OBJECTIVE: To explore the expression level of PLK1 in mantle cell lymphoma(MCL), and the effect of silencing PLK1 gene by RNA interference on the cell proliferation, apoptosis, and cell cycle.
METHODS: S-P immunohistochemistry technique was used to detect the expression of PLK1 in tissues of 42 patients with MCL and 30 patients with reactive proliferative lymphodenitis(RPL), their expression levels were compared and analyzed. The Jeko-1 cells were transfected with lentivirus contaiming PLK-1 shRNA, then the mRNA and protein expression of PLK-1 was detected by real-time guantitative PCR and Western blot nespectively, and the silencing efficacy of PLK-1 shRNA was identificd. The cell proliferation was detected by CCK method, the cell apoptosis was detected by Annexin V/PI double staining, the cell cycle was detected by PI single staining, the changes of apoptosis-related proteins BAX, BCL-2 and Caspase 3 were detected by Western blot.
RESULTS: The positive expression rate of PLK-1 in tissue of MCL patients was 66.67%(28/42), which was significanfly higher than 20%(6/30) in tissue of RPL patients (P＜0.05). The PLK-1 positive expression correlated with B symptom, IPI score, Ann-Arbor stage(P＜0.05). After infection of Jeko-1 cells with lentivirus containing PLK-1 shRNA for 72 hours, the mRNA and protein expressions of PLK-1 were significantly down-regulated(P＜0.05), the proliferation rate of cells in group of PLK-1 shRNA was significanly lower than that in control and Neg shRNA groups(P＜0.05); the apoptosis rate of cells in PLK-1 shRNA group was (27.42±3.44)%, which was significantly higher than that in control group (1.23±0.42)% and Neg shRNA group (2.07±0.58) % (P＜0.05). The cell cycle analysis showed that the cell ratio in G
CONCLUSION: The PLK-l overexpression appears in tissue of MCL patients. The silencing PLK-1 gene can inhibit the proliferation of Jeko-1 cells, induce the apopotosis of Jeko-1 cells and arrestes cell cycle in G

OBJECTIVE: To investigate the expression of miR-101 and EZH2 in patients with mantle cell lymphoma(MCL) and to analyze its correlation with clinical prognosis of MCL patients.
METHODS: RQ-PCR and S-P immunohistochemistry were used to detect the expressions of miR-101 and EZH2 in tissue of MCL patients. CCK-8 was used to assay the effect of miR-100 minics on the proliferation of Jeko-1 and Mino cells; the flow cytometry with Annexin V/PI double staining was used to assay the apoptosis; Western blot was used to assay the effect of miR-101 minics on the expression of EZH2 protein in Jeko-1 and Mino cells.
RESULTS: Compared with control group, miR-101 lowly expressed, and EZH2 protein highly expressed in MCL group, with very statistically significant difference(P<0.01).There was negative correlation between miR-101 and EZH2 expression(r=-0.638，P<0.05). The expression of miR-101 and EZH2 significantly correlated with B symptoms, International Prognostic Index(IPI) and Ann Arbor stage, respectively. Survival analysis showed that the overall survival(OS) rate of patients with low expression of miR-101 were significantly lower than that of patients with high miR-101 expression (P=0.0014), the OS rate of patients with EZH2 high expression were significantly lower than that of patients with EZH2 low expression (P=0.0093). The miR-100 minics could inhibit the proliferation of Jeko-1 and Mino cells, and increase the apoptotic rate. The expression of EZH2 protein was markedly suppressed by the miR-100 minics.
CONCLUSION: The expression of miR-101 and EZH2 is different in MCL patients with different clinical stage and prognosis. The miR-101 can inhibit the cell proliferation and induce cell apoptosis of mantle cell lymphoma by targeting EZH2.

