SAVI: a statistical algorithm for variant frequency identification

BACKGROUND: Many problems in biomedical research can be posed as a comparison between related samples (healthy vs. disease, subtypes of the same disease, longitudinal data representing the progression of a disease, etc). In the cases in which the distinction has a genetic or epigenetic basis, next-generation sequencing technologies have become a major tool for obtaining the difference between the samples. A commonly occurring application is the identification of somatic mutations occurring in tumor tissue samples driving a single cell to expand clonally. In this case, the progression of the disease can be traced through the trajectory of the frequency of the oncogenic alleles. Thus obtaining precise estimates of the frequency of abnormal alleles at various stages of the disease is paramount to understanding the processes driving it. Although the procedure is conceptually simple, technical difficulties arise due to inhomogeneous samples, existence of competing subclonal populations, and systematic and non-systematic errors introduced by the sequencing technologies. RESULTS: We present a method, Statistical Algorithm for Variant Frequency Identification (SAVI), to estimate the frequency of alleles in a set of samples. The method employs Bayesian analysis and uses an iterative procedure to derive empirical priors. The approach allows for the comparison of allele frequencies across several samples, e.g. normal/tumor pairs and more complex experimental designs comparing multiple samples in tumor progression, as well as analyzing sequencing data from RNA sequencing experiments. CONCLUSIONS: Analyzing sequencing data through estimating allele frequencies using empirical Bayes methods is a powerful complement to the ever-increasing throughput of the sequencing technologies

Consensus opinion from an international group of experts on measurable residual disease in hairy cell leukemia

A significant body of literature has been generated related to the detection of measurable residual disease (MRD) at the time of achieving complete remission (CR) in patients with hairy cell leukemia (HCL). However, due to the indolent nature of the disease as well as reports suggesting long-term survival in patients treated with a single course of a nucleoside analog albeit without evidence of cure, the merits of detection of MRD and attempts to eradicate it have been debated. Studies utilizing novel strategies in the relapse setting have demonstrated the utility of achieving CR with undetectable MRD (uMRD) in prolonging the duration of remission. Several assays including immunohistochemical analysis of bone marrow specimens, multi-parameter flow cytometry and molecular assays to detect the mutant BRAF V600E gene or the consensus primer for the immunoglobulin heavy chain gene (IGH) rearrangement have been utilized with few comparative studies. Here we provide a consensus report on the available data, the potential merits of MRD assessment in the front-line and relapse settings and recommendations on future role of MRD assessment in HCL

Origin and pathogenesis of nodular lymphocyte–predominant Hodgkin lymphoma as revealed by global gene expression analysis

The pathogenesis of nodular lymphocyte–predominant Hodgkin lymphoma (NLPHL) and its relationship to other lymphomas are largely unknown. This is partly because of the technical challenge of analyzing its rare neoplastic lymphocytic and histiocytic (L&H) cells, which are dispersed in an abundant nonneoplastic cellular microenvironment. We performed a genome-wide expression study of microdissected L&H lymphoma cells in comparison to normal and other malignant B cells that indicated a relationship of L&H cells to and/or that they originate from germinal center B cells at the transition to memory B cells. L&H cells show a surprisingly high similarity to the tumor cells of T cell–rich B cell lymphoma and classical Hodgkin lymphoma, a partial loss of their B cell phenotype, and deregulation of many apoptosis regulators and putative oncogenes. Importantly, L&H cells are characterized by constitutive nuclear factor {kappa}B activity and aberrant extracellular signal-regulated kinase signaling. Thus, these findings shed new light on the nature of L&H cells, reveal several novel pathogenetic mechanisms in NLPHL, and may help in differential diagnosis and lead to novel therapeutic strategies

Gene Expression Profiling of Hairy Cell Leukemia Reveals a Phenotype Related to Memory B Cells with Altered Expression of Chemokine and Adhesion Receptors

