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Background The diagnosis of CLL is supported by a typical morphology and immunophenotype and usually does not present difficulties. Nevertheless, some patients with CLL can show an atypical phenotype, this raising the possibility of a lymphoproliferative disorder other than CLL. It has been recently shown that the expression of CD200 could be a rather consistent marker for CLL. Methods The expression of CD200 was investigated in 120 consecutive patients with B-cell chronic lymphoproliferative disorders (B-CLPD) (65 cases diagnosed as typical CLL, 16 atypical CLL, and 39 non-CLL before entering the study) by using multiparametric flow cytometry with four color combinations. CD200 was analyzed as percentage of positive cells (>= 30%) and MFIR expression. ROC curves were used to determine the cut-off for the CD200 MFIR. Matutes score (MS) was used as comparator. Results All 81 (100%) patients classified as CLL and 25 of 39 (64.1%) classified as non-CLL expressed high CD200 expression (>= 30%). CD200 expression showed a high sensitivity (100%) and a low specificity (35.9%), and the accuracy was similar to that of Matutes score markers (range: 79.2%-86.7%); except SmIg that was 59.1%. The addition of CD200 to the Matutes score correctly identified 74 of 81 (91.4%) CLL cases including 9 of 16 atypical CLL cases. As per non CLL cases, 37 of 39 (94.9%) were correctly diagnosed by the modified system. Altogether, CD200 improved the diagnostic accuracy of Matutes score from 86.7% to 92.5% (P < .01). Conclusion These results show that CD200 is a valuable, albeit not specific, CLL diagnostic marker. (c) 2018 International Clinical Cytometry Society

Chemoimmunotherapy (CIT) and targeted therapy with single‐agent ibrutinib are both recommended first‐line treatments for chronic lymphocytic leukemia (CLL), although their outcomes have not been directly compared. Using ibrutinib data from the RESONATE‐2 (PCYC‐1115/1116) study conducted in patients ≥65 years without del(17p), we performed a cross‐trial comparison with CIT data from published phase 3 studies in first‐line treatment of CLL. Progression‐free survival (PFS), overall survival (OS), and safety data for ibrutinib (median follow‐up 35.7 months) were evaluated alongside available CIT data. CIT regimens included: fludarabine + cyclophosphamide + rituximab (CLL8, CLL10), bendamustine + rituximab (CLL10), obinutuzumab + chlorambucil and rituximab + chlorambucil (CLL11), and ofatumumab + chlorambucil (COMPLEMENT‐1). Median age across studies was 61‐74 years, with older populations receiving ibrutinib, obinutuzumab + chlorambucil, or rituximab + chlorambucil. Median follow‐up varied across studies/regimens (range 14.5‐37.4 months). Among all patients, PFS appeared longer with ibrutinib relative to CIT and OS appeared comparable. Relative to CIT studies that similarly excluded patients with del(17p) (CLL10) or enrolled older/less‐fit patients (CLL11), PFS appeared favorable for ibrutinib in high‐risk subgroups, including advanced disease, bulky lymph nodes, unmutated IGHV status, and presence of del(11q). Grade ≥ 3 infections ranged from 9% (ofatumumab + chlorambucil) to 40% (fludarabine + cyclophosphamide + rituximab), and was 25% with ibrutinib. Grade ≥ 3 neutropenia was 12% for ibrutinib and 26%‐84% for CIT. Although definitive conclusions cannot be made due to inherent limitations of cross‐trial comparisons, this report suggests that ibrutinib has a favorable benefit/risk profile and may potentially eliminate the need for chemotherapy in some patients. Randomized, comparative studies are needed to support these findings.

Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. We have established a simple, nonviral protocol in three important biomedical model organisms frequently used in physiological studies. The protocol is based on the hyperactive Sleeping Beauty transposon system, SB100X, which reproducibly promoted generation of transgenic founders at frequencies of 50–64, 14–72, and 15% in mice, rats, and rabbits, respectively. The SB100X-mediated transgene integrations are less prone to genetic mosaicism and gene silencing as compared to either the classical pronuclear injection or to lentivirus-mediated transgenesis. The method was successfully applied to a variety of transgenes and animal models, and can be used to generate founders with single-copy integrations. The transposon vector also allows the generation of transgenic lines with tissue-specific expression patterns specified by promoter elements of choice, exemplified by a rat reporter strain useful for tracking serotonergic neurons. As a proof of principle, we rescued an inborn genetic defect in the fawn-hooded hypertensive rat by SB100X transgenesis. A side-by-side comparison of the SB100X- and piggyBac-based protocols revealed that the two systems are complementary, offering new opportunities in genome manipulation.—Katter, K., Geurts, A. M., Hoffmann, O., Mátés, L., Landa,V., Hiripi, L., Moreno, C., Lazar, J., Bashir, S., Zidek, V., Popova, E., Jerchow, B., Becker, K., Devaraj, A., Walter, I., Grzybowksi, M., Corbett, M., Rangel Filho, A., Hodges, M. R., Bader, M., Ivics, Z., Jacob, H. J., Pravenec, M., Bősze, Z., Rülicke, T., Izsvák, Z. Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits.

