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Plasmodium falciparum, the parasite responsible for severe malaria, develops within erythrocytes. Merozoite invasion and subsequent egress of intraerythrocytic parasites are essential for this erythrocytic cycle, parasite survival and pathogenesis. In the present study, we report the essential role of a novel protein, P. falciparum Merozoite Surface Antigen 180 (PfMSA180), which is conserved across Plasmodium species and recently shown to be associated with the P. vivax merozoite surface. Here, we studied MSA180 expression, processing, localization and function in P. falciparum blood stages. Initially we examined its role in invasion, a process mediated by multiple ligand-receptor interactions and an attractive step for targeting with inhibitory antibodies through the development of a malaria vaccine. Using antibodies specific for different regions of PfMSA180, together with a parasite containing a conditional pfmsa180-gene knockout generated using CRISPR/Cas9 and DiCre recombinase technology, we demonstrate that this protein is unlikely to play a crucial role in erythrocyte invasion. However, deletion of the pfmsa180 gene resulted in a severe egress defect, preventing schizont rupture and blocking the erythrocytic cycle. Our study highlights an essential role of PfMSA180 in parasite egress, which could be targeted through the development of a novel malaria intervention strategy.

The heterogeneous nature of human CD34+ hematopoietic stem cells (HSCs) has hampered our understanding of the cellular and molecular trajectories that HSCs navigate during lineage commitment. Using various platforms including single cell RNA-sequencing and extensive xenotransplantation, we have uncovered an uncharacterized human CD34+ HSC population. These CD34+EPCR+(CD38/CD45RA)− (simply as EPCR+) HSCs have a high repopulating and self-renewal abilities, reaching a stem cell frequency of ~1 in 3 cells, the highest described to date. Their unique transcriptomic wiring in which many gene modules associated with differentiated cell lineages confers their multilineage lineage output both in vivo and in vitro. At the single cell level, EPCR+ HSCs are the most transcriptomically and functionally homogenous human HSC population defined to date and can also be easily identified in post-natal tissues. Therefore, this EPCR+ population not only offers a high human HSC resolution but also a well-structured human hematopoietic hierarchical organization at the most primitive level.

Idiopathic aplastic anemia (AA) has 2 key characteristics: an autoimmune response against hematopoietic stem/progenitor cells and regulatory T-cells (Tregs) deficiency. We have previously demonstrated reduction in a specific subpopulation of Treg in AA, which predicts response to immunosuppression. The aims of the present study were to define mechanisms of Treg subpopulation imbalance and identify potential for therapeutic intervention. We have identified 2 mechanisms that lead to skewed Treg composition in AA: first, FasL-mediated apoptosis on ligand interaction; and, second, relative interleukin-2 (IL-2) deprivation. We have shown that IL-2 augmentation can overcome these mechanisms. Interestingly, when high concentrations of IL-2 were used for in vitro Treg expansion cultures, AA Tregs were able to expand. The expanded populations expressed a high level of p-BCL-2, which makes them resistant to apoptosis. Using a xenograft mouse model, the function and stability of expanded AA Tregs were tested. We have shown that these Tregs were able to suppress the macroscopic clinical features and tissue manifestations of T-cell–mediated graft-versus-host disease. These Tregs maintained their suppressive properties as well as their phenotype in a highly inflammatory environment. Our findings provide an insight into the mechanisms of Treg reduction in AA. We have identified novel targets with potential for therapeutic interventions. Supplementation of ex vivo expansion cultures of Tregs with high concentrations of IL-2 or delivery of IL-2 directly to patients could improve clinical outcomes in addition to standard immunosuppressive therapy.