Hairy cell leukemia (HCL) is a chronic B cell malignancy characterized by the diffuse infiltration of bone marrow and spleen by cells displaying a typical “hairy” morphology. However, the nature of the HCL phenotype and its relationship to normal B cells and to other lymphoma subtypes remains unclear. Using gene expression profiling, we show here that HCL displays a homogeneous pattern of gene expression, which is clearly distinct from that of other B cell non-Hodgkin lymphomas. Comparison with the gene expression profiles of purified normal B cell subpopulations, including germinal center (GC), pre-GC (naive), and post-GC (memory) B cells, shows that HCL cells are more related to memory cells, suggesting a derivation from this B cell population. Notably, when compared with memory cells, HCL cells displayed a remarkable conservation in proliferation, apoptosis, and DNA metabolism programs, whereas they appeared significantly altered in the expression of genes controlling cell adhesion and response to chemokines. Finally, these analyses have identified several genes that are specifically expressed in HCL and whose expression was confirmed at the protein level by immunocytochemical analysis of primary HCL cases. These results have biological implications relevant to the pathogenesis of this malignancy as well as clinical implications for its diagnosis and therapy

Origin and pathogenesis of nodular lymphocyte–predominant Hodgkin lymphoma as revealed by global gene expression analysis

The pathogenesis of nodular lymphocyte–predominant Hodgkin lymphoma (NLPHL) and its relationship to other lymphomas are largely unknown. This is partly because of the technical challenge of analyzing its rare neoplastic lymphocytic and histiocytic (L&H) cells, which are dispersed in an abundant nonneoplastic cellular microenvironment. We performed a genome-wide expression study of microdissected L&H lymphoma cells in comparison to normal and other malignant B cells that indicated a relationship of L&H cells to and/or that they originate from germinal center B cells at the transition to memory B cells. L&H cells show a surprisingly high similarity to the tumor cells of T cell–rich B cell lymphoma and classical Hodgkin lymphoma, a partial loss of their B cell phenotype, and deregulation of many apoptosis regulators and putative oncogenes. Importantly, L&H cells are characterized by constitutive nuclear factor κB activity and aberrant extracellular signal-regulated kinase signaling. Thus, these findings shed new light on the nature of L&H cells, reveal several novel pathogenetic mechanisms in NLPHL, and may help in differential diagnosis and lead to novel therapeutic strategies

Expression of the IRTA1 receptor identifies intraepithelial and subepithelial marginal zone B cells of the mucosa-associated lymphoid tissue (MALT)

AbstractIRTA1 (immunoglobulin superfamily receptor translocation-associated 1) is a novel surface B-cell receptor related to Fc receptors, inhibitory receptor superfamily (IRS), and cell adhesion molecule (CAM) family members and we mapped for the first time its distribution in human lymphoid tissues, using newly generated specific antibodies. IRTA1 was selectively and consistently expressed by a B-cell population located underneath and within the tonsil epithelium and dome epithelium of Peyer patches (regarded as the anatomic equivalents of marginal zone). Similarly, in mucosa-associated lymphoid tissue (MALT) lymphomas IRTA1 was mainly expressed by tumor cells involved in lympho-epithelial lesions. In contrast, no or a low number of IRTA1+ cells was usually observed in the marginal zone of mesenteric lymph nodes and spleen. Interestingly, monocytoid B cells in reactive lymph nodes were strongly IRTA1+. Tonsil IRTA1+ cells expressed the memory B-cell marker CD27 but not mantle cell-, germinal center-, and plasma cell-associated molecules. Polymerase chain reaction (PCR) analysis of single tonsil IRTA1+ cells showed they represent a mixed B-cell population carrying mostly mutated, but also unmutated, IgV genes. The immunohistochemical finding in the tonsil epithelial areas of aggregates of IRTA1+ B cells closely adjacent to plasma cells surrounding small vessels suggests antigen-triggered in situ proliferation/differentiation of memory IRTA1+ cells into plasma cells. Collectively, these results suggest a role of IRTA1 in the immune function of B cells within epithelia. (Blood. 2003;102: 3684-3692

Identification of novel STAT5B mutations and characterization of TCR beta signatures in CD4+T-cell large granular lymphocyte leukemia

