Flow Cytometry of CD5-Positive Hairy Cell Leukemia.

BACKGROUND AND OBJECTIVE: Hairy cell leukemia (HCL) is a chronic lymphoproliferative disorder for which diagnosis is typically straightforward, based on bone marrow morphology and flow cytometry (FC) or immunohistochemistry. Nevertheless, variants present atypical expressions of cell surface markers, as is the case of CD5, for which the differential diagnosis can be more difficult. The aim of the current paper was to describe diagnosis of HCL with atypical CD5 expression, with an emphasis on FC. METHODS: The detailed diagnostic methodology for HCL with atypical CD5 expression is presented, including differential diagnosis from other lymphoproliferative diseases with similar pathologic features, by FC analysis of the bone marrow aspirate. RESULTS: Diagnosis of HCL by means of FC started by gating all events based on side scatter (SSC) versus CD45 and B lymphocytes were selected from the lymphocytes gate as CD45/CD19 positive. The gated cells were positive for CD25, CD11c, CD20, and CD103, while CD10 proved to be dim to negative. Moreover, cells positive for CD3, CD4, and CD8, the three pan-T markers, as well as CD19, showed a bright expression of CD5. The atypical CD5 expression is usually correlated with a negative prognosis and thus chemotherapy with cladribine should be initiated. CONCLUSION: HCL is an indolent chronic lymphoproliferative disorder and diagnosis is usually straightforward. However, atypical expression of CD5 renders its differential diagnosis more difficult, but FC is a useful tool that allows an optimal classification of the disease and allows initiation of timely satisfactory therapy.

Cyclin D1 expression, cell proliferation, and clonal persistence characterize primary cutaneous CD4+ small or medium T-cell lymphoproliferative disorder.

AIMS: The aim was to gain insight into the biology of primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder (PCSM-LPD). METHODS: We describe the histopathological and clinical characteristics of 177 PCSM-LPD diagnosed at our consultation centre. We performed immunohistochemical multistaining in a subset of cases (n = 46) including PD1, Cyclin D1, and multiple markers of proliferation. We evaluated clonal T-cell-receptor-(TCR) rearrangements and used tissue microdissection to analyse TCR-clonality of PD1(+) cells. RESULTS: The cohort of n = 177 PCSM-LPD included 84 males and 93 females (median age 57, range 13-85). Clinical presentation was as a solitary nodule or plaque (head and neck > trunk > extremities). Most patients were treated by local excision or steroids (96%, 69/72); relapses occurred in 12/65 (18%) of patients with follow up. Histopathology revealed the predominance of a nodular pattern (75%, 134/177) and frequent clustering of PD1(+) large cells (70%, 103/147). We detected Cyclin D1 and PD1 coexpression (>10% of PD1(+)-cells) in 26/46 (57%), which was not associated with CCND1 breaks or amplifications. PD1(+)-cells in PCSM-LPDs showed a significantly higher expression of proliferation-associated proteins compared to PD1(-)-cells. A clonal TCR-rearrangement was present in 176/177 (99%), with a clonal persistence in 7/8 patients at relapse including distant sites. Tissue-microdissection revealed PD1(+)-cells as the source of clonality, whilst PD1(-)-cells remained polyclonal. CONCLUSION: PCSM-LPD is a clinically indolent, albeit neoplastic, disease driven by clonal expansion of PD1(+)-cells. We demonstrate Cyclin D1-expression associated with accelerated proliferation as a surprising new biological feature of the disease.

CCL22 mutations drive natural killer cell lymphoproliferative disease by deregulating microenvironmental crosstalk.

Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is characterized by clonal expansion of natural killer (NK) cells where the underlying genetic mechanisms are incompletely understood. In the present study, we report somatic mutations in the chemokine gene CCL22 as the hallmark of a distinct subset of CLPD-NK. CCL22 mutations were enriched at highly conserved residues, mutually exclusive of STAT3 mutations and associated with gene expression programs that resembled normal CD16dim/CD56bright NK cells. Mechanistically, the mutations resulted in ligand-biased chemokine receptor signaling, with decreased internalization of the G-protein-coupled receptor (GPCR) for CCL22, CCR4, via impaired ß-arrestin recruitment. This resulted in increased cell chemotaxis in vitro, bidirectional crosstalk with the hematopoietic microenvironment and enhanced NK cell proliferation in vivo in transgenic human IL-15 mice. Somatic CCL22 mutations illustrate a unique mechanism of tumor formation in which gain-of-function chemokine mutations promote tumorigenesis by biased GPCR signaling and dysregulation of microenvironmental crosstalk.