ZAP-70 is a tyrosine kinase that participates in early B-cell differentiation and is a prognostic factor in chronic lymphocytic leukaemia (CLL), where it is associated with an unmutated configuration of the IgV(H) genes. In this study ZAP-70 expression was studied by immunohistochemistry in a spectrum of B-cell lymphoid neoplasms; this staining method was compared with flow cytometry, and the relationship of ZAP-70 expression to mutational status and prognosis was assessed. 242 tissue samples from 225 patients with B-cell lymphoid neoplasms arising at different maturational stages were included. Flow cytometry was performed in all CLL cases (n = 52). IgV(H) mutational status was determined in 25 CLL and 12 mantle cell lymphoma (MCL) patients. ZAP-70 was positive in 34/52 (65%) CLL, 9/31 (31%) Burkitt's lymphoma, 2/7 (29%) lymphoblastic lymphomas, 3/36 (8%) MCL, 1/23 (4%) marginal zone lymphoma, and 1/45 (2%) diffuse large B-cell lymphomas, but in none of the 19 follicular lymphomas or the 14 Hodgkin lymphomas. An identical ZAP-70 pattern was obtained in six patients with simultaneous biopsies from different sites and in 12 patients with sequential biopsies. Immunohistochemistry and flow cytometry gave identical results in 48 the 52 CLLs. All but one ZAP-70-positive CLL had IgV(H) gene in an unmutated configuration, whereas all but one ZAP-70-negative CLL had somatically hypermutated IgV(H). The 12 MCLs analysed were ZAP-70 negative regardless of IgV(H) mutational status (4 mutated, 8 unmutated). ZAP-70 positive CLL was associated with a shorter overall survival (median time 103 months vs. 293 months, p = 0.01) and a shorter time to disease progression (median time 26 months vs. 60 months, p = 0.01). In conclusion, ZAP-70 is expressed in several types of B-cell neoplasm and is easily detected by immunohistochemistry, providing a useful prognostic marker in patients with CLL from whom no other material is available or when other techniques for its assessment cannot be performed.

The two tumour necrosis factor family proteins BAFF (TNFSF13B) and APRIL (TNFSF13) and their receptors [BAFF-R (TNFRSF13C), TACI (TNFRSF13B), BCMA (TNFRSF17)] play a critical role in the survival of normal B cells. The sensitivity of normal B cells to BAFF and APRIL can be modulated by signals regulated by their receptors. This modulation, however, has not been extensively investigated in chronic lymphocytic leukaemia (CLL) cells. We evaluated the expression, regulation and signalling of BAFF and APRIL receptors in normal and in CLL cells upon stimulation through CD40+IL4R and BCR. We further analysed the prognostic value of BAFF and APRIL receptors expression in patients with CLL. BCMA expression was significantly higher on CLL cells than on normal B cells. BCR and CD40+IL4R stimulation promoted an increase in TACI and BCMA expression, cell viability and activation in normal B cells. A similar effect was observed in CLL cells after CD40+IL4R but not BCR stimulation. BCMA expression correlated with unmutated IGHV genes, poor-risk cytogenetics, and short progression-free survival. These findings further characterize the link between CD40+IL4R regulatory signals, BAFF, APRIL and their receptors and the survival of leukaemic cells and clinical features of CLL.

Objective: To develop an easy-to-use system for initiating and monitoring enteral and parenteral nutrition support for pediatric inpatients. Design: A multidisciplinary pediatric nutrition support team developed a system to deliver appropriate nutrition support to pediatric inpatients. The package was devised and implemented to improve timeliness, safety, and efficacy of pediatric inpatient nutrition support. The goals included an easy to complete nutrition assessment, a system for early appropriate initiation of nutrition, advancement toward projected goal calories, and monitoring parameters for improved outcomes. Results The comprehensive evidence based package consisted of a four-sided laminated pocket reference with flow charts and tables; concise enteral and parenteral clinical nutrition support pathways; and computerized parenteral nutrition order form, a lab order set, and four one-page information sheets for initiation and advancement of age specific parenteral nutrition. A lecture summarizing this manuscript with handouts and bibliography heightened awareness in each department primarily responsible for the care for pediatric patients (Pediatrics, Family Practice, and Surgery) in our hospital. Conclusion: This package is a comprehensive educational tool for our housestaff. We believe it increases the likelihood of appropriate nutrition assessment and delivery by physicians working with pediatric inpatients at our hospital. It is easily adaptable to fit the needs of any inpatient pediatric nutrition support practice setting.