Postoperative RNFL-Changes after Successful Trabeculectomy: 2-Year Outcomes. / Zwei-Jahres-Ergebnisse postoperativer RNFL-Entwicklung nach erfolgreicher Trabekulektomie.

BACKGROUND: The most important tool in glaucoma therapy is to lower the intraocular pressure to slow down the apoptosis of retinal ganglion cells. Trabeculectomy (TE) is considered the gold standard in glaucoma surgery. The aim of this study was to analyse the postoperative changes in retinal nerve fibre layer (RNFL) using optical coherence tomography (OCT) after TE. MATERIAL AND METHODS: We examined 40 patients naïve to prior glaucoma surgery retrospectively, who received a TE for medically uncontrolled primary open-angle glaucoma (POAG). Intraocular pressure (IOP), IOP-lowering medication, mean deviation of perimetry, visual acuity and peripapillary RNFL-thickness using OCT were evaluated during the first 24 month after TE. RESULTS: In total 40 eyes from 40 patients were treated with TE. Mean IOP decreased from 25.0 ± 0,9 to 13.9 ± 0.6 (p < 0.01), and the mean number of IOP-lowering eye drops from 3.3 ± 0.2 to 0.5 ± 0.2 (p < 0.01). Visual acuity and mean deviation in perimetry remained stable while mean global RNFL-thickness decreased from 67.8 ± 2.9 to 63.7 ± 2.9 (p < 0.01) and 63.4 ± 2.9 µm (p < 0.01) 12 and 24 months after TE. CONCLUSION: The TE is an effective method to reduce the IOD and the amount of IOP-lowering medication. Nevertheless, a significant further loss in RNFL thickness was observed in the first 12 months after TE. Thus, RNFL changes seem to stabilise only after a protracted period.

Idelalisib (PI3Kδ inhibitor) therapy for patients with relapsed/refractory chronic lymphocytic leukemia: A Swedish nation-wide real-world report on consecutively identified patients.

OBJECTIVES: We examined the efficacy and toxicity of the PI3Kδ inhibitor idelalisib in combination with rituximab salvage therapy in consecutively identified Swedish patients with chronic lymphocytic leukemia (CLL). METHODS AND RESULTS: Thirty-seven patients with relapsed/refractory disease were included. The median number of prior lines of therapy was 3 (range 1-11); the median age was 69 years (range 50-89); 22% had Cumulative Illness Rating Scale (CIRS) >6 and 51% had del(17p)/TP53 mutation. The overall response rate was 65% (all but one was partial response [PR]). The median duration of therapy was 9.8 months (range 0.9-44.8). The median progression-free survival was 16.4 months (95% CI: 10.4-26.3) and median overall survival had not been reached (75% remained alive at 24 months of follow-up). The most common reason for cessation of therapy was colitis (n = 8, of which seven patients experienced grade ≥3 colitis). The most common serious adverse event was grade ≥3 infection, which occurred in 24 patients (65%). CONCLUSIONS: Our real-world results suggest that idelalisib is an effective and relatively safe treatment for patients with advanced-stage CLL when no other therapies exist. Alternative dosing regimens and new PI3K inhibitors should be explored, particularly in patients who are double-refractory to inhibitors of BTK and Bcl-2.

Implantation of XEN After Failed Trabeculectomy: an Efficient Therapy? / Die XEN-Microstent-Implantation nach erfolgloser Trabekulektomie: eine sinnvolle Behandlungsoption?

BACKGROUND: Trabeculectomy (TE) has been the standard procedure in glaucoma surgery for a long time. This study examined the efficacy and safety profile of XEN45 Gel Stent (XEN) after failed and/or scarred trabeculectomy. MATERIAL AND METHODS: We analysed all files of patients, who received a XEN after insufficient TE and examined changes in intraocular pressure (IOP), IOP-lowering medication, best corrected visual acuity, visual field tests as well as the intra- and postoperative complications recorded within a 12-month follow-up period. RESULTS: 31 eyes of 28 patients were analysed in our study (mean age: 66,2 ± 13,4 years; 39% female; 48% right eye; mean follow-up after TE: 70,3 ± 64,9 months). The mean IOP decreased from 23,5 ± 6,5 to 18,0 ± 5,3 mmHg (- 23,5% compared to baseline-IOP; p = 0,01) while the mean IOP-lowering medication could be reduced from 2,8 ± 1,1 to 1,1 ± 1,5 (p < 0,01) 12 months after XEN-implantation. The mean visual acuity did not change significantly (pre-op: 0,5 ± 0,6 logMAR; 12 months post-op: 0,5 ± 0,6 logMAR). The most common complications postoperatively were choroideal detachment due to postoperative hypotony in 4 eyes (13%), a needling procedure in 9 eyes (29%), a Re-XEN-Implantation in 4 eyes (13%), an open revision of the conjunctiva in 3 eyes (10%), and a Re-TE in 1 eye (3%) as well as an Ahmed-Valve implantation in 2 eyes (6%). Overall, neither needling procedure nor further glaucoma surgery was necessary in 19 eyes (61%). In 10 of 22 evaluable eyes (45%) an IOP reduction of > 20% was achieved 12 months after XEN implantation. CONCLUSION: XEN could be an effective method to reduce IOP after failed TE. The rate of complications seems to be low and the rate of needling procedures and/or revisions is acceptable.

