LYmphoid NeXt-Generation Sequencing (LYNX) Panel: A Comprehensive Capture-Based Sequencing Tool for the Analysis of Prognostic and Predictive Markers in Lymphoid Malignancies.

B-cell neoplasms represent a clinically heterogeneous group of hematologic malignancies with considerably diverse genomic architecture recently endorsed by next-generation sequencing (NGS) studies. Because multiple genetic defects have a potential or confirmed clinical impact, a tendency toward more comprehensive testing of diagnostic, prognostic, and predictive markers is desired. This study introduces the design, validation, and implementation of an integrative, custom-designed, capture-based NGS panel titled LYmphoid NeXt-generation sequencing (LYNX) for the analysis of standard and novel molecular markers in the most common lymphoid neoplasms (chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma). A single LYNX test provides the following: i) accurate detection of mutations in all coding exons and splice sites of 70 lymphoma-related genes with a sensitivity of 5% variant allele frequency, ii) reliable identification of large genome-wide (≥6 Mb) and recurrent chromosomal aberrations (≥300 kb) in at least 20% of the clonal cell fraction, iii) the assessment of immunoglobulin and T-cell receptor gene rearrangements, and iv) lymphoma-specific translocation detection. Dedicated bioinformatic pipelines were designed to detect all markers mentioned above. The LYNX panel represents a comprehensive, up-to-date tool suitable for routine testing of lymphoid neoplasms with research and clinical applicability. It allows a wide adoption of capture-based targeted NGS in clinical practice and personalized management of patients with lymphoproliferative diseases.

Eosinophilia/Hypereosinophilia in the Setting of Reactive and Idiopathic Causes, Well-Defined Myeloid or Lymphoid Leukemias, or Germline Disorders.

OBJECTIVES: To report the findings of the 2019 Society for Hematopathology/European Association for Haematopathology Workshop within the categories of reactive eosinophilia, hypereosinophilic syndrome (HES), germline disorders with eosinophilia (GDE), and myeloid and lymphoid neoplasms associated with eosinophilia (excluding entities covered by other studies in this series). METHODS: The workshop panel reviewed 109 cases, assigned consensus diagnosis, and created diagnosis-specific sessions. RESULTS: The most frequent diagnosis was reactive eosinophilia (35), followed by acute leukemia (24). Myeloproliferative neoplasms (MPNs) received 17 submissions, including chronic eosinophilic leukemia, not otherwise specified (CEL, NOS). Myelodysplastic syndrome (MDS), MDS/MPN, and therapy-related myeloid neoplasms received 11, while GDE and HES received 12 and 11 submissions, respectively. CONCLUSIONS: Hypereosinophilia and HES are defined by specific clinical and laboratory criteria. Eosinophilia is commonly reactive. An acute leukemic onset with eosinophilia may suggest core-binding factor acute myeloid leukemia, blast phase of chronic myeloid leukemia, BCR-ABL1-positive leukemia, or t(5;14) B-lymphoblastic leukemia. Eosinophilia is rare in MDS but common in MDS/MPN. CEL, NOS is a clinically aggressive MPN with eosinophilia as the dominant feature. Bone marrow morphology and cytogenetic and/or molecular clonality may distinguish CEL from HES. Molecular testing helps to better subclassify myeloid neoplasms with eosinophilia and to identify patients for targeted treatments.

Doporucené postupy pro diagnostiku, terapii a následnou péci u pacientu s chronickou lymfocytární leukémií (CLL) / Recommended practices for diagnosis, therapy and follow-up care in patients with chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukemia is a disease with an extremely diverse clinical course. It is a disease of older people, the median age of the patient at the time of diagnosis is between 65-70 years. CLL is the most common leukemia in the Western world, accounting for over 30% of all cases of the disease. It is the most common leukemia among adults in the Czech Republic.

Chronická lymfocytární leukémie je onemocnení s mimorádne ruznorodým klinickým prubehem. Jde o onemocnení starsích lidí, medián veku pacienta v dobe stanovení diagnózy se pohybuje mezi 65­70 lety. CLL je nejcastejsí leukemií v západním svete, tvorí pres 30 % vsech prípadu onemocnení. Jde o nejcastejsí leukemii dospelých v Ceské republice.

SHOC2 scaffold protein modulates daunorubicin-induced cell death through p53 modulation in lymphoid leukemia cells.