Identification of Germline Non-coding Deletions in XIAP Gene Causing XIAP Deficiency Reveals a Key Promoter Sequence.

PURPOSE: X-linked inhibitor of apoptosis protein (XIAP) deficiency, also known as the X-linked lymphoproliferative syndrome of type 2 (XLP-2), is a rare immunodeficiency characterized by recurrent hemophagocytic lymphohistiocytosis, splenomegaly, and inflammatory bowel disease. Variants in XIAP including missense, non-sense, frameshift, and deletions of coding exons have been reported to cause XIAP deficiency. We studied three young boys with immunodeficiency displaying XLP-2-like clinical features. No genetic variation in the coding exons of XIAP was identified by whole-exome sequencing (WES), although the patients exhibited a complete loss of XIAP expression. METHODS: Targeted next-generation sequencing (NGS) of the entire locus of XIAP was performed on DNA samples from the three patients. Molecular investigations were assessed by gene reporter expression assays in HEK cells and CRISPR-Cas9 genome editing in primary T cells. RESULTS: NGS of XIAP identified three distinct non-coding deletions in the patients that were predicted to be driven by repetitive DNA sequences. These deletions share a common region of 839 bp that encompassed the first non-coding exon of XIAP and contained regulatory elements and marks specific of an active promoter. Moreover, we showed that among the 839 bp, the exon was transcriptionally active. Finally, deletion of the exon by CRISPR-Cas9 in primary cells reduced XIAP protein expression. CONCLUSIONS: These results identify a key promoter sequence contained in the first non-coding exon of XIAP. Importantly, this study highlights that sequencing of the non-coding exons that are not currently captured by WES should be considered in the genetic diagnosis when no variation is found in coding exons.

Reactive Intralymphovascular Immunoblastic Proliferations Mimicking Aggressive Lymphomas.

Reactive intralymphovascular immunoblastic proliferations (ILVIPs) may mimic aggressive lymphomas and are rarely reported. Herein, we characterize the clinicopathologic features of 8 patients with ILVIPs. No patients had lymphadenopathy, hepatosplenomegaly, or other findings suggestive of lymphoma. The ILVIPs involved the small or large intestine (n=5) and appendix (n=3). Patients were evaluated for abdominal pain, suspected appendicitis, intestinal obstruction, diverticulitis, volvulus, or tumor resection. Histologic sections showed expanded lymphovascular spaces filled by intermediate to large immunoblasts, positive for CD38, CD43, CD45, CD79a, and MUM1/IRF4 in all cases tested. Five of 6 (83%) cases were positive for CD30. CD20 was weakly positive in a subset of cells in 2 (25%) cases, and PAX5 was weakly positive in 4 (50%) cases. The immunoblasts expressed polytypic light chains in all cases tested. In 1 case, a subset of immunoblasts expressed T-cell markers indicating the presence of a T-cell component. The immunoblasts were negative for ALK, BCL-2, BCL-6, CD10, CD56, CD138, and Epstein-Barr virus-encoded small RNA in all cases assessed. The proliferation index shown by Ki-67 was high with a median of 80%. In all 6 cases tested, the immunoblasts were shown within lymphatic channels highlighted by D2-40. In conclusion, ILVIPs can be rarely observed in patients with inflammatory or infectious conditions, especially in gastrointestinal tract surgical specimens. The immunoblasts are predominantly of B-lineage with a postgerminal center immunophenotype and are located within lymphatic channels. It is essential to distinguish reactive ILVIPs from aggressive lymphomas to avoid unnecessary therapy.

Diacylglycerol Kinase alpha in X Linked Lymphoproliferative Disease Type 1.

