlamm J Baens M Wlodarska I Stefanova-Ouzounova M Hernandez JM Hossfeld DK De Wolf-Peeters C Hagemeijer A Van den Berghe H Marynen P The apoptosis inhibitor gene API2 and a novel 18q gene, MLT, are recurrently rearranged in the t(11;18)(q21;q21)p6ssociated with mucosa-associated lymphoid tissue lymphomas.Blood. 1999; 93: 3601-3609Crossref PubMed Google Scholar, 7Akagi T Motegi M Tamura A Suzuki R Hosokawa Y Suzuki H Ota H Nakamura S Morishima Y Taniwaki M Seto M A novel gene, MALT1 at 18q21, is involved in t(11;18) (q21;q21) found in low-grade B-cell lymphoma of mucosa-associated lymphoid tissue.Oncogene. 1999; 18: 5785-5794Crossref PubMed Scopus (301) Google Scholar Subsequently, API2-MALT1 fusion has been demonstrated exclusively in MALT lymphomas with varying frequency depending on the anatomical site.9Remstein ED James CD Kurtin PJ Incidence and subtype specificity of API2-MALT1 fusion translocations in extranodal, nodal, and splenic marginal zone lymphomas.Am J Pathol. 2000; 156: 1183-1188Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 12Motegi M Yonezumi M Suzuki H Suzuki R Hosokawa Y Hosaka S Kodera Y Morishima Y Nakamura S Seto M API2-MALT1 chimeric transcripts involved in mucosa-associated lymphoid tissue type lymphoma predict heterogeneous products.Am J Pathol. 2000; 156: 807-812Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 13Baens M Maes B Steyls A Geboes K Marynen P De Wolf-Peeters C The product of the t(11;18), an API2-MLT fusion, marks nearly half of gastric MALT type lymphomas without large cell proliferation.Am J Pathol. 2000; 156: 1433-1439Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar On the other hand, its involvement was rare in nodal and splenic MZBLs and in diffuse large B-cell lymphomas.8Rosenwald A Ott G Stilgenbauer S Kalla J Bredt M Katzenberger T Greiner A Ott MM Gawin B Dohner H Muller-Hermelink HK Exclusive detection of the t(11;18)(q21;q21) in extranodal marginal zone B cell lymphomas (MZBL) of MALT type in contrast to other MZBL and extranodal large B cell lymphomas.Am J Pathol. 1999; 155: 1817-1821Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 9Remstein ED James CD Kurtin PJ Incidence and subtype specificity of API2-MALT1 fusion translocations in extranodal, nodal, and splenic marginal zone lymphomas.Am J Pathol. 2000; 156: 1183-1188Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 10Inagaki H Okabe M Seto M Nakamura S Ueda R Eimoto T API2-MALT1 fusion transcripts involved in mucosa-associated lymphoid tissue lymphoma: multiplex RT-PCR detection using formalin-fixed paraffin-embedded specimens.Am J Pathol. 2001; 158: 699-706Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 11Yonezumi M Suzuki R Suzuki H Yoshino T Oshima K Hosokawa Y Asaka M Morishima Y Nakamura S Seto M Detection of API2-MALT1 chimaeric gene in extranodal and nodal marginal zone B-cell lymphoma by reverse transcription polymerase chain reaction (PCR) and genomic long and accurate PCR analyses.Br J Haematol. 2001; 115: 588-594Crossref PubMed Scopus (41) Google Scholar, 12Motegi M Yonezumi M Suzuki H Suzuki R Hosokawa Y Hosaka S Kodera Y Morishima Y Nakamura S Seto M API2-MALT1 chimeric transcripts involved in mucosa-associated lymphoid tissue type lymphoma predict heterogeneous products.Am J Pathol. 2000; 156: 807-812Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 13Baens M Maes B Steyls A Geboes K Marynen P De Wolf-Peeters C The product of the t(11;18), an API2-MLT fusion, marks nearly half of gastric MALT type lymphomas without large cell proliferation.Am J Pathol. 