Title: Molecular Diagnostic Approach to Non-Hodgkin's Lymphoma
Abstract: The evaluation of hematopoietic neoplasms now requires a variety of methods to use the modern classification systems. Morphological features remain the cornerstone of the evaluation of leukemias and malignant lymphomas, but ancillary studies are needed in many, if not most, cases. Immunophenotyping is helpful in both the diagnosis and classification of these tumors and is essential for the proper use of recently described classifications of malignant lymphomas.1Harris 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, 2Harris NL Jaffe ES Diebold J Flandrin G Müller-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 1997.J Clin Oncol. 1997; 17: 3835-3849Google Scholar The vast majority of leukemias and lymphomas can be diagnosed without the use of molecular genetic or cytogenetic studies. However, some cytogenetic abnormalities define a disease. For example, detection of the Philadelphia chromosome is an essential part of the diagnosis of chronic myelogenous leukemia. In the acute leukemias, cytogenetic and molecular genetic findings have marked prognostic significance, but they are not usually necessary to determine whether a proliferation is neoplastic or reactive. Most of the significant acute leukemia abnormalities are detectable by routine karyotype analysis. In contrast, the molecular genetic abnormalities of malignant lymphoma are often not easily detectable by routine karyotype analysis, and molecular diagnostic tests are necessary for evaluation. In addition, the detection of specific chromosomal translocations has helped to define clinically relevant lymphoma entities.1Harris 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, 2Harris NL Jaffe ES Diebold J Flandrin G Müller-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 1997.J Clin Oncol. 1997; 17: 3835-3849Google Scholar This is particularly true in the low-grade lymphomas. The molecular genetic associations have resulted in improved recognition of the morphological and immunophenotypic features of these lymphomas. Despite these improved criteria for diagnosis, however, some cases still require molecular testing for proper classification. In lymphoid proliferations, molecular diagnostic tests have two primary uses: to demonstrate a clonal abnormality when the differential diagnosis is between a reactive or neoplastic proliferation, and to identify a disease-associated finding, such as an associated virus or specific chromosomal translocation, that is useful in subclassification of the lymphoma. A variety of methods can be used for molecular diagnostic testing, and no one methodology is ideal for all tests. A detailed review of the different methods used for testing is beyond the scope of this review, but a brief summary of some of the methods will be given. In some instances, karyotype analysis is of limited use, because obtaining adequategrowth of low-grade lymphoma cells may be difficult and a normal karyotype, from non-neoplastic cells, may result. In addition, immunoglobulin heavy and light chain and T cell receptor chain gene rearrangements of malignant lymphomas are not detectable by karyotype analysis. Southern blot analysis has been the traditional gold standard for most molecular diagnostic testing. This procedure requires fresh tissue in fairly large amounts and is a labor-intensive, time-consuming method. A large percentage of the cells in the sample (5–10%) must harbor the suspected abnormality for this method to detect it. Despite these limitations, Southern blot analysis remains a useful methodology for some testing. Procedures using the polymerase chain reaction (PCR) have replaced many of the traditional Southern blot tests. This methodology requires only a small amount of DNA or RNA, is relatively rapid, and can detect abnormalities at a very low level. Direct PCR amplifies genomic DNA, and this method can be used for many of the common lymphoma translocations. When a translocation site is variable, requiring a larger area of DNA to be amplified, reverse transcriptase (RT) PCR can be used. RT-PCR amplifies complementary DNA (cDNA), usually made from an RNA fusion product that does not contain all of the regions of the original genomic DNA. Direct PCR tests can usually be performed on paraffin-embedded tissues, as well as fresh and frozen tissues. Due to RNA degradation, most RT-PCR tests do not work on paraffin-embedded tissue unless the RT-PCR product is very small. In situ hybridization studies allow for probing of tissue on a glass slide or cell suspension so that the intact positive cells can be directly visualized. This methodology is particularly useful in determining a viral association with a specific cell type. Fluorescence in situ hybridization (FISH) also allows for direct visualization of a specific chromosomal abnormality. FISH studies are less sensitive than PCR-based methods, but can detect abnormalities, such as monosomies and trisomies, that cannot be studied by PCR analysis. In situ PCR is a method in which the polymerase chain reaction actually takes place in the cell on a slide, and the product can be visualized in the same way as in traditional in situhybridization. The methodology is technically difficult, is often inconsistent, and is not used in most diagnostic laboratories. Microarray technology allows for a large number of genetic abnormalities to be screened on a single chip that is then scanned and analyzed by a computer. Although recent studies have shown the power of this methodology in recognizing prognostically significant trends in large cell lymphoma, it currently remains a research tool.3Alizadeh AA Eisen MB Davis RE Ma C Lossos IS Rosenwald A Boldrick JC Sabet H Tran T Yu X Powell JI Yang L Marti GE Moore T Hudson Jr, J Lu L Lewis DB Tibshirani R Sherlock G Chan WC Greiner TC Weisenburger DD Armitage JO Warnke R Levy R Wilson W Grever MR Byrd JC Botstein D Brown PO Staudt LM Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.Nature. 2000; 403: 503-511Crossref PubMed Scopus (7948) Google Scholar, 4Berns A Gene expression in diagnosis.Nature. 2000; 403: 491-492Crossref PubMed Scopus (59) Google Scholar The best method for testing depends on the question that is being asked and the abnormality that is being tested for. The advantages and limitations of the commonly used techniques will be discussed below in the context of the abnormality being evaluated. The most common abnormalities are listed in Table 1.Table 1Most Common Molecular Abnormalities Studied in Non-Hodgkin's LymphomaGene studiedChromosomal siteMost common disease associationsImmunoglobulin heavy chain (IgH) rearrangements14q32B cell neoplasms*Lineage infidelity may occur in some neoplasms, particularly lymphoblastic leukemias and lymphomas, which may result in detection of aberrant gene rearrangements (see text).Immunoglobulin kappa light chain (Igκ) rearrangements2p11B cell neoplasmsJH/BCL-1t(11;14)(q13;q32)Mantle cell lymphomaJH/BCL-2t(14;18)(q32;q21)Follicular lymphoma, some diffuse large B cell lymphomasPAX5/IgHt(9;14)(p13;q32)Lymphoplasmacytic lymphomaAPI2/MLTt(11;18)(q21;q21)Extranodal marginal zone lymphomaBCL-6 translocationst(3;n)(q27;n)Some diffuse large B cell lymphomasC-MYC translocationst(8;n)(q24;n)Burkitt's lymphomaT cell receptor β chain (TCRβ) rearrangements7q34T cell neoplasms*Lineage infidelity may occur in some neoplasms, particularly lymphoblastic leukemias and lymphomas, which may result in detection of aberrant gene rearrangements (see text).T cell receptor γ chain (TCRγ) rearrangements7q15T cell neoplasms*Lineage infidelity may occur in some neoplasms, particularly lymphoblastic leukemias and lymphomas, which may result in detection of aberrant gene rearrangements (see text).NPM/ALKt(2;5)(p23;q35)Anaplastic large cell lymphoma* Lineage infidelity may occur in some neoplasms, particularly lymphoblastic leukemias and lymphomas, which may result in detection of aberrant gene rearrangements (see text). Open table in a new tab Rearrangement of the immunoglobulin heavy chain region on chromosome region 14q32 occurs in all normal developing B lymphocytes.5Korsmeyer SJ Hieter PA Revetch JV Poplack DG Waldmann TA Leder P Developmental hierarchy of immunoglobulin gene rearrangements in human leukemic pre-B-cells.Proc Natl Acad Sci USA. 1981; 78: 7096-7100Crossref PubMed Scopus (383) Google Scholar, 6Cossman J Uppenkamp M Sundeen J Coupland R Raffeld M Molecular genetics and the diagnosis of lymphoma.Arch Pathol Lab Med. 1988; 112: 117-127PubMed Google Scholar, 7Pascual V Capra JD Human immunoglobulin heavy-chain variable region genes: organization, polymorphism, and expression.Adv Immunol. 