SHOC2 scaffold protein has been mainly related to oncogenic ERK signaling through the RAS-SHOC2-PP1 phosphatase complex. In leukemic cells however, SHOC2 upregulation has been previously related to an increased 5-year event-free survival of pediatric pre-B acute lymphoid leukemia, suggesting that SHOC2 could be a potential prognostic marker. To address such paradoxical function, our study investigated how SHOC2 impact leukemic cells drug response. Our transcriptome analysis has shown that SHOC2 can modulate the DNA-damage mediated by p53. Notably, upon genetic inhibition of SHOC2 we observed a significant impairment of p53 expression, which in turn, leads to the blockage of key apoptotic molecules. To confirm the specificity of DNA-damage related modulation, several anti-leukemic drugs has been tested and we did confirm that the proposed mechanism impairs cell death upon daunorubicin-induced DNA damage of human lymphoid cells. In conclusion, our study uncovers new insights into SHOC2 function and reveals that this scaffold protein may be essential to activate a novel mechanism of p53-induced cell death in pre-B lymphoid cells.

Application of flow cytometry in the analysis of lymphoid disease in the lung and pleural space.

Various types of lymphoid neoplasms can occur in the lung. Lung parenchyma, the pleura or the pleural cavity can be the primary site of a lymphoid neoplasm or can be involved secondarily as a result of systemic dissemination from a separate primary site. Recognition of pulmonary lymphoid neoplasms (PLN) has increased secondary to technological advances in the medical field. Multiparameter flow cytometry (FC) is a one of the diagnostic tools that serves an essential role in the detecting and categorizing PLNs. FC allows for rapid identification and immunophenotypic characterization of PLN. In this article, we discuss the role of FC in the diagnosis of the most commonly encountered PLNs as well as their basic clinicopathologic features. We briefly discuss the role of FC in identifying non-hematolymphoid neoplasms in lung specimens as well.

Changes in long term survival after diagnosis with common hematologic malignancies in the early 21st century.

Five-year survival has increased for many hematologic malignancies in the 21st century. However, whether this has translated into greater long-term survival is unknown. Here, we examine 10- and 20-year survival for patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), chronic lymphoid leukemia (CLL), chronic myeloid leukemia (CML), non-Hodgkin lymphoma (NHL), and Hodgkin lymphoma (HL). Data were extracted from the Surveillance, Epidemiology, and End Results-9 database. Patients age 15+ with the above malignancies were included. The newly developed boomerang method was used to examine 10- and 20-year relative survival (RS) for patients in 2002-2006 and 2012-16. Ten and 20-year RS increased for each malignancy examined, with increases ranging from +4.4% units for 20-year RS for AML to +23.1% units for 10-year RS for CML. Ten year RS was >50% in 2012-16 for patients with CLL, CML, HL, NHL, and DLBCL, at 77.1%, 62.1%, 63.9%, 64.5%, and 63.0%, respectively. Survival dropped between 10 and 20 years after diagnosis for most malignancies. Long-term survival is increasing for common hematologic malignancies, but late mortality is an ongoing issue. Further study of long-term outcomes in curable malignancies to determine the reason for these later decreases in survival is indicated.

Assessment of immunoglobulin heavy chain, immunoglobulin light chain, and T-cell receptor clonality testing in the diagnosis of feline lymphoid neoplasia.

BACKGROUND: Differentiation between neoplastic and reactive lymphocytic proliferations can be challenging in cats. PCR for antigen receptor rearrangements (PARR) testing is a useful diagnostic tool to assess clonality of a lymphoid population. Previous feline PARR studies evaluated clonality of complete immunoglobulin heavy chain V-D-J (IGH-VDJ) and T-cell receptor gamma (TRG) gene rearrangements. OBJECTIVES: We aimed to evaluate the sensitivity and specificity of feline PARR primers targeting complete IGH-VDJ and TRG rearrangements, as well as incomplete IGH-DJ, kappa deleting element (Kde), and immunoglobulin lambda light chain (IGL) gene rearrangements in defined feline neoplasms and nonneoplastic controls. METHODS: Fluorescently labeled PCR primers were designed to amplify complete IGH-VDJ, incomplete IGH-DJ, Kde, IGL, and TRG gene rearrangements in two multiplexed PCR reactions, and PCR products were analyzed by fragment analysis. Fresh tissue samples from 12 flow cytometrically confirmed B-cell lymphomas, 26 cytologically confirmed gastric and renal lymphomas of presumed B-cell origin, 30 flow cytometrically confirmed T-cell leukemias, and 11 negative control cats were tested. RESULTS: Using four immunoglobulin primer sets (IGH-VDJ, IGH-DJ, Kde, and IGL), clonal immunoglobulin rearrangements were detected in 87% (33/38) of the presumed B-cell neoplasms. The IGH-VDJ reaction alone only detected clonality in 50% (19/38) of these cases. TRG rearrangements were clonal in 97% (29/30) of the T-cell leukemia cases. All negative control samples had polyclonal immunoglobulin and TRG rearrangements. CONCLUSIONS: The PARR assay developed in this study is useful for assessing clonality in feline lymphoid neoplasms. Clonality assessment of incomplete IGH-DJ, Kde, and IGL rearrangements helped identify clonal B-cell neoplasms not detected with complete IGH-VDJ PARR alone.