Megakaryocytes harbour the del(5q) abnormality despite complete clinical and cytogenetic remission induced by lenalidomide treatment.

The mechanisms underlying lenalidomide-resistance of del(5q) MDS stem cells remain to be elucidated and may include cell-intrinsic as well as microenvironmental causes. Abnormal hypolobated megakaryocytes constitute one of the hallmarks of del(5q) MDS. We hypothesized that these cells have potential implications for the regulation of haematopoietic stem cells (HSC) similarly to what has recently been described for megakaryocytes in the murine system. Therefore, we conducted a study to determine the response of abnormal hypolobated megakaryocytes to lenalidomide therapy. We studied lenalidomide-treated patients in the MDS-004 trial as well as a cohort seen at our institution. Morphological evaluation at time of complete cytogenetic remission (CCyR) demonstrated the persistence of hypolobated megakaryocytes in all evaluable patients (n = 9). Furthermore, we provide evidence that the abnormal hypolobated morphology is restricted to del(5q) megakaryocytes, both at diagnosis and during CCyR. Using fluorescence in situ hybridisation analysis on flow-sorted stem- and progenitor populations, we observed a similar degree of clonal involvement in megakaryocyte-erythroid-progenitors as in HSC. Taken together, our findings suggest that megakaryocyte morphology might aid in the evaluation of patients where discontinuation of lenalidomide is considered and offers interesting hypotheses for further investigation of lenalidomide resistance.

Progression in patients with low- and intermediate-1-risk del(5q) myelodysplastic syndromes is predicted by a limited subset of mutations.

A high proportion of patients with lower-risk del(5q) myelodysplastic syndromes will respond to treatment with lenalidomide. The median duration of transfusion-independence is 2 years with some long-lasting responses, but almost 40% of patients progress to acute leukemia by 5 years after starting treatment. The mechanisms underlying disease progression other than the well-established finding of small TP53-mutated subclones at diagnosis remain unclear. We studied a longitudinal cohort of 35 low- and intermediate-1-risk del(5q) patients treated with lenalidomide (n=22) or not (n=13) by flow cytometric surveillance of hematopoietic stem and progenitor cell subsets, targeted sequencing of mutational patterns, and changes in the bone marrow microenvironment. All 13 patients with disease progression were identified by a limited number of mutations in TP53, RUNX1, and TET2, respectively, with PTPN11 and SF3B1 occurring in one patient each. TP53 mutations were found in seven of nine patients who developed acute leukemia, and were documented to be present in the earliest sample (n=1) and acquired during lenalidomide treatment (n=6). By contrast, analysis of the microenvironment, and of hematopoietic stem and progenitor cells by flow cytometry was of limited prognostic value. Based on our data, we advocate conducting a prospective study aimed at investigating, in a larger number of cases of del(5q) myelodysplastic syndromes, whether the detection of such mutations before and after lenalidomide treatment can guide clinical decision-making.

Refractive Changes after Glaucoma Surgery-A Comparison between Trabeculectomy and XEN Microstent Implantation.