1991; 49: 1-74Crossref PubMed Scopus (201) Google Scholar This chromosomal region contains over 100 variable (V), 30 diversity (D), and 6 joining (J) regions. When the B cell undergoes immunoglobulin heavy chain gene rearrangement (Figure 1), one V, one D, and one J region move into close proximity to each other. Because each normal B cell undergoes a unique rearrangement, there are differences among each cell resulting in a polyclonal B cell population. Following rearrangement of the immunoglobulin heavy chain gene, the immunoglobulin kappa light chain region of chromosome 2p11 rearranges in a similar fashion with the exception that it does not contain diversity (D) regions. If this rearrangement is not productive in either allele (approximately one third of cases), the kappa light chain constant region locus is deleted and the immunoglobulin lambda light chain region on chromosome 22q11 undergoes rearrangement. Because mature B cell lymphomas are clonal neoplasms, immunoglobulin heavy chain and kappa light chain rearrangements are detectable in essentially all cases. Many precursor B cell malignancies (lymphoblastic lymphomas and leukemias), however, will demonstrate only immunoglobulin heavy chain rearrangements because the neoplastic transformation occurs before rearrangement of the immunoglobulin kappa light chain region. Because lambda light chain rearrangements do not always occur and occur later in B cell development when present, this region is not a good initial target for clonality testing. Immunoglobulin gene rearrangements are usually detected by Southern blot analysis or by use of the polymerase chain reaction. The Southern blot procedure requires a large amount (at least 10 μg) of high quality DNA and requires fresh or frozen tissue. The DNA is cut with restriction enzymes, size electrophoresed, transferred to a membrane, and then probed for a specific portion of the immunoglobulin heavy chain or kappa light chain joining regions. If the B cells in the specimen are polyclonal, the restriction enzymes will cut different sized segments that are too few in number to be detected by the probe. The remaining non-rearranged cells (non-B cells) will not have undergone gene rearrangements for the area probed and will show bands of expected sizes (germline) on the probed membrane or radiograph. If a large number of polyclonal B cells is present in the sample, a weak smear without distinct rearranged bands may occur. Specimens with a monoclonal B cell population will have a prominent cell population that cuts to a specific size with the restriction enzymes, usually different from the non-rearranged germline cells, and will demonstrate additional bands on the membrane or radiograph. Criteria are published for the interpretation of Southern blots; generally, they require exclusion of bands due to partial digestion of DNA and require that rearrangements be seen with two of the three enzymes, or that two rearrangements be observed with a single enzyme for an interpretation of a clonal gene rearrangement.8Cossman J Zehnbauer B Garrett CT Smith LJ Williams M Jaffe ES Hanson LO Love J Gene rearrangements in the diagnosis of lymphoma/leukemia. Guidlines for use based on a multiinstitutional study.Am J Clin Pathol. 1991; 95: 347-354Crossref PubMed Scopus (82) Google Scholar, 9O'Leary TJ Brindza L Kant JA Kaul K Sperry L Stetler-Stevenson M Immunoglobulin and T-cell receptor gene rearrangement assays; approved guideline.NCCLS. 1995; 15: 1-30Google Scholar Very detailed and useful guidelines for specimen collection, transport, performance, and interpretation of immunoglobulin and T cell receptor gene rearrangement assays are published by National Committee for Critical Laboratory Standards (document MM2-A).9O'Leary TJ Brindza L Kant JA Kaul K Sperry L Stetler-Stevenson M Immunoglobulin and T-cell receptor gene rearrangement assays; approved guideline.NCCLS. 