Ellen D. Remstein

Incidence and Subtype Specificity of API2-MALT1 Fusion Translocations in Extranodal, Nodal, and Splenic Marginal Zone Lymphomas

The t(11;18)(q21;q21) is thought to represent an important primary event in the development of marginal zone lymphomas, although an accurate estimation of the frequency and distribution of this genetic alteration among nodal, splenic, and extranodal marginal zone lymphoma types has yet to be determined. Recently, molecular genetic studies have shown that this translocation results in the fusion of the API2 gene on chromosome 11 and a novel gene termed MALT1 on chromosome 18. To investigate the incidence of API2-MALT1 fusion transcripts among marginal zone lymphomas and to determine possible marginal zone lymphoma subtype associations, we used reverse transcriptase-polymerase chain reaction to analyze RNAs extracted from frozen tissue samples of 99 marginal zone lymphomas. Fifty-seven involved diverse extranodal sites including 14 stomach, 11 lung, 7 orbit, 7 parotid, 5 thyroid, 5 lacrimal gland, 3 small intestine, 2 large intestine, 1 kidney, 1 paraspinal region and 1 skin. Twenty-one primary splenic and twenty-one primary nodal marginal zone lymphomas were also studied. API2-MALT1 fusion transcripts were detected in 12 of 57 extranodal marginal zone lymphomas (21%), but in none of the nodal or splenic cases. The cDNA sequences of the fusion transcripts were determined, revealing variation in the coding sequence fusion point for both API2 and MALT1. The findings suggest that t(11;18)(q21;q21) is restricted to extranodal marginal zone lymphomas and that these tumors have distinct genetic etiologies in comparison with their splenic and nodal counterparts.

Splenic marginal zone lymphoma proposals for a revision of diagnostic, staging and therapeutic criteria.

Since the initial description of splenic marginal zone lymphoma (SMZL) in 1992, an increasing number of publications have dealt with multiple aspects of SMZL diagnosis, molecular pathogenesis and treatment. This process has identified multiple inconsistencies in the diagnostic criteria and lack of clear guidelines for the staging and treatment. The authors of this review have held several meetings and exchanged series of cases with the objective of agreeing on the main diagnostic, staging and therapeutic guidelines for patients with this condition. Specific working groups were created for diagnostic criteria, immunophenotype, staging and treatment. As results of this work, guidelines are proposed for diagnosis, differential diagnosis, staging, prognostic factors, treatment and response criteria. The guidelines proposed here are intended to contribute to the standardization of the diagnosis and treatment of these patients, and should facilitate the future development of clinical trials that could define more precisely predictive markers for histological progression or lack of response, and evaluate new drugs or treatments.

The incidence and anatomic site specificity of chromosomal translocations in primary extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) in North America.

Several balanced translocations have been identified in extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) but there are few data regarding their frequency in different anatomic sites or the frequency of translocations involving BCL6 or κ or λ immunoglobulin light chain genes (IGK or IGL), particularly in patients from geographic regions other than Europe and Japan. One hundred thirty-three paraffin-embedded North American primary MALT lymphoma specimens from diverse anatomic sites were studied by fluorescence in situ hybridization (FISH) using probes for API2-MALT1, IGH-MALT1, IGH-BCL10, IGH-FOXP1, IGH, +/− centromeres 3, 7, 12, and 18, and a subset (n=74) were analyzed using FISH probes for IGK, IGL, and BCL6. Translocations were mutually exclusive and were detected in 26% of cases (17% API2-MALT1, 5% IGH-MALT1, 3% IGH-unknown translocation partner, and 1% IGH-BCL10). Aneuploidy was associated with IGH-MALT1 and IGH-BCL10 but only rarely with API2-MALT1. There was striking site specificity, with API2-MALT1 showing a marked predilection for lung and intestine, and IGH-MALT1 and IGH-BCL10 occurring almost exclusively in lung. Twenty-three percent of translocation-negative primary MALT lymphomas from diverse sites showed complete/partial trisomy 18. No MALT lymphomas with translocations involving IGK, IGL, BCL6, or FOXP1 were identified. This FISH panel detected cytogenetic abnormalities in half of all MALT lymphomas, and translocations arose preferentially in MALT lymphomas of the lung and gastrointestinal tract. Differences in incidence and anatomic site specificity of translocations between North American and non-North American cases may reflect geographic variability of infectious or other etiologic factors.

