David W. Bahler

Whole-genome sequencing identifies recurrent somatic NOTCH2 mutations in splenic marginal zone lymphoma

NOTCH2 mutations in splenic marginal zone lymphoma are associated with poor prognosis.

Indolent mantle cell lymphoma with nodal involvement and mutated immunoglobulin heavy chain genes

Mantle cell lymphoma (MCL) is typically considered an aggressive but incurable neoplasm composed of cyclin D1+ monoclonal B-cells with a t(11;14)(q13;q32) and usually unmutated immunoglobulin (Ig) genes. Although it has been suggested that a more indolent leukemic disorder exists with the same phenotype and genotype but with mutated Ig genes, others have considered these cases to be variants of chronic lymphocytic leukemia. We present a case of an indolent MCL that was documented with cyclin D1 expression in a lymph node biopsy performed more than 12 years ago. The patient has peripheral blood involvement with a lymphocyte count in the reference range, variable thrombocytopenia, and minimal adenopathy but is otherwise well, never having received any antineoplastic therapy. Study of peripheral blood samples from 2002 revealed a CD5-variable B-cell monoclonal proliferation with a t(11;14)(q13;q32) plus other karyotypic abnormalities, positive fluorescence in situ hybridization studies for the CCND1/IgH translocation, and clonal Ig gene rearrangement with mutated Ig genes (95.7% homology to VH 4-31). The subtle but diagnostic lymph node biopsy in this case helps to further support that an indolent t(11;14) monoclonal lymphocytosis with mutated Ig genes can represent an MCL variant rather than chronic lymphocytic leukemia.

Whole-Genome Scanning by Array Comparative Genomic Hybridization as a Clinical Tool for Risk Assessment in Chronic Lymphocytic Leukemia

Array-based comparative genomic hybridization (array CGH) provides a powerful method for simultaneous genome-wide scanning and prognostic marker assessment in chronic lymphocytic leukemia (CLL). In the current study, commercially available bacterial artificial chromosome and oligonucleotide array CGH platforms were used to identify chromosomal alterations of prognostic significance in 174 CLL cases. Tumor genomes were initially analyzed by bacterial artificial chromosome array CGH followed by confirmation and breakpoint mapping using oligonucleotide arrays. Genomic changes involving loci currently interrogated by fluorescence in situ hybridization (FISH) panels were detected in 155 cases (89%) at expected frequencies: 13q14 loss (47%), trisomy 12 (13%), 11q loss (11%), 6q loss (7.5%), and 17p loss (4.6%). Genomic instability was the second most commonly identified alteration of prognostic significance with three or more alterations involving loci not interrogated by FISH panels identified in 37 CLL cases (21%). A subset of 48 CLL cases analyzed by six-probe FISH panels (288 total hybridizations) was concordant with array CGH results for 275 hybridizations (95.5%); 13 hybridizations (4.5%) were discordant because of clonal populations that comprised less than 30% of the sample. Array CGH is a powerful, cost-effective tool for genome-wide risk assessment in the clinical evaluation of CLL.

T-Cell Large Granular Lymphocyte Leukemias Have Multiple Phenotypic Abnormalities Involving Pan-T-Cell Antigens and Receptors for MHC Molecules

T-cell large granular lymphocyte (T-LGL) leukemias represent monoclonal T-cell expansions that express CD16, CD56, or CD57 and cause cytopenias. The identification of T-LGL leukemias can be difficult because reactive T-LGL cells also can express CD16, CD56, and CD57, and many leukemia cases show only mild lymphocytoses. In this study, 23 T-LGL leukemia cases were analyzed by 3- and 4-color flow cytometry to identify markers that could aid in discriminating leukemic from normal T-LGL. In most cases (18/23), abnormalities (bright, dim, or negative expression) of 2 or more pan-T-cell antigens were identified, with all cases showing abnormal CD5 levels. Abnormal expression of CD94 was identified in 22 of 23 cases, and 15 of 21 cases also showed abnormal expression of class 1 MHC receptor molecules identified by antibodies against CD158a, CD158b, CD158e, CD158i, CD158k, and CD94. These studies help define abnormal phenotypic features typical of T-LGL leukemia that may have important diagnostic value.

Splenic Marginal Zone Lymphomas Appear to Originate from Different B Cell Types

Splenic marginal zone lymphomas (SMZLs) have been proposed to originate from postgerminal center memory B cells that usually have mutated immunoglobulin heavy-chain variable (VH) genes. However, the majority of SMZLs are thought to express both IgD and IgM, which is more typical of naïve B cells that have unmutated VH genes. To better define the SMZL cell of origin and pathogenesis, we studied the histological and immunophenotypic features of eight cases and also sequenced their rearranged VH genes. Half of the cases had unmutated VH genes consistent with a naïve B-cell origin and half had mutated VH genes consistent with a memory B-cell origin. Most of the unmutated cases (three of four) were positive for IgD, which further supports a naïve B-cell origin, whereas the others were negative. In addition, VH gene segment use seems to be nonrandom because seven of eight cases used genes from the VH1 or VH4 families and repetitive use of the V1-2, V1-69, and V4-34 gene segments was observed. Our results suggest there are two types of SMZLs, one that originates from naïve marginal zone B cells in addition to one that originates from memory marginal zone B cells, and that antigen selection may be occurring during lymphomagenesis.