Plasmodium falciparum cysteine-rich protective antigen (CyRPA) is a conserved component of an essential erythrocyte invasion complex (RH5/Ripr/CyRPA) and a target of potent cross-strain parasite-neutralizing antibodies. While naturally acquired human RH5 antibodies have been functionally characterized, there are no similar reports on CyRPA. Thus, we analyzed the parasite-neutralizing activity of naturally acquired human CyRPA antibodies. In this regard, CyRPA human antibodies were measured and purified from malaria-infected plasma obtained from patients in central India and analyzed for their parasite neutralizing activity via in vitro growth inhibition assays (GIA). We report that, despite being susceptible to antibodies, CyRPA is a highly conserved antigen that does not appear to be under substantial immune selection pressure, as a very low acquisition rate for anti-CyRPA antibodies was reported in malaria-exposed Indians. We demonstrate for the first time that the small amounts of natural CyRPA antibodies exhibited functional parasite-neutralizing activity and that a CyRPA-based vaccine formulation induces highly potent antibodies in rabbits. Importantly, the vaccine-induced CyRPA antibodies exhibited a robust 50% inhibitory concentration (IC(50)) of 21.96 μg/ml, which is comparable to the IC(50) of antibodies against the leading blood-stage vaccine candidate, reticulocyte-binding-like homologous protein 5 (RH5). Our data support CyRPA as a unique vaccine target that is highly susceptible to immune attack but is highly conserved compared to other leading candidates such as MSP-1 and AMA-1, further substantiating its promise as a leading blood-stage vaccine candidate.

Kinase domain mutations in BCR-ABL1 are associated with resistance to tyrosine kinase inhibitors in patients with chronic myeloid leukaemia. Next-generation sequencing (NGS) allows detection of low-level kinase domain mutations, but its relevance in clinical practice remains debated. We aimed to examine the clinical effects of low-level kinase domain mutations identified using NGS in patients with chronic myeloid leukaemia.In this population-based study, we included consecutive patients newly diagnosed with chronic myeloid leukaemia treated with first-line tyrosine kinase inhibitors, and patients identified at the time of resistance to first-line treatment with imatinib at six institutions (teaching hospitals and district hospitals) in southeast England. We screened patients for BCR-ABL1 kinase domain mutations using NGS, irrespective of patient response to tyrosine kinase inhibitor therapy. When we detected a mutation with NGS, we retrospectively analysed all previous samples to establish the date of first occurrence and subsequent kinetics of the mutant subclone (or subclones). The primary endpoints of this study were progression-free and event-free survival at 5 years.Between Feb 1, 2007, and Dec 31, 2014, we screened 121 patients with chronic myeloid leukaemia for BCR-ABL1 kinase domain mutation. 99 consecutive patients were newly diagnosed, with available sequential RNA stored. The remaining 22 patients were diagnosed between June 1, 1999, and June 30, 2006, and were screened at the time of resistance to first-line treatment with imatinib. Imatinib was the first-line treatment for 111 patients, nilotinib for seven patients, and dasatinib for three patients. We detected a kinase domain mutation in 25 (21%) of 121 patients. Low-level kinase domain mutations were first identified in 17 (68%) of 25 patients with mutation. For patients with a complete cytogenetic response, 13 (14%) of 93 patients screened had a mutation. Five (71%) of the seven patients with a clinically relevant mutation lost complete cytogenetic response compared with 15 (17%) of 86 patients without a clinically relevant mutation (80 patients without mutation and six patients with a tyrosine kinase inhibitor-sensitive mutation, p=0·0031). Patients harbouring a mutant clone had poorer 5-year progression-free survival (65·3% [95% CI 40·5-81·8] vs 86·9% [75·8-93·2]; p=0·0161) and poorer 5-year event-free survival (22·2% [CI 5·6-45·9] vs 62·0% [50·4-71·6]; p0·0001) than did patients without a mutation. We identified a kinase domain mutation in four (10%) of 41 patients with samples available at 3 months after starting first-line tyrosine kinase inhibitor treatment; all four subsequently progressed to accelerated phase disease compared with only three (8%) of 37 without a mutation (p0·0001).NGS reliably and consistently detected early appearance of kinase domain mutations that would not otherwise be detected by Sanger sequencing. For the first time, to our knowledge, we report the presence of kinase domain mutations after only 3 months of therapy, which could have substantial clinical implications. NGS will allow early clinical intervention and our findings will contribute to the establishment of new recommendations on the frequency of kinase domain mutation analysis to improve patient clinical care.None.

Over the last 20 years, significant progress has been made in the development of immunodeficient mouse models that now represents the gold standard tool in stem cell biology research. The latest major improvement has been the use of biomaterials in these xenogeneic mouse models to generate human "bone marrow like" tissues, which not only provides a more relevant xenograft model but can also potentially enable us to delineate the interactions that are specific between human bone marrow cells. There are a number of biomaterials and strategies to create humanized niches in immunodeficient mouse models, and the methods can also differ significantly among various research institutes. Here, we describe a protocol to create a humanized 3D collagen-based scaffold human niche in immunodeficient mouse model(s). This humanized in vivo model provides a powerful technique for understanding the human BM microenvironment and the role it plays in the regulation of normal as well as malignant hematopoiesis.