MALT lymphoma with t(14;18)(q32;q21)/IGH-MALT1 is characterized by strong cytoplasmic MALT1 and BCL10 expression

Mucosa‐associated lymphoid tissue (MALT) lymphoma is specifically associated with t(11;18)(q21;q21), t(1;14)(p22;q32) and t(14;18)(q32;q21). t(11;18)(q21;q21) fuses the N‐terminus of the API2 gene to the C‐terminus of the MALT1 gene and generates a functional API2‐MALT1 product. t(1;14)(p22;q32) and t(14;18)(q32;q21) bring the BCL10 and MALT1 genes respectively to the IGH locus and deregulate their expression. The oncogenic activity of the three chromosomal translocations is linked by the physiological role of BCL10 and MALT1 in antigen receptor‐mediated NFκB activation. In this study, MALT1 and BCL10 expression was examined in normal lymphoid tissues and 423 cases of MALT lymphoma from eight sites, and their expression was correlated with the above translocations, which were detected by molecular and molecular cytogenetic methods. In normal B‐cell follicles, both MALT1 and BCL10 were expressed predominantly in the cytoplasm, high in centroblasts, moderate in centrocytes and weak/negative in mantle zone B‐cells. In MALT lymphoma, MALT1 and BCL10 expression varied among cases with different chromosomal translocations. In 9/9 MALT lymphomas with t(14;18)(q32;q21), tumour cells showed strong homogeneous cytoplasmic expression of both MALT1 and BCL10. In 12/12 cases with evidence of t(1;14)(p22;q32) or variants, tumour cells expressed MALT1 weakly in the cytoplasm but BCL10 strongly in the nuclei. In all 67 MALT lymphomas with t(11;18)(q21;q21), tumour cells expressed weak cytoplasmic MALT1 and moderate nuclear BCL10. In MALT lymphomas without the above translocations, both MALT1 and BCL10, in general, were expressed weakly in the cytoplasm. Real‐time quantitative RT‐PCR showed a good correlation between MALT1 and BCL10 mRNA expression and underlining genetic changes, with t(14;18)(q32;q21)‐ and t(1;14)(p22;q32)‐positive cases displaying the highest MALT1 and BCL10 mRNA expression respectively. These results show that MALT1 expression pattern is identical to that of BCL10 in normal lymphoid tissues but varies in MALT lymphomas, with high cytoplasmic expression of both MALT1 and BCL10 characterizing those with t(14;18)(q32;q21). Copyright

Recurrent translocations involving the IRF4 oncogene locus in peripheral T-cell lymphomas

Oncogenes involved in recurrent chromosomal translocations serve as diagnostic markers and therapeutic targets in hematopoietic tumors. In contrast to myeloid and B-cell neoplasms, translocations in peripheral T-cell lymphomas (PTCLs) are poorly understood. Here, we identified recurrent translocations involving the multiple myeloma oncogene-1/interferon regulatory factor-4 (IRF4) locus in PTCLs. IRF4 translocations exist in myeloma and some B-cell lymphomas, but have not been reported earlier in PTCLs. We studied 169 PTCLs using fluorescence in situ hybridization and identified 12 cases with IRF4 translocations. Two cases with t(6;14)(p25;q11.2) had translocations between IRF4 and the T-cell receptor-alpha (TCRA) locus. Both were cytotoxic PTCLs, unspecified (PTCL-Us) involving bone marrow and skin. In total, 8 of the remaining 10 cases were cutaneous anaplastic large-cell lymphomas (ALCLs) without TCRA rearrangements (57% of cutaneous ALCLs tested). These findings identified IRF4 translocations as a novel recurrent genetic abnormality in PTCLs. Cytotoxic PTCL-Us involving bone marrow and skin and containing IRF4/TCRA translocations might represent a distinct clinicopathologic entity. Translocations involving IRF4 but not TCRA appear to occur predominantly in cutaneous ALCLs. Detecting these translocations may be useful in lymphoma diagnosis. Further, due to its involvement in translocations, MUM1/IRF4 protein may play an important biologic role in some PTCLs, and might represent a possible therapeutic target.

