Standard Morphological Criteria Research Articles

Composition and diversity of Culicoides biting midges (Diptera: Ceratopogonidae) in rural and suburban environments of Algeria

Culicoides biting midges are well known biological vectors of several arboviruses causing more than 100 veterinary and medical diseases worldwide. In Algeria, bluetongue virus, which is transmitted by Culicoides midges, is responsible for one of the most critical insect-borne diseases of sheep. For example, this disease caused clinically severe morbidity of about 2,661 confirmed cases out of 21,175 susceptible sheep in Algeria. This study compared the abundance, richness, and diversity of Culicoides species in rural and suburban environments of semi-arid regions in North Africa. It examined the potential influence of the bio-climatic factors on the variation of population sizes and the elevation distribution of biting midges. Specimen collection was carried out from June to September during two successive years (2018 and 2019) using CDC light traps installed at 15 sites in different environments. Culicoides specimens were dissected, slide-mounted, and morphologically identified using the interactive identification key IIKC and various standard morphological criteria. A total of 1,046 Culicoides specimens (1,024 females and 22 males) were trapped and classified into 22 species, belonged to nine distinct subgenera. Two new species records for Algeria and even North Africa are reported: Culicoides albicans (Winnertz, 1852) and Culicoides nubeculosus (Meigen, 1830). Culicoides newsteadi Austen, 1921 (51.6%) was the dominant species, and it was followed by Culicoides punctatus (Meigen, 1804) (16.3%) and Culicoides odiatus Austen, 1921 (11.5%). These three species, comprising 80% of the collected Culicoides, were the most abundant both outside and inside livestock stables in rural and suburban environments. Species diversity was similar in the two settings, with a slight increase in suburban environments. None of the Culicoides species encountered correlated significantly with the climatic factors (mean temperature, precipitation, and relative humidity). Elevation was the most determinant environmental parameter that affected the abundance and distribution of Culicoides midges in the semi-arid and sub-humid areas studied. The maximum distribution of Culicoides species was detected at mid elevations (400‒800 m). Using a modeling approach, we explored for the first time the variation of composition and diversity in Culicoides communities within different climatic regions, environments and livestock settings in Algeria. This survey deepens our understanding of the relationships among environmental factors, abundance, diversity, and geographic distribution of Culicoides. This is a crucial step to assess the epidemiological situation of the diseases transmitted by these biting midges and to allow mitigation of the associated risks.

OP37 Bacterial suppression of intestinal fungi via activation of human gut Vδ2+ T-cells

Abstract Background The human gut is colonized by diverse microbes including fungi such as Candida albicans, which has been implicated in the pathogenesis of Crohn’s disease (CD). Intestinal C. albicans typically exists as commensal yeast, but can also form tissue-invasive filaments that secrete Candidalysin toxin to disrupt epithelial barriers. Commensal gut bacteria produce molecules that induce host immunity, including phosphoantigen HMB-PP which stimulates Vδ2+ T-cell homing to intestine and mucosal expression of IL-22 to promote barrier integrity. We aimed to identify mechanisms of gut barrier defence against fungal invasion in health and CD. Methods Colonic biopsies / resected tissue were stimulated or not with HMB-PP in the presence or absence of anti-IL-22 blocking antibody and analysed by flow-cytometry / microscopy to determine extent of fungal outgrowth. Fungal isolates were identified using standard morphological and biochemical criteria before assessment of filamentation, cell wall composition, and NETosis induction. Results Mucosal Vδ2+ T-cell activation with bacterial phosphoantigen HMB-PP, in the presence of epithelial damage-associated cytokine IL-15, potently suppressed growth of endogenous fungi in human colonic organ cultures. Vδ2-mediated fungal suppression required IL-22 and was sufficient to restrict the growth of multiple fungal species, including a range of emerging pathogenic moulds as well as archetypal pathobiont C. albicans. In Vδ2-deficient CD patients, mucosal biopsy stimulation with HMB-PP failed to control fungal growth. CD-derived C. albicans strains also displayed rapid filamentation ex vivo and higher levels of NOD2 ligand chitin than were observed in isolates from healthy controls. Comparing hypoxic with oxygenated cultures that mimic inflamed intestine, pSILAC proteomic analysis of a representative CD-derived C. albicans strain confirmed features of invasive growth (e.g. DAO1, IHD1), whereas a healthy control isolate exhibited metabolic hallmarks of symbiosis (Jen1p, SCH9). These divergent characteristics directly impacted on host anti-fungal immune responses, since the CD-derived strain rapidly induced neutrophil extracellular traps in vitro, whereas the healthy control isolate did not. Conclusion These data suggest that commensal bacteria can activate host Vδ2 T-cells to suppress fungal invasion of the gut epithelium via an IL22-dependent mechanism that is deficient in CD patients.