2000; 156: 1433-1439Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar API2is a member of the IAP (inhibitor of apoptosis) genefamily,14Rothe M Pan MG Henzel WJ Ayres TM Goeddel DV The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins.Cell. 1995; 83: 1243-1252Abstract Full Text PDF PubMed Scopus (1062) Google Scholar, 15Hofmann K Bucher P Tschopp J The CARD domain: a new apoptotic signalling motif.Trends Biochem Sci. 1997; 22: 155-156Abstract Full Text PDF PubMed Scopus (452) Google Scholar and is essential for the suppression of apoptosis.16Roy N Deveraux QL Takahashi R Salvesen GS Reed JC The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases.EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1145) Google Scholar MALT1 is identical to a novel caspase-like protein termed paracaspase,17Uren AG O'Rourke K Aravind LA Pisabarro MT Seshagiri S Koonin EV Dixit VM Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma.Mol Cell. 2000; 6: 961-967Abstract Full Text Full Text PDF PubMed Google Scholar although its biological function remains unclear. Our previous data suggest that API2-MALT1 chimeric transcripts may lead to an increased inhibition of apoptosis and thereby help MALT lymphomas to survive.10Inagaki H Okabe M Seto M Nakamura S Ueda R Eimoto T API2-MALT1 fusion transcripts involved in mucosa-associated lymphoid tissue lymphoma: multiplex RT-PCR detection using formalin-fixed paraffin-embedded specimens.Am J Pathol. 2001; 158: 699-706Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar BCL10 was cloned from another translocation of t(1;14)(p22;q32) in MALT lymphomas; through this rare translocation, the entire BCL10 is juxtaposed to the IgH enhancer region, and activates nuclear factor kappa B (NF-κB), a transcription factor for several survival-related genes, via interaction with MALT1 protein.18Willis TG Jadayel DM Du MQ Peng H Perry AR Abdul-Rauf M Price H Karran L Majekodunmi O Wlodarska I Pan L Crook T Hamoudi R Isaacson PG Dyer MJ Bcl10 is involved in t(1;14)(p22;q32) of MALT B cell lymphoma and mutated in multiple tumor types.Cell. 1999; 96: 35-45Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar, 19Zhang Q Siebert R Yan M Hinzmann B Cui X Xue L Rakestraw KM Naeve CW Beckmann G Weisenburger DD Sanger WG Nowotny H Vesely M Callet-Bauchu E Salles G Dixit VM Rosenthal A Schlegelberger B Morris SW Inactivating mutations and overexpression of BCL10, a caspase recruitment domain-containing gene, in MALT lymphoma with t(1;14)(p22;q32).Nat Genet. 1999; 22: 63-68Crossref PubMed Scopus (337) Google Scholar, 20Lucas PC Yonezumi M Inohara N McAllister-Lucas LM Abazeed ME Chen FF Yamaoka S Seto M Nunez G Bcl10 and MALT1, independent targets of chromosomal translocation in MALT lymphoma, cooperate in a novel NF-κ B signaling pathway.J Biol Chem. 2001; 276: 19012-19019Crossref PubMed Scopus (380) Google Scholar Moreover, the API2-MALT1 fusion protein also strongly activates NF-κB through a common signaling pathway.20Lucas PC Yonezumi M Inohara N McAllister-Lucas LM Abazeed ME Chen FF Yamaoka S Seto M Nunez G Bcl10 and MALT1, independent targets of chromosomal translocation in MALT lymphoma, cooperate in a novel NF-κ B signaling pathway.J Biol Chem. 2001; 276: 19012-19019Crossref PubMed Scopus (380) Google Scholar Recently, several researchers have shown that these two genetic alterations, t(1;14) and t(11;18), which both result in NK-κB activation, are associated with an aberrant nuclear expression of BCL10 protein on paraffin section immunohistochemistry of gastric MALT lymphoma.21Ye H Dogan A Karran L Willis TG Chen L Wlodarska I Dyer MJ Isaacson PG Du MQ BCL10 expression in normal and neoplastic lymphoid tissue: nuclear localization in MALT lymphoma.Am J Pathol. 2000; 157: 1147-1154Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 22Liu H Ye H Dogan A Ranaldi R Hamoudi RA Bearzi I Isaacson PG Du MQ T(11;18)(q21;q21) is associated with advanced mucosa-associated lymphoid tissue lymphoma that expresses nuclear BCL10.Blood. 2001; 98: 1182-1187Crossref PubMed Scopus (230) Google Scholar, 23Maes B Demunter A Peeters B De Wolf-Peeters C BCL10 mutation does not represent an important pathogenic mechanism in gastric MALT-type lymphoma, and the presence of the API2-MLT fusion is associated with aberrant nuclear BCL10 expression.Blood. 