Yuanxue Huang

A practical strategy for the routine use of BIOMED‐2 PCR assays for detection of B‐ and T‐cell clonality in diagnostic haematopathology

BIOMED‐2 polymerase chain reaction (PCR) assays for clonality analysis of immunoglobulin (IG) and T‐cell receptor (TCR) gene rearrangements were evaluated in routine haematopathological practice where paraffin‐embedded tissues constitute the majority of specimens. One hundred and twenty‐five fresh/frozen and 316 paraffin specimens were analysed for DNA quality and clonality. Seventy‐nine per cent of paraffin specimens yielded PCR products of over 300 bp. These specimens and all fresh/frozen specimens were analysed with the complete set of BIOMED‐2 reactions for IG (8 reactions) and/or TCR (6 reactions) gene rearrangements. The rate of detection of clonality was 96% in mature B‐cell neoplasms and 98% in mature T‐cell neoplasms and there were no significant differences in these rates between paraffin and fresh/frozen specimens. As the value of sole use of any individual BIOMED‐2 reaction in clonality detection was limited, we assessed combinations of reactions that gave the greatest sensitivity with fewest reactions and were applicable for both fresh/frozen and paraffin specimens. For IG gene rearrangements, three reactions combining one targeting the IG heavy chain framework‐2 region and two targeting the IG kappa locus achieved a 91% detection rate. For TCR gene rearrangements, the two TCR gamma reactions gave a 94% detection rate. We therefore recommend this strategy as the first‐line assays for routine B‐ and T‐cell clonality analysis in diagnostic haematopathology.

Splenic marginal zone lymphoma: characterization of 7q deletion and its value in diagnosis

The diagnosis of splenic marginal zone lymphoma (SMZL) is frequently a challenge, due to its lack of specific histological features and immunophenotypic markers, and the existence of other poorly characterized splenic lymphomas defying classification. Moreover, the clinical outcome of SMZL is variable, with 30% of cases pursuing an aggressive clinical course, the prediction of which remains problematic. Thus, there is a real need for biomarkers in the diagnosis and prognostication of SMZL. To search for genetic markers, we comprehensively investigated the genomic profile, TP53 abnormalities, and immunoglobulin heavy gene (IGH) mutation in a large cohort of SMZLs. 1 Mb resolution array comparative genomic hybridization (aCGH) on 25 SMZLs identified 7q32 deletion (44%) as the most frequent copy number change, followed by gains of 3q (32%), 8q (20%), 9q34 (20%), 12q23–24 (8%), and chromosome 18 (12%), and losses of 6q (16%), 8p (12%), and 17p (8%). High‐resolution chromosome 7 tile‐path aCGH on 17 SMZLs with 7q32 deletion identified by 1 Mb aCGH or interphase FISH screening mapped the minimal common deletion to a 3 Mb region at 7q32.1–32.2. Although it is not yet possible to identify the genes targeted by the deletion, interphase FISH screening showed that the deletion was seen in SMZL (19/56 = 34%) and splenic B‐cell lymphoma/leukaemia unclassifiable (3/9 = 33%), but not in 39 cases of other splenic lymphomas including chronic lymphocytic leukaemia (n = 14), hairy cell leukaemia (4), mantle cell lymphoma (12), follicular lymphoma (6), and others. In SMZL, 7q32 deletion was inversely correlated with trisomy 18, but not associated with other copy number changes, TP53 abnormalities, or IGH mutation status. None of the genetic parameters examined showed significant and independent association with overall or event‐free survival. In conclusion, 7q32 deletion is a characteristic feature of SMZL, albeit seen in isolated cases of splenic B‐cell lymphoma/leukaemia unclassifiable, and its detection may help the differential diagnosis of splenic B‐cell lymphomas. Copyright

Mantle cell lymphoma with aberrant expression of CD10

Aims:  Morphological, immunophenotypic and genetic heterogeneity amongst mantle cell lymphomas (MCLs) can lead to difficulties in diagnosis and management. The aim was to describe the clinical and pathological features of MCLs with aberrant expression of CD10.