Myelodysplastic syndromes (MDS) are clonal stem cell diseases characterized mainly by ineffective hematopoiesis. Here, we present an approach that enables robust long-term engraftment of primary MDS stem cells (MDS-SC) in mice by implantation of human mesenchymal cell–seeded scaffolds. Critically for modeling MDS, where patient sample material is limiting, mononuclear bone marrow cells containing as few as 104 CD34+ cells can be engrafted and expanded by this approach with the maintenance of the genetic make-up seen in the patients. Noninvasive high-resolution ultrasound imaging shows that these scaffolds are fully perfused. Our data show that the human microenvironment but not mouse is essential to MDS-SC homing and engraftment. Notably, the alternative niche provided by healthy donor mesenchymal stromal cells enhances engraftment of MDS-SCs. This study characterizes a new tool to model MDS human disease with the level of engraftment previously unattainable in mice and offers insights into human-specific determinants of the MDS-SC microenvironment. Significance: These findings are significant for understanding the niche dependence of MDS. This report provides the evidence of the migratory behavior of hematopoietic stem cells in myeloid cancers. Our model offers a unique opportunity to study the clonal behavior of the myeloid/lymphoid cancers and delineate how cancer cells interact with different niches.

Immunotherapy has established itself as a promising tool for cancer treatment. There are many challenges that remain including lack of targets and some patients across various cancers who have not shown robust clinical response. One of the major problems that have hindered the progress in the field is the dearth of appropriate mouse models that can reliably recapitulate the complexity of human immune-microenvironment as well as the malignancy itself. Immunodeficient mice reconstituted with human immune cells offer a unique opportunity to comprehensively evaluate immunotherapeutic strategies. These immunosuppressed and genetically modified mice, with some overexpressing human growth factors, have improved human hematopoietic engraftment as well as created more functional immune cell development in primary and secondary lymphoid tissues in these mice. In addition, several new approaches to modify or to add human niche elements to further humanize these immunodeficient mice have allowed a more precise characterization of human hematopoiesis. These important refinements have opened the possibility to evaluate not only human immune responses to different tumor cells but also to investigate how malignant cells interact with their niche and most importantly to test immunotherapies in a more preclinically relevant setting, which can ultimately lead to better success of these drugs in clinical trials.

Background Targeting multiple key antigens that mediate distinct Plasmodium falciparum erythrocyte invasion pathways is an attractive approach for the development of blood-stage malaria vaccines. However, the challenge is to identify antigen cocktails that elicit potent strain-transcending parasite-neutralizing antibodies efficacious at low immunoglobulin G concentrations feasible to achieve through vaccination. Previous reports have screened inhibitory antibodies primarily against well adapted laboratory parasite clones. However, validation of the parasite-neutralizing efficacy against clinical isolates with minimal in vitro cultivation is equally significant to better ascertain their prospective in vivo potency. Methods We evaluated the parasite-neutralizing activity of different antibodies individually and in combinations against laboratory adapted clones and clinical isolates. Clinical isolates were collected from Central India and Mozambique, Africa, and characterized for their invasion properties and genetic diversity of invasion ligands. Results In our portfolio, we evaluated 25 triple antibody combinations and identified the MSP-Fu+CyRPA+RH5 antibody combination to elicit maximal parasite neutralization against P. falciparum clinical isolates with variable properties that underwent minimal in vitro cultivation. Conclusions The MSP-Fu+CyRPA+RH5 combination exhibited highly robust parasite neutralization against P. falciparum clones and clinical isolates, thus substantiating them as promising candidate antigens and establishing a proof of principle for the development of a combinatorial P. falciparum blood-stage malaria vaccine.