1995; 15: 1-30Google Scholar The use of PCR for the detection of immunoglobulin heavy chain gene rearrangements allows for the use of smaller amounts of DNA and even DNA from paraffin-embedded tissue. This method uses consensus primer pairs that anneal to the V and J regions of the rearranged chromosome 14.10Segal GH Jorgensen T Masih AS Braylan RC Optimal primer selection for clonality assessment by polymerase chain reaction analysis: I. Low grade B-cell lymphoproliferative disorders of nonfollicular center cell type.Hum Pathol. 1994; 25: 1269-1275Abstract Full Text PDF PubMed Scopus (148) Google Scholar Certain nucleotide sequences are similar among the different V and J regions, and the consensus primers are made to anneal to these sequences even if they are not a perfect match. Because different, polyclonal rearrangements result in slightly different-sized PCR products, a smear or ladder is seen on the gel in polyclonal specimens, and one or two discrete bands on a gel (or peaks on a capillary electrophoresis instrument printout) are seen with a monoclonal proliferation (Figure 2). The primers with the highest detection rate for the immunoglobulin heavy chain gene rearrangements are directed against a region termed the framework (FR) III region of the various VHgenes. FRIII-directed primers detect approximately 60% of clonal B cell malignancies.11Abdel-Reheim FA Edwards E Arber DA Utility of a rapid polymerase chain reaction panel for the detection of molecular changes in B-cell lymphoma.Arch Pathol Lab Med. 1996; 120: 357-363PubMed Google Scholar The addition of other framework regions, particularly FRII primers, will increase the detection rate of this test. Framework I is composed of multiple families of regions, which require multiple PCR reactions to detect reliably. A combination of FRII and FRIII primers will detect 70 to 90% of B cell neoplasms depending on the type of disease. In one study using only FRIII primers, 35% of follicular lymphomas were positive, compared to 82% of non-follicular B cell lymphomas (including 72% of diffuse large B cell lymphomas, 86% of small lymphocytic lymphomas and 100% of mantle cell and Burkitt's/Burkitt-like lymphomas).11Abdel-Reheim FA Edwards E Arber DA Utility of a rapid polymerase chain reaction panel for the detection of molecular changes in B-cell lymphoma.Arch Pathol Lab Med. 1996; 120: 357-363PubMed Google Scholar Somatic mutations of the immunoglobulin heavy chain gene of some mature B disorders, especially follicular lymphomas and plasma cell malignancies, alter the sequence of the region amplified by the primers so that primer hybridization is suboptimal or does not occur, resulting in false negative PCR results.10Segal GH Jorgensen T Masih AS Braylan RC Optimal primer selection for clonality assessment by polymerase chain reaction analysis: I. Low grade B-cell lymphoproliferative disorders of nonfollicular center cell type.Hum Pathol. 1994; 25: 1269-1275Abstract Full Text PDF PubMed Scopus (148) Google Scholar Therefore, a negative PCR result does not exclude the presence of a monoclonal B cell proliferation. In addition, consensus primers are not a perfect match to the sequence being amplified and result in less efficient amplification. Therefore, they are less sensitive in the detection of minimal residual disease than PCR primers specific to a region of a translocation or primers made specifically against a patient's gene rearrangement. This limits the use of the immunoglobulin heavy chain PCR test in the evaluation of minimal residual disease. Most tests that employ consensus primers can detect only one clonal cell in 100 polyclonal cells. PCR tests directed against rearrangement of the kappa light chain gene or the kappa-deleting segment are also useful in the detection of B cell clonality in mature B cell proliferations and are reported to detect clonality in up to 50% of B cell lymphomas.12Gong JZ Zheng S Chiarle R De Wolf-Peeters C Palestro G Frizzera G Inghirami G Detection of immnoglobulin κ light chain rearrangements by polymerase chain reaction. An improved method for detecting clonal B-cell lymphoproliferative disorders.Am J Pathol. 