Hyper-activated STAT5B variants are high value oncology targets for pharmacologic intervention. STAT5B

Mature T- and NK-cell neoplasms are a heterogeneous hematological malignancy. The current treatment of mature T- and NK-cell lymphoma depends on combination chemotherapy with or without auto/allogeneic hematopoietic stem cell transplantation. Recent comprehensive, integrated genetic analyses have revealed distinct genetic and molecular subgroups, which are related to different therapeutic responses. Thus, the genetic landscape of mature T- and NK-cell neoplasms is essential for the development of novel treatment modalities and offers opportunities for individualized therapy. This review aims to discuss the recent progress regarding genetic and molecular analyses of mature T- and NK-cell neoplasms toward stratified therapy.

Among the hematologic tumors, lymphoid malignancies, especially B-cell lymphomas (BLs), are categorized according to the WHO classification into a large number of entities, which are aggregates of clinically and biologically diverse diseases. It is thought that various molecular genetic abnormalities are involved in lymphoid tumorigenesis. Recent advances in genetic analysis technology have resulted in the discovery of many novel and recurrent genetic abnormalities. In particular, those associated with diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma have been identified utilizing molecular pathology techniques, which is important aspect for the future development of novel therapeutic drugs and indications for clinical trials. In this section, the possibility of patient-stratification based on genetic abnormalities of DLBCL and FL, which are particularly prevalent among mature BLs, are described. I also introduce the genetic features and clinical importance of BL and MCL identified in a small number of patients in Japan.

Immunoglobulin heavy chain variable region (IGHV) gene mutation status is a biomarker for the prognosis of chronic lymphocytic leukemia, whether it is associated with the diagnosis, staging, and prognosis of patients with mantle cell lymphoma (MCL) remains to be determined.The IGHV gene mutations of 52 MCL patients were determined by DNA sequencing and compared with published IGHV germline sequences.DNA sequence alignment of IGHV variable regions with published IGHV germline sequences showed that the coincidence rate was 94% to 100%. Ten cases (21%) were significantly mutated with the rate of 96.9% to 94.0%. The overall survival time of patients was negatively correlated with the degree of IGHV gene mutation. Further survival analysis with log-rank test demonstrated that the patients with significant IGHV gene mutations showed a trend towards poor survival.The mutation rate of the IGHV variant region may be determined to assess the prognosis and overall survival time of MCL patients.

Patients transplanted from solid organs have an increased risk of cancer, especially lymphomas. Lymphomas correspond to 4 to 5% of malignant neoplasms in the general population and in solid organ transplant patients it reaches an incidence of 21%. The incidence of non-Hodgkin lymphomas is 10 times higher than in the non-transplanted population. We report the case of a 68-year-old man with a kidney transplant who 6 years after transplantation, developed a non-Hodgkin diffuse large cells B lymphoma with lymph node and pulmonary involvement, with markers of very poor prognosis (triple MYC expressor, BCL2 and BCL6). and its evolution with chemotherapy with DA R EPOCH.

The association of HCV-infection with B-lymphomas is supported by the regression of most indolent/low-grade lymphomas following anti-viral therapy. Studies on direct and indirect oncogenic mechanisms have elucidated the pathogenesis of HCV-associated B-lymphoma subtypes. These include B-lymphocyte proliferation and sustained clonal expansion by HCV-envelope protein stimulation of B-cell receptors, and prolonged HCV-infected B-cell growth by overexpression of an anti-apoptotic BCL-2 oncogene caused by the increased frequency of t(14;18) chromosomal translocations in follicular lymphomas. HCV has been implicated in lymphomagenesis by a "hit-and-run" mechanism, inducing enhanced mutation rate in immunoglobulins and anti-oncogenes favoring immune escape, due to permanent genetic damage by double-strand DNA-breaks. More direct oncogenic mechanisms have been identified in cytokines and chemokines in relation to NS3 and Core expression, particularly in diffuse large B-cell lymphoma. By reviewing genetic alterations and disrupted signaling pathways, we intend to highlight how mutually non-contrasting mechanisms cooperate with environmental factors toward progression of HCV-lymphoma.