Primary effusion lymphoma: genomic profiling revealed amplification of SELPLG and CORO1C encoding for proteins important for cell migration

Primary effusion lymphoma (PEL) is associated with Kaposi sarcoma herpesvirus (KSHV) but its pathogenesis is poorly understood. Many KSHV‐associated products can deregulate cellular pathways commonly targeted in cancer. However, KSHV infection alone is insufficient for malignant transformation. PEL also lacks the chromosomal translocations seen in other lymphoma subtypes. We investigated 28 PELs and ten PEL cell lines by 1 Mb resolution array comparative genomic hybridization (CGH) and found frequent gains of 1q21–41 (47%), 4q28.3‐35 (29%), 7q (58%), 8q (63%), 11 (32%), 12 (61%), 17q (29%), 19p (34%), and 20q (34%), and losses of 4q (32%), 11q25 (29%), and 14q32 (63%). Recurrent focal amplification was seen at several regions on chromosomes 7, 8, and 12. High‐resolution chromosome‐specific tile‐path array CGH confirmed these findings, and identified selectin‐P ligand (SELPLG) and coronin‐1C (CORO1C) as the targets of a cryptic amplification at 12q24.11. Interphase FISH and quantitative PCR showed SELPLG/CORO1C amplification (>4 extra copies) and low levels of copy number gain (1–4 extra copies) in 23% of PELs, respectively. Immunohistochemistry revealed strong expression of both SELPLG and coronin‐1C in the majority of PELs, irrespective of their gene dosage. SELPLG is critical for cell migration and chemotaxis, while CORO1C regulates actin‐dependent processes, thus important for cell motility. Their overexpression in PEL is expected to play an important role in its pathogenesis. Copyright

Continual monitoring of intraepithelial lymphocyte immunophenotype and clonality is more important than snapshot analysis in the surveillance of refractory coeliac disease

Objective An aberrant immunophenotype and monoclonality of intraepithelial lymphocytes (IELs) are frequently found in refractory coeliac disease (RCD). However, the utility of continual monitoring of IEL immunophenotype and clonality in the surveillance of RCD remains to be studied. Design The diagnostic and follow-up biopsies from 33 patients with CD, 7 with suspected RCD, 41 with RCD and 20 with enteropathy-associated T cell lymphoma (EATL) (including 11 evolved from RCD) were investigated by CD3ɛ/CD8 double immunohistochemistry and PCR-based clonality analysis of the rearranged T cell receptor (TCR) genes. Results An aberrant immunophenotype (CD3ɛ+CD8− IELs ≥40%) and monoclonality were detected occasionally in CD biopsies, either transiently in patients with CD not compliant with a gluten-free diet or in those who subsequently developed suspected RCD, RCD or EATL. In contrast, the aberrant immunophenotype and monoclonality were found in 30 of 41 (73%) and 24 of 37 (65%) biopsies, respectively, at the time of RCD diagnosis. Among the patients with RCD who did not show these abnormalities in their diagnostic biopsies, 8 of 10 (80%) and 5 of 11 (45%) cases gained an aberrant immunophenotype and monoclonality, respectively, during follow-up. Irrespective of whether detected in diagnostic or follow-up biopsies, persistence of both abnormalities was characteristic of RCD. Importantly, the presence of concurrent persistent monoclonality and aberrant immunophenotype, especially ≥80% CD3ɛ+CD8− IELs, was a strong predictor of EATL development in patients with RCD (p=0.001). Conclusions Continual monitoring of both immunophenotype and clonality of IELs is more important than snapshot analysis for RCD diagnosis and follow-up, and could provide a useful tool for surveillance of patients at risk of EATL.

Cyclin D1‐positive diffuse large B‐cell lymphoma

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An integrated genomic and expression analysis of 7q deletion in splenic marginal zone lymphoma.

Splenic marginal zone lymphoma (SMZL) is an indolent B-cell lymphoproliferative disorder characterised by 7q32 deletion, but the target genes of this deletion remain unknown. In order to elucidate the genetic target of this deletion, we performed an integrative analysis of the genetic, epigenetic, transcriptomic and miRNomic data. High resolution array comparative genomic hybridization of 56 cases of SMZL delineated a minimally deleted region (2.8 Mb) at 7q32, but showed no evidence of any cryptic homozygous deletion or recurrent breakpoint in this region. Integrated transcriptomic analysis confirmed significant under-expression of a number of genes in this region in cases of SMZL with deletion, several of which showed hypermethylation. In addition, a cluster of 8 miRNA in this region showed under-expression in cases with the deletion, and three (miR-182/96/183) were also significantly under-expressed (P<0.05) in SMZL relative to other lymphomas. Genomic sequencing of these miRNA and IRF5, a strong candidate gene, did not show any evidence of somatic mutation in SMZL. These observations provide valuable guidance for further characterisation of 7q deletion.