Diacylglycerol kinases are intracellular enzymes that control the balance between the secondary messengers diacylglycerol and phosphatidic acid. DGKα and DGKζ are the prominent isoforms that restrain the intensity of T cell receptor signalling by metabolizing PLCγ generated diacylglycerol. Thus, their activity must be tightly controlled to grant cellular homeostasis and refine immune responses. DGKα is specifically inhibited by strong T cell activating signals to allow for full diacylglycerol signalling which mediates T cell response. In X-linked lymphoproliferative disease 1, deficiency of the adaptor protein SAP results in altered T cell receptor signalling, due in part to persistent DGKα activity. This activity constrains diacylglycerol levels, attenuating downstream pathways such as PKCθ and Ras/MAPK and decreasing T cell restimulation induced cell death. This is a form of apoptosis triggered by prolonged T cell activation that is indeed defective in CD8+ cells of X-linked lymphoproliferative disease type 1 patients. Accordingly, inhibition or downregulation of DGKα activity restores in vitro a correct diacylglycerol dependent signal transduction, cytokines production and restimulation induced apoptosis. In animal disease models, DGKα inhibitors limit CD8+ expansion and immune-mediated tissue damage, suggesting the possibility of using inhibitors of diacylglycerol kinase as a new therapeutic approach.

Simiao Qingwen Baidu decoction inhibits Epstein-Barr virus-induced B lymphoproliferative disease and lytic viral replication.

CONTEXT: Simiao Qingwen Baidu decoction (SQBD), a traditional Chinese medicine prescription, can ameliorate Epstein-Barr virus (EBV) induced disease. However, its mechanism still remains unknown. OBJECTIVE: To detect the mechanism of SQBD in EBV-induced B lymphoproliferative disease in vitro. MATERIALS AND METHODS: Sprague-Dawley (SD) rats (n = 20) were given SQBD (10 mL/kg) by gavage once a day for 7 d. SQBD-containing serum was obtained from abdominal aortic blood of rats, and diluted with medium to obtain 5%, 10% or 20%-medicated serum. SD rats (n = 10) were given normal saline, and normal serum was collected as a control. EBV-transformed B cells (CGM1) were cultured in medium containing 5%, 10% or 20%-medicated serum. CGM1 cells were treated with normal serum as a control. Cell viability and apoptosis were examined. The expression and activity of proteins were assessed. RESULTS: We found that IC50 (83 ± 26.07%, 24 h; 69.88 ± 4.69%, 48 h) of 10% medicated serum was higher than that of 5% (25.47 ± 6.98%, 24 h; 21.62 ± 7.30%, 48 h) and 20%-medicated serum (51 ± 7.25%, 24 h; 56.03 ± 2.56%, 48 h). Moreover, SQBD promoted apoptosis of CGM1 cells by regulating EBV latency proteins expression. SQBD inhibited EBV-induced lytic viral replication. CONCLUSIONS: Our data confirmed that SQBD inhibits EBV-induced B lymphoproliferative disease and lytic viral replication. This work provides a theoretical basis for the mechanism of SQBD in EBV-induced B lymphoproliferative disease, and SQBD may be an effectively therapeutic drug for EBV-induced B lymphoproliferative disease.

Calcium flux control by Pacs1-Wdr37 promotes lymphocyte quiescence and lymphoproliferative diseases.

Endoplasmic reticulum (ER) calcium (Ca2+ ) stores are critical to proteostasis, intracellular signaling, and cellular bioenergetics. Through forward genetic screening in mice, we identified two members of a new complex, Pacs1 and Wdr37, which are required for normal ER Ca2+ handling in lymphocytes. Deletion of Pacs1 or Wdr37 caused peripheral lymphopenia that was linked to blunted Ca2+ release from the ER after antigen receptor stimulation. Pacs1-deficient cells showed diminished inositol triphosphate receptor expression together with increased ER and oxidative stress. Mature Pacs1-/- B cells proliferated and died in vivo under lymphocyte replete conditions, indicating spontaneous loss of cellular quiescence. Disruption of Pacs1-Wdr37 did not diminish adaptive immune responses, but potently suppressed lymphoproliferative disease models by forcing loss of quiescence. Thus, Pacs1-Wdr37 plays a critical role in stabilizing lymphocyte populations through ER Ca2+ handling and presents a new target for lymphoproliferative disease therapy.

Lichen aureus with pseudolymphomatous infiltrate.