Discriminating between IgG4-related orbital disease and other causes of orbital inflammation with intra voxel incoherent motion (IVIM) MR imaging at 3T

PurposeThe purpose of this prospective study was to determine the capabilities of intravoxel incoherent motion (IVIM) MRI at 3 Tesla in discriminating between IgG4-related orbital disease (IgG4-ROD) and other causes of orbital inflammation. Materials and methodsMain selection criteria for the patients enrolled in this prospective study were age over 18 years and histopathologicaly proven orbital inflammatory lesion. MRI examinations were performed prior to surgery and treatment in all patients with suspected orbital inflammation. Two neuroradiologists, blinded to clinical data, independently analyzed structural MRI examinations and IVIM sequences obtained with 15 b values ranging from 0 to 2000 s/mm². Apparent diffusion coefficient (ADC), “true” diffusion coefficient (D), perfusion fraction (f) and pseudodiffusion coefficient (D*) values were calculated from all orbital lesions. Diagnostic capabilities of IVIM parameters were assessed using receiver operating-characteristic (ROC) curves and area under the curve (AUC). Sensitivity, specificity, and accuracy of IVIM parameters were calculated for the best threshold values and reported with their corresponding 95% confidence intervals (CI). ResultsThirty-five patients (21 women and 14 men; mean age, 49.2 ± 13.75 [SD] years; age range: 23–77 years) with 48 orbital lesions were enrolled in the study. Fifteen patients (15/35; 43%) had IgG4-ROD and 20 (20/35; 57%) had other causes of orbital inflammation. Median D value was significantly greater in patients with IgG4-ROD (1 × 10-3 mm2/s; interquartile range [IQR]: 0.9 × 10-3; 1.2 × 10-3) as compared to patients with non IgG4-ROD (0.80 × 10-3 mm2/s; IQR: 0.7 × 10-3; 1 × 10-3) (P = 0.04). There was no significant difference for ADC, f or D*. Area under the curve were of 0.54, 0.73, 0.63 and 0.56 for ADC, D, f and D*, respectively. Optimal threshold derived from ROC curves for D was 0.87 × 10-3 mm2/s, yielding 92% sensitivity (95% CI: 62–100%) and 71% specificity (95% CI: 44–90%) for the diagnosis of IgG4-ROD. No differences in standard morphological MRI criteria were found between IgG4-ROD and non IgG4-ROD. ConclusionOur study shows that IVIM MRI is a useful imaging technique to distinguish IgG4-ROD from other causes of orbital inflammation.

MITOCHONDRIAL DNA CONTENT IS NOT A USEFUL METHOD TO PREDICT THE REPRODUCTIVE OUTCOME OF EUPLOID BLASTOCYSTS

To evaluate if the assessment of mitochondrial DNA (mtDNA) content is a useful tool to predict implantation and pregnancy performance in frozen euploid blastocysts transfer cycles. Retrospective comparative study. One hundred and eighty-two elective single frozen blastocyst transfers after preimplantation genetic testing for aneuploidy (PGT-A) and mtDNA content assessment via next generation sequencing (NGS) were performed between May 2016 and October 2019. Euploid blastocysts for transfer were selected based on standard morphological criteria and independently of the mtDNA content result. Blastocysts transferred were grouped according to the value of a mtDNA content score (MS) as following: group A (MS < 18.19, N = 30), group B (MS = 18.19 to 24.15, N = 55) and group C (MS = 24.15 to 50.58, N = 94). Three of the studied blastocysts had MS > 50.58 and were excluded from the study. Study groups were comparable regarding patients' age, indication for PGT-A and proportion of good quality blastocysts. Student's t and Chi-squared tests were used as appropriate. No differences were observed between groups regarding implantation, clinical and ongoing pregnancies and miscarriage rates (table 1).Table 1Outcomes results between groupsStudy groupsABCImplantation43.3 (13/30)45.4 (25/55)37.2 (35/94)Clinical Pregnancies43.3 (13/30)43.6 (24/55)35.1 (33/94)Miscarriages15.3 (2/13)12.5 (3/24)18.2 (6/33)Ongoing pregnancies36.7 (11/30)38.2 (21/55)28.7 (27/94) Open table in a new tab Our study did not find any differences in mtDNA content with regards to embryo implantation or pregnancy outcomes. A bigger sample size would be crucial to confirm these preliminary results.

Diagnostic value of 3D dynamic contrast-enhanced magnetic resonance imaging in lymph node metastases of head and neck tumors: a correlation study with histology.

BackgroundAccurate staging of cervical lymph nodes (LN) is pivotal for further clinical management of patients with head and neck cancer. Functional magnetic resonance imaging (MRI) such as three-dimensional (3D) dynamic contrast-enhanced (DCE) acquisition might improve the diagnosis of cervical LN metastases.PurposeTo evaluate the additional diagnostic value of high-resolution 3D T1-weighted DCE in detecting LN metastasis compared to standard morphological imaging criteria in patients with head and neck tumors as correlated to histopathology.Material and MethodsStandard MRI with 3D DCE acquisition at voxel sizes of 1 × 1×1 mm was performed in 15 patients before surgery; 92 LN of the head and neck were histopathologically analyzed. A logistic regression analysis of semi-quantitative DCE parameters, time-intensity curve (TIC) shapes, and morphological criteria was performed to differentiate benign from malignant LN.ResultsStandard MRI was sufficient for diagnosis of malignancy in LN with a short-axis diameter ≥ 15 mm (n = 17). For LN metastases with a short-axis diameter <15 mm (n = 12), however, the combination of 3D DCE MRI parameters, TIC shapes, and LN diameter significantly increased the sensitivity and specificity of diagnosing metastases (DCE + TIC shape + LN diameter: 92% and 88% vs. DCE only: 83% and 68% (P < 0.01) vs. LN diameter only: 83% and 77% (P = 0.04).ConclusionMRI including isotropic high-resolution 3D DCE acquisition combined with morphological criteria allows an accurate assessment of small cervical LN metastases in patients with head and neck cancer. For LN ≥ 15 mm diameter, morphologic imaging may suffice to diagnose metastatic disease to the LN.