A decision-tree approach for the differential diagnosis of chronic lymphoid leukemias and peripheral B-cell lymphomas.

BACKGROUND AND OBJECTIVE: Here we propose a decision-tree approach for the differential diagnosis of distinct WHO categories B-cell chronic lymphoproliferative disorders using flow cytometry data. Flow cytometry is the preferred method for the immunophenotypic characterization of leukemia and lymphoma, being able to process and register multiparametric data about tens of thousands of cells per second. METHODS: The proposed decision-tree is composed by logistic function nodes that branch throughout the tree into sets of (possible) distinct leukemia/lymphoma diagnoses. To avoid overfitting, regularization via the Lasso algorithm was used. The code can be run online at https://codeocean.com/2018/03/08/a-decision-tree-approach-for-the-differential-diagnosis-of-chronic-lymphoid-leukemias-and-peripheral-b-cell-lymphomas/ or downloaded from https://github.com/lauramoraes/bioinformatics-sourcecode to be executed in Matlab. RESULTS: The proposed approach was validated in diagnostic peripheral blood and bone marrow samples from 283 mature lymphoid leukemias/lymphomas patients. The proposed approach achieved 95% correctness in the cross-validation test phase (100% in-sample), 61% giving a single diagnosis and 34% (possible) multiple disease diagnoses. Similar results were obtained in an out-of-sample validation dataset. The generated tree reached the final diagnoses after up to seven decision nodes. CONCLUSIONS: Here we propose a decision-tree approach for the differential diagnosis of mature lymphoid leukemias/lymphomas which proved to be accurate during out-of-sample validation. The full process is accomplished through seven binary transparent decision nodes.

Pathogenic B-cell receptor signaling in lymphoid malignancies: New insights to improve treatment.

Signals emanating from the B-cell receptor (BCR) promote proliferation and survival in diverse forms of B-cell lymphoma. Precision medicine strategies targeting the BCR pathway have been generally effective in treating lymphoma, but often fail to produce durable responses in diffuse large B-cell lymphoma (DLBCL), a common and aggressive cancer. New insights into DLBCL biology garnered from genomic analyses and functional proteogenomic studies have identified novel modes of BCR signaling in this disease. Herein, we describe the distinct roles of antigen-dependent and antigen-independent BCR signaling in different subtypes of DLBCL. We highlight mechanisms by which the BCR cooperates with TLR9 and mutant isoforms of MYD88 to drive sustained NF-κB activity in the activated B-cell-like (ABC) subtype of DLBCL. Finally, we discuss progress in detecting and targeting oncogenic BCR signaling to improve the survival of patients with lymphoma.

Cutaneous B-Cell Lymphoblastic Lymphoma.

B-cell lymphoblastic lymphoma (B-LBL) is a malignant neoplasm of immature B cells that accounts for only 10% of all cases of lymphoblastic lymphoma. Most commonly, B-LBL presents as bony lesions, but in rare cases, the disease manifests cutaneously. We present a case of simultaneous cutaneous and systemic presentation of B-LBL in an otherwise healthy 28-year-old man in which the lymphoblastic infiltrate stained positive for CD79a, Tdt, CD10, and CD20. A diagnosis of cutaneous B-LBL was made, and systemic work-up revealed widespread involvement of the skin, bone, and lymph nodes. Review of all currently described cases of cutaneous B-LBL with or without systemic involvement revealed that the most frequently positive tumor markers were CD79a (92.3%), Tdt (90.6%), and CD10 (83.3%). Systemic involvement of B-LBL was found in nearly half of all cases with cutaneous presentation.