BIOMED‐2 PCR assays for IGK gene rearrangements are essential for B‐cell clonality analysis in follicular lymphoma

B‐cell clonality analysis is commonly performed by polymerase chain reaction (PCR) targeting the IGH genes although a high false‐negative rate is recognized for germinal centre/post‐germinal centre B‐cell malignancies, especially follicular lymphoma. We assessed the diagnostic value of BIOMED‐2 IGK assays and investigated the cause of IGH PCR failure in 77 patients with follicular lymphoma. Using the full set of BIOMED‐2 reactions, clonal immunoglobulin gene rearrangements were detected in 74 (96%) cases. The clonality detection rate was 86% by two IGK reactions but only 68% by five IGH reactions (P < 0·001). Sequencing of the clonal PCR products showed significantly fewer somatic mutations in the rearranged IGKV (9/27 cases, 33%, mean mutation rate 0·5%) than IGHV (17/17 cases, 100%, rate 11·0%) (P < 0·01). All IGHV‐IGHJ PCR failures occurred in cases with at least one mutation at the corresponding IGHV primer binding sites. t(14:18)(q32:q21)/IGH‐BCL2 was detected in 50 of 71 (70%) cases and the presence of the translocation was not associated with the poor performance of IGH assays. Our results showed that BIOMED‐2 IGK assays are significantly more sensitive than IGH assays in follicular lymphoma due to the fact that the rearranged IGKV is less frequently targeted by somatic hypermutation than IGHV, and therefore, are essential in routine clonality analysis of these lymphomas.

The Lymphoma-associated Fusion Tyrosine Kinase ITK-SYK Requires Pleckstrin Homology Domain-mediated Membrane Localization for Activation and Cellular Transformation

ITK-SYK, a novel fusion tyrosine kinase (FTK) resulting from a recurrent t(5;9)(q33;q22), was recently identified in a poorly understood subset of peripheral T-cell lymphomas. However, the biochemical and functional properties of ITK-SYK are unknown. Here we demonstrate that ITK-SYK is a catalytically active tyrosine kinase that is sensitive to an established inhibitor of SYK. The expression of ITK-SYK, but not SYK, transformed NIH3T3 cells, inducing loss of contact inhibition and formation of anchorage-independent colonies in soft agar, in a kinase activity-dependent manner. ITK-SYK is unusual among FTKs in having an N-terminal phosphatidylinositol 3,4,5-trisphosphate-binding pleckstrin homology (PH) domain. Introduction of a well characterized loss-of-function mutation (R29C) into the PH domain of ITK-SYK inhibited its phosphorylation, markedly reduced its catalytic activity, and abrogated its ability to activate the ERK signaling pathway and to transform NIH3T3 cells. Although ITK-SYK was membrane-associated, ITK-SYK-R29C was not. However, each of these properties could be recovered by retargeting ITK-SYK-R29C back to the plasma membrane by the addition of an N-terminal myristylation sequence. Consistent with a model in which ITK-SYK requires PH domain-mediated binding to phosphatidylinositol 3,4,5-trisphosphate generated by phosphatidylinositol 3-kinase (PI3K), ITK-SYK activity was reduced by pharmacological inhibition of PI3K and increased by co-expression with a constitutively active form of PI3K. Together, these findings identify ITK-SYK as an active, transforming FTK dependent upon PH domain-mediated membrane localization, identify a novel mechanism for activation of an oncogenic FTK, and suggest ITK-SYK as a rational therapeutic target for t(5;9)(q33;q22)-positive lymphomas.

TNFAIP3 abnormalities in MALT lymphoma with autoimmunity

Ann F. Mohrbacher Stephen J. Forman Paul Frankel Helen X. Chen James H. Doroshow David R. Gandara Departments of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, Duarte, CA, Hematology and Hematopoietic Cell Transplantation, City of Hope Cancer Center, Duarte, CA, Department of Pathology, University of Arkansas, Little Rock, AR, Department of Hematology, University of Chicago, Chicago, IL, Division of Hematology and Oncology, University of California Davis Cancer Center, Sacramento, CA, Division of Hematology, University of Southern California, Los Angeles, CA, Department of Information Sciences, City of Hope, Duarte, CA, and Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD, USA. E-mail: gsomlo@coh.org