BACKGROUND: Various subsets of diffuse large B-cell lymphoma(DLBCL) are distinguished based on molecular and immunohistochemical features. As we know, CD5 is a pan-T-cell surface marker and is seldom expressed in DLBCL. Large-scale studies of de novo CD5+ DLBCL are lacking in Chinese patients.
METHOD: A total of 139 patients with DLBCL (30 CD5+ DLBCL and 109 CD5- DLBCL) who were immunophenotyped and treated with chemotherapy were subjected to this analysis. There were 85 males and 54 females. Their age ranged from 17 to 84 years old, and the median age was 58 years old.
RESULTS: In this study CD5+ DLBCL was associated with higher IPI scores (>2), bone marrow involvement, higher probability of >1 ECOG performance status, non-germinal center B-cell like(non-GCB), BCL2 overexpression, whereas seldom expressed CD10 or BCL6, and unconspicuous higher expression of Ki67. With standard chemotherapy, CD5+ DLBCL patients had significantly worse overall survival (OS, median, 29.5 months vs. not reached, P = 0.0004) and progression-free survival (PFS, median, 18.0 months vs. not reached, P = 0.0002) than CD5- DLBCL patients, which had independent prognostic significance of the International Prognostic Index (IPI), and subtype of the non-GCB DLBCL. For CD5+ DLBCL, the addition of rituximab to chemotherapy may not significantly improve the OS (median, 14 months vs. 29.5 months, P = 0.72) and PFS (median, 10 months vs. 12 months, P = 0.92).
CONCLUSION: CD5+ DLBCL patients have the distinctive clinical and biological features, they should be provided with clinic individualized treatment and important pathways with therapeutic implications should be underscored.

Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of lymphoid malignancies. Several recent comprehensive genomic studies characterize the genomic alterations of each PTCL type and reveal the complexity and heterogeneity. The updated World Health Organization classification has precisely distinguished "lymphoma" from "lymphoproliferative disorder" and has included a new entity based on the tumor phenotypes. Although establishing the classifications based on genomic alterations has been difficult because of heterogeneity, the genomic alterations may support the diagnosis. Establishing genomic alterations that act as predictive markers for clinical courses and/or therapeutic targets is needed in future studies using genomic alteration information. Since patients with PTCL generally have poor prognosis, establishing the target and standard therapies is one of the major issues to be addressed. In addition to clinicopathological and genomic analyses, patient-derived xenograft models can provide these therapeutic strategies. Integration of analyzed data is considered to promote future PTCL studies, leading to improved PTCL prognosis.

Non-coding RNAs (ncRNAs) are essential regulators of gene expression. In recent years, it has become more and more evident that the different classes of ncRNAs, such as micro RNAs, long non-coding RNAs and circular RNAs are organized in tightly controlled networks. It has been suggested that deregulation of these networks can lead to disease. Several studies show a contribution of these so-called competing-endogenous RNA networks in various cancer entities. In this review, we highlight the involvement of ncRNA networks in anaplastic-large cell lymphoma (ALCL), a T-cell neoplasia. A majority of ALCL cases harbor the molecular hallmark of this disease, a fusion of the anaplastic lymphoma kinase (ALK) gene with the nucleophosmin (NPM, NPM1) gene leading to a permanently active kinase that promotes the malignant phenotype. We have focused especially on ncRNAs that are regulated by the