Adult B-cell lymphomas with burkitt-like morphology are phenotypically and genotypically heterogeneous with aggressive clinical behavior.

Adult, de novo B-cell lymphomas meeting the WHO morphologic criteria for atypical Burkitt/Burkitt-like lymphoma cause diagnostic difficulty for pathologists because the genetic and clinical characteristics of this group of lymphomas have not been clearly defined. Thirty-one such lymphomas, designated as Burkitt-like lymphomas (BLL), were selected based on morphologic features and evaluated for immunophenotype, MYC and BCL2 status, and clinical features. Nine childhood Burkitt lymphomas (BL) and 87 adult, de novo diffuse large B-cell lymphomas (DLBL) were similarly evaluated for comparison. The BL group demonstrated uniform characteristics: all had Burkitt lymphoma morphology, an identical immunophenotype (positive for CD20, CD10, bcl-6, CD43, and p53; negative for CD138, CD23, bcl-2), high MIB-1 positivity, IGH/MYC translocation, no IGH/BCL2 translocation, and all patients were alive at the last follow-up. The BLL and DLBL groups were heterogeneous. Burkitt-like morphology alone correlated with decreased survival. IGH/MYC or IGL/MYC fusion was identified in 11 of 27 (41%) BLL and 4 of 76 (5%) DLBL and was associated with decreased survival in both groups. MIB-1 positivity did not correlate with morphology, MYC abnormalities, or survival. We propose that adult B-cell lymphomas with BLL morphology are a phenotypically and genetically heterogeneous group of aggressive lymphomas, biologically distinct from childhood BL. Until biologically accurate subgroups within this morphologically defined group are identified, it is appears that both recognition of BLL morphology and direct evaluation for the presence of MYC fusion to immunoglobulin genes are important for identification of adult patients with poorer prognosis than those with DLBL.

Overexpression of Syk tyrosine kinase in peripheral T-cell lymphomas

Peripheral T-cell lymphomas (PTCLs) are fatal in the majority of patients and novel treatments, such as protein tyrosine kinase (PTK) inhibition, are needed. The recent finding of SYK/ITK translocations in rare PTCLs led us to examine the expression of Syk PTK in 141 PTCLs. Syk was positive by immunohistochemistry (IHC) in 133 PTCLs (94%), whereas normal T cells were negative. Western blot on frozen tissue (n=6) and flow cytometry on cell suspensions (n=4) correlated with IHC results in paraffin. Additionally, western blot demonstrated that Syk-positive PTCLs show tyrosine (525/526) phosphorylation, known to be required for Syk activation. Fluorescence in situ hybridization showed no SYK/ITK translocation in 86 cases. Overexpression of Syk, phosphorylation of its Y525/526 residues and the availability of orally available Syk inhibitors suggest that Syk merits further evaluation as a candidate target for pharmacologic PTK inhibition in patients with PTCL.

Expression of CXCL13, a chemokine highly upregulated in germinal center T-helper cells, distinguishes angioimmunoblastic T-cell lymphoma from peripheral T-cell lymphoma, unspecified