2002; 99: 1398-1404Crossref PubMed Scopus (70) Google Scholar The clinicopathologic and biological significance of API2-MALT1 fusion has not fully been appreciated. In the stomach, approximately 60–80% of MALT lymphomas eventually regress by H. pylori eradication therapy alone.24Wotherspoon AC Doglioni C Diss TC Pan L Moschini A de Boni M Isaacson PG Regression of primary low-grade B-cell gastric lymphoma of mucosa-associated lymphoid tissue type after eradication of Helicobacter pylori.Lancet. 1993; 342: 575-577Abstract PubMed Scopus (2022) Google Scholar, 25Parsonnet J Hansen S Rodriguez L Gelb AB Warnke RA Jellum E Orentreich N Vogelman JH Friedman GD Helicobacter pylori infection and gastric lymphoma.N Engl J Med. 1994; 330: 1267-1271Crossref PubMed Scopus (1749) Google Scholar, 26Sackmann M Morgner A Rudolph B Neubauer A Thiede C Schulz H Kraemer W Boersch G Rohde P Seifert E Stolte M Bayerdoerffer E Regression of gastric MALT lymphoma after eradication of Helicobacter pylori is predicted by endosonographic staging: MALT Lymphoma Study Group.Gastroenterology. 1997; 113: 1087-1090Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 27Nakamura S Matsumoto T Suekane H Takeshita M Hizawa K Kawasaki M Yao T Tsuneyoshi M Iida M Fujishima M Predictive value of endoscopic ultrasonography for regression of gastric low grade and high grade MALT lymphomas after eradication of Helicobacter pylori.Gut. 2001; 48: 454-460Crossref PubMed Scopus (222) Google Scholar However, it has been indicated that the API2-MALT1 fusion transcript serves as a molecular marker for those not responding to H. pylori eradication.28Nakamura T Nakamura S Yonezumi M Suzuki T Matsuura A Yatabe Y Yokoi T Ohashi K Seto M Helicobacter pylori and the t(11;18)(q21;q21) translocation in gastric low-grade B-cell lymphoma of mucosa-associated lymphoid tissue type.Jpn J Cancer Res. 2000; 91: 301-309Crossref PubMed Scopus (71) Google Scholar, 29Liu H Ruskon-Fourmestraux A Lavergne-Slove A Ye H Molina T Bouhnik Y Hamoudi RA Diss TC Dogan A Megraud F Rambaud JC Du MQ Isaacson PG Resistance of t(11;18) positive gastric mucosa-associated lymphoid tissue lymphoma to Helicobacter pylori eradication therapy.Lancet. 2001; 357: 39-40Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 30Sugiyama T Asaka M Nakamura T Nakamura S Yonezumi S Seto M API2-MALT1 chimeric transcript is a predictive marker for the responsiveness of H. pylori eradication treatment in low-grade gastric MALT lymphoma.Gastroenterology. 2001; 120: 1884-1885Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 31Liu H Ye H Ruskone-Fourmestraux A De Jong D Pileri S Thiede C Lavergne A Boot H Caletti G Wundisch T Molina T Taal BG Elena S Thomas T Zinzani PL Neubauer A Stolte M Hamoudi RA Dogan A Isaacson PG Du MQ T(11;18) is a marker for all stage gastric MALT lymphomas that will not respond to H. pylori eradication.Gastroenterology. 2002; 122: 1286-1294Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar Nakamura et al recently demonstrated that gastric MALT lymphomas that were negative for H. pylori, as determined from serological and culture findings, possessed API2-MALT1 fusion, suggesting that this genetic alteration has pathogenetic significance independent of precursor lesions.32Nakamura T Nakamura S Yokoi T Ohashi K Seto M Clinicopathologic comparison between the API2-MALT1 chimeric transcript-positive and -negative gastric low-grade B-cell lymphoma of mucosa-associated lymphoid tissue type.Jpn J Cancer Res. 2002; 93: 677-684Crossref PubMed Scopus (34) Google Scholar Interestingly, MALT lymphomas primarily involving the lung have a higher frequency of this fusion gene than those located anywhere else.8Rosenwald A Ott G Stilgenbauer S Kalla J Bredt M Katzenberger T Greiner A Ott MM Gawin B Dohner H Muller-Hermelink HK Exclusive detection of the t(11;18)(q21;q21) in extranodal marginal zone B cell lymphomas (MZBL) of MALT type in contrast to other MZBL and extranodal large B cell lymphomas.Am J Pathol. 