Blastocyst collapse as an embryo marker of low implantation potential: a time-lapse multicentre study.

Spontaneous blastocyst collapse during in vitro embryo development has been suggested as a novel marker of embryo quality. Therefore, the aim of this multicentre study was to carry out a retrospective multicentre analysis to investigate the correlation between blastocyst collapse and pregnancy outcome. Here, 1297 intracytoplasmic sperm injection (ICSI)/in vitro fertilization (IVF) fresh cycles, with an elective single blastocyst transfer (eSET) were included in this study. Embryos were cultured individually in 6.0% CO2, 5.0% O2, 89.0% N2, using single step medium (GTLTM VitroLife, Sweden) or sequential medium (CookTM, Cook Medical, Australia) and selected for transfer using standard morphological criteria. With the use of time-lapse monitoring (TLM), blastocysts were analyzed by measuring the maximum volume reduction and defined as having collapsed, if there was ≥ 50% volume reduction from the expanded blastocyst and the collapse event. Following embryo replacement, each blastocyst was retrospectively allocated to one of two groups (collapsed or not collapsed). Here, 259 blastocysts collapsed once or more during development (19.9%) and the remaining 1038 either contracted minimally or not collapsed (80.1%). A significantly higher ongoing pregnancy rate (OPR) of 51.9% (95% CI 48.9-59.9%) was observed when blastocysts that had not collapsed were replaced compared with cycles in which collapsed blastocysts were transferred 37.5% (95% CI 31.6-43.4%). This study suggests that human blastocysts that collapse spontaneously during development are less likely to implant and generate a pregnancy compared with embryos that do not. Although this is a retrospective study, the results demonstrated the utility of collapse episodes as new marker of embryo selection following eSET at blastocyst stage.

Spontaneous blastocyst collapse as an embryo marker of low pregnancy outcome: A Time-Lapse study.

Objective:In this study we investigate the correlation between spontaneous blastocyst collapse and pregnancy outcome.Methods:This is a retrospective study performed at Edinburgh Assisted Conception Programme, EFREC, Royal Infirmary of Edinburgh, UK. Embryos were cultured individually in 6.0% CO2, 5.0% O2, 89.0% N2, using single step medium (GTL™ Vitrolife, Göteborg, Sweden) and selected for transfer using standard morphological criteria. Using the EmbryoScope™ time-lapse monitoring (TLM), blastocysts collapse was analyzed by measuring the maximum volume reduction and defined as having collapsed if there was >50% volume reduction. Couples undergoing IVF/ICSI treatment and having an elective single embryo transfer (eSET) at blastocyst stage were included in this study. After the embryo transfer, retrospectively, each blastocyst was allocated to one of two groups (collapsed or not collapsed). 62 blastocysts collapsed once or more during development (17.4%), the remaining 294 showed no collapse (82.6%).Results:A significantly higher implantation rate (IR) of 61.2% and ongoing pregnancy rate (OPR) of 53.7% was observed when blastocysts which had not collapsed were replaced compared to cycles in which collapsed blastocysts were replaced (IR rate 22.6% and OPR 17.7%).Conclusion:This study demonstrated that human blastocysts which collapse spontaneously during in vitro development are less likely to implant and generate a pregnancy compared with embryos which do not. Although this is a retrospective study, the results establish the utility of collapse episodes as new marker of embryo selection following eSET at blastocyst stage.

Comparative Transcriptome Analyses of Laser-Captured Human Primary Megakaryocytes with Platelets from the Same Healthy Donors and from Cord Blood Derived Megakaryocytes

