Epstein-Barr virus-associated large B-cell lymphoma transformation in marginal zone B-cell lymphoma: a series of four cases.

AIMS: To present four examples of clonally related Epstein-Barr virus (EBV)-associated large-cell transformation of marginal zone lymphoma (MZL) (of nodal, extranodal and splenic types), occurring 120, 11 and 5 months after the initial diagnosis in three instances, and concurrently in one case; and to discuss several interesting features of EBV infection. METHODS AND RESULTS: Somatic mutations were detected by use of a customised panel for next-generation sequencing and polymerase chain reaction studies of IgH in both low-grade and high-grade components of each case. In case 1, the initial biopsy of nodal MZL showed scattered EBV-positive cells, which might constitute an indication of EBV-induced progression. Case 2 showed heterogeneous EBV expression, a phenomenon attributable to loss of the EBV episomes during cell division, or to a secondary superinfection or reactivation of the virus. In case 3, p53 overexpression related to gene mutation and EBV-encoded small RNAs were identified in the same neoplastic component. In case 4, the mucosa-associated lymphoid tissue-type MZL and the high-grade component were identified concurrently in a patient previously treated with methotrexate for an autoimmune disorder. CONCLUSION: These data suggest that the presence of EBV should be added to the list of potential markers to be analysed for MZL prognosis.

Bone marrow infiltration by flow cytometry at diffuse large B-cell lymphoma NOS diagnosis implies worse prognosis without considering bone marrow histology.

BACKGROUND: The significance of discrepant findings between histology (BMB) and flow cytometry (FC) in bone marrow (BM) examination at diffuse large B-cell lymphoma (DLBCL) diagnosis is uncertain. METHODS: We performed a 5-year retrospective single-center study of patients diagnosed by DLBCL not otherwise specified (n = 82), divided into three groups according to BM infiltration at diagnosis: BMB-/FC- (75.6%), BMB+/FC+ (13.4%), and BMB-/FC+ (11%). RESULTS: Median infiltration by FC analysis of the BMB-/FC+ group was 0.8% and if we considered BM infiltration as positive in all cases, 4/9 would be upstaged. Median follow was 33 months. Event-free survival (EFS) after 18 months was 82, 23, and 27% for BMB-/FC-, BMB-/FC+, and BMB+/FC+, respectively (p < .001). After 18 months of observation, OS was 87, 46, and 55% for BMB-/FC-, BMB-/FC+, and BMB+/FC+, respectively (p = .001). In multivariate analysis (BM infiltration vs. cell-of-origin according to Hans algorithm and standard IPI), BM infiltration was independently associated with EFS (HR: 1.94, 95% CI: 1.3-2.9) and overall survival (HR: 1.69, 95% CI: 1.1-2.7). CONCLUSION: In summary, minimal BM infiltration, detected by FC but not by BMB, has same prognostic implications than overt BM infiltration and should be considered as extranodal involvement regardless the infiltration quantity.

The utility of multiparametric flow cytometry in the detection of primary effusion lymphoma (PEL).

Primary effusion lymphoma (PEL) is a rare B cell lymphoproliferative disorder that arises predominantly in body cavities causing malignant effusions. The incidence of PEL is very low, accounting for approximately 4% of all HIV-associated non-Hodgkin lymphomas. PEL has also been described in elderly patients and after solid-organ transplantation. It is associated in all cases with human herpes virus 8 (HHV8). We describe a case of PEL in a 88-year-old HIV-negative woman who presented with progressive dyspnea and moderate right-sided pleural effusion without significant lymphadenopathies or other effusions. The cytological study of the pleural fluid revealed a dense proliferation of large plasmablastic cells. A six-color multiparametric flow cytometry immunophenotyping study was carried out, and revealed 45% of large in size and high cellular complexity cells positive for CD45 (dim), CD38, CD138, CD30 and HLA-DR; and negative for CD19, CD20, cytoplasmatic CD79a, surface and cytoplasmic light chains Kappa and Lambda, CD3, CD4, CD5, CD7, CD8, CD28, CD56, CD81, and CD117. In situ hybridization for EBV-encoded smalI RNA was negative and immunohistochemistry for Kaposi sarcoma herpesvirus (HHV8) confirmed the diagnosis of PEL. Our results confirm that flow cytometry bring useful data in the diagnosis of large-cell lymphomas involving body cavities. © 2018 International Clinical Cytometry Society.

Immunomodulatory effect of Polypodium leucotomos (Anapsos) in child palatine tonsil model.