Lichen aureus is a variant of pigmented purpuric dermatoses. The usual histopathology of lichen aureus is characterized by a subepidermal dense, band-like lymphocytic infiltrate, extravasated erythrocytes, and hemosiderin deposits. We report three patients with lichen aureus on the extremities with similar clinical, dermoscopic, and histopathological findings characterized by a dense band-like relatively deep dermal infiltrate accompanied by extravasation of erythrocytes and hemosiderin deposits occasioning a resemblance to a lymphoproliferative disorder.

A review of CD30 expression in cutaneous neoplasms.

BACKGROUND: The surface protein CD30 is a therapeutic target of monoclonal antibody therapy. Knowledge of the frequency of CD30 expression and its prognostic relevance is therefore interesting, not only in lymphoproliferative disorders (LPD) but also in solid tumors of the skin. METHODS: A review was completed in PubMed for all published reports of CD30 expression in cutaneous lymphomas, mastocytosis, epithelial tumors and sarcomas from 1982 to April 2019. Only accessible articles in English and German were considered. Entities with an expected CD30 expression, such as CD30-positive LPD, were not evaluated. RESULTS: The electronic research identified 1091 articles and a further 34 articles were obtained from manual bibliographic reference. Overall 91 articles were included that examined CD30 expression in various entities of cutaneous neoplasms and matched the inclusion criteria. CONCLUSION: Apart from cutaneous CD30-positive LPD, the best-studied group for CD30 expression was mycosis fungoides (MF). CD30 positivity was found in 32% of classical (patch and plaque stage) and in 59.4% cases of transformed MF. CD30 was also frequently expressed in cutaneous mastocytosis (96.5%). In solid tumors, some single reports describe CD30 expression by tumor cells, but CD30-reactive lymphocytes were frequently observed in the tumor microenvironment (TME), especially in keratoacanthoma (KA).

Low-dose total skin electron beam therapy for refractory cutaneous CD30 positive lymphoproliferative disorders.

We describe a case of a 48-year-old woman with a refractory cutaneous CD30 positive lymphoproliferative disorder treated successfully with total skin electron beam radiotherapy (TSEBT).

Evaluation of Positive B- and T-Cell Gene Rearrangement Studies in Patients With Negative Morphology, Flow Cytometry, and Immunohistochemistry.

CONTEXT.­: The significance of positive immunoglobulin (IG) or T-cell receptor (TCR) gene rearrangement studies in the context of otherwise normal ancillary findings is unknown. OBJECTIVE.­: To examine long-term hematologic outcomes of individuals with positive gene rearrangement studies with otherwise unremarkable blood or bone marrow studies in parallel. DESIGN.­: Data from patients who underwent IG or TCR gene rearrangement testing at the authors' affiliated Veterans Affairs Hospital January 1, 2013 to July 6, 2018 were extracted from medical records. Date of testing, specimen source, and morphologic, flow cytometric, immunohistochemical, and cytogenetic characterization of the tissue source were recorded. Gene rearrangement results were categorized as test positive/phenotype positive (T+/P+), test positive/phenotype negative (T+/P-), test negative/phenotype negative (T-/P-), or test negative/phenotype positive (T-/P+) based on comparison to other studies and/or final diagnosis. Patient records were reviewed for subsequent diagnosis of hematologic malignancy for patients with positive gene rearrangements but no other evidence for a disease process. RESULTS.­: A total of 136 patients with 203 gene rearrangement studies were analyzed. For TCR studies, there were 2 T+/P- and 1 T-/P+ results in 47 peripheral blood assays, as well as 7 T+/P- and 1 T-/P+ results in 54 bone marrow assays. Regarding IG studies, 3 T+/P- and 12 T-/P+ results in 99 bone marrow studies were identified. None of the 12 patients with T+/P- TCR or IG gene rearrangement studies later developed a lymphoproliferative disorder. CONCLUSIONS.­: Positive IG/TCR gene rearrangement studies in the context of otherwise negative bone marrow or peripheral blood findings are not predictive of lymphoproliferative disorders.

Acral lymphomatoid papulosis: Report of five cases, differential diagnosis, and review.