Anucleated platelets have a rich repertoire of RNAs, many of which have provided insights into megakaryocyte-platelet biology and have been proposed as disease biomarkers. Although it is generally believed that the platelet transcriptome is a reflection of the megakaryocyte (MK) RNA content and is transported into platelets during pro-platelet production, this is a poorly studied concept. Healthy human bone marrow (BM) is difficult to access and because MKs represent <0.05% of nucleated cells in BM, making it technically challenging to obtain a pure population. Cultured MKs from CD34-positive umbilical vein cord blood have advantages of being human and initially primary cells, but it is unknown how well the transcriptomes of differentiating MKs in culture correlate with primary BM MKs. Microdissection of BM aspirates by laser capture scanning microscopy can overcome purity limitations by isolating virtually 100% MKs. The goals of this study were to assess the transcriptome correlations between laser-captured primary human MKs (LC-MKs) and both (1) platelets from the same individuals who donated the BM and (2) cord blood derived MKs (CB-MKs).BM aspirates and peripheral blood were collected from three healthy donors. Mature BM MKs were identified by a Board certified Hematologist using standard morphologic criteria, and approximately 200 MKs were microdissected by laser capture scanning microscopy from each aspirate. Peripheral blood platelets were isolated from the same donors followed by leukocyte and erythrocyte depletion. Separately, CD34-positive cells were isolated from umbilical vein cord blood, cultured under standard MK-producing conditions with thrombopoietin and stem cell factor, and CD61-positive CB-MKs were isolated at day 6, 9 and 13 in three independent experiments. Total RNA was extracted from LC-MKs, platelets and CB-MKs and subjected to next-generation RNA-sequencing (total of 15 samples: 5 cell types from 3 independent experiments). The Table summarizes the RNA-seq read numbers generated and the number of protein-coding genes determined for each sample. Notably, the LC-MK RNA yielded fewer reads because the input (estimated in the low picogram range) was less than the other samples. Several known platelet transcripts, including PPBP, PF4, B2M, TMSB4X and OST4 were abundant in our LC-MKs RNA-sequencing analysis. All samples exhibited high expression (1,721-74,923 FPKM) of mitochondrial transcripts.To assess RNA correlations across cell types, we only considered transcripts expressed in both comparison groups. mRNAs from LC-MKs were statistically significantly correlated with mRNAs from both platelets and day 13 CD41a-CD42a-positive CB-MKs. These correlations were highest for the most abundantly expressed (FPKM>50) transcripts (Spearman rank correlation Rho = 0.71 for LC-MKs vs. platelets, and Rho = 0.67 for LC-MKs vs. day 13 CB-MKs). However, even the lower expressed transcripts (using the standard threshold of FPKM>0.3) showed moderate correlation (Rho = 0.37-0.42, p value <E-13). Regardless of transcript abundance, the platelet transcriptome displayed higher correlations with LC-MKs than did CB-MKs. Of the 400 mRNAs common to all LC-MKs samples, 23 were not identified in any of the platelet samples and 9 were not identified in any of the d13 CB-MKs. A secondary goal was to compare transcriptome profiles of CB-MKs during differentiation. Not surprisingly, mRNAs from cultured CB-MKs showed higher correlation when their time in culture was shorter than longer (Rho = 0.87 for d13 vs. d9; 0.81 for d9 vs. d6; and 0.64 for d13 vs. d6). During CB-MK differentiation, 825 transcripts were up-regulated whereas 2725 transcripts were down-regulated.In summary, we provide the first report of transcript profiling from normal, primary human MKs. Peripheral blood platelet RNA transcript levels were representative of MK transcripts from the same subjects, especially if abundantly expressed; cultured MK mRNAs correlated strongly with LC-MKs, while platelet RNAs from the same donor showed an even stronger correlation. mRNA expression differences between platelets and BM MKs could provide insights into thrombopoiesis and may be due to (1) regulated transcript partitioning from MK to platelets, (2) differential RNA endocytosis or exocytosis or (3) RNA degradation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Trematode diversity in the freshwater snail Bithynia siamensis goniomphalos sensu lato from Thailand and Lao PDR.

In order to obtain a comprehensive understanding of trematode diversity in Bithynia siamensis goniomphalos sensu lato, the first intermediate host of the liver fluke Opisthorchis viverrini s.l., the prevalence of larval trematode species was investigated in different localities in Thailand and Lao People's Democratic Republic (Lao PDR). In Thailand, snail samples were collected from 29 localities in the nine provinces: Buri Ram, Surin, Chaiya Phum, Maha Sarakham, Khon Kaen, Kalasin, Mukdahan, Sakon Nakhon and Nakhon Phanom. In Lao PDR, snail samples were collected from 21 localities in Vientiane Province and six localities in Savannakhet Province. Snails were identified by standard morphological criteria and then examined for trematode infection using the cercarial shedding method. Twenty different types of cercariae were detected and identified, based on morphological criteria. Virgulate type 1 emerged as the most common cercaria, with an average prevalence of 10.90% (range 0.26-54.22%) in Thailand and 6.58% (range 1.15-89.77%) in Lao PDR. Opisthorchis viverrini s.l. cercariae were the fourth most common in Thailand, with an average prevalence of 1.59% (0.15-6.93), while in Lao PDR their prevalence was 0.96% (0.08-8.37). The high diversity of trematode cercariae observed in this study indicates that B. s. goniomphalos s.l. is highly susceptible to infection with a variety of trematode species. However, the role of non-opisthorchiid trematodes as fish-borne parasites in human health is not fully known and further molecular identification is required.

Prevalence of rheumatic heart disease in young adults from New Caledonia

Rheumatic heart disease (RHD) is an important public health issue, particularly in the Pacific region, but its true burden is unknown. To evaluate the prevalence of rheumatic heart disease (RHD) in young adults from New Caledonia, based on echocardiography, and to evaluate the accuracy of dynamic criteria, focusing on mitral valve (MV) leaflet motion. Blind analysis of echocardiography by three cardiologists; diagnosis of RHD required at least one dynamic criterion (exaggerated or restricted MV leaflet motion); subjects with morphological criteria (MV leaflet thickening), but without dynamic criteria, were considered as borderline. There were 834 subjects from three socioeconomic groups, aged 18-22 years: 699 had normal echocardiography; 93 (11.5%) had physiological regurgitation; nine (0.9%) had borderline RHD; and five (0.59%) had RHD. The prevalence of RHD in New Caledonia was thus estimated at 5.9 per 1000 (95% confidence interval 2.6-12.2). The RHD cases were of Pacific ethnicity. Physiological regurgitation was more frequent in Pacific subjects (13.7%) than in non-Pacific subjects (6.9%; P<0.0001). RHD was more prevalent in the lowest socioeconomic group. No disagreement occurred between the three reviewers concerning analysis of dynamic criteria; all disagreements were related to morphological criteria. The prevalence of echocardiographically diagnosed RHD in adults in New Caledonia is estimated at 5.9 per 1000; it occurs most frequently in Pacific subjects and those with low incomes. Dynamic criteria were more accurate and reproducible than standard morphological criteria.

