Decreased ratio of FOXP3+/FOXP3-CD45RA+CD4+ T cells in peripheral blood is associated with unexplained infertility and ART failure.

Unexplained infertility has a huge social impact and is a significant challenge for both clinicians and researchers. Previous studies have shown the involvement of multiple factors in infertility. Among these, the subset of regulatory T cells is of particular interest for the maternal tolerance towards the semi-allogenic fetus. We investigated circulating CD45RA+ regulatory and non-regulatory CD4+ T cells in healthy women and patients with unexplained infertility in the context of thymic output and peripheral proliferation. The proportion of FOXP3+ and FOXP3-CD45RA+CD4+ T cells in peripheral blood was studied in control groups of healthy parous and nulliparous (never-pregnant) women and in patients with unexplained infertility. In the same groups thymic output and peripheral proliferation were defined by the sj/ßTREC ratio, and signal joint T-cell receptor excision circles (sjTREC) and Ki67 expression, respectively. In parous women a decrease in sjTREC/105 cells and CD45RA+ T lymphocytes, compared to nulliparous group was found. At the same time, the proportion of FOXP3-CD45RA+CD4+ cells, but not FOXP3+CD45RA+ Tregs was reduced. In contrast, in patients with unsuccessful pregnancy, proportions of both regulatory and non-regulatory T cell counterparts were lower. Taken together, our results provide evidence for group-specific properties in the CD45RA+ T cell compartment between healthy parous, nulliparous and women with unexplained infertility.

Secondary acute myeloid leukemia and de novo acute myeloid leukemia with myelodysplasia-related changes - close or complete strangers?

AIM: To compare the main features of patients with secondary acute myeloid leukemias (AMLs) after post-myelodysplastic syndrome (AML-post-MDS) or post-myeloproliferative neoplasms (AML-post-MPN) and myeloid blast crisis of chronic myeloid leukemia (CML-BC) vs. de novoAMLs with myelodysplastic characteristics (dn-AML-MDS).

Bone Marrow Microenvironment Interplay and Current Clinical Practice in Multiple Myeloma: A Review of the Balkan Myeloma Study Group.

The course of multiple myeloma (MM) is influenced by a variety of factors, including the specificity of the tumour microenvironment (TME). The aim of this review is to provide insight into the interplay of treatment modalities used in the current clinical practice and TME. Bortezomib-based triplets are the standard for MM first-line treatment. Bortezomib is a proteasome inhibitor (PI) which inhibits the nuclear factor kappa B (NF-κB) pathway. However, bortezomib is decreasing the expression of chemokine receptor CXCR4 as well, possibly leading to the escape of extramedullary disease. Immunomodulatory drugs (IMiDs), lenalidomide, and pomalidomide downregulate regulatory T cells (Tregs). Daratumumab, anti-cluster of differentiation 38 (anti-CD38) monoclonal antibody (MoAb), downregulates Tregs CD38+. Bisphosphonates inhibit osteoclasts and angiogenesis. Sustained suppression of bone resorption characterises the activity of MoAb denosumab. The plerixafor, used in the process of stem cell mobilisation and harvesting, block the interaction of chemokine receptors CXCR4-CXCL12, leading to disruption of MM cells' interaction with the TME, and mobilisation into the circulation. The introduction of several T-cell-based immunotherapeutic modalities, such as chimeric-antigen-receptor-transduced T cells (CAR T cells) and bispecific antibodies, represents a new perspective in MM treatment affecting TME immune evasion. The optimal treatment approach to MM patients should be adjusted to all aspects of the individual profile including the TME niche.

Absolute Monocyte and Platelet Counts May Provide Additional Prognostic Information in Primary Gastric Diffuse Large B-cell Lymphoma Patients Treated with Rituximab and CHOP.