Monosomy 7 [-7] and/or partial loss of chromosome 7 [del(7q)] are associated with poor and intermediate prognosis, respectively, in myelodysplastic syndromes (MDS), but somatic mutations may also play a key complementary role. We analyzed the impact on the outcomes of deep targeted mutational screening in 280 MDS patients with -7/del(7q) as isolated cytogenetic abnormality (86 with del(7q) and 194 with -7). Patients with del(7q) or -7 had similar demographic and disease-related characteristics. Somatic mutations were detected in 79% (93/117) of patients (82% in -7 and 73% in del(7q) group). Median number of mutations per patient was 2 (range 0-8). There was no difference in mutation frequency between the two groups. Patients harbouring >= 2 mutations had a worse outcome than patients with

Abarrategi et al. summarize the methods used for human hematopoietic cell xenotransplantation and their milestones. They also discuss the latest approaches in generating humanized BM tissues in mice to study human normal and malignant hematopoiesis., Xenotransplantation of patient-derived samples in mouse models has been instrumental in depicting the role of hematopoietic stem and progenitor cells in the establishment as well as progression of hematological malignancies. The foundations for this field of research have been based on the development of immunodeficient mouse models, which provide normal and malignant human hematopoietic cells with a supportive microenvironment. Immunosuppressed and genetically modified mice expressing human growth factors were key milestones in patient-derived xenograft (PDX) models, highlighting the importance of developing humanized microenvironments. The latest major improvement has been the use of human bone marrow (BM) niche–forming cells to generate human–mouse chimeric BM tissues in PDXs, which can shed light on the interactions between human stroma and hematopoietic cells. Here, we summarize the methods used for human hematopoietic cell xenotransplantation and their milestones and review the latest approaches in generating humanized BM tissues in mice to study human normal and malignant hematopoiesis.

Cytarabine (AraC) represents the most effective single agent treatment for AML. Nevertheless, overriding AraC resistance in AML remains an unmet medical need. Here we show that the CHK1 inhibitor (CHK1i) GDC-0575 enhances AraC-mediated killing of AML cells both in vitro and in vivo, thus abrogating any potential chemoresistance mechanisms involving DNA repair. Importantly, this combination of drugs does not affect normal long-term hematopoietic stem/progenitors. Moreover, the addition of CHK1i to AraC does not generate de novo mutations and in patients’ samples where AraC is mutagenic, addition of CHK1i appears to eliminate the generation of mutant clones. Finally, we observe that persistent residual leukemic cells are quiescent and can become responsive to the treatment when forced into cycle via granulocyte colony-stimulating factor (G-CSF) administration. This drug combination (AraC+CHK1i+G-CSF) will open the doors for a more efficient treatment of AML in the clinic., Overriding cytarabine resistance in AML remains an unmet medical need. Here, the authors show that the CHK1 inhibitor GDC-0575 in combination with cytarabine and G-CSF has a significant anti-leukemic effect without toxicity to normal marrow stem and progenitor cells.

The BM niche comprises a tightly controlled microenvironment formed by specific tissue and cells that regulates the behavior of hematopoietic stem cells (HSCs). Here, we have provided a 3D model that is tunable in different BM niche components and useful, both in vitro and in vivo, for studying the maintenance of normal and malignant hematopoiesis. Using scaffolds, we tested the capacity of different stromal cell types to support human HSCs. Scaffolds coated with human mesenchymal stromal cells (hMSCs) proved to be superior in terms of HSC engraftment and long-term maintenance when implanted in vivo. Moreover, we found that hMSC-coated scaffolds can be modulated to form humanized bone tissue, which was also able to support human HSC engraftment. Importantly, hMSC-coated humanized scaffolds were able to support the growth of leukemia patient cells in vivo, including the growth of samples that would not engraft the BM of immunodeficient mice. These results demonstrate that an s.c. implantation approach in a 3D carrier scaffold seeded with stromal cells is an effective in vivo niche model for studying human hematopoiesis. The various niche components of this model can be changed depending on the context to improve the engraftment of nonengrafting acute myeloid leukemia (AML) samples.

Bone marrow (BM) genetic abnormalities in myelodysplastic syndrome (MDS) have provided important biological and prognostic information; however, frequent BM sampling in older patients has been associated with significant morbidity. Utilizing single-nucleotide polymorphism array (SNP-A) and targeted gene sequencing (TGS) of 24 frequently mutated genes in MDS, we assessed the concordance of genetic abnormalities in BM and peripheral blood (PB) samples concurrently from 201 MDS patients. SNP-A karyotype in BM was abnormal in 108 (54%) and normal in 93 (46%) patients, with 95% (190/201) having an identical PB karyotype. The median copy number (CN) for deletions was significantly lower in BM (CN:1.4 (1-1.9)) than in PB (CN:1.5 (1-1.95), P