1999; 155: 355-363Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 13Seriu T Hansen-Hagge TE Stark Y Bartram CR Immunoglobulin κ gene rearrangements between the κ deleting element and Jκ recombination signal sequences in acute lymphoblastic leukemia and normal hematopoiesis.Leukemia. 2000; 14: 671-674Crossref PubMed Scopus (14) Google Scholar Although this method does not detect as many B cell neoplasms as the immunoglobulin heavy chain PCR test, Igκ PCR is useful as a second line test. It is particularly helpful in detecting a clonal population in plasma cell disorders that give false negative results for the IgH PCR test due to somatic hypermutation of the immunoglobulin heavy chain gene. Igκ PCR testing also uses consensus primers that limit the ability to detect minimal residual disease at a level below one clonal cell in 100 polyclonal cells. T cell receptor gene rearrangements (see below) may also be detectable in B cell malignancies.14Pelicci P-G Knowles DMII Dalla Favera R Lymphoid tumors displaying rearrangements of both immunoglobulin and T cell receptor genes.J Exp Med. 1985; 162: 1015-1024Crossref PubMed Scopus (174) Google Scholar This occurs most commonly in the precursor B cell lymphoblastic malignancies, and in these cases the gene rearrangement studies are not helpful in assigning lineage. Immunophenotyping studies, however, are usually adequate to resolve the lineage of most of these neoplasms. In mature B cell tumors, the addition of immunoglobulin kappa light chain Southern blot analysis or PCR analysis can aid in confirming the B-lineage of the tumor, as this locus is uncommonly rearranged in T cell malignancies. Specific cytogenetic translocations are also associated with some types of malignant lymphoma. Unlike the translocations of acute leukemia, many of the more common lymphoma translocations do not involve large introns and can be reliably amplified at the DNA level. Therefore, PCR tests for these can be performed on paraffin-embedded tissues. Molecular changes, other than gene rearrangements, seen with specific disease types will be discussed below. Due to somatic hypermutation of the immunoglobulin heavy chain gene in follicular center cells, only 35 to 50% of follicular lymphomas will have a detectable immunoglobulin heavy chain rearrangement by PCR analysis.11Abdel-Reheim FA Edwards E Arber DA Utility of a rapid polymerase chain reaction panel for the detection of molecular changes in B-cell lymphoma.Arch Pathol Lab Med. 1996; 120: 357-363PubMed Google Scholar, 15Segal GH Jorgensen T Scott M Braylan RC Optimal primer selection for clonality assessment by polymerase chain reaction analysis: II. Follicular lymphomas.Hum Pathol. 1994; 25: 1276-1282Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 16Ashton-Key M Diss TC Isaacson PG Smith MEF A comparative study of the value of immunohistochemistry and the polymerase chain reaction in the diagnosis of follicular lymphoma.Histopathology. 1995; 27: 501-508Crossref PubMed Scopus (43) Google Scholar Because these mutations do not affect the overall gene rearrangement, virtually all follicular lymphomas will show a rearrangement by Southern blot analysis. Despite the relatively high false negative rate for immunoglobulin heavy chain gene rearrangement by PCR analysis, most (70–80%) follicular lymphomas will demonstrate t(14;18)(q32;q21) involving the immunoglobulin heavy chain gene on chromosome 14 and the BCL-2 gene on chromosome 18 (Figure 3),17Weiss LM Warnke RA Sklar J Cleary ML Molecular analysis of the t(14;18) chromosomal translocation in malignant lymphomas.N Engl J Med. 1987; 317: 1185-1189Crossref PubMed Scopus (587) Google Scholar and 70 to 90% of these translocations are detectable by PCR analysis.18Turner GE Ross FM Krajewski AS Detection of t(14;18) in British follicular lymphoma using cytogenetics, Southern blotting and polymerase chain reaction.Br J Haematol. 