Best-corrected visual acuity often decreases temporarily or permanently after trabeculectomy (TE). The purpose of this study was to compare visual acuity and refractive changes after TE or XEN microstent implantation (XEN) in primary open-angle glaucoma (POAG) or pseudoexfoliation glaucoma (PEX) cases naïve to prior glaucoma surgery over a 24-month follow-up period. We analyzed 149 consecutive glaucoma patients who received either TE or XEN because of medically uncontrollable POAG or PEX. Intraocular pressure (IOP), IOP-lowering medication use, subjective and objective refraction and best-corrected visual acuity were evaluated. In addition, surgically induced astigmatism (SIA) was calculated and compared using the vector analysis method described by Jaffe and Clayman. A total of 93 eyes (85 POAG; 8 PEX) were treated with TE and 56 eyes (50 POAG; 6 PEX) with XEN. After 24 months, the mean IOP and number of IOP-lowering medications used decreased significantly after TE (p < 0.01) and XEN (p < 0.01). In the TE group, mean best-corrected visual acuity (BCVA) changed from 0.16 ± 0.26 to 0.23 ± 0.28 logMAR (p < 0.01) after 24 months, while mean BCVA did not change significantly in the XEN group (preoperative: 0.40 ± 0.50 logMAR, postoperative: 0.36 ± 0.49 logMAR; p = 0.28). SIA was almost the same in both groups at the end of the 24-month follow-up period (0.75 ± 0.60 diopters after TE and 0.81 ± 0.56 diopters after XEN; p = 0.57). In addition, there was no significant correlation between SIA and the observed BCVA changes or SIA and IOP reduction 12 or 24 months after TE or XEN. Our results demonstrate that TE and XEN are effective methods for reducing IOP and IOP-lowering medication use. The SIA was nearly similar in both groups. The SIA does not seem responsible for the decreased visual acuity after TE.

Two Year Functional and Structural Changes-A Comparison between Trabeculectomy and XEN Microstent Implantation Using Spectral Domain Optical Coherence Tomography.

The aim of this study was to analyze retinal nerve fiber layer (RNFL) thickness after trabeculectomy (TE) versus XEN microstent implantation (XEN) in primary open-angle glaucoma (POAG) cases naïve to prior incisional glaucoma surgery. We examined 119 consecutive glaucoma patients retrospectively, who received a TE or XEN for medically uncontrolled POAG. Intraocular pressure (IOP), amount of IOP-lowering medication, mean deviation of standard automated perimetry and peripapillary RNFL thickness were evaluated during the first 24 months after surgery. Fifty eyes were treated with TE and 69 eyes with XEN. Mean IOP decreased from 25.1 ± 0.8 to 13.3 ± 0.6 mm Hg (p < 0.01) and mean number of IOP-lowering eye drops from 3.2 ± 0.2 to 0.4 ± 0.1 (p < 0.01) 24 months after TE. In 69 eyes undergoing XEN, mean IOP dropped from 24.8 ± 0.6 to 15.0 ± 0.4 mm Hg (p < 0.01) and medication from 3.0 ± 0.1 to 0.6 ± 0.1 (p < 0.01) during the 24 months follow-up. Mean deviation of standard automated perimetry remained stable in TE (8.5 ± 0.7 to 8.1 ± 0.8 dB; p = 0.54) and XEN group (11,0 ± 0.5 to 11.5 ± 0.5 dB; p = 0.12) after 24 months, while mean RNFL thickness further deteriorated in the TE (−2.28 ± 0.65 µm/year) and XEN (−0.68 ± 0.34 µm/year) group. Postoperative RNFL loss develops after TE and XEN despite effective and significant lowering of IOP and amount of IOP-lowering medication. RNFL loss was more pronounced in the first year after glaucoma surgery.

Fusion of hematopoietic cells with Purkinje neurons does not lead to stable heterokaryon formation under noninvasive conditions.

Transplanted hematopoietic cells have previously been shown to contribute to cells of other tissues by cell fusion. We wanted to elucidate whether this phenomenon of cell fusion also occurs under physiological conditions. Using a transgenic mouse reporter system to irreversibly label cells of the hematopoietic lineage, we were able to test their contribution to other tissues in the absence of any additional and potentially confounding factors such as irradiation or chemoablation. We found genetically marked, fused Purkinje neurons as well as hepatocytes in numbers comparable to previous bone marrow transplantation studies. The number of fused Purkinje neurons increased after intrathecal administration of bacterial lipopolysaccharide, suggesting that cell fusion can be induced by inflammation. In contrast to previous studies, however, genetically labeled Purkinje neurons never contained more than one nucleus, and we found only a single cell containing two Y-chromosomes in a male mouse. Consistent with results from the mouse model and unlike human bone marrow transplant recipients, postmortem adult human cerebelli of nontransplanted individuals were devoid of binucleated or polyploid Purkinje neurons. Therefore, our data suggests that fusion of hematopoietic cells with Purkinje neurons is only transient and does not lead to stable heterokaryon formation under noninvasive conditions.

Comparison of ATP-binding cassette transporter interactions with the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib.