1999; 155: 1817-1821Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 9Remstein ED James CD Kurtin PJ Incidence and subtype specificity of API2-MALT1 fusion translocations in extranodal, nodal, and splenic marginal zone lymphomas.Am J Pathol. 2000; 156: 1183-1188Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 10Inagaki H Okabe M Seto M Nakamura S Ueda R Eimoto T API2-MALT1 fusion transcripts involved in mucosa-associated lymphoid tissue lymphoma: multiplex RT-PCR detection using formalin-fixed paraffin-embedded specimens.Am J Pathol. 2001; 158: 699-706Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 11Yonezumi M Suzuki R Suzuki H Yoshino T Oshima K Hosokawa Y Asaka M Morishima Y Nakamura S Seto M Detection of API2-MALT1 chimaeric gene in extranodal and nodal marginal zone B-cell lymphoma by reverse transcription polymerase chain reaction (PCR) and genomic long and accurate PCR analyses.Br J Haematol. 2001; 115: 588-594Crossref PubMed Scopus (41) Google Scholar, 12Motegi M Yonezumi M Suzuki H Suzuki R Hosokawa Y Hosaka S Kodera Y Morishima Y Nakamura S Seto M API2-MALT1 chimeric transcripts involved in mucosa-associated lymphoid tissue type lymphoma predict heterogeneous products.Am J Pathol. 2000; 156: 807-812Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar In this study, we examined 51 cases of pulmonary MALT lymphoma from the viewpoint of API2-MALT1 fusion, which is now detectable by a multiplex reverse transcription (RT)-polymerase chain reaction (PCR) using formalin-fixed, paraffin-embedded sections,10Inagaki H Okabe M Seto M Nakamura S Ueda R Eimoto T API2-MALT1 fusion transcripts involved in mucosa-associated lymphoid tissue lymphoma: multiplex RT-PCR detection using formalin-fixed paraffin-embedded specimens.Am J Pathol. 2001; 158: 699-706Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar to verify this finding in a large cohort and to further clarify the clinicopathologic significance of this genetic abnormality. We also investigated the possible relationship between the aberrant expression of BCL10 protein and the detection of the API2-MALT1 fusion transcript. From the pathology files of Nagoya City University Medical School, Aichi Cancer Center Hospital, Fukuoka University Medical School, Okayama University Medical School, and other collaborating institutions, 84 cases of MALT lymphoma involving the lung were retrieved. Specimens were obtained at the initial presentation of the patients, fixed in formalin, and embedded in paraffin. All cases were carefully reviewed by three independent pathologists (H. I., T. E., and S. N.), and the diagnosis of MALT lymphoma was made according to the criteria of the WHO Classification for Tumors of Hematopoietic and Lymphoid Tissues.1Jaffe E Harris NL Stein H Vardiman JW Tumours of Haematopoietic and Lymphoid Tissues. World Health Organization Classification of Tumors, Pathology, and Genetics. IARC Press, Lyon2001Google Scholar The immunophenotype of the tumor cells was CD20+, CD79a+, cyclin D1-, CD5-, CD10-, CD3-, CD45RO-, CD23-, and CD56-. Monoclonality was examined by molecular and/or immunophenotypic techniques. Briefly, DNA extracted from paraffin sections was amplified by PCR with primers (FR2A, FR3A, LJH, and VLJH) to detect rearrangement occurring between framework regions and the joining region of the immunoglobulin heavy chain gene.33Inagaki H Nonaka M Nagaya S Tateyama H Sasaki M Eimoto T Monoclonality in gastric lymphoma detected in formalin-fixed, paraffin-embedded endoscopic biopsy specimens using immunohistochemistry, in situ hybridization, and polymerase chain reaction.Diagn Mol Pathol. 1995; 4: 32-38Crossref PubMed Scopus (29) Google Scholar, 34Inagaki H Chan JK Ng JW Okabe M Yoshino T Okamoto M Ogawa H Matsushita H Yokose T Matsuno Y Nakamura N Nagasaka T Ueda R Eimoto T Nakamura S Primary thymic extranodal marginal-zone B-cell lymphoma of mucosa-associated lymphoid tissue type exhibits distinctive clinicopathological and molecular features.Am J Pathol. 