BACKGROUND: Mycosis fungoides (MF) is indolent, but may disseminate to leukemia. We reported that C-C motif chemokine ligand 21 (CCL21) is associated with MF invasion and progression. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long noncoding RNA, is associated with several cancer types, however, how it interacts with CCL21 to regulate MF progression, remains unclear.
MATERIALS AND METHODS: Expression of long noncoding RNAs MALAT1, antisense noncoding RNA in the INK4 locus (ANRIL), Hox antisense intergenic RNA (HOTAIR), highly up-regulated in liver cancer RNA (HULC), and leukemia-associated non-coding insulin-like growth factor 1 receptor activator RNA 1 (LUNAR1) in tissues from MF was studied using polymerase chain reaction and RNA interference in MF cell line MyLa were used to address this question.
RESULTS: Expression of MALAT1 was selectively increased in MF tissues. C-C Chemokine receptor type 7 (CCR7) expression was found to be increased in MyLa cells. CCL21 was found not only to mediate migration, but also to enhance MALAT1 and mammalian target of rapamycin (mTOR) activation in MyLa cells. Knockdown of MALAT1 abrogated CCL21-mediated migration, but not mTOR activation. In contrast, mTOR inhibition reduced CCL21-mediated migration and MALAT1 expression.
CONCLUSION: CCL21 induced mTOR activation in MyLa cells, followed by expression of MALAT1, causing cell migration. MALAT1 and mTOR are potential therapeutic targets for MF.

OBJECTIVE: To investigate the expression of miR-155 in patients with diffuse large B-cell lymphoma (DLBCL), and to explore the effect of transfection of miR-155 inhibitor on the biological characteristics of DLBCL cells.
METHODS: A total of 76 patients with DLBCL treated in our hospital were selected from April 2013 to December 2017. In the same time, 40 cases of lymph node reactive hyperplasia (LNRH) were selected as control group. DB cells were cultured and divided into miR-155 inhibitor, negative control and blank groups. The expressions of miR-155 in DLBCL, negative and blank control groups were detected by using real-time PCR, the cell proliferation was detected by MTT assay, the apoptosis was detected by flow cytometry, and the cell migration and invasion were detected by Transwell assay.
RESULTS: The relative expression level of miR-155 in tissues of DLBCL patients was significantly higher than that in tissne of controls (1.93±0.16 vs 1.01±0.09) (t=33.991, P=0.000). The expression level of miR-155 increased (P＜0.05) in DLBCL patients with LDH level abnormarity, BCL-2
CONCLUSION: The miR-155 highly expresses in DLBCL tissue, which relates with tumor malignancy and invasion progression. The specific inhibition of miR-155 expression in DB cells can reduce cell proliferation, accelerate cell apoptosis, and inhibit cell migration and invasion.

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A), expressed in EBV latency, contributes to Burkitt lymphoma (BL) development in a murine model by acting as a constitutively active B cell receptor (BCR) mimic. Mice expressing both LMP2A and

BACKGROUND Mantle cell lymphoma (MCL) is a high-grade B-cell lymphoma with poor prognosis. Fludarabine is used alone or in combination for relapsed and advanced-stage MCL. The expression of the signal transducer and activator of transcription 5B (STAT5B) gene is associated with tumorigenesis in solid tumors, but its role in MCL remains unknown. The aims of this study were to investigate the role of STAT5B in GRANTA-519 human mantle cell lymphoma cells and drug resistance. MATERIAL AND METHODS GRANTA-519 human mantle cell lymphoma cells were cultured with and without 10 μM fludarabine dephosphorylated 9-ß-D-arabinofuranosyl-2-fluoroadenine, (2-F-araA) or 10 μM 4-hydroperoxycyclophosphamide (4-HC). The MTT assay assessed cell proliferation. Flow cytometry was used to investigate the cell cycle in MCL cells treated with the specific inhibitor of the Akt pathway, LY294002, and assessed cell cycle and cell apoptosis. Western blot was used to detect the expression levels of p-Akt/Akt and STAT5B/p-STAT5B. The gene expression profiles of lymph node (LN)-derived MCL cells were compared with peripheral blood (PB)-derived lymphocytes using bioinformatics and hierarchical cluster analysis. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed to determine the expression of the marker of proliferation Ki-67 (MKI67) gene. RESULTS STAT5B was significantly upregulated in LN-derived MCL cells compared with PB lymphocytes. Increased expression of STAT5B was associated with increased MCL cell proliferation and reduced cell apoptosis and was associated with drug resistance and activation of Akt. CONCLUSIONS STAT5B promoted cell proliferation and drug resistance in human MCL cells by activating the Akt signaling pathway.