Myelodysplastic syndromes (MDS) represent a heterogeneous group of hematological clonal disorders. Here, we have tested the bone marrow (BM) cells from 38 MDS patients covering all risk groups in two immunodeficient mouse models: NSG and NSG-S. Our data show comparable level of engraftment in both models. The level of engraftment was patient specific with no correlation to any specific MDS risk group. Furthermore, the co-injection of mesenchymal stromal cells (MSCs) did not improve the level of engraftment. Finally, we have developed an in vitro two-dimensional co-culture system as an alternative tool to in vivo. Using our in vitro system, we have been able to co-culture CD34+ cells from MDS patient BM on auto- and/or allogeneic MSCs over 4 weeks with a fold expansion of up to 600 times. More importantly, these expanded cells conserved their MDS clonal architecture as well as genomic integrity.

To retrospectively analyze the potential sources of error for IOL power calculation in patients with X-linked related megalocornea (XLMC).Case report and comparative analysis of refractive outcomes in previously reported phacoemulsification procedures in XLMC cases.A 52-year-old patient with XLMC and cataracts underwent bilateral clear corneal phacoemulsification, capsule tension ring (CTR) insertion, and in the bag intraocular lens (IOL) implantation. Two years after the procedure the IOL remained centrally located and stable in both eyes. In the postoperative refraction, the patient had a large hyperopic refractive error in the right eye, and a moderate hyperopic refractive error in the left eye. A similar pattern was observed in previously reported cases. Pooling all cases together we observed that the Holladay II formula produced more accurate IOL power calculations than the SRK-T formula. Still, both formulas diverged from the ideal IOL power by approximately 1 diopter per mm of axial length in subjects with axial lengths larger than 24 mm.Axial length seems to be the main source of IOL power calculation error in XLMC patients. Compared to SRK-T the Holladay II formula provides better refractive results, yet both formulas may require further adjustment depending on the axial length.

The recent identification of acquired mutations in key components of the spliceosome machinery strongly implicates abnormalities of mRNA splicing in the pathogenesis of myelodysplastic syndromes. However, questions remain as to how these aberrations functionally combine with the growing list of mutations in genes involved in epigenetic modification and cell signaling/transcription regulation identified in these diseases. In this study, amplicon sequencing was used to perform a mutation screen in 154 myelodysplastic syndrome patients using a 22-gene panel, including commonly mutated spliceosome components (SF3B1, SRSF2, U2AF1, ZRSR2), and a further 18 genes known to be mutated in myeloid cancers. Sequencing of the 22-gene panel revealed that 76% (n=117) of the patients had mutations in at least one of the genes, with 38% (n=59) having splicing gene mutations and 49% (n=75) patients harboring more than one gene mutation. Interestingly, single and specific epigenetic modifier mutations tended to coexist with SF3B1 and SRSF2 mutations (P

Erythrocyte invasion by Plasmodium falciparum merozoites is central to blood-stage infection and malaria pathogenesis. This intricate process is coordinated by multiple parasite adhesins that bind erythrocyte receptors and mediate invasion through several alternate pathways. P. falciparum expresses 2700 genes during the blood-stages, of which the identity and function of many remains unknown. Here, we have identified and characterized a novel P. falciparum rhoptry associated adhesin (PfRA) that mediates erythrocyte invasion through the sialic-acid dependent pathway. PfRA appears to play a significant functional role as it is conserved across different Plasmodium species. It is localized in the rhoptries and further translocated to the merozoite surface. Both native and recombinant PfRA specifically bound erythrocytes in a sialic-acid dependent, chymotrypsin and trypsin resistant manner, which was abrogated by PfRA antibodies confirming a role in erythrocyte invasion. PfRA antibodies inhibited erythrocyte invasion and in combination with antibodies against other parasite ligands produced an additive inhibitory effect, thus validating its important role in erythrocyte invasion. We have thus identified a novel P. falciparum adhesin that binds with a sialic acid containing erythrocyte receptor. Our observations substantiate the strategy to block P. falciparum erythrocyte invasion by simultaneously targeting multiple conserved merozoite antigens involved in alternate invasion pathways.