2002; 160: 1435-1443Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar Amplification of a β-globin DNA fragment (268 bp) was used to indicate satisfactory preservation of DNA in the sample. Monotypic immunoglobulin light chain was detected by immunohistochemistry for κ and λ light chains. In addition to verifying the tumor monoclonality, we carried out a preliminary examination of the extent of RNA preservation for detection of the API2-MALT1 fusion transcript in all cases by means of RT-PCR amplification of β-actin mRNA.10Inagaki H Okabe M Seto M Nakamura S Ueda R Eimoto T API2-MALT1 fusion transcripts involved in mucosa-associated lymphoid tissue lymphoma: multiplex RT-PCR detection using formalin-fixed paraffin-embedded specimens.Am J Pathol. 2001; 158: 699-706Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar Excluded were those cases whose monoclonality was not established by either molecular or immunohistochemical means and those where the RNA was not of satisfactory quality. Finally, 51 cases of the present series were selected for this study. The following clinical factors were analyzed: age, sex, chief complaints, autoimmune disease, lactate dehydrogenase (LDH), paraproteinemia, radiographical tumor distribution, regional lymph node involvement, clinical stage, B-symptoms, treatment, and follow–up. Staging of the disease was performed according to the Ann Arbor Classification System for Extranodal Lymphomas35Musshoff K Stadieneneinteilung der nicht-Hodgkin lymphome.Strahlentherapie. 1977; 153: 218-221PubMed Google Scholar by examining the clinical status, chest X-ray, CT scan of the mediastinum and the abdomen, 67Ga scintigraphy, and a bone marrow aspiration. The API2-MALT1 fusion transcript was detected using archival paraffin sections according to the method we recently reported.10Inagaki H Okabe M Seto M Nakamura S Ueda R Eimoto T API2-MALT1 fusion transcripts involved in mucosa-associated lymphoid tissue lymphoma: multiplex RT-PCR detection using formalin-fixed paraffin-embedded specimens.Am J Pathol. 2001; 158: 699-706Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar All eight variant fusion transcripts that have been reported can be detected with this assay (Figure 1, accession number L49432 for API2 and accession number AF130356 for MALT1). Briefly, total RNA was extracted from the paraffin sections by proteinase K (Roche Diagnostics, Mannheim, Germany) digestion. RNA was subjected to first-round multiplex one-tube RT-PCR, then to second-round nested multiplex PCRs (three parallel; Second PCR-A, Second PCR-B, and Second PCR-C). The final PCR products were run on 8% polyacrylamide gels and stained with ethidium bromide. The band size ranged from 80 to 179 bp. RNA samples known to possess API2-MALT1 fusion were used as positive controls. As an internal control for RNA quality, the ubiquitously expressed β-actin mRNA fragment (190 bp) was amplified. In all cases positive for API2-MALT1 fusion transcripts, the breakpoints were confirmed by direct sequencing. In short, fragments obtained in the second round PCR were separated and purified. They were then directly sequenced by means of cycle sequencing with dye-labeled terminators (BigDye Terminators, Applied Biosystems, Foster City, CA) and analyzed on a DNA sequencer (Model 310, Applied Biosystems). API2 primers required for the second round PCR were used as sequencing primers. Both freshly frozen samples and formalin-fixed, paraffin-embedded samples were obtained from 23 MALT lymphomas of various anatomical sites (pulmonary origin, 13; gastric, 9; and salivary, 1). These cases were examined by RT-PCR for API2-MALT1 fusion using high quality RNA extracted from frozen lymphoma materials as described previously;12Motegi M Yonezumi M Suzuki H Suzuki R Hosokawa Y Hosaka S Kodera Y Morishima Y Nakamura S Seto M API2-MALT1 chimeric transcripts involved in mucosa-associated lymphoid tissue type lymphoma predict heterogeneous products.Am J Pathol. 