BACKGROUND: Recurrent tonsillitis might reduce the immunological capability of fighting against the infection of tonsil tissue. Polypodium leucotomos (Anapsos) immunomodulating effect has been subject of research in the last years. The aim of this research is to test the in vitro immunomodulating capacity of Anapsos in a child palatine tonsil explants model. METHODS: Palatine tonsils explants of children undergoing amigdalectomy were stimulated with mononuclear cells obtained from their own blood by density gradient centrifugation. Some were then treated with Anapsos while others rest untreated. Cytokines were measured by ELISA, immune cells activation was measured by flow cytometry and activation of immunoglobulins was appreciated by indirect immunofluorescence in tonsils tissue. RESULTS: Anapsos activates Natural Killers cells. It increases IL-2 and IFN-γ levels by the activation of Th2 lymphocytes, and IL-10, by the Th1 lymphocytes. Anapsos also increases immunoglobulins IgM, IgD and IgG4 by B-lymphocyte activation in tonsils tissue. CONCLUSION: Anapsos has an immunomodulating effect, both in humoral and cellular responses, which might benefit children suffering of recurrent tonsillitis as it could enhance their immune system. This effect might reduce the number of episodes suffered and therefore the number of children undergoing surgery.

Enfermedad residual mínima en la leucemia linfática crónica / Minimal residual disease in chronic lymphocytic leukaemia

La evaluación de la enfermedad mínima residual (EMR) es un objetivo importante en el tratamiento de la leucemia linfocítica crónica (LLC). Su obtención es predictiva de una supervivencia libre de progresión (SLP) y una supervivencia global prolongadas y podría considerarse una variable subrogada de la SLP en el contexto del tratamiento con quimioinmunoterapia. La evaluación de la EMR mediante citometría de flujo o técnicas moleculares en la era de los nuevos inhibidores de BCR o Bcl-2 podría identificar la secuencia de tratamiento más coste-efectiva y la duración de la misma. Una aproximación terapéutica guiada por el nivel de EMR también podría determinar qué pacientes se beneficiarían de un tratamiento de consolidación o de una finalización precoz del mismo. En esta revisión discutimos los diferentes métodos de análisis de la EMR, qué muestras deben ser analizadas, el papel futuro de la detección del ADN tumoral circulante y el papel potencial de la negatividad de la EMR en la práctica clínica en la era moderna del tratamiento de la LLC (AU)

Minimal residual disease (MRD) assessment is an important endpoint in the treatment of chronic lymphocytic leukaemia (CLL). It is highly predictive of prolonged progression-free survival (PFS) and overall survival and could be considered a surrogate for PFS in the context of chemoimmunotherapy based treatment. Evaluation of MRD level by flow cytometry or molecular techniques in the era of the new BCR and Bcl-2 targeted inhibitors could identify the most cost-effective and durable treatment sequencing. A therapeutic approach guided by the level of MRD might also determine which patients would benefit from an early stop or consolidation therapy. In this review, we discuss the different MRD methods of analysis, which source of tumour samples must be analysed, the future role of the detection of circulating tumour DNA, and the potential role of MRD negativity in clinical practice in the modern era of CLL therapy (AU)

Minimal residual disease in chronic lymphocytic leukaemia. / Enfermedad residual mínima en la leucemia linfática crónica.

Minimal residual disease (MRD) assessment is an important endpoint in the treatment of chronic lymphocytic leukaemia (CLL). It is highly predictive of prolonged progression-free survival (PFS) and overall survival and could be considered a surrogate for PFS in the context of chemoimmunotherapy based treatment. Evaluation of MRD level by flow cytometry or molecular techniques in the era of the new BCR and Bcl-2 targeted inhibitors could identify the most cost-effective and durable treatment sequencing. A therapeutic approach guided by the level of MRD might also determine which patients would benefit from an early stop or consolidation therapy. In this review, we discuss the different MRD methods of analysis, which source of tumour samples must be analysed, the future role of the detection of circulating tumour DNA, and the potential role of MRD negativity in clinical practice in the modern era of CLL therapy.

Case report of a primary effusion lymphoma successfully treated with oral valganciclovir after failing chemotherapy.

Primary effusion lymphoma is a rare non-Hodgkin lymphoma that presents with pleural effusions and lacking of tumour mass. It is universally associated with human herpesvirus 8 (HHV8) and is more frequent among immunosuppressed patients. There is no standard treatment, chemotherapy and anti-HIV therapy have been used with poor results, but there is still no strong evidence supporting the use of valganciclovir. We present the case of a HIV positive man that presented with pleural effusion compatible with primary effusion lymphoma and positivity for HHV8 DNA in blood. Bortezomib-containing treatment protocol was started, but the disease progressed within the chemotherapy. Therefore, treatment with oral valganciclovir was decided and the patient achieved a sustained radiological complete response. HHV8 DNA turned negative 6 months after starting the treatment with valganciclovir.