The Role of PI3K Inhibition in Lymphoid Malignancies.

PURPOSE OF REVIEW: The outcome of patients with lymphoid malignancies has markedly improved in recent years which is likely due to a combination of advances in supportive care, and therapeutic options. In this article, we will provide an overview over the role PI3-kinase signalling, one of the most important dysregulated pathways in cancer, and its successful inhibition in lymphoma. RECENT FINDINGS: PI3-kinase inhibitors have shown remarkable activity in an increasing subset of patients with non-Hodgkin lymphomas. The first drug to be approved was idelalisib for patients with relapsed/refractory follicular lymphoma and CLL/SLL as monotherapy, or in combination with rituximab, respectively. After an initial setback related to increased toxicity including deaths observed in several upfront studies, there has been a resurgence in interest in this pathway following the promising efficacy of second-generation PI3K inhibitors including in patients with T cell lymphomas. PI3K inhibition continues to be an invaluable tool in the therapy of patients with lymphoid malignancies if managed cautiously. Preclinical models are helpful in predicting possible side effects and identifying new lymphoma subtypes that may be susceptible to this class of agents. The future will likely involve rationally designed combinatorial approaches to deepen the response rate and prevent the emergence of resistance.

Role of CD71 in acute leukemia- An immunophenotypic marker for erythroid lineage or proliferation?

BACKGROUND: CD71 or Transferrin receptor is expressed on the surface of erythroid lineage cells. CD71 expression has been found to be significantly increased in rapidly proliferating cells. METHODS: This cross-sectional study included 37 bone marrow samples of acute leukemia cases diagnosed between October 2016 to April 2018. The samples were analysed on BD FACS Canto II. We evaluated the expression of CD71 on leukemic blasts and compared median fluorescent intensities (MFI) of blasts in different types of acute leukemias. RESULTS: The 37 cases comprised of 21 Acute Myeloid Leukemia (AML), 13 B-Acute Lymphoblastic Leukemia (B-ALL), 2 T- Acute Lymphoblastic Leukemia (T-ALL) and 1 mixed phenotypic acute leukemia (MPAL), T/Myeloid. CD 71 expression was noted in 70.3% (n= 26/37) of acute leukemia cases. CD71 expression was most commonly observed in AML (n= 15/21;71.4%), followed by B-ALL (n= 9/13;69.2%) and T-ALL (n= 1/2;50%). Single case of MPAL revealed blasts positive for CD71. MFI of leukemic blasts of single CD71 positive T-ALL was found to be highest, followed by AML, MPAL (T/Myeloid) and least in B ALL. Of the AML cases, the blasts of AML-M6, acute promyelocytic leukemia and AML-M1 showed higher CD71 expression in terms of MFI. CONCLUSIONS: Surface CD71 expression is not only found in erythroid lineage cells, but also in proliferating cells. CD71 MFI is highest in T lymphoblasts followed by leukemic erythroblasts, myeloblasts and least in B lymphoblasts.

ClonoSEQ assay for the detection of lymphoid malignancies.

Introduction: Immunoglobulin rearrangement studies by molecular methods are routinely used to detect clonality and to follow up patients with lymphoid malignancies. The design of a next-generation sequencing (NGS) panel using a comprehensive pool of primers, the establishment of a straightforward analytical protocol, a bioinformatic pipeline and the availability of the results of clinical studies have allowed the Clonoseq platform to be licensed as the first assay to measure minimal residual disease (MRD) both in acute lymphoblastic leukemia (ALL) and in multiple myeloma (MM). Areas covered: An extensive literature review (Pubmed search) on the applicability of the high-throughput sequencing (HTS) approach in lymphoid malignancies was conducted. This review discusses recent data in the field and compares this emerging molecular technique with standardized technologies. Expert Opinion: Real-time quantitative (RQ)-PCR and multiparametric flow cytometry (MPFC) are still the gold standard methods of minimal residual disease assessment. New HTS methods as Clonoseq show a high concordance with the above-mentioned techniques and at the same time it provides potential advantages to detect clonal changes. Clonoseq could be helpful to optimize risk-stratification and adjusting treatments in lymphoid malignancies. Moreover, HTS could also be applied to the detection of circulating tumor DNA (ctDNA) on the plasma in lymphomas.