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From October 2022 through January 2023, nine patients with NDM-5/OXA-48-carbapenemase-producing Enterobacter hormaechei ST79 were detected in Denmark and subsequently one patient in Iceland. There were no nosocomial links between patients, but they had all been treated with dicloxacillin capsules. An NDM-5/OXA-48-carbapenemase-producing E. hormaechei ST79, identical to patient isolates, was cultured from the surface of dicloxacillin capsules in Denmark, strongly implicating them as the source of the outbreak. Special attention is required to detect the outbreak strain in the microbiology laboratory. From October 2022 through January 2023, nine patients with NDM-5/OXA-48-carbapenemase-producing Enterobacter hormaechei ST79 were detected in Denmark and subsequently one patient in Iceland. There were no nosocomial links between patients, but they had all been treated with dicloxacillin capsules. An NDM-5/OXA-48-carbapenemase-producing E. hormaechei ST79, identical to patient isolates, was cultured from the surface of dicloxacillin capsules in Denmark, strongly implicating them as the source of the outbreak. Special attention is required to detect the outbreak strain in the microbiology laboratory.

lgangur: Að taka saman gögn um sýklalyfjaávísanir íslenskra tannlækna árin 2010 til 2021, þar með talið fjölda ávísana, hvaða sýklalyf eru mest notuð og hver þróunin hefur verið síðustu ár. Efniviður og aðferðir: Ópersónugreinanlegar upplýsingar um ávísanir sýklalyfja utan sjúkrahúsa voru unnar úr lyfjagagnagrunni embættis landlæknis. Sótt voru gögn um ávísanir tannlækna fyrir sýklalyf í ATC flokkum J01 og P01AB01 árin 2010 til 2021. Niðurstöður: Hlutdeild tannlækna af sýklalyfjaávísunum var um 7% af notkun á Íslandi. Fjöldi sýklalyfjaávísana tannlækna á hverja 1000 íbúa var á bilinu 43,8 til 49,7 á tímabilinu 2010 til 2021, eða 47,3 að meðaltali. Ávísanir til barna (

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) both belong to the group of mature B-cell malignancies. However, MCL is typically clinically aggressive while the clinical course of CLL varies. CLL can be divided into prognostic subgroups based on IGHV mutational status and into multiple subsets based on closely homologous (stereotyped) B-cell receptors. In paper I we investigated 31 MCL cases using high-density 250K single-nucleotide polymorphism arrays and gene expression arrays. Although most copy-number aberrations (CNAs) were previously reported in MCL, a novel deletion was identified at 20q (16%) containing the candidate tumor suppressor gene ZFP64. A high proliferation gene expression signature was associated with poor prognosis, large CNAs, 7p gains and 9q losses. Losses at 1p/8p/13q/17p were associated with increased genomic complexity. In paper II we sequenced exons 4 to 8 of the TP53 gene in 119 MCL cases. 17p copy-number status was known from previous studies or determined by real-time quantitative polymerase chain reaction. TP53 mutations were detected in 14% of cases and were strongly associated with poor survival while 17p deletions were more common (32%) but did not predict survival. In papers III and IV we applied high-resolution genomic 27K methylation arrays to 20 MCL and 39 CLL samples. In paper III MCL displayed a homogenous methylation profile without correlation with the proliferation signature whereas MCL was clearly separated from CLL. Gene ontology analysis revealed enrichment of developmental genes, in particular homeobox transcription factor genes, among targets methylated in MCL. In paper IV we compared three different stereotyped CLL subsets: #1 (IGHV unmutated), #2 (IGHV3-21) and #4 (IGHV mutated). Many genes were differentially methylated between each two subsets and immune response genes (e.g. CD80 and CD86) were enriched among genes methylated in subset #1 but not in subsets #2/#4. In summary, CNAs were frequent and not random in MCL. Specific CNAs correlated with a high proliferation gene expression signature or genomic complexity. TP53 mutations predicted short survival whereas 17p deletions did not. A high proliferation signature was not associated with differential DNA methylation in MCL, which demonstrated a homogeneous methylation pattern. In contrast, genomic methylation patterns differed between MCL and CLL and between stereotyped CLL subsets.

Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) and numerous copy number aberrations (CNAs). Recently, gene expression profiling defined a proliferation gene expression signature in MCL where high scores predict shorter survival. We investigated 31 MCL cases using high-density single nucleotide polymorphism arrays and correlated CNA patterns with the proliferation signature and with clinical data. Many recurrent CNAs typical of MCL were detected, including losses at 1p (55%), 8p (29%), 9q (29%), 11q (55%), 13q (42%) and 17p (32%), and gains at 3q (39%), 8q (26%), 15q (23%) and 18q (23%). A novel deleted region at 20q (16%) contained only one candidate gene, ZFP64, a putative tumor suppressor. Unsupervised clustering identified subgroups with different patterns of CNAs, including a subset (19%) characterized by the presence of 11q loss in all cases and by the absence of 13q loss, and 3q and 7p gains. Losses at 1p, 8p, 13q and 17p were associated with