BACKGROUND: MYC is a heterogeneously expressed transcription factor that plays a multifunctional role in many biological processes such as cell proliferation and differentiation. It is also associated with many types of cancer including the malignant lymphomas. There are two types of aggressive B-cell lymphoma, namely Burkitt lymphoma (BL) and a subgroup of diffuse large cell lymphoma (DLBCL), which both carry MYC translocations and overexpress MYC but both differ significantly in their clinical outcome. In DLBCL, MYC translocations are associated with an aggressive behavior and poor outcome, whereas MYC-positive BL show a superior outcome.
METHODS: To shed light on this phenomenon, we investigated the different modes of actions of MYC in aggressive B-cell lymphoma cell lines subdivided into three groups: (i) MYC-positive BL, (ii) DLBCL with MYC translocation (DLBCLpos) and (iii) DLBCL without MYC translocation (DLBCLneg) for control. In order to identify genome-wide MYC-DNA binding sites a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) was performed. In addition, ChIP-Seq for H3K4me3 was used for determination of genomic regions accessible for transcriptional activity. These data were supplemented with gene expression data derived from RNA-Seq.
RESULTS: Bioinformatics integration of all data sets revealed different MYC-binding patterns and transcriptional profiles in MYC-positive BL and DLBCL cell lines indicating different functional roles of MYC for gene regulation in aggressive B-cell lymphomas. Based on this multi-omics analysis we identified ADGRE5 (alias CD97) - a member of the EGF-TM7 subfamily of adhesion G protein-coupled receptors - as a MYC target gene, which is specifically expressed in BL but not in DLBCL regardless of MYC translocation.
CONCLUSION: Our study describes a diverse genome-wide MYC-DNA binding pattern in BL and DLBCL cell lines with and without MYC translocations. Furthermore, we identified ADREG5 as a MYC target gene able to discriminate between BL and DLBCL irrespectively of the presence of MYC breaks in DLBCL. Since ADGRE5 plays an important role in tumor cell formation, metastasis and invasion, it might also be instrumental to better understand the different pathobiology of BL and DLBCL and help to explain discrepant clinical characteristics of BL and DLBCL.

Burkitt lymphoma (BL) is the most common B-cell lymphoma in children. Within the International Cancer Genome Consortium (ICGC), we performed whole genome and transcriptome sequencing of 39 sporadic BL. Here, we unravel interaction of structural, mutational, and transcriptional changes, which contribute to MYC oncogene dysregulation together with the pathognomonic IG-MYC translocation. Moreover, by mapping IGH translocation breakpoints, we provide evidence that the precursor of at least a subset of BL is a B-cell poised to express IGHA. We describe the landscape of mutations, structural variants, and mutational processes, and identified a series of driver genes in the pathogenesis of BL, which can be targeted by various mechanisms, including IG-non MYC translocations, germline and somatic mutations, fusion transcripts, and alternative splicing.

B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.

Translocation (11;18)(q21;q21) is found in mucosa-associated lymphoid tissue (MALT) lymphoma, resulting in API2/MALT1 gene fusion. It is known that t(11;18)-positive MALT lymphoma shows a tendency to disseminate and be resistant to Helicobacter pylori eradication by antibiotics. However, the prognostic features including recurrence and histological transformation (HT) remain unknown. We conducted a single-institute retrospective analysis of 464 patients with newly diagnosed MALT lymphoma, evaluating the impact of t(11;18) on clinical outcomes. One hundred and six patients were screened for the translocation by fluorescence in situ hybridization and/or reverse transcriptase-polymerase chain reaction. Of these patients, 26 patients (25%) were diagnosed as MALT lymphoma with t(11;18). The patients had a significantly shortened progression-free survival (PFS at 10 years; 26% v 57%; P = 0.004) compared to those without t(11;18). However, this did not translate into overall survival or incidence of HT. We confirmed previous reports stating that t(11;18)-positive MALT lymphoma showed disseminated disease and refractoriness to H. pylori eradication therapy. Patients with t(11;18) had more frequent monoclonal gammopathy, especially of IgM subtype (31% v 8%; P = 0.008), some of which developed class switch. These findings characterize the features of t(11;18)-positive MALT lymphoma, suggesting that it comprises a distinct clinical entity of MALT lymphoma.