INTRODUCTION: Primary gastric diffuse large B cell lymphoma (PG-DLBCL) is the most common histological subtype of primary gastric lymphoma. The standard of care of PG-DLBCL patients is the combination rituximab-based immunochemotherapy (R-CHOP). Re-cently, different host-related factors have been shown to have significant prognostic significance in non-Hodgkin lymphoma. However, data regarding their prognostic contribution to PG-DLBCL are limited. AIM: To assess the prognostic impact of a panel of simple, cost-effective laboratory variables which are easy to apply in routine labora-tory use for R-CHOP-treated PG-DLBCL patients in an attempt to identify those among them that are high-risk category. MATERIALS AND METHODS: We retrospectively assessed the possible prognostic impact of different laboratory markers in 42 R-CHOP treated PG-DLBCL patients treated between 2004 and 2014 and followed at a single institution. RESULTS: The estimated 5-year overall (OS) and progression-free survival (PFS) of the whole group were 80.9% and 78%, respectively. The absolute monocyte and platelet counts in univariate analysis predicted PFS and OS when analyzed as continuous and dichotomized variables. On multivariate analysis performed with factors included in the stage-modified International Prognostic Index (m-IPI), the absolute monocyte and platelet counts remained independent predictors of PFS and OS. Therefore, the absolute monocyte and platelet counts were combined to generate a prognostic index that identified patients with an especially poor overall survival. CONCLUSIONS: This prognostic index was independent of the m-IPI and could provide additional prognostic information for better stratification of these patients.

Independent Continuing Medical Education (CME)/Continuing Professional Development (CPD) Must Deliver Unbiased Information.

Physicians commit themselves always to act in the best interests of their patients, and this includes their approach to continuing medical education (CME) as well as continuing professional development (CPD). For many years professional codes, and in some countries also the civil law, have defined that CME/CPD must be independent of commercial interests. Over the last few decades, numerous bodies have introduced CME/CPD accreditation to ensure that the planning and conduct of CME/CPD follows a set of defined standards, with independence of commercial interests as one of the leading principles. Recently industry has proposed that it be accepted by accrediting bodies as a direct provider of accredited CME-CPD. Such a move would not only open the door to the introduction of an inevitable bias in CME/CPD but would jeopardise the professional standing of physicians. Accreditation of CME/CPD currently serves several different purposes, but its credibility depends on whether it can retain its ability to differentiate independent CME/CPD from the provision of commercially framed information.

Late Postpregnancy Modifications in the Subset of Peripheral Natural T Regulatory Cells in Healthy Women.

The establishment of a relevant regulatory T cell (Treg) pool in the periphery is of importance to ensure immune homoeostasis. Finely tuned signaling pathways in Tregs control the immune response during extreme endocrine changes in pregnancy and afterward. In this study, we investigate the population of Tregs and, in particular, the natural Tregs (nTregs) in healthy women divided into three groups according to the number of previous pregnancies, if any (Gr.1-one pregnancy, Gr.2-≥2 pregnancies, and Gr.0-no pregnancy). The overall analysis showed similar proportions in the entire Treg pool and nTregs (FoxP3+CD45RA+) in all the three groups (p > 0.05). However, the age-related trend of CD25+ nTregs was found to be different in parous and nonparous women. Analysis of phosphorylated ERK1/2, an important signaling molecule in T cell maintenance, showed a significantly higher percentage in CD25+ nTregs in the group of nonparous compared with parous women (p < 0.05). Thus, our results provide evidence that pregnancy may exert a long-lasting impact on the subset of nTregs due to the extreme changes in the hormonal status, which in turn, influences pre- and post-thymic maturation.

Differences in ex-vivo Chemosensitivity to Anthracyclines in First Line Acute Myeloid Leukemia.