1995; 89: 223-225Crossref PubMed Scopus (27) Google Scholar, 19Ladanyi M Wang S Detection of rearrangements of the BCL2 major breakpoint region in follicular lymphomas. Correlation of polymerase chain reaction results with Southern blot analysis.Diagn Mol Pathol. 1992; 1: 31-35Crossref PubMed Scopus (33) Google Scholar Over expression of bcl-2 protein, which results from this translocation, is associated with a loss of apoptosis. This translocation is detectable by either Southern blot or by PCR (JH/BCL-2) analysis.18Turner GE Ross FM Krajewski AS Detection of t(14;18) in British follicular lymphoma using cytogenetics, Southern blotting and polymerase chain reaction.Br J Haematol. 1995; 89: 223-225Crossref PubMed Scopus (27) Google Scholar Most translocations involve the major breakpoint region (MBR) of BCL-2, but 5 to 10% involve a minor cluster region (MCR) that requires the use of different PCR primers and Southern blot probes to detect.19Ladanyi M Wang S Detection of rearrangements of the BCL2 major breakpoint region in follicular lymphomas. Correlation of polymerase chain reaction results with Southern blot analysis.Diagn Mol Pathol. 1992; 1: 31-35Crossref PubMed Scopus (33) Google Scholar, 20Ngan B-Y Nourse J Cleary ML Detection of chromosomal translocation t(14;18) within the minor cluster region of bcl-2 by polymerase chain reaction and direct genomic sequencing of the enzymatically amplified DNA in follicular lymphomas.Blood. 1989; 73: 1759-1762PubMed Google Scholar, 21Liu J Johnson RM Traweek ST Rearrangement of the BCL-2 gene in follicular lymphoma. Detection by PCR in both fresh and fixed tissue samples.Diagn Mol Pathol. 1993; 2: 241-247Crossref PubMed Scopus (83) Google Scholar Although most JH/BCL-2 translocations can be detected from paraffin-embedded tissues, some breakpoints result in PCR products that are very large and may not be detectable after fixation.22Wang YL Addya K Edwards RH Rennert H Dodson L Leonard DGB Wilson RB Novel bcl-2 breakpoints in patients with follicular lymphoma.Diagn Mol Pathol. 1998; 7: 85-89Crossref PubMed Scopus (11) Google Scholar A recent study has suggested an improved prognosis in patients with follicular lymphoma with the MCR translocation,23McNelis FL McDonnell TI McLaughlin P Smith T Pugh W Hagemeister F Rodríguez MA Romaguera JE Younes A Sarris AH Preti HA Lee M-S Corrleation of bcl-2 rearrangement with clinical characterstics and outcome in indolent follicular lymphoma.Blood. 1999; 93: 3081-3087PubMed Google Scholar but this test is not used as a prognostic marker in most laboratories at this time. A variable cluster region (VCR) of the BCL-2 gene is also present approximately 225 kb 5′ to the MBR region. The VCR is occasionally involved in translocations involving the kappa light chain or lambda light chain genes on chromosomes 2 and 22, respectively, in cases of small lymphocytic lymphoma/chronic lymphocytic leukemia.24Merup M Spasokoukotskaja T Einhorn S Smith CIE Gahrton G Juliussin G Bcl-2 rearrangements with breakpoints in both vcr and mbr in non-Hodgkin's lymphomas and chronic lymphocytic leukaemia.Br J Haematol. 1996; 92: 647-652Crossref PubMed Scopus (25) Google Scholar The t(14;18) has also been reported to be detected by JH/BCL-2 PCR analysis in normal peripheral blood and in reactive lymph nodes.25Limpens J de Jong D van Krieken JHJM Price CGA Young BD van Ommen G-JB Kluin PM Bcl -2/JH rearrangements in benign lymphoid tissues with follicular hyperplasia.Oncogene. 1991; 6: 2271-2276PubMed Google Scholar, 26Ohshima K Masahiro K Kobari S Masuda Y Eguchi F Kimura N Amplified bcl-2/JH rearrangements in reactive lymphadenopathy.Virchows Arch (Cell Pathol). 1993; 63: 197-198Crossref Scopus (13) Google Scholar, 27Limpens J Stad R Vos C de Vlaam C de Jong D van Ommen G-JB Schurring E Kluin PM Lymphoma-assocaited translocation t(14;18) in blood B cells of normal individuals.Blood. 