Idiopathic aplastic anemia (AA) is an immune-mediated and serious form of bone marrow failure. Akin to other autoimmune diseases, we have previously shown that in AA regulatory T-cells (Tregs) are reduced in number and function. The aim of this study was to further characterize Treg subpopulations in AA and investigate the potential correlation between specific Treg subsets and response to immunosuppressive therapy (IST) as well as their in-vitro expandability for potential clinical use. Using mass cytometry (CyTOF) and an unbiased multidimensional analytical approach, we identified two specific human Treg subpopulations (Treg A and Treg B) with distinct phenotypes, gene-expression, expandability and function. Treg subpopulation B, predominates in IST responder patients, has a memory/activated phenotype (with higher expression of CD95, CCR4 and CD45RO within FOXP3hi, CD127lo Tregs), expresses the IL- 2/STAT5 pathway and cell-cycle commitment genes. Furthermore, in-vitro expanded Tregs become functional and with the characteristics of Treg subpopulation B. Collectively, this study identifies human Treg subpopulations that can be used as predictive biomarkers for response to IST in AA and potentially other autoimmune diseases. We also show that Tregs from AA patients are IL-2 sensitive and expandable in-vitro, suggesting novel therapeutic approaches such as low dose IL-2 therapy and/or expanded autologous Tregs and meriting further exploration.

BACKGROUND: The customized mandible reconstruction has been a challenging task in maxillofacial surgery. Designing an implant taking into considering the surrounding bone contours is really critical. Various computer aided design techniques have been used in the designing the customized reconstruction implants, but nevertheless study on the comparison between these techniques is rarely used. OBJECTIVE: The objective of this study is to compare the mirroring and anatomical reconstruction design techniques used in the maxillofacial surgery and select the best design technique. METHODS: The three mandible bone defects-small (< 20 mm), medium (20 to 40 mm) and large (41 to 53 mm) tumors are reconstructed using the two reconstruction design techniques and compared to their accuracy, using a 3 dimensional (3D) implant design evaluation and part to Computer aided design (CAD) comparison using a Co-ordinate measuring machine (CMM). RESULTS: The analysis results indicate that the mirroring technique provides higher accuracy for the implant design as compared to the anatomical technique for the medium and large tumors at maxillofacial regions. In case of implant design in small tumors, the anatomical design provides perfect implant fitting. CONCLUSIONS: Based on the results, it is recommended to select the anatomy design technique only for small tumor regions and mirroring technique for medium and large tumors.

The role of CD4+ T cells in the pathogenesis of aplastic anemia (AA) is not well characterized. We investigate CD4+ T-cell subsets in AA. Sixty-three patients with acquired AA were studied. Th1 and Th2 cells were significantly higher in AA patients than in healthy donors (HDs; P = .03 and P = .006). Tregs were significantly lower in patients with severe AA than in HDs (P < .001) and patients with non-severe AA (P = .01). Th17 cells were increased in severe AA (P = .02) but normal in non-severe AA. Activated and resting Tregs were reduced in AA (P = .004; P = .01), whereas cytokine-secreting non-Tregs were increased (P = .003). Tregs from AA patients were unable to suppress normal effector T cells. In contrast, AA effector T cells were suppressible by Tregs from HDs. Th1 clonality in AA, investigated by high-throughput sequencing, was greater than in HDs (P = .03). Our results confirm that Th1 and Th2 cells are expanded and Tregs are functionally abnormal in AA. The clonally restricted expansion of Th1 cells is most likely to be antigen-driven, and induces an inflammatory environment, that exacerbate the functional impairment of Tregs, which are reduced in number.

Mutations in the TET2 gene are frequent in myeloid disease, although their biologic and prognostic significance remains unclear. We analyzed 355 patients with myelodysplastic syndromes using “next-generation” sequencing for TET2 aberrations, 91 of whom were also subjected to single-nucleotide polymorphism 6.0 array karyotyping. Seventy-one TET2 mutations, with a relative mutation abundance (RMA) ≥ 10%, were identified in 39 of 320 (12%) myelodysplastic syndrome and 16 of 35 (46%) chroni myelomonocytic leukemia patients (P < .001). Interestingly, 4 patients had multiple mutations likely to exist as independent clones or on alternate alleles, suggestive of clonal evolution. “Deeper” sequencing of 96 patient samples identified 4 additional mutations (RMA, 3%-6.3%). Importantly, TET2 mutant clones were also found in T cells, in addition to CD34+ and total bone marrow cells (23.5%, 38.5%, and 43% RMA, respectively). Only 20% of the TET2-mutated patients showed loss of heterozygosity at the TET2 locus. There was no difference in the frequency of genome-wide aberrations, TET2 expression, or the JAK2V617F 46/1 haplotype between TET2-mutated and nonmutated patients. There was no significant prognostic association between TET2 mutations and World Health Organization subtypes, International Prognostic Scoring System score, cytogenetic status, or transformation to acute myeloid leukemia. On multivariate analysis, age (> 50 years) was associated with a higher incidence of TET2 mutation (P = .02).