BACKGROUND: Induction schedules in acute myeloid leukemia (AML) are based on combinations of cytarabine and anthracyclines. The choice of the anthracycline employed has been widely studied in multiple clinical trials showing similar complete remission rates. MATERIALS AND METHODS: Using an ex vivo test we have analyzed if a subset of AML patients may respond differently to cytarabine combined with idarubicin, daunorubicin or mitoxantrone. Bone marrow (BM) samples of 198 AML patients were incubated for 48 hours in 96 well plates, each well containing different drugs or drug combinations at different concentrations. Ex vivo drug sensitivity analysis was made using the PharmaFlow platform maintaining the BM microenvironment. Drug response was evaluated as depletion of AML blast cells in each well after incubation. Annexin V-FITC was used to quantify the ability of the drugs to induce apoptosis, and pharmacological responses were calculated using pharmacokinetic population models. RESULTS: Similar dose-respond graphs were generated for the three anthracyclines, with a slight decrease in EC50 with idarubicin (p=1.462E-06), whereas the interpatient variability of either drug was large. To identify those cases of selective sensitivity to anthracyclines, potency was compared, in terms of area under the curve. Differences in anthracycline monotherapy potency greater than 30% from 3 pairwise comparisons were identified in 28.3% of samples. Furthermore, different sensitivity was detected in 8.2% of patients comparing combinations of cytarabine and anthracyclines. DISCUSSION: A third of the patients could benefit from the use of this test in the first line induction therapy selection, although it should be confirmed in a clinical trial specifically designed.

A Rare Case of Primary Histiocytic Sarcoma of the Stomach.

Histiocytic sarcoma is a rare lymphohematopoietic malignancy with aggressive clinical course and poor therapy response. The diagnosis relies on the confirmation of its histiocytic lineage and exclusion of other poorly differentiated tumors. Most of the cases present in extranodal sites, but primary gastric involvement is exceptional. We report a case of a 69-year-old woman with epigastric pain and systemic symptoms. Gastroscopy findings and biopsy report suggested a malignant neoplasm. The patient underwent distal subtotal gastrectomy with a 6-cm tumor in the body and antrum of the stomach and ten associated enlarged perigastric lymph nodes. Microscopically they were infiltrated with atypical tumor cells and immunohistochemical staining was positive for CD68, lysozyme, CD45, and CD4; 45% of the cells stained for Ki-67. The pathologic diagnosis was histiocytic sarcoma. CT body scans showed only enlarged retroperitoneal and abdominal lymph nodes. The patient received six cycles of CHOEP chemotherapy with complete therapeutic response, but three months later she experienced an aggressive systemic sarcoma recurrence and although salvage chemotherapy was initiated she died of progressive disease. The presented case widens the differential diagnosis of gastric malignancies, and emphasizes the significance of immunohistochemical examination for histiocytic sarcoma diagnosis. The collection and evaluation of cases of gastric histiocytic sarcoma are important to obtain further progress in prognosis and treatment.

The future of accreditation of continuing medical education (CME)-continuing professional development (CPD) in Europe: harmonisation through dialogue and consensus.

In Europe, there are currently some 30 different jurisdictions and no overarching legislation regarding CME-CPD accreditation, since legislative competency related to national health-care systems lies with national authorities. Thus, public demonstration of professional agreement regarding the principles, rules and practice of CME-CPD as well as its accreditation is a highly desirable professional and political objective in Europe, where free movement and freedom to offer professional (medical) services is a key feature of the EU vision of the single market. The newly formed association of independent European accreditors, Continuing Medical Education - European Accreditors (CME-EA) is committed to offering a platform for dialogue between individuals and organisations involved in definition of professional codes in general, and accreditation of CME-CPD in particular on the national level. The aim is to reach a European consensus on principles and rules applied in planning and delivery of CME-CPD. This includes consensus on constituent characteristics of accreditors as well as principles and practice of accreditation.

Calorimetric markers for monitoring of multiple myeloma and Waldenström's macroglobulinemia patients.

The blood proteome has been studied extensively for identification of novel reliable disease biomarkers. In recent years, differential scanning calorimetry has emerged as a new tool for characterization of the thermodynamic properties of the major serum/plasma proteins and for the establishment of calorimetric markers for a variety of diseases. Here we applied calorimetry to monitor the effect of treatment of patients diagnosed with multiple myeloma and Waldenström's macroglobulinemia on the calorimetric profiles of patients' blood sera. The parameters derived from the calorimetric profiles were compared with the primary serum biomarkers, monoclonal immunoglobulin (M protein) concentration, and κ/λ free light chain ratio. For the secretory cases, the calorimetric parameters thermogram's shape similarity and weighted average center strongly depended on the M protein level but had lower sensitivity and specificity. By contrast, for non-secretory cases, the calorimetric parameters did not depend on the κ/λ free light chains ratio and exhibited significantly higher sensitivity and specificity than M protein levels. A combination of the immunological and calorimetric tests was found to greatly improve the sensitivity and specificity of the clinical status evaluation. The pronounced differences in blood sera thermograms before and during monitoring reflected the individual patients' response to treatment received and showed maintenance of heterogeneity during the disease course.