Acral lymphomatoid papulosis (a-LyP) is a rare clinical variant of LyP whose diagnosis may be challenging. A case series of a-LyP was studied clinically, histopathologically, immunohistochemically, and from molecular point of view. Including ours, 25 cases of a-LyP have so far been reported. Clinically, a-LyP may present as acral involvement exclusively, in combination with mucosal lesions, (in itself a rare presentation), or in association with conventional LyP. The age of presentation was slightly higher than that of conventional LyP (55 vs 45 years) and a male predominance has been observed, as usually reported. Histopathologically, no morphological differences exclusively from conventional LyP were observed. LyP types A and E were the main variants. We describe for the first time one case of type D a-LyP. Acral LyP is a rare entity and correct diagnosis can only be reached with clinical and histopathological correlation, to avoid aggressive treatment of this indolent lymphoproliferative disorder.

A Higher Level of Expression of the Nucleolar Protein SURF6 in Human Normal Activated Lymphocytes and in Lymphocytes of Patients with Lymphoproliferative Disorders.

Proliferation of mammalian cells is often accompanied by an increase in the content of the nucleolar proteins, which allows researchers to consider such proteins as potential activation markers. To test this assumption experimentally, we examined the expression pattern of the nucleolar rRNA processing factor SURF6 in normal (resting) peripheral blood lymphocytes, lymphocytes activated for proliferation in vitro, and in blood samples from patients with lymphoproliferative diseases. Using two methods (immunofluorescence and immunoblotting), we for the first time showed that the SURF6 protein is not detected in normal lymphocytes but can easily be visualized in lymphocytes after PHA activation and in lymphocytes of lymphocytic leukemia patients. The level of SURF6 expression in patients correlated with the aggressiveness of the disease development determined by the content of Ki-67-positive lymphocytes. These results allow the SURF6 nucleolar protein to be considered as a putative activation marker of lymphocytes in human blood disorders.

Latest perspectives on glucocorticoid-induced apoptosis and resistance in lymphoid malignancies.

Glucocorticoids are essential drugs in the treatment protocols of lymphoid malignancies. These steroidal hormones trigger apoptosis of the malignant cells by binding to the glucocorticoid receptor (GR), which is a member of the nuclear receptor superfamily. Long term glucocorticoid treatment is limited by two major problems: the development of glucocorticoid-related side effects, which hampers patient quality of life, and the emergence of glucocorticoid resistance, which is a gradual process that is inevitable in many patients. This emphasizes the need to reevaluate and optimize the widespread use of glucocorticoids in lymphoid malignancies. To achieve this goal, a deep understanding of the mechanisms governing glucocorticoid responsiveness is required, yet, a recent comprehensive overview is currently lacking. In this review, we examine how glucocorticoids mediate apoptosis by detailing GR's genomic and non-genomic action mechanisms in lymphoid malignancies. We continue with a discussion of the glucocorticoid-related problems and how these are intertwined with one another. We further zoom in on glucocorticoid resistance by critically analyzing the plethora of proposed mechanisms and highlighting therapeutic opportunities that emerge from these studies. In conclusion, early detection of glucocorticoid resistance in patients remains an important challenge as this would result in a timelier treatment reorientation and reduced glucocorticoid-instigated side effects.

Cold agglutinin disease: where do we stand, and where are we going?

Primary cold agglutinin disease (CAD) is characterized by a very indolent bone marrow clonal B-cell lymphoproliferative disorder that initiates an autoimmune hemolytic anemia. The clonal B cells produce a monoclonal autoantibody termed cold agglutinin, most often of the immunoglobulin (Ig) Mκ class. After binding to its antigen, the IgM initiates a complement classical pathway-driven erythrocyte destruction, predominantly mediated by opsonization with complement protein C3b and extravascular hemolysis in the liver. We review the molecular biology, histopathology, clinical features, and diagnostic procedures in CAD. Some patients are only slightly anemic and do not require treatment, but moderate or severe anemia frequently occurs, and the disease burden has been underestimated. CAD should not be treated with corticosteroids. Several B-cell-directed treatment options are available, and complement-directed approaches are being rapidly developed. Current and possible future therapies are reviewed.

Chronic active Epstein-Barr virus infection: A heterogeneous entity requiring a high index of suspicion for diagnosis.

Chronic active Epstein-Barr virus infection of T- and NK-cell type, systemic form, is a rare entity within the spectrum of EBV-driven T- and NK-cell lymphoproliferative disorders. Established diagnostic criteria and a characteristic clinical course help to differentiate it from other closely related EBV-positive neoplasms and clinical states. We present a patient and review the natural history, pathologic features, pathogenesis, and differential diagnosis of this entity.