Although the development of tyrosine kinase inhibitors (TKIs) to control the unregulated activity of BCR-ABL revolutionized the therapy of chronic myeloid leukemia, resistance to TKIs is a clinical reality. Among the postulated mechanisms of resistance is the overexpression of ATP-binding cassette (ABC) transporters, such as P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2), which mediate reduced intracellular drug accumulation. We compared the interactions of the TKIs imatinib, nilotinib, and dasatinib with ABCB1 and ABCG2 in ex vivo and in vitro systems. The TKIs inhibited rhodamine 123 and Hoechst 33342 efflux mediated by endogenous expression of the transporters in murine and human hematopoietic stem cells with potency order nilotinib >> imatinib >> dasatinib. Studies with ABCB1-, ABCG2-, and ABCC1-transfected human embryonic kidney 293 cells verified that nilotinib was the most potent inhibitor of ABCB1 and ABCG2. Cytotoxicity assays in stably transduced K562-ABCG2 and K562-ABCB1 cells confirmed that the TKIs were also substrates for the two transporters. Like imatinib, both nilotinib and dasatinib decreased ABCG2 surface expression in K562-ABCG2 cells. Finally, we found that all TKIs were able to compete labeling of ABCB1 and ABCG2 by the photo-cross-linkable prazosin analog [(125)I]iodoarylazidoprazosin, suggesting interaction at the prazosin-binding site of both proteins. Our experiments support the hypothesis that all three TKIs are substrates of ABC transporters and that, at higher concentrations, TKIs overcome transporter function. Taken together, the results suggest that therapeutic doses of imatinib and nilotinib may diminish the potential of ABCB1 and ABCG2 to limit oral absorption or confer resistance. Clinical data are required to definitively answer the latter question.

Symptomatic marginal ulcer after pancreatoduodenectomy.

BACKGROUND: Marginal ulcer is a well-known complication after pancreatoduodenectomy. In light of increasing long-term survival after pancreatoduodenectomy, the identification of risk factors and preventive strategies are of utmost importance. We assessed the incidence, clinical impact, and potential risk factors of marginal ulcer after pancreatoduodenectomy. METHODS: A prospectively maintained database of patients undergoing pancreatoduodenectomy was analyzed retrospectively. Univariate and bivariate competing-risk Cox regression analyses were performed to identify risk factors for marginal ulcer. RESULTS: Two hundred and fifty-five consecutive patients underwent pancreatoduodenectomy. The median follow-up was 35.7 months. Marginal ulcer was diagnosed in 19 patients (7.5%), and the median time from pancreatoduodenectomy to marginal ulcer diagnosis was 450 days. Thirteen of these 19 patients presented with abdominal pain, melena, or anemia. In all these 13 patients, marginal ulcer resolved with proton pump inhibitor therapy and endoscopic surveillance. Six patients with marginal ulcer presented with an acute abdomen and underwent emergency laparotomy for marginal ulcer perforation and peritonitis. There was no marginal ulcer-related mortality. Univariate and bivariate competing-risk analyses showed an increased risk for marginal ulcer with discontinuation of proton pump inhibitor therapy, smoking, alcohol intake, and the use of non-steroidal anti-inflammatory drugs. Discontinuation of proton pump inhibitor therapy was an independent risk factor for marginal ulcer development. CONCLUSION: Marginal ulcer is a relevant long-term complication after pancreatoduodenectomy that occurs more frequently after proton pump inhibitor therapy is discontinued. Based on our data, permanent use of proton pump inhibitor after pancreatoduodenectomy is strongly recommended especially for those patients who smoke, consume alcohol, or use non-steroidal anti-inflammatory drugs.

ABCG2 is expressed in late spermatogenesis and is associated with the acrosome.

An increasingly exploited strategy for the isolation of stem cells is based on the increased efflux of Hoechst 33342 lipophilic dye mediated by ABCG2, an ATP-binding cassette transporter which is highly expressed in various stem cells. We found ABCG2 expression to be present at later stages of spermatogenesis. Western blot analysis using an anti-ABCG2 antibody revealed expression of a 72kDa band in mature sperm obtained from mice, rats, bulls or humans. Immunocytochemistry studies revealed acrosomal staining pattern of ABCG2 in spermatozoa. Experiments using the Hoechst 33342 ABCG2 substrate and the ABCG2-specific inhibitor FTC demonstrated efflux activity of ABCG2 in mature sperm. Incubation of sperm in capacitating medium in the presence of the ABCG2-inhibitor FTC resulted in decreased cholesterol depletion compared to sperm incubated in the absence of FTC. Our results demonstrate that ABCG2 is expressed at the acrosome in mature sperm. ABCG2 may thus serve to mediate cholesterol removal.

The age and genomic integrity of neurons after cortical stroke in humans.