Position paper on current aspects of sponsoring in accredited CME.

This position paper is the result of a collaborative approach of several European Specialty Accreditation Boards (ESABs) and, has been stimulated by their current experience in accreditation regarding roles and responsibilities assumed by sponsors of accredited continuing medical education (CME). The suggestions made in this paper aim to preserve the fundamental principle in CME accreditation that the physician in charge of the programme has sole responsibility for the selection of topics, speakers, content and format, as well as mode of presentation, and that sponsors will under no circumstances interfere with this principle. This is considered as a responsibility of an individual physician (or physicians), which cannot be delegated, even in part, to third parties. This responsibility has been extended to include all communication before and after the event. The paper also identifies undecided issues, about which ESABs are committed to elaborate proposals in the future.

Apoptotic mechanisms of the biotechnologically produced arylnaphtalene lignan justicidin B in the acute myeloid leukemia-derived cell line HL-60.

BACKGROUND: The present study aimed at optimization of the biotechnological production of the lignan justicidin B by genetically transformed cultures of Linum leonii and the pharmacological evaluation of the pro-apoptotic effects of the compound in HL-60 cells. METHODS: A rapidly growing selected root line of L. leonii was grown in 2-L bioreactor for period of 40 days and the protocols for obtaining of the compound have been optimized. The pharmacological study included evaluation of the cytotoxicity of the compound in HL-60 cells (MTT-assay), its apoptogenic effects and its effects on caspase 3,8 and 9 activation. RESULTS: After 40 days of sterile run scale up of hairy root culture in bioreactor, 27.2g/L dry weight of root biomass was harvested from the bioreactor culture vessel, recording about nine times increase over initial inoculum (3.0g), with 1.55%±0.07 Justicidin B, greater than yields from 300ml flasks. Our findings are the first work toward the scale up of L. leonii hairy roots-based biotechnological production of Justicidin B, employing bioreactors for high biomass production to meet the industrial requirement. The results from the pharmacological evaluation have shown that the tested arylnaphtalene lignan is a potent cytotoxic and proapoptotic agent against HL-60. The induction of apoptosis proceeds via activation of the intrinsic mitochondrial cell-death signaling pathways. CONCLUSION: The potent activity at low micromolar concentration and the feasibility of biotechnological production of justicidin B implies that there is enormous scope in its further evaluation as possible antineoplastic drug candidate.

NPM1 Gene Type A Mutation in Bulgarian Adults with Acute Myeloid Leukemia: A Single-Institution Study.