Purpose of review The femtosecond laser has potentially improved the safety and precision of creating corneal flaps in laser in situ keratomileusis (LASIK). This review focuses on recent advances in applying femtosecond laser technology to other corneal procedures. Recent findings The femtosecond laser is capable of cutting tissue at various depths and patterns with minimal collateral tissue injury. The laser can now be used to perform corneal pockets for ring segments, arcuate wedge-shaped resection in correction of high astigmatism, lamellar dissections in anterior lamellar keratoplasty, donor tissue preparation in Descemet's stripping endothelial keratoplasty, and shaped full-thickness keratoplasty. Summary Femtosecond laser-assisted corneal surgery may provide a more precise and safer approach to corneal surgery.

Despite the recent evidence of the existence of myelodysplastic syndrome (MDS) stem cells in 5q-MDS patients, it is unclear whether haematopoietic stem cells (HSCs) could also be the initiating cells in other MDS subgroups. Here we demonstrate that SF3B1 mutation(s) in our cohort of MDS patients with ring sideroblasts can arise from CD34+CD38−CD45RA−CD90+CD49f+ HSCs and is an initiating event in disease pathogenesis. Xenotransplantation of SF3B1 mutant HSCs leads to persistent long-term engraftment restricted to myeloid lineage. Moreover, genetically diverse evolving subclones of mutant SF3B1 exist in mice, indicating a branching multi-clonal as well as ancestral evolutionary paradigm. Subclonal evolution in mice is also seen in the clinical evolution in patients. Sequential sample analysis shows clonal evolution and selection of the malignant driving clone leading to AML transformation. In conclusion, our data show SF3B1 mutations can propagate from HSCs to myeloid progeny, therefore providing a therapeutic target., Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders with diverse phenotypes and can derive from hematopietic stem cells after the acquisition of specific somatic aberrations. In this study, the authors show that MDS initiating cells in some cases of sideroblastic anemia with SF3B1 mutations, can arise from hematopoietic stem cells.

Summary Background A mechanism for clonal growth advantage in isolated del(5q) disease remains elusive. CSNK1A1 resides on the critically deleted region, and deletion of this gene has been shown in mouse knockout and transplantation studies to produce some characteristics of bone marrow failure, including a proliferative advantage. We aimed to establish the frequency, nature, and clinical association of CSNK1A1 mutations in patients with myelodysplastic syndrome and associated myeloid neoplasms. Methods Between June 1, 2004, and May 31, 2014, in King's College (London, UK), we did whole-exome sequencing of five patients with isolated del(5q) followed by targeted screening for CSNK1A1 mutations and 20 myelodysplastic syndrome-associated mutations in 245 additional patients with myeloid neoplasms. All patients met present WHO diagnostic criteria for myelodysplastic syndrome and other related myeloid neoplasms. Findings 39 (16%) of 250 patients with myeloid neoplasms had isolated del(5q), of whom seven (18%) had CSNK1A1 mutations. All these mutations were missense and presented in a highly conserved region that is implicated in ATP catalysis. Serial sampling and response to lenalidomide treatment showed that CSNK1A1 mutations were highly associated with the del(5q) clone. Only one patient with a CSNK1A1 mutation showed complete cytogenetic response to lenalidomide. Four (57%) of the seven patients carrying a CSNK1A1 mutation showed disease progression coupled with an increase in mutant allele burden (all four were on lenalidomide). We detected coexisting myelodysplastic syndrome-related gene mutations in patients with CSNK1A1 mutations, including TP53 . Interpretation Similar to the effect of TP53 mutations on progression of del(5q) abnormality, mutant CSNK1A1 also gives rise to a poor prognosis in del(5q) abnormality, for which a coupled increase in P53 activation is suggested. CSNK1A1 mutations in del(5q) disease are important in the context of therapeutic manipulation and need incorporation into future prospective studies. Funding Leukaemia and Lymphoma Research.