Prostatic-Like Syndrome in a Woman with Chronic Lymphocytic Leukemia: Sequential Kinase Inhibitor Therapy.

Chronic lymphocytic leukemia (CLL) is an incurable lymphoproliferative disorder with a heterogeneous genetic and clinical course. Two kinase inhibitors, ibrutinib and idelalisib, have demonstrated achievement of complete and durable remissions in relapse/refractory genetically unselected CLL patients. We present a case of relapsed CLL with extensive disease and hourglass deformity of urinary bladder as a result of the compression of two extraperitoneal paravesical soft tissue bulky masses, with excellent response to sequential kinase inhibitor therapy.

The use of adipose mesenchymal stem cells and human umbilical vascular endothelial cells on a fibrin matrix for endothelialized skin substitute.

In recent years, the reconstruction of human skin by tissue engineering represents a clinical challenge and has offered a therapeutic alternative. Avascular engineered skin equivalents have been available for several years and used to treat wounds due to burns, nonhealing ulcers, and surgical excisions. They are constituted by different types of cultured cells included in a three-dimensional structure that permits cellular proliferation to create tissue substitutes. The major drawback of these artificial skin substitutes is their lack of blood supply, since the endurance and cell proliferation of the substitute depend on an adequate oxygen and nutrient supply and on toxin removal. These functions are served by the vascular system. We have produced a new model of endothelialized skin substitute that promotes the formation of capillary-like structures by seeding human umbilical vein endothelial cells (HUVECs) with dermal fibroblasts and human adipose-derived mesenchymal stem cells (hADMSCs) in a fibrin matrix. Dermal fibroblasts and hADMSCs produce extracellular matrix that stimulates cellular growth and proliferation. hADMSCs secrete significant quantities of angiogenic and antiapoptotic factors (vascular endothelial growth factor and hepatocyte growth factor), which induce in vitro differentiation of these cells into endothelial cells promoting angiogenesis and participating in tissue repair and skin regeneration processes. We obtained the artificial skin substitute with similar structure to native skin, including dermis and epidermis. We demonstrated that endothelial cells (CD31 and von Willebrand factor positive) proliferated and organized themselves into capillary-like structures within the fibrin matrix. The epidermis showed a complete epithelization by squamous cells (AE1/AE3 cytokeratin positive) with intracytoplasmic keratohyalin granules, hyperkeratosis, and parakeratosis. We have established a novel artificial skin substitute that facilitates the formation of capillary-like structures that may provide a novel therapeutic approach to different skin defects and prove to be a useful tool for regenerative medicine.

Vascular endothelial growth factor A (VEGFA) gene polymorphisms have an impact on survival in a subgroup of indolent patients with chronic lymphocytic leukemia.

Vascular endothelial growth factor (VEGF)-mediated angiogenesis contributes to the pathogenesis of B-cell chronic lymphocytic leukaemia (CLL). We investigated the impact of VEGFA gene diversity on the clinical outcome of patients with this disease. A VEGFA haplotype conformed by positions rs699947 (-1540C>A), rs833061 (-460T>C) and rs2010963 (405C>G) and two additional single-nucleotide polymorphisms (SNPs), rs3025039 (936C>T) and rs25648 (1032C>T), were analysed in 239 patients at the time of their CLL diagnosis. Here, we showed that homozygosity for rs699947/rs833061/rs2010963 ACG haplotype (ACG+/+ genotype) correlated with a reduced survival in CLL patients (ACG+/+ vs other genotypes: HR = 2.3, p = 0.002; recessive model). In multivariate analysis, the ACG+/+ genotype was identified as a novel independent prognostic factor (HR = 2.1, p = 0.005). Moreover, ACG homozygosity subdivided patients with CLL with otherwise indolent parameters into prognostic subgroups with different outcomes. Specifically, patients carrying the ACG+/+ genotype with mutated IgVH, very low and low-risk cytogenetics, initial clinical stage, CD38 negative status or early age at diagnosis showed a shorter survival (ACG+/+ vs other genotypes: HR = 3.5, p = 0.035; HR = 3.4, p = 0.001; HR = 2.2, p = 0.035; HR = 3.4, p = 0.0001 and HR = 3.1, p = 0.009, respectively). In conclusion, VEGFA ACG+/+ genotype confers an adverse effect in overall survival in CLL patients with an indolent course of the disease. These observations support the biological and prognostic implications of VEGFA genetics in CLL.

Clinical significance of occult cerebrospinal fluid involvement assessed by flow cytometry in non-Hodgkin's lymphoma patients at high risk of central nervous system disease in the rituximab era.