2000; 156: 807-812Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar 16 cases (pulmonary, 8; gastric, 7; and salivary, 1) were positive for the fusion and the remaining seven were negative. RNA extracted from paraffin-embedded tissues of these 23 lymphomas was analyzed for API2-MALT1 fusion using multiplex RT-PCR as described above. When analyzed using paraffin sections as a source of RNA, 15 (pulmonary, 7; gastric, 7; and salivary, 1) of 16 positive controls had the API2-MALT1 fusion transcript (sensitivity, 94%) and all of the seven negative controls did not (specificity 100%), indicating the high efficacy of multiplex RT-PCR assay using paraffin tissues. Direct sequencing confirmed identical breakpoints of API2-MALT1 fusion products between frozen and paraffin materials. Tissue sections cut at a thickness of three micrometers were deparaffinized and rehydrated. After antigen retrieval by heat treatment, endogenous peroxidase activity was blocked by 3% H2O2. Immunohistochemistry was performed by an automated immunostainer (OptiMax Plus, BioGenex, San Ramon, CA) using monoclonal antibody against BCL10 (clone 151,22Liu H Ye H Dogan A Ranaldi R Hamoudi RA Bearzi I Isaacson PG Du MQ T(11;18)(q21;q21) is associated with advanced mucosa-associated lymphoid tissue lymphoma that expresses nuclear BCL10.Blood. 2001; 98: 1182-1187Crossref PubMed Scopus (230) Google Scholar Zymed, South San Francisco, CA). MALT lymphomas harboring BCL10 gene rearrangement, as detected by Southern blot analysis, were used as positive controls, and normal lymph nodes and spleens, as normal controls. When most lymphoma cells of a patient showed nuclear BCL10 expression, the case was considered positive. Statistical evaluation of data from two groups was performed using the Fischer's exact test, Mann-Whitney U-test, and Student's t-test with the statistical package StatView (Abacus Concepts, Berkley, CA). All analyses were two-tailed. A value of P < 0.05 for each test was regarded as statistically significant, and 0.05 < P < 0.1 as marginally significant. Among the 51 pulmonary MALT lymphoma cases with a sufficient quality of RNA, 21 (41%) were positive for the API2-MALT1 fusion transcript with RT-PCR products varying in size depending on the breakpoints and primer sets used (Figure 2). The fusion types were estimated with 8% polyacrylamide electrophoresis and confirmed by direct sequencing. All but one case (20 of 21 cases, 95%) had an API2 breakpoint at 1446, and the one exception had a breakpoint at 1701 (accession number L49432). MALT1 breakpoints (accession number AF130356) varied as follows: seven cases (33%) at 814, 10 cases (48%) at 1123, and four cases (19%) at 1150 (Figure 1). All fusion transcripts were fused in-frame, and none of the positive cases showed an atypical transcript such as an insertion or deletion. All API2-MALT1 fusion variants were in keeping with those that have been reported previously.10Inagaki H Okabe M Seto M Nakamura S Ueda R Eimoto T API2-MALT1 fusion transcripts involved in mucosa-associated lymphoid tissue lymphoma: multiplex RT-PCR detection using formalin-fixed paraffin-embedded specimens.Am J Pathol. 2001; 158: 699-706Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar There were 22 males and 29 females with ages ranging from 31 to 81 years (mean, 62 years and median, 64 years). Radiological detection of a pulmonary lesion in an asymptomatic patient was the most common route of discovery (38 of 51 cases). The remaining 13 patients presented with chest-related symptoms such as cough, chest pain, fever, and hemoptysis. Six of the 51 patients had an underlying autoimmune disorder: three with Sjogren's syndrome, two with rheumatoid arthritis, and one with progressive systemic sclerosis. An elevated serum LDH was observed in 5 of 51 patients and paraproteinemia in 11 of 30. Multiple pulmonary lesions were recognized in 16 of 51 patients, both lungs were involved in 7 of 51, and regional lymph node involvement was detected in 11 of 51. Twenty-nine patients had St
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