The germinal center T-helper cell has been proposed as the cell of origin for angioimmunoblastic T-cell lymphoma. Our recent report of expression of CXCL13, a chemokine critical for germinal center formation and one of the most highly upregulated genes in the germinal center T-helper cell subset, in the majority of angioimmunoblastic T-cell lymphoma cases, provided further support for this theory. To determine the specifity of this marker for angioimmunoblastic T-cell lymphoma, we evaluated CXCL13 expression in 26 nodal-based peripheral T-cell lymphomas and 14 lymph nodes showing paracortical lymphoid hyperplasia. No significant paracortical CXCL13 staining was seen in the reactive lymph nodes. By WHO classification criteria, 20 of the lymphoma cases were considered peripheral T-cell lymphoma, unspecified, and six were reclassified as angioimmunoblastic T-cell lymphoma after immunohistochemical detection of disorganized follicular dendritic cell meshworks. Combining the results of our studies, 31 of 35 angioimmunoblastic T-cell lymphoma cases (89%) showed CXCL13 expression, in contrast to two out of 20 peripheral T-cell lymphoma, unspecified cases (10%). The two peripheral T-cell lymphoma, unspecified cases that were positive for CXCL13 showed a Lennert lymphoma-like histology. While these cases did not meet all histologic criteria for angioimmunoblastic T-cell lymphoma, they did show an increase in EBV-positive B cells, suggesting they may be histologic variants of angioimmunoblastic T-cell lymphoma. In conclusion, CXCL13 expression is a distinctive feature of angioimmunoblastic T-cell lymphoma, providing further support for the germinal center T-helper cell as the cell of origin for this neoplasm. Given its specificity when compared to cases of peripheral T-cell lymphoma, unspecified as well as paracortical lymphoid hyperplasia, it may be a useful marker in the diagnosis of angioimmunoblastic T-cell lymphoma.

Diagnostic utility of fluorescence in situ hybridization in mantle-cell lymphoma

Mantle‐cell lymphoma (MCL) has a poorer prognosis than other small B‐cell lymphomas, thus a definitive diagnosis is essential. The t(11;14)(q13;q32) associated with MCL juxtaposes portions of CCND1 (11q13) and IGH (14q32), resulting in over‐expression of cyclin D1. In this study, a highly sensitive two‐colour fluorescence in situ hybridization (FISH) method was developed to detect t(11;14)(q13;q32) in nuclei isolated from paraffin‐embedded tissue. Twenty‐three MCLs, 13 normal controls and nine small B‐cell lymphomas other than MCL were studied by FISH. Each MCL had been previously investigated to detect genomic IGH–CCND1 fusion by polymerase chain reaction (PCR) using DNA extracted from frozen tissue. The IGH–CCND1 fusion detection rate in the MCLs was 96% by FISH compared with 35% by PCR. By FISH, one MCL and three small B‐cell lymphomas other than MCL harboured abnormalities involving only IGH. Less than 1% of cells showed false‐positive IGH–CCND1 fusion in normal specimens by FISH. Thus, this highly sensitive FISH assay is very useful in confirming the diagnosis of MCL, has wide applicability as it may be performed on both paraffin‐embedded and fresh tissue, and may also facilitate detection of translocations involving these loci in tumours other than MCL.

Mucosa-Associated Lymphoid Tissue Lymphomas with t(11;18)(q21;q21) and Mucosa-Associated Lymphoid Tissue Lymphomas with Aneuploidy Develop Along Different Pathogenetic Pathways

t(11;18)(q21;q21) and aneuploidy are recurrent chromosomal aberrations in mucosa-associated lymphoid tissue (MALT) lymphomas. To investigate their relationship and clinical significance, we developed a two-color fluorescence in situ hybridization (FISH) technique to detect t(11;18) and aneuploidy in nuclei isolated from paraffin-embedded tissue. Thirty-seven MALT lymphomas (all previously evaluated for t(11;18) by reverse transcriptase-polymerase chain reaction), 1 large cell lymphoma (LCL) arising subsequent to MALT lymphoma, and 16 controls were tested by FISH using the t(11;18) probe set and multiple centromeric probes. t(11;18)(q21;q21) was present by FISH in 11 of 12 polymerase chain reaction-positive MALT lymphomas (92%). The LCL and its clonally identical antecedent MALT lymphoma both showed t(11;18). The LCL had trisomy 12, and a small subset of MALT lymphoma cells had trisomy 3 and/or 12. Only one other MALT lymphoma with t(11;18) showed aneuploidy (trisomy 3) in a small clone, whereas 15 of 25 t(11;18)-negative MALT lymphomas (60%) showed trisomy of chromosomes 18 (n = 12), 3 (n = 8), 7 (n = 2), and/or 11 (n = 1). t(11;18) and aneuploidy are primarily mutually exclusive events, suggesting different pathogenetic pathways in the development of MALT lymphomas. Both t(11;18) and aneuploidy were seen disproportionately in lung, and both were associated with recurrent disease.