BACKGROUND AND AIM: Flow cytometry (FCM) analysis of cerebrospinal fluid (CSF) is more sensitive than conventional cytology (CC) for diagnosis of lymphomatous meningeosis, but the clinical significance of occult central nervous system (CNS) disease (positive FCM with negative CC) remains unknown. PATIENTS AND METHODS: CSF samples from 105 patients with newly diagnosed aggressive lymphomas at high risk of CNS involvement were prospectively studied by both CC and FCM, and results were correlated with cumulative incidence of CNS relapse and overall survival (OS). Patients were divided into three groups: 1) patients without CNS involvement (CC-/FCM-; n=83); 2) individuals with occult CNS disease (FCM+/CC-; n=15); and 3) cases with CNS disease (CC+/FCM+; n=7). RESULTS: Six cases showed CNS relapse or progression: two in Group 1 (2.4%), two in Group 2 (13%) and two in Group 3 (28.5%) (Group 2 vs. 1, P=0.04; Group 3 vs. 1, P<0.001). Patients from Groups 2 (P=0.05) and 3 (P<0.001) also showed a higher cumulative incidence of CNS relapse than those from Group 1. Significant differences were observed in OS between FCM-/CC- and FCM+/CC+ cases (P=0.02), while patients with occult CNS disease (FCM+/CC-) displayed intermediate OS rates, although differences did not reach statistical significance. CONCLUSIONS: The presence of occult CNS involvement at diagnosis in patients with NHL at high risk of CNS disease is associated with a higher probability of CNS relapse.

Prospective study on the practice of central nervous system prophylaxis and treatment in non-Hodgkin's lymphoma in Spain.

BACKGROUND AND OBJECTIVE: Central nervous system (CNS) involvement in patients diagnosed with non-Hodgkin's lymphoma (NHL) or other lymphoproliferative disorders is an infrequent complication with a poor prognosis. The prophylaxis and treatment of CNS involvement in these patients are not homogenous. The aim of this prospective longitudinal study was to report the current practice of CNS prophylaxis and treatment in patients with lymphoproliferative disorders in Spain. METHODS: Prospective study conducted from June 2005 to June 2006. Adult patients (> or = 18 yr) diagnosed with NHL or other lymphoproliferative disorders who received CNS prophylaxis or treatment were consecutively included through online registration. RESULTS: 228 patients from 33 hospitals were included. The mean (SD) age was 52 (16) yr and 144 (63%) were males. CNS therapy was given to 41 cases and consisted of triple intrathecal (IT) therapy (TIT, methotrexate, cytarabine and hydrocortisone) in 22, liposomal depot cytarabine in 18 and methotrexate in one. In addition, 4 patients received cranial radiotherapy. CNS prophylaxis (n = 187) consisted of TIT (166 cases), IT methotrexate (17), IT liposomal depot cytarabine (3) and IT cytarabine (1), whereas cranial or craniospinal radiotherapy was administered to 2 patients. The main reasons for CNS prophylaxis cited by the investigators included extranodal involvement (89 patients), raised serum lactate dehydrogenase level (87), IPI score > 2 (62), bulky mass (43), extranodal involvement in more than one organ (33), age over 60 yr (28) and human immunodeficiency virus infection (13). CONCLUSIONS: The results of this study point out the generalized use of TIT therapy both for CNS prophylaxis and therapy in patients with lymphoproliferative disorders in Spain. The introduction of the new formulations of drugs, especially liposomal depot cytarabine for CNS involvement, and the scarce use of radiotherapy are also of note. Similar to other studies, the absence of homogeneous criteria for CNS prophylaxis is of note.

Flow cytometric DNA analysis on fine needle aspiration biopsies of liver lesions.

Flow cytometric DNA analysis on fine needle aspiration biopsies of liver lesions The DNA cell content of 39 fine needle aspiration biopsies (FNAs) from five benign liver lesions, nine hepatocellular carcinomas (HCCs), and 25 metastatic tumours was analysed in a prospective fashion by flow cytometry (FCM). All benign lesions were diploid. Aneuploidy was found in five (55.6%) HCCs and in nine (36%) metastatic tumours. DNA index (DI) differences were not significant. The S-phase fraction (SPF) was higher in the malignant tumours, both combined (P < 0.02) and separated primary and metastatic (P < 0.05). We could not demonstrate an association between diploidy and percentage of benign hepatocytes in the smears of malignant tumours. The serum alpha-fetoprotein (AFP) level did not correlate with ploidy, DI, or SPF in the HCCs. In conclusion, ploidy and DI do not discriminate between benign and malignant liver lesions, but the SPF is higher in malignant tumours. DNA analysis does not help to distinguish primary from metastatic liver tumours. The presence of benign hepatocytes in samples from malignant tumours does not seem to influence the analysis of ploidy by FCM.