It has been unclear whether ischemic stroke induces neurogenesis or neuronal DNA rearrangements in the human neocortex. Using immunohistochemistry; transcriptome, genome and ploidy analyses; and determination of nuclear bomb test-derived (14)C concentration in neuronal DNA, we found neither to be the case. A large proportion of cortical neurons displayed DNA fragmentation and DNA repair a short time after stroke, whereas neurons at chronic stages after stroke showed DNA integrity, demonstrating the relevance of an intact genome for survival.

Myelodysplastic syndromes are propagated by rare and distinct human cancer stem cells in vivo.

Evidence for distinct human cancer stem cells (CSCs) remains contentious and the degree to which different cancer cells contribute to propagating malignancies in patients remains unexplored. In low- to intermediate-risk myelodysplastic syndromes (MDS), we establish the existence of rare multipotent MDS stem cells (MDS-SCs), and their hierarchical relationship to lineage-restricted MDS progenitors. All identified somatically acquired genetic lesions were backtracked to distinct MDS-SCs, establishing their distinct MDS-propagating function in vivo. In isolated del(5q)-MDS, acquisition of del(5q) preceded diverse recurrent driver mutations. Sequential analysis in del(5q)-MDS revealed genetic evolution in MDS-SCs and MDS-progenitors prior to leukemic transformation. These findings provide definitive evidence for rare human MDS-SCs in vivo, with extensive implications for the targeting of the cells required and sufficient for MDS-propagation.

The transporter ABCB7 is a mediator of the phenotype of acquired refractory anemia with ring sideroblasts.

Refractory anemia with ring sideroblasts (RARS) is characterized by mitochondrial ferritin (FTMT) accumulation and markedly suppressed expression of the iron transporter ABCB7. To test the hypothesis that ABCB7 is a key mediator of ineffective erythropoiesis of RARS, we modulated its expression in hematopoietic cells. ABCB7 up and downregulation did not influence growth and survival of K562 cells. In normal bone marrow, ABCB7 downregulation reduced erythroid differentiation, growth and colony formation, and resulted in a gene expression pattern similar to that observed in intermediate RARS erythroblasts, and in the accumulation of FTMT. Importantly, forced ABCB7 expression restored erythroid colony growth and decreased FTMT expression level in RARS CD34+ marrow cells. Mutations in the SF3B1 gene, a core component of the RNA splicing machinery, were recently identified in a high proportion of patients with RARS and 11 of the 13 RARS patients in this study carried this mutation. Interestingly, ABCB7 exon usage differed between normal bone marrow and RARS, as well as within the RARS cohort. In addition, SF3B1 silencing resulted in downregulation of ABCB7 in K562 cells undergoing erythroid differentiation. Our findings support that ABCB7 is implicated in the phenotype of acquired RARS and suggest a relation between SF3B1 mutations and ABCB7 downregulation.

The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors.

A promising and increasingly exploited property of hematopoietic stem cells is their ability to efflux the fluorescent dye Hoechst 33342. The Hoechst-negative cells are isolated by fluorescence-activated cell sorting as a so-called side "population" (SP) of bone marrow. This SP from bone marrow, as well as other tissues, is reported to contain immature stem cells with considerable plasticity. Some cell lines also efflux Hoechst and generate SP profiles. Reverse transcription-polymerase chain reaction (RT-PCR) and efflux inhibition studies with the lung carcinoma cell line, A549, implicated the ABCG2 transporter as a Hoechst efflux pump. Furthermore, it is shown that transient expression of ABCG2 generates a robust SP phenotype in human embryonic kidney (HEK293) cells. The results allow the conclusion that ABCG2 is a potent Hoechst efflux pump. Semiquantitative RT-PCR was used to characterize the developmental pattern of expression of ABCG2 in hematopoiesis. It is expressed at relatively high levels in putative hematopoietic stem cells (isolated as SP, 34+/38- or 34+/KDR+ populations) and drops sharply in committed progenitors (34+/38+, 34+/33+, or 34+/10+). Expression remains low in most maturing populations, but rises again in natural killer cells and erythroblasts. Comparison of messenger RNA (mRNA) levels for the 3 major multidrug-resistant efflux pumps, MDR1, MRP1, and ABCG2, in bone marrow SP cells reveals that ABCG2 is the predominant form in these cells. These data suggest that ABCG2 contributes significantly to the generation of the SP phenotype in hematopoietic stem cells. Furthermore, the sharp down-regulation of ABCG2 at the stage of lineage commitment suggests that this gene may play an important role in the unique physiology of the pluripotent stem cell.