OBJECTIVE: Mutations of the nucleophosmin (NPM1) gene are considered as the most frequent acute myeloid leukemia (AML)-associated genetic lesion, reported with various incidences in different studies, and type A (NPM1-A) is the most frequent type. However, since most series in the literature report on the features of all patients regardless of the type of mutation, NPM1-A(+) cases have not been well characterized yet. Therefore, we evaluated the prevalence of NPM1-A in Bulgarian AML patients and searched for an association with clinical and laboratory features. MATERIALS AND METHODS: One hundred and four adults (51 men, 53 women) were included in the study. NPM1-A status was determined using allele-specific reverse-transcription polymerase chain reaction with co-amplification of NPM1-A and ß-actin and real-time quantitative TaqMan-based polymerase chain reaction. Patients received conventional induction chemotherapy and were followed for 13.2±16.4 months. RESULTS: NPM1-A was detected in 26 (24.8%) patients. NPM1-A mutation was detected in all AML categories, including in one patient with RUNX1-RUNX1T1. There were no differences associated with the NPM1-A status with respect to age, sex, hemoglobin, platelet counts, percentage of bone marrow blasts, splenomegaly, complete remission rates, and overall survival. NPM1-A(+) patients, compared to NPM1-A(-) patients, were characterized by higher leukocyte counts [(75.4±81.9)x109/L vs. (42.5±65.9)x109/L; p=0.049], higher frequency of normal karyotype [14/18 (77.8%) vs. 26/62 (41.9%); p=0.014], higher frequency of FLT3-ITD [11/26 (42.3%) vs. 8/77 (10.4%); p=0.001], and lower incidence of CD34(+) [6/21 (28.8%) vs. 28/45 (62.2%); p=0.017]. Within the FLT3-ITD(-) group, the median overall survival of NPM1-A(-) patients was 14 months, while NPM1-A(+) patients did not reach the median (p=0.10). CONCLUSION: The prevalence of NPM1-A mutation in adult Bulgarian AML patients was similar to that reported in other studies. NPM1-A(+) patients were characterized by higher leukocyte counts, higher frequency of normal karyotypes and FLT3-ITD, and lower incidence of CD34(+), supporting the idea that the specific features of type A mutations might contribute to the general clinical and laboratory profile of NPM1(+) AML patients.

Alkylphosphocholines and curcumin induce programmed cell death in cutaneous T-cell lymphoma cell lines.

While most patients with early-stage cutaneous T-cell lymphomas (CTCL) have a very good prognosis, the survival of patients with extensive tumour stage and visceral involvement remains extremely poor and necessitates the development of more effective treatment modalities. In this study, we evaluated the in vitro effects of two alkylphosphocholines (APCs, miltefosine and erufosine) and the polyphenolic compound curcumin on 5 human CTCL cell lines (Hut-78, HH, MJ, My-La CD4+ and My-La CD8+). All tested drugs showed considerable cytotoxic activity, as determined by the MTT dye reduction assay. The IC50 values of both APCs ranged from the low micromolar level (Hut-78 cells) to 60-80µM (HH cells). The IC50 values of curcumin ranged from 12 to 24µM. All tested drugs induced apoptosis, as ascertained by morphological changes, DNA fragmentation and activation of caspase cascades. Miltefosine and erufosine induced dephosphorylation of Akt in My-La CD8+ cells and phosphorylation of JNK in Hut-78 and My-La CD8+ cells. APCs increased the level of the autophagic marker LC3B in Hut-78 and MJ cells. Results from co-treatment with autophagy modulators suggested that the cytotoxicity of APCs in CTCL cells is mediated, at least in part, by induction of autophagy.

Simultaneous occurrence of ETV6-RUNX1 and BCR-ABL1 (e1a2) transcripts in a child with B-cell acute lymphoblastic leukemia.

We report on a rare case of a 3-year-old boy with B-cell acute lymphoblastic leukemia (B-ALL), which was characterized simultaneously with two different fusion transcripts: ETV6-RUNX1 and BCR-ABL1 (e1a2). The patient presented with fever, diarrhea, normal white blood cell counts of 5.9×10(9)/L without circulating abnormal cells, anemia, and thrombocytopenia, as well as an enlarged liver without splenomegaly. The bone marrow was markedly hypercellular with a total infiltration of agranular lymphoid blast cells with a B-II (pre-B) lymphoblastic phenotype: cyCD79α(+), CD19(+), sCD22(+), CD10(+), CD20(-), CD34(+), and sIgM(-), with dim aberrant co-expression of the myeloid-associated markers CD13(+) and CD33(+). Conventional cytogenetic analysis was unsuccessful; however, molecular analysis revealed the BCR-ABL1 (p190) and ETV6-RUNX1 transcripts. A diagnosis of BCR-ABL1 (p190)-positive and ETV6-RUNX1-positive B-ALL was made, and treatment was initiated according to the AIEOP-BFM-ALL2000 protocol. A complete remission was achieved after the first induction course of chemotherapy. Twelve months after the diagnosis, the child is alive with levels of residual disease of <0.05% estimated both by 8-color flow cytometry and real-time quantitative reverse transcription polymerase chain reaction.