Circular RNAs (circRNAs) act as pivotal functions in tumor progression. Nevertheless, the functions and mechanism of circRNAs in T-cell lymphoblastic lymphoma (T-LBL) remain unclear. In this work, we first screened the differentially expressed circRNAs between T-LBL tissues and normal infantile thymus and circ-LAMP1 was identified the highest expressed circRNA in cancerous tissues. qRT-PCR further verified its upregulation in T-LBL tissues and cell lines. Cell counting kit-8 (CCK-8) experiment proved the cell proliferation-promoting role of circ-LAMP1. This effect is partially dependent on its inhibition on cell apoptosis proved by flow cytometric assay. Dual-luciferase reporter system further identified that miR-615-5p could be sponged by circ-LAMP1 and discoidin domain receptor tyrosine kinase 2 (DDR2) 3'-UTR is the direct target of miR-615-5p. Rescue assays demonstrated that the biological function of circ-LAMP1 is partly attributed to the modulation of miR-615-5p/DDR2 signaling. In summary, these findings documented that circ-LAMP1 might be an oncogene in T-LBL, which might be useful in developing promising therapies for T-LBL.

BACKGROUND: Eukaryotes compact chromosomes densely and non-randomly, forming three-dimensional structures. Alterations of the chromatin structures are often associated with diseases. In particular, aggressive cancer development from the disruption of the humoral immune system presents abnormal gene regulation which is accompanied by chromatin reorganizations. How the chromatin structures orchestrate the gene expression regulation is still poorly understood. Herein, we focus on chromatin dynamics in normal and abnormal B cell lymphocytes, and investigate its functional impact on the regulation of gene expression.
METHODS: We conducted an integrative analysis using publicly available multi-omics data that include Hi-C, RNA-seq and ChIP-seq experiments with normal B cells, lymphoma and ES cells. We processed and re-analyzed the data exhaustively and combined different scales of genome structures with transcriptomic and epigenetic features.
RESULTS: We found that the chromatin organizations are highly preserved among the cells. 5.2% of genes at the specific repressive compartment in normal pro-B cells were switched to the permissive compartment in lymphoma along with increased gene expression. The genes are involved in B-cell related biological processes. Remarkably, the boundaries of topologically associating domains were not enriched by CTCF motif, but significantly enriched with Prdm1 motif that is known to be the key factor of B-cell dysfunction in aggressive lymphoma.
CONCLUSIONS: This study shows evidence of a complex relationship between chromatin reorganization and gene regulation. However, an unknown mechanism may exist to restrict the structural and functional changes of genomic regions and cognate genes in a specific manner. Our findings suggest the presence of an intricate crosstalk between the higher-order chromatin structure and cancer development.

Chromosomal microdeletions frequently cause loss of prognostically relevant tumor suppressor genes in hematologic malignancies; however, detection of minute deletions by conventional methods for chromosomal analysis, such as G-banding and fluorescence in situ hybridization (FISH), is difficult due to their low resolution. Here, we describe a new diagnostic modality that enables detection of chromosomal microdeletions, using CDKN2A gene deletion in B cell lymphomas (BCLs) as an example. In this method, which we refer to as amplified-FISH (AM-FISH), a 31-kb fluorescein isothiocyanate (FITC)-conjugated DNA probe encoding only CDKN2A was first hybridized with the chromosome, and then labeled with Alexa Fluor 488-conjugated anti-FITC secondary antibody to increase sensitivity. CDKN2A signals were equally identifiable by AM-FISH and conventional FISH in normal mononuclear blood cells. In contrast, when two BCL cell lines lacking CDKN2A were analyzed, CDKN2A signals were not detected by AM-FISH, whereas conventional FISH yielded false signals. Furthermore, AM-FISH detected CDKN2A deletions in two BCL patients with 9p21 microdeletions, which were not detected by conventional FISH. These results suggest that AM-FISH is a highly sensitive, specific, and simple method for diagnosis of chromosomal microdeletions.

Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a rare T-cell lymphoproliferative disorder occurring in patients with breast implants. Genomic characterization performed in BIA-ALCL to date has demonstrated qualitatively similar molecular abnormalities to those seen in its more common counterpart [ALK-negative systemic anaplastic large cell lymphoma (sALCL)] including JAK/STAT activation and MYC/TP53 dysregulation. Despite these observed similarities at the molecular level, the outcomes of sALCL and BIA-ALCL are markedly different with sALCL typically associated with an aggressive course and inferior outcomes compared with BIA-ALCL. This review describes the findings of high-throughput sequencing and other genomic characterization to date in BIA-ALCL and the insights these studies have given into the molecular drivers of this rare lymphoma subtype.

High-throughput mRNA sequencing (RNA-Seq) provides both qualitative and quantitative evaluation of the transcriptome. This method uses complementary DNA (cDNA) to generate several millions of short sequence reads that are aligned to a reference genome allowing the comprehensive characterization of the transcripts in a cell. RNA-Seq has a wide variety of applications which lead to a pervasive adoption of this method well beyond the genomics community and a deployment of this technique as a standard part of the toolkit applied in life sciences. This chapter describes a protocol to perform mRNA sequencing using the Illumina NextSeq or MiSeq platforms, presents sequencing data quality metrics, and outlines a bioinformatic pipeline for sequence alignment, digital gene expression, identification of gene fusions, detection of transcript isoforms, description and annotation of genetic variants, and de novo immunoglobulin gene assembly.

MicroRNAs (miRNAs) play important roles in development, differentiation, and homeostasis by regulating protein translation. In B-cell lymphoma, many miRNAs have altered expression levels, and for a limited subset of them, experimental data supports their functional relevance in lymphoma pathogenesis. This chapter describes an unbiased next-generation sequencing (NGS)-based high-throughput screening approach to identify miRNAs that are involved in the control of cell growth. First, we provide a protocol for performing high-throughput screening for miRNA inhibition and overexpression. Second, we describe the procedure for next-generation sequencing library preparation. Third, we provide a workflow for data analysis.

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell non-Hodgkin lymphomas (B-NHL) and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. MRD diagnostics performed by real-time quantitative PCR (RQ-PCR) is still the gold standard and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). Alternatively, droplet digital PCR (ddPCR) can be used for MRD monitoring in multiple myeloma, mantle cell lymphoma, and follicular lymphoma with comparable sensitivity, accuracy, and reproducibility.The most broadly applicable MRD target in B-NHL is the junctional regions of the rearranged immunoglobulin heavy chain gene (IGHV). Chromosomal translocations like the t(14;18) translocation in FL and t(11;14) translocation in MCL can be used as MRD target in selected lymphoma subtypes. In patients with B-cell chronic lymphocytic leukemia, both flow-cytometry and RQ-PCR are equally suited for MRD assessment as long as a sensitivity of 10