The distinction between acquired aplastic anemia (AA) and hypocellular myelodysplastic syndrome (hMDS) is often difficult, especially nonsevere AA. We postulated that somatic mutations are present in a subset of AA, and predict malignant transformation. From our database, we identified 150 AA patients with no morphological evidence of MDS, who had stored bone marrow (BM) and constitutional DNA. We excluded Fanconi anemia, mutations of telomere maintenance, and a family history of BM failure (BMF) or cancer. The initial cohort of 57 patients was screened for 835 known genes associated with BMF and myeloid cancer; a second cohort of 93 patients was screened for mutations in ASXL1, DNMT3A, BCOR, TET2, and MPL. Somatic mutations were detected in 19% of AA, and included ASXL1 (n = 12), DNMT3A (n = 8) and BCOR (n = 6). Patients with somatic mutations had a longer disease duration (37 vs 8 months, P.04), and shorter telomere lengths (median length, 0.9 vs 1.1, P.001), compared with patients without mutations. Somatic mutations in AA patients with a disease duration of6 months were associated with a 40% risk of transformation to MDS (P.0002). Nearly one-fifth of AA patients harbor mutations in genes typically seen in myeloid malignancies that predicted for later transformation to MDS.

This study aimed to determine the incidence/prognostic impact of TP53 mutation in 318 myelodysplastic syndrome (MDS) patients, and to correlate the changes to cytogenetics, single nucleotide polymorphism array karyotyping and clinical outcome. The median age was 65 years (17-89 years) and median follow-up was 45 months [95% confidence interval (CI) 27-62 months]. TP53 mutations occurred in 30 (9.4%) patients, exclusively in isolated del5q (19%) and complex karyotype (CK) with -5/5q-(72%), correlated with International Prognostic Scoring System intermediate-2/high, TP53 protein expression, higher blast count and leukaemic progression. Patients with mutant TP53 had a paucity of mutations in other genes implicated in myeloid malignancies. Median overall survival of patients with TP53 mutation was shorter than wild-type (9 versus 66 months, P < 0.001) and it retained significance in multivariable model (Hazard Ratio 3.8, 95%CI 2.3-6.3,P < 0.001). None of the sequentially analysed samples showed a disappearance of the mutant clone or emergence of new clones, suggesting an early occurrence of TP53 mutations. A reduction in mutant clone correlated with response to 5-azacitidine, however clones increased in non-responders and persisted at relapse. The adverse impact of TP53 persists after adjustment for cytogenetic risk and is of practical importance in evaluating prognosis. The relatively common occurrence of these mutations in two different prognostic spectrums of MDS, i.e. isolated 5q- and CK with -5/5q-, possibly implies two different mechanistic roles for TP53 protein.

Purpose Cryptic chromosomal aberrations, such as regions of uniparental disomy (UPD), have been shown to harbor homozygous mutations and are a common feature in myelodysplastic syndrome (MDS). We investigated the sequence integrity of 4q24 candidate tumor suppressor gene TET2 in MDS patients with UPD on chromosome 4. Patients and Methods The coding exons of TET2 were analyzed by 454 deep sequencing and Sanger sequencing in nine patients with UPD on 4q. Four patients had refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS) and UPD4q24, and five patients (refractory anemia with excess blasts-II, n = 1; 5q– syndrome, n = 1; RCMD-RS, n = 1; refractory anemia, n = 1; refractory cytopenia with multilineage dysplasia, n = 1) had no UPD4q24. Results Mutations on TET2 were identified in all four patients with UPD4q24. These were localized to exons 3, 6, and 9 and resulted in two premature stop codons, one frameshift mutation, and one cysteine to glycine amino acid change. Mutant clone size varied between 30% and 85%. One patient with UPD outside of q24 (UPD4q28.3) displayed additional TET2 mutations, but these were at low clonal levels (13%, 4%, and 4% for a silent mutation, a 180-base pair deletion in exon 3, and a lysine to phenylalanine substitution in exon 11, respectively). The other patients who did not have UPD4q24 did not have verifiable TET2 mutations. Conclusion Our data identify novel TET2 mutations in a dominant clone in patients with UPD4q24. The presence of UPD4q24 and mutations in RCMD-RS patients may suggest specificity to this subtype. Our preliminary results need to be confirmed in a large cohort of all MDS subtypes.