Laboratory Practice Guidelines for Detecting and Reporting BCR-ABL Drug Resistance Mutations in Chronic Myelogenous Leukemia and Acute Lymphoblastic Leukemia: A Report of the Association for Molecular Pathology

The BCR-ABL tyrosine kinase produced by the t(9;22)(q34;q11) translocation, also known as the Philadelphia chromosome, is the initiating event in chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). Targeting of BCR-ABL with tyrosine kinase inhibitors (TKIs) has resulted in rapid clinical responses in the vast majority of patients with CML and Philadelphia chromosome+ ALL. However, long-term use of TKIs occasionally results in emergence of therapy resistance, in part through the selection of clones with mutations in the BCR-ABL kinase domain. We present here an overview of the current practice in monitoring for such mutations, including the methods used, the clinical and laboratory criteria for triggering mutational analysis, and the guidelines for reporting BCR-ABL mutations. We also present a proposal for a public database for correlating mutational status with in vitro and in vivo responses to different TKIs to aid in the interpretation of mutation studies.

Genomic analyses reveal recurrent mutations in epigenetic modifiers and the JAK–STAT pathway in Sézary syndrome

Sézary syndrome (SS) is an aggressive leukaemia of mature T cells with poor prognosis and limited options for targeted therapies. The comprehensive genetic alterations underlying the pathogenesis of SS are unknown. Here we integrate whole-genome sequencing (n=6), whole-exome sequencing (n=66) and array comparative genomic hybridization-based copy-number analysis (n=80) of primary SS samples. We identify previously unknown recurrent loss-of-function aberrations targeting members of the chromatin remodelling/histone modification and trithorax families, including ARID1A in which functional loss from nonsense and frameshift mutations and/or targeted deletions is observed in 40.3% of SS genomes. We also identify recurrent gain-of-function mutations targeting PLCG1 (9%) and JAK1, JAK3, STAT3 and STAT5B (JAK/STAT total ∼11%). Functional studies reveal sensitivity of JAK1-mutated primary SS cells to JAK inhibitor treatment. These results highlight the complex genomic landscape of SS and a role for inhibition of JAK/STAT pathways for the treatment of SS.

Hairy Cell Leukemia Variant Fact or Fiction

Hairy cell leukemia variant (HCL-V) is a poorly described, rare B-cell lymphoproliferative disorder typically positive for CD103 and CD11c, while lacking CD25. Splenic marginal zone lymphomas (SMZL) also have this unusual phenotype in 15% to 25% of cases, have other overlapping clinical or morphologic features, and are more common than HCL-V. The purpose of our study was to better characterize HCL-V and determine whether most cases could be distinguished from SMZL. Cases with an HCL-V phenotype were identified from our flow cytometry service, and 10 were selected for further study based on bone marrow or splenic tissue availability. All cases had cytologic features consistent with HCL-V, and 9 of 10 patients had lymphocytosis. Bone marrow involvement was mostly interstitial and/or sinusoidal without lymphoid nodules. Coexpression of preswitched with postswitched heavy chain isotypes, an unusual feature of HCL, was seen in 2 of 4 cases. This study better defines HCL-V and establishes that most cases do not represent SMZL.

Array CGH analysis of chronic lymphocytic leukemia reveals frequent cryptic monoallelic and biallelic deletions of chromosome 22q11 that include the PRAME gene

We used BAC array-based CGH to detect genomic imbalances in 187 CLL cases. Submicroscopic deletions of chromosome 22q11 were observed in 28 cases (15%), and the frequency of these deletions was second only to loss of the 13q14 region, the most common genomic aberration in CLL. Oligonucleotide-based array CGH analysis showed that the 22q11 deletions ranged in size from 0.34 Mb up to approximately 1 Mb. The minimally deleted region included the ZNF280A, ZNF280B, GGTLC2, and PRAME genes. Quantitative real-time PCR revealed that ZNF280A, ZNF280B, and PRAME mRNA expression was significantly lower in the 22q11 deletion cases compared to non-deleted cases.

Analysis of CD10+ Hairy Cell Leukemia

Hairy cell leukemia (HCL) has been reported to sometimes express CD10. However, the reported frequencies have been quite variable and the significance of CD10 expression has not been addressed. Cases of HCL submitted to our flow cytometry service during a 2-year period were evaluated for CD10 expression. Information regarding demographics, clinical manifestations, tissue morphologic features, and response to treatment was reviewed. Of the 97 HCL cases identified, 10 expressed CD10. The level of CD10 staining was typically well above control levels and also could be detected easily by immunohistochemical analysis. All cases analyzed were negative for bcl-6. Our study suggests that approximately 10% of otherwise typical cases of HCL show aberrant CD10 expression. CD10+ HCL cases seem to be morphologically and clinically similar to CD10-HCL cases. Appreciating that HCL can express CD10 may be especially important when evaluating specimens with suboptimal morphologic features and/or limited immunophenotyping panels.