CD4+ T-cell large granular lymphocyte leukemia (T-LGLL) is a rare subtype of T-LGLL with unknown etiology. In this study, we molecularly characterized a cohort of patients (n = 35) by studying their T-cell receptor (TCR) repertoire and the presence of somatic STAT5B mutations. In addition to the previously described gain-of-function mutations (N642H, Y665F, Q706L, S715F), we discovered six novel STAT5B mutations (Q220H, E433K, T628S, P658R, P702A, and V712E). Multiple STAT5B mutations were present in 22% (5/23) of STAT5B mutated CD4+ T-LGLL cases, either coexisting in one clone or in distinct clones. Patients with STAT5B mutations had increased lymphocyte and LGL counts when compared to STAT5B wild-type patients. TCR beta sequencing showed that, in addition to large LGL expansions, non-leukemic T cell repertoires were more clonal in CD4+ T-LGLL compared to healthy. Interestingly, 25% (15/59) of CD4+ T-LGLL clonotypes were found, albeit in much lower frequencies, in the non-leukemic CD4+ T cell repertoires of the CD4+ T-LGLL patients. Additionally, we further confirmed the previously reported clonal dominance of TRBV6-expressing clones in CD4+ T-LGLL. In conclusion, CD4+ T-LGLL patients have a typical TCR and mutation profile suggestive of aberrant antigen response underlying the disease.Peer reviewe

Dactinomycin induces complete remission associated with nucleolar stress response in relapsed/refractory NPM1-mutated AML

Acute myeloid leukemia (AML) with mutated NPM1 accounts for one-third of newly diagnosed AML. Despite recent advances, treatment of relapsed/refractory NPM1-mutated AML remains challenging, with the majority of patients eventually dying due to disease progression. Moreover, the prognosis is particularly poor in elderly and unfit patients, mainly because they cannot receive intensive treatment. Therefore, alternative treatment strategies are needed. Dactinomycin is a low-cost chemotherapeutic agent, which has been anecdotally reported to induce remission in NPM1-mutated patients, although its mechanism of action remains unclear. Here, we describe the results of a single-center phase 2 pilot study investigating the safety and efficacy of single-agent dactinomycin in relapsed/refractory NPM1-mutated adult AML patients, demonstrating that this drug can induce complete responses and is relatively well tolerated. We also provide evidence that the activity of dactinomycin associates with nucleolar stress both in vitro and in vivo in patients. Finally, we show that low-dose dactinomycin generates more efficient stress response in cells expressing NPM1 mutant compared to wild-type cells, suggesting that NPM1-mutated AML may be more sensitive to nucleolar stress. In conclusion, we establish that dactinomycin is a potential therapeutic alternative in relapsed/refractory NPM1-mutated AML that deserves further investigation in larger clinical studies

B-cell receptor reactivity against Rothia mucilaginosa in nodular lymphocyte-predominant Hodgkin lymphoma

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a Hodgkin lymphoma expressing functional B-cell receptors (BCR). Recently, we described a dual stimulation model of IgD+ lymphocyte-predominant cells by Moraxella catarrhalis antigen RpoC and its superantigen MID/hag, associated with extralong CDR3 and HLA-DRB1*04 or HLADRB1*07 haplotype. The aim of the present study was to extend the antigen screening to further bacteria and viruses. The fragment antibody-binding (Fab) regions of seven new and 15 previously reported cases were analyzed. The reactivity of non-Moraxella spp.-reactive Fab regions against lysates of Rothia mucilaginosa was observed in 5/22 (22.7%) cases. Galactofuranosyl transferase (Gltf) and 2,3-butanediol dehydrogenase (Bdh) of R. mucilaginosa were identified by comparative silver- and immuno-staining in two-dimensional gels, with subsequent mass spectrometry and validation by western blots and enzyme-linked immunosorbent assay. Both R. mucilaginosa Gltf and Bdh induced BCR pathway activation and proliferation in vitro. Apoptosis was induced by recombinant Gltf/ETA’-immunotoxin conjugates in DEV cells expressing recombinant R. mucilaginosa-reactive BCR. Reactivity against M. catarrhalis RpoC was confirmed in 3/7 newly expressed BCR (total 10/22 reactive to Moraxella spp.), resulting in 15/22 (68.2%) cases with BCR reactivity against defined bacterial antigens. These findings strengthen the hypothesis of bacterial trigger contributing to subsets of NLPHL