Hypoplastic MDS Is a Distinct Clinico-Pathological Entity with Somatic Mutations Frequent in Patients with Prior Aplastic Anaemia with Favorable Clinical Outcome

Introduction:Hypocellular myelodysplastic syndrome (hMDS) is characterized by decreased marrow cellularity, and is often difficult to distinguish from aplastic anemia (AA) based on standard morphological criteria. It represents around 10-15% of patients diagnosed with MDS, but is not currently considered a separate entity by WHO. Hypocellularity is defined as bone marrow cellularity of less than 30% in patients younger than 70 years or less than 20% in those older than 70 years.Patients and Methods:We retrospectively evaluated the demographic, clinical features, bone marrow aspirate/trephine, treatment characteristics and outcomes of 100 patients with hMDS. The bone marrow aspirates and trephines were also analysed for dysplasia, blast percentage, cellularity, reticulin, p53 by Immunohistochemistry (IHC), CD34 and CD117. An MiSeq based targeted gene panel comprising of 24 frequently mutated MDS genes was used in a subset of patients.Results:The median age was 51 years (18-87), with only a third of patients >60 years. WHO subtypes RA (n=2), MDS 5q- (n=1), RCMD (n=95) and RAEB (n=2), IPSS risk groups low risk (n=20), Int1 (n=61), int2 (n=11) and high risk (n=2). 23% had evolved from previous AA.For the hMDS group IPSS cytogenetic categories comprised good risk 67% (n=62), intermediate 14% (n=13), including 6 cases of trisomy 8, and poor 18% (n=17), with 7 cases of monosomy 7. Of the normal cytogenetics, 15/62 (24%) had positive (moderate to strong) p53 by IHC, 30/62 (48%) with CD117 positivity (1-20%), 48/62 (77%) have fibrosis grade 1-2. Only 2/62 with normal cytogenetics had normal p53, CD34, CD117 and reticulin and these have evidence of dysplasia on morphology. The presence of cytogenetic abnormalities and other features such as p53, CD117 and fibrosis reflect a distinct population differing from the hypocellular marrow of aplastic anemia.A higher incidence (18%) of autoimmune disorders was seen, including ITP (n=2), thyroid dysfunction (n=3), alopecia (n=2), inflammatory bowel disease (n=4), coeliac (n=2), SLE/SjogrenÕs (n=2), others (n=6).Forty percent (n=32) had a PNH clone, all except 2 were subclinical. Progression of disease to AML (n=3), upstaging of disease (RCMD to RAEB, n=6) or cytogenetic evolution (n=3) was seen in 15% of hMDS, including 3 cases with both increase in blasts and karyotypic evolution.A subset (n=33) of hMDS were evaluated for recurrently mutated genes in MDS; 7/33 (21%) of patients harbored somatic mutations in ASXL1 (n=4), DNMT3A (n=2) and BCOR (n=2). All except one patient with somatic mutation had a prior history of aplastic anaemia.As the predominant group was hypocellular RCMD (95%), we compared the clinical features, treatment and prognosis with cellular RCMD cohort during the same time period.Median ages of hMDS and non-hypocellular MDS (nhMDS) were 51 and 60.3 years respectively. Patients with hMDS presented with more significant thrombocytopenia (median platelet count at presentation 43 vs 93), neutropenia (median ANC at presentation 1.13 vs 1.3), anaemia (median Hb 94g/L vs 104g/L), transfusion dependency, and more intermediate-2/high-risk disease than the nhMDS group (p=0.0257). Among hMDS patients, 30% (28/95) had chromosomal abnormalities, an incidence similar to that of nhMDS, 25% (23/94).Treatments received by hMDS cohort were: single agent Cyclosporin (n=27), anti-thymocyte globulin (n=7), erythropoietin and GCSF (n=13), 5-azacytidine (n=6), intensive chemotherapy (n=4), HSCT (n=26), including 9 patients who underwent upfront HSCT. As expected the use of IST was infrequent (8%) in the nhMDS cohort compared to 35% in the hMDS (p<0.03).The rate of progression to acute myeloid leukaemia was lower in hMDS compared to nhMDS group (3% vs 13%, p<0.02). The median overall survival was longer for patients with hMDS compared with the nhMDS (11.1 years vs 4.3 years p<0.001).Conclusion:This large study of hMDS identifies a specific subgroup of MDS with lower median age, severe cytopenia, high risk disease and a good prognosis. The increased incidence of trisomy 8 and autoimmune disorders is indicative of immunological dysfunction in this group of patients. The presence of specific genomic abnormalities (ASXL1, DNMT3A, BCOR), especially in patients with prior history of aplastic anaemia needs further comprehensive evaluation in a prospective study. [Display omitted] Hypocellular MDS vs Normo/hypercellular MDS (median OS 11.1 vs 4.3 years, p<0.001) DisclosuresNo relevant conflicts of interest to declare.