1995; 85: 2528-2536Crossref PubMed Google Scholar These reports suggest that this translocation can occur in small numbers of cells without the development of malignant lymphoma. Non-nested PCR tests for JH/BCL-2 that do not amplify over 45 cycles do not usually get these “false positive” results.28Segal GH Scott M Jorgensen T Braylan RC Standard polymerase chain reaction analysis does not detect t(14;18) in reactive lymphoid hyperplasia.Arch Pathol Lab Med. 1994; 118: 791-794PubMed Google Scholar The t(14;18)(q32;q21), identical to the translocations of follicular lymphomas, is identified in 17 to 38% of diffuse large B cell lymphoma, and the detection methods are identical to those described above.11Abdel-Reheim FA Edwards E Arber DA Utility of a rapid polymerase chain reaction panel for the detection of molecular changes in B-cell lymphoma.Arch Pathol Lab Med. 1996; 120: 357-363PubMed Google Scholar, 29Lipford E Wright JJ Urba W Whang-Peng J Kirsch IR Raffeld M Cossman J Longo DL Bakhshi A Korsmeyer SJ Refinement of lymphoma cytogenetics by the chromosme 18q21 major breakpoint region.Blood. 1987; 70: 1816-1823PubMed Google Scholar, 30Yunis JJ Mayer MG Arnesen MA Aeppli DP Oken MM Frizzera G bcl -2 and other genomic alterations in the prognosis of large-cell lymphoma.N Engl J Med. 1989; 320: 1047-1054Crossref PubMed Scopus (270) Google Scholar, 31Hill ME MacLennan KA Cunningham DC Hudson BV Burke M Clarke P Di Stefano F Anderson L Hudson GV Mason D Selby P Linch DC Prognostic significance of BCL-2 expression and bcl -2 major breakpoint region rearrangement in diffuse large cell non-Hodgkin's lymphoma: a British National Lymphoma Investigation study.Blood. 1996; 88: 1046-1051PubMed Google Scholar Some studies have suggested that the presence of t(14;18) in large cell lymphoma is an indicator of a poor prognosis.30Yunis JJ Mayer MG Arnesen MA Aeppli DP Oken MM Frizzera G bcl -2 and other genomic alterations in the prognosis of large-cell lymphoma.N Engl J Med. 1989; 320: 1047-1054Crossref PubMed Scopus (270) Google Scholar, 31Hill ME MacLennan KA Cunningham DC Hudson BV Burke M Clarke P Di Stefano F Anderson L Hudson GV Mason D Selby P Linch DC Prognostic significance of BCL-2 expression and bcl -2 major breakpoint region rearrangement in diffuse large cell non-Hodgkin's lymphoma: a British National Lymphoma Investigation study.Blood. 1996; 88: 1046-1051PubMed Google Scholar In both follicular lymphomas and diffuse large B cell lymphomas, detection of this translocation does not correlate completely with BCL-2 protein expression. Detection of t(14;18) by molecular methods is not necessary for the diagnosis of most cases of follicular lymphoma. However, such testing may be valuable in the detection of minimal residual disease, such as in bone marrow material aspirated after chemotherapy or bone marrow transplantation for follicular lymphoma (see below). The t(11;14)(q13;q32), which involves the immunoglobulin heavy chain gene of chromosome 14 and the BCL-1/PRAD1 gene of chromosome 11, is detected in approximately 60% of mantle cell lymphoma cases.32Rosenberg CL Wong E Petty EM Bale AE Tsujimoto Y Harris NL Arnold A PRAD1, a candidate BCL1 oncogene: mapping and expression in centrocytic lymphoma.Proc Natl Acad Sci USA. 1991; 88: 9638-9642Crossref PubMed Scopus (419) Google Scholar, 33Rimokh R Berger F Delsol G Digonnet I Rouault JP Tigaud JD Gadoux M Coiffier B Bryon PA Magaud JP Detection of the chromosomal translocation t(11;14) by polymerase chain reaction in mantle cell lymphomas.Blood. 1994; 83: 1871-1875PubMed Google Scholar The BCL-1 gene encodes a cell cycle protein (termed cyclin D1, PRAD1, or BCL-1) and over expression is associated with the aggressive behavior of this tumor, and has been useful in further defining this disease. The major translocation cluster (MTC) region is involved in 40 to 50% of cases, but the remaining translocations involve a multitude of different sites that are not easily detectable by PCR analysis.34Raynaud SD Bekri S Leroux D Grosgeorge J Klein B Bastard C Gaudray P Simon M-P Expanded range of 11q23 breakpoints with differeng patterns of cyclin D1 expression in B-cell malignancies.Genes Chromosom Cancer. 1993; 8: 80-87Crossref PubMed Scopus (108) Google Scholar Methods for detection of BCL-1 mRNA are described that detected over 95% of cases of mantle cell lymphoma, and the mRNA expression presumably occurs with translocations that involve the MTC as well as other breakpoints.35de