Comprehensive analysis of the clonotypic B cell receptor immunoglobulin (BcR IG) gene rearrangement sequences in patients with mature B cell neoplasms has led to the identification of significant repertoire restrictions, culminating in the discovery of subsets of patients expressing highly similar, stereotyped BcR IG. This finding strongly supports selection by common epitopes or classes of structurally similar epitopes in the ontogeny of these tumors. BcR IG stereotypy was initially described in chronic lymphocytic leukemia (CLL), where the stereotyped fraction of the disease accounts for a remarkable one-third of patients. However, subsequent studies showed that stereotyped BcR IG are also present in other neoplasms of mature B cells, including mantle cell lymphoma (MCL) and splenic marginal zone lymphoma (SMZL). Subsequent cross-entity comparisons led to the conclusion that stereotyped IG are mostly "disease-specific," implicating distinct immunopathogenetic processes. Interestingly, mounting evidence suggests that a molecular subclassification of lymphomas based on BcR IG stereotypy is biologically and clinically relevant. Indeed, particularly in CLL, patients assigned to the same subset due to expressing a particular stereotyped BcR IG display remarkably consistent biological background and clinical course, at least for major and well-studied subsets. Thus, the robust assignment to stereotyped subsets may assist in the identification of mechanisms underlying disease onset and progression, while also refining risk stratification. In this book chapter, we provide an overview of the recent BcR IG stereotypy studies in mature B cell malignancies and outline previous and current methodological approaches used for the identification of stereotyped IG.

Normal and malignant B cells carry rearranged immunoglobulin (Ig) variable region genes, which due to their practically limitless diversity represent ideal clonal markers for these cells. We describe here an approach to isolate single cells from frozen tissue sections by microdissection using a laser-based method. From the isolated cells, rearranged IgH and Igκ genes are amplified in a semi-nested PCR approach, using a collection of V gene subgroup-specific primers recognizing nearly all V genes together with primers for the J genes. By sequence analysis of V region genes from distinct cells, the clonal relationship of the B lineage cells can unequivocally be determined and related to the histological distribution of the cells. The approach is also useful to determine V, D, and J gene usage. Moreover, the presence and pattern of somatic Ig V gene mutations give valuable insight into the stage of differentiation of the B cells.

BACKGROUND: Long non-coding RNAs (lncRNAs) exhibit remarkable cell-type specificity and disease association. LncRNA's functional versatility includes epigenetic modification, nuclear domain organization, transcriptional control, regulation of RNA splicing and translation, and modulation of protein activity. However, most lncRNAs remain uncharacterized due to a shortage of predictive tools available to guide functional experiments.
RESULTS: To address this gap for lymphoma-associated lncRNAs identified in our studies, we developed a new computational method, Predicting LncRNA Activity through Integrative Data-driven 'Omics and Heuristics (PLAIDOH), which has several unique features not found in other methods. PLAIDOH integrates transcriptome, subcellular localization, enhancer landscape, genome architecture, chromatin interaction, and RNA-binding (eCLIP) data and generates statistically defined output scores. PLAIDOH's approach identifies and ranks functional connections between individual lncRNA, coding gene, and protein pairs using enhancer, transcript cis-regulatory, and RNA-binding protein interactome scores that predict the relative likelihood of these different lncRNA functions. When applied to 'omics datasets that we collected from lymphoma patients, or to publicly available cancer (TCGA) or ENCODE datasets, PLAIDOH identified and prioritized well-known lncRNA-target gene regulatory pairs (e.g., HOTAIR and HOX genes, PVT1 and MYC), validated hits in multiple lncRNA-targeted CRISPR screens, and lncRNA-protein binding partners (e.g., NEAT1 and NONO). Importantly, PLAIDOH also identified novel putative functional interactions, including one lymphoma-associated lncRNA based on analysis of data from our human lymphoma study. We validated PLAIDOH's predictions for this lncRNA using knock-down and knock-out experiments in lymphoma cell models.
CONCLUSIONS: Our study demonstrates that we have developed a new method for the prediction and ranking of functional connections between individual lncRNA, coding gene, and protein pairs, which were validated by genetic experiments and comparison to published CRISPR screens. PLAIDOH expedites validation and follow-on mechanistic studies of lncRNAs in any biological system. It is available at https://github.com/sarahpyfrom/PLAIDOH .