Prognostic and predictable value of COX2 expression in Russian women with stage I breast cancer.

80 Background: Some previous studies found worse prognosis among cyclooxygenase-2 (COX2)-expressing breast cancers; we study prognostic and predictiable value of COX2 expression in breast cancer stage I. Methods: our study included Russian women with breast cancer stage I (n=315) treated in RCRC, RMAPE (1985-2009). A Tissue Micro Array (TMA) with triplicate 1 mm tumor tissue punches taken from tumor blocks was constructed in LUMC; sections were immunohistochemically stained for ER, PR, HER2-status, Ki67 (by standard morphological criteria); COX2-expression were evaluated as positive (&gt;median; 31/309 cases, 10%) or negative (≤median; 278/209 cases, 90%). Also the histological type, grade, age and adjuvant endocrine therapy were examined. We analyzed the clinic and morphological data of COX2-positive tumors, prognostic value for survival (relapses free- RFS, overall- OS and cancer specific- CSS) and predictable value for endocrine therapy. Results: COX2-positive tumor were associated with ductal histological type (p=0,018), PR-negative status (p=0,027) and high Ki67 (p&lt;0,0001), but not correlated (p&gt;0,05) with age, grade, ER, HER2 status or biological subtype. In women with ER-negative tumors (104 patients, 34,1%) COX2-expression did not associate with worse survival (p&gt;0,05). In contrast to this, in patients with ER-positive tumors (201 women, 65,9%) COX2-expression strongly correlated with worse RFS in univariate (HR 2,829, 95% CI 1,366-5,860, p=0,005) and multivariate analyses (HR=2,972, 95% CI 1,190-7,423, p=0,02). The same value of COX2-expression in women with ER-positive tumors was found for CSS (univariate: HR 3,421, 95% CI 1,436-8,149, p=0,005; multivariate: HR 4,260, 95% CI 1,344-13,504, p=0,014), but not for OS (p&gt;0,05). In women who did not receive adjuvant endocrine therapy (145 patients, 46%) COX2 expression did not have any prognostic value for RFS, OS and CSS (p&gt;0,05) but in patients that used adjuvant endocrine therapy (170 women, 54%) COX2-expression strongly associated with worse cancer-specific survival (HR 5,614 95% CI 1,165-27,059, p=0,032), but not with RFS and OS (p&gt;0,05). Conclusions: COX2 expression plays a role in hormonal pathways and sensitivity for endocrine therapy.

Fusarium napiforme systemic infection: case report with molecular characterization and antifungal susceptibility tests.

IntroductionDuring the last decades, Fusarium spp. has been reported as a significant cause of disease in humans, especially in immunocompromised patients, who have high risk of invasive life-threatening disease. Fusarium species usually reported as cause of human disease are F. solani, F. oxysporum and F. verticillioides.Case descriptionWe describe the second case in the literature of disseminated fusariosis caused by Fusarium napiforme, that occurred in a 60-year-old woman with multiple myeloma after subsequent cycles of chemotherapy.Discussion and EvaluationWe identified the F. napiforme not only by standard morphologic criteria by macroscopic and microscopic characteristics, but also confirmed by molecular biology methods, including sequencing. The antifungal susceptibility of the F. napiforme isolates were tested to seven antifungal drugs; the azoles were the most active drug against all the isolates tested.ConclusionsFusarium spp. are of relevance in medical mycology, and their profiles of low susceptibility to antifungal drugs highlight the importance for faster and more accurate diagnostic tests, what can contribute to an earlier and precise diagnosis and treatment.

CD10 is useful to identify gastrointestinal contamination in endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) cytology from pancreatic ductal adenocarcinoma.

Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) cytology is an effective tool to diagnose pancreatic ductal adenocarcinoma (PDA). Standard morphological criteria are usually reliable. When contaminating gastrointestinal (GI) epithelial cells are prevalent among neoplastic cells, these can be highlighted by carcinoembryonic antigen (CEA) staining. CD10 is a cell-surface metallopeptidase normally expressed by the GI epithelial apical border, whose expression is decreased or lost in PDA. We included CD10 in a panel, together with CEA, to discriminate the GI contaminant cells from PDA cells on cell blocks. Eight cases of EUS-FNA of PDA, featuring both contaminating GI cells and neoplastic cells, whose corresponding cell blocks were available for immunostaining, were selected. CD10 and CEA were stained on cell blocks by standard methods. CD10 strongly labelled only the GI cells, with a well-defined apical membrane signal; conversely, GI cells did not show CEA staining; benign duodenal cells were faintly labelled in only one case. Malignant cells were positive for CEA and negative for CD10, with the exception of one case with labelled neoplastic cells with weak diffuse cytoplasmic positivity. CD10 apical membrane staining was a feature only seen in benign GI cells. As a loss of CD10 is a consistent feature of PDA, this marker can be useful, together with CEA, to aid the cytopathologist to identify neoplastic cells in a background rich in GI contaminant cells.

Enumeration and identification of dust fungal elements from the weather inversion phenomenon in Isfahan, Iran.

Background:Fungi are the major pathogens or allergens for which the air is the natural medium of their dispersal. Since the air pollution is associated with a wide range of adverse health outcomes, then identification of the type and population of fungi in these conditions will help the management of hygienic and control of fungal disease.Materials and Methods:A total of 103 dust samples were collected from glass surfaces of different places by sedimentation method. Pollution standard indexes were provided by Environmental Protection Agency in Isfahan. All dust samples were mixed and homogenized in distilled water containing antibacterial agents. Serial cultures were done in 5 times experiments on two standard culture media. Isolated fungal colonies were identified by their standard morphologic and physiologic criteria. The analysis was performed by Mann-Whitney test calculating by SPSS version 20 (SPSS Inc., Chicago, IL, USA).Results:The real mean of total culture-able fungi in 1 g of sedimentation dust were account about 44800 colonies of different fungi. More than half of the viable fungi (62.8%) could grow out of 1 g of dust on Mycosel agar were the genera of Aspergillus, Penicillium and Cladosporium with 28.8%, 23.4% and 10.6% respectively. The dominant genus could grow on Sabouraud dextrose agar with chloramphenicol medium were the genera of Aspergillus, Cladosporium and Penicillium with 23.7%, 21.1% and 14.5% respectively.Conclusions:Our data show the amount and variety of viable colony-forming fungi, which we are faced with in Isfahan during the air pollution condition. The real abundance of fungal particles and non-cultivable fungi in dust are still poorly understood and remain for further study in the future.

The Relation of Clinical Response and Minimal Residual Disease and Their Prognostic Impact On Outcome in Acute Myeloid Leukemia

Abstract 1418▪▪This icon denotes a clinically relevant abstract Introduction:Presence of minimal residual disease (MRD) after induction therapy predicts risk of relapse in AML (San Miguel JF et al 2001). Furthermore, achievement of complete remission (CR) rather than CR with incomplete platelet recovery (CRp) (defined using standard morphologic criteria) after induction therapy is independently associated with shorter relapse-free survival (Walter RB et al 2010). These results suggest a correlation between peripheral blood count recovery at the time of morphologic remission and MRD; however, this has not been documented. Here we address this question and examine whether response and MRD have independent effects on relapse. Patients and methods:We retrospectively analyzed data from 340 adults with AML who achieved CR, CRp or CRi (CR with incomplete peripheral count recovery; cellular marrow with <5% blasts but neutrophil count < 1,000/mL) after induction therapy at University of Washington/Fred Hutchinson Cancer Research Center since 2008. The bone marrows were collected on or closest to the first date of response and MRD was determined by ten-color multiparametric flow cytometry. The impact of MRD and response on relapse was analyzed using Cox proportional regression models. Cytogenetics were classified according to SWOG criteria. Results:Among the 340 patients, 23% of the 247 with CR, 37% of the 65 with CRp and 50% of the 28 with CRi had MRD (p =0.002). Amounts of MRD differed among the 3 groups (p=0.015) and in particular was higher with CRi median 2.6 (interquartile range [IQR] 1.3–3.4), than with CRp median 0.7 (IQR 0.3–1.3) and CR median 0.7 (IQR 0.3–2.4). By univariable Cox proportional regression analysis with death before relapse considered a competing risk, patients with MRD were more likely to relapse than patients without MRD regardless of whether MRD was considered as 0 vs. > 0 (hazard ratio [HR] 1.85; 95% CI 1.25–2.72; p=0.002) or as a numerical variable (HR 1.15 for each 1% increase; 95% CI 1.06–1.25; p <0.001). As expected, the risk of relapse was also higher in patients achieving only CRi (vs. CR: HR 2.95; 95% CI 1.63–5.36; p <0.001) or CRp (vs. CR: HR 2.17; 95% CI 1.37–3.42; p<0.001). In multivariable Cox proportional regression analyses including cytogenetic risk factors, MRD 0 vs. >0 (HR 1.50; 95% CI 1.00–2.23; p=0.047), response (CR vs. CRp and CRi: HR 2.11; 95% CI 1.40–3.17; p<0.001), and cytogenetic factors (intermediate vs. favorable: HR 4.67; 95% CI 1.13–19.2; p=0.033 or unfavorable/unknown prognostic significance vs. favorable: HR 4.59; 95% CI 1.11–19.0; p=0.035) were each independently associated with relapse. Conclusion:CRp and CRi are associated with increased frequency of MRD (0 vs. >0) in comparison to CR while CRi is characterized by higher levels of MRD than CRp or CR. This indicates that failure of blood count recovery is often due to persistent AML rather than toxic effects on normal progenitors and thus should often call for further therapy rather than delay. Clinical response, MRD and cytogenetic factors are independent predictors for relapse in AML patients achieving remission. Disclosures:No relevant conflicts of interest to declare.

Expression of calretinin by breast carcinoma and the potential for misdiagnosis of mesothelioma

Calretinin and cytokeratin (CK)5/6 are frequently used to differentiate between metastatic breast cancer and primary malignant mesothelioma in pleural biopsies, but both tumours may express these markers. This study was aimed at evaluating the frequency of calretinin expression in primary breast carcinomas, and assessing the characteristics of the calretinin-positive tumours. Fifty-three primary breast adenocarcinomas were analysed for immunohistochemical expression of calretinin. CK5/6 and epidermal growth factor receptor (EGFR) immunostaining were performed on the calretinin-positive subset. Tumours were classified as basal-like if they met standard morphological and immunohistochemical criteria. Fifteen per cent (8/53) of the breast tumours were positive for calretinin. Eighty-eight per cent (7/8) of the calretinin-positive tumours were grade 3, as compared with 20% (9/45) of the calretinin-negative tumours (P<0.001). Only 13% (1/8) of the calretinin-positive tumours were also oestrogen receptor (ER)-positive, as compared with 87% (39/45) of the calretinin-negative tumours (P<0.001). Eleven per cent (6/53) of the tumours were classified as basal-like. Of these, four were positive for calretinin and two were negative (P=0.003). Fifteen per cent of breast carcinomas stain with calretinin. These tumours are more likely to be high-grade, ER-negative, and display a basal-like phenotype. These tumours may be misdiagnosed as malignant mesothelioma when they metastasize to the pleura.

Does meiotic spindle normality predict improved blastocyst development, implantation and live birth rates?

To investigate the relationship between spindle normality and: 1) blastocyst formation; 2) implantation rates; and 3) live birth rates. Prospective observational study. A private fertility clinic. One hundred patients ≤40 years old undergoing intracytoplasmic sperm injection (ICSI). Meiotic spindles were imaged before ICSI with the use of Oosight microscopy. With the use of specific criteria, spindle normality was independently assessed by two researchers. Blastocysts were chosen for transfer by standard light microscopic morphologic criteria by researchers who were blinded to the spindle assessment. 1) Blastocyst formation; 2) implantation; and 3) live birth rates. A total of 808/920 oocytes were metaphase II. Of those, 711 (88%) had a visible spindle: 205 (29%) were normal spindles (NS) and 506 (71%) abnormal spindles (AS). Fertilization rates were significantly higher in NS oocytes. Although NS and AS oocytes both formed morphologically good-quality blastocysts, implantation and live birth rates were higher from NS oocytes. All ongoing pregnancies resulted from NS oocytes. Spindle assessment with polarized light microscopy provides an early predictor of the pregnancy potential of that oocyte. AS oocytes can form good-quality blastocysts, but these appear to have little chance of live birth. Therefore, spindle assessment should improve the selection of the best embryo for single embryo transfer.

The Rate of In Vitro Maturation of Primary Follicles From Adult Mice and the Quality of Oocytes is Improved in the Absence of Anti-Müllerian Hormone

Anti-Müllerian hormone (AMH) inhibits the recruitment of primordial follicles into the growing pool, but its role in primary and secondary follicles is not clear. We isolated primary follicles from the ovaries of 9- to 10-week old mice and examined whether AMH affected follicular development. Follicles were matured in media that was prepared using unsexed fetal bovine serum (FBS) or female FBS (FFBS) with or without added AMH for approximately 2 weeks and maturation rates to secondary follicles and metaphase II (MII) oocytes were measured by standard morphological criteria. Rates of parthenogenetic activation and in vitro fertilization (IVF) were assessed by cleavage and blastocyst development, respectively. Whereas addition of AMH blocked primary to secondary follicle transition, the primary to secondary and secondary to MII follicle maturation rates was significantly improved with FFBS. Folliculogenesis resumed once AMH was removed from the media of the arrested primary follicles. The rates of IVF and parthenogenesis of oocytes after in vitro maturation (IVM) without AMH were also improved compared to controls. The results indicate that removal of AMH from culture conditions during IVM from primary follicular stages should be considered to improve outcome.

Immunohistochemical and molecular markers in breast phyllodes tumors.

Phyllodes tumors of the breast are diagnostically and managerially enigmatic, as their malignant potential is difficult to predict based on the standard morphologic criteria. Thus, there is a need for additional markers of biologic potential. Although a number of ancillary tests have been reported, consensus in the literature is lacking. We studied 38 cellular fibroadenomas and phyllodes tumors of various grade (World Health Organization benign, borderline, and malignant) with a panel of immunohistochemical stains (p53, CD117, phospho-Histone3, mdm2, cdk4) and screened 26 of the tumors for mutations across 30 cancer-related genes using PCR and mass-spectrometry based methods. p53 and phospho-Histone3 (mitotic marker) showed increased staining in higher grade phyllodes tumors. CD117, mdm2, and cdk4 showed no difference in expression across different grades of phyllodes tumors. Mutational analysis revealed an S8R substitution in FBX4 (an E3 ubiquitin ligase) in 3 cases: 1 benign and 2 borderline. The S8R substitution seems to be more common in phyllodes tumors (11.5%) as compared with other cancers. FBX4 S8R cases had high cyclin D1 expression, but this finding was not specific. Our data support earlier studies showing that p53 has potential use in pathologic assessment of phyllodes tumors, and we newly characterized phospho-Histone3 for this application. Further studies are needed to characterize the molecular pathogenesis of the phyllodes tumors, as we were unable to identify activating mutations despite screening for a large panel of activating hotspot mutations. The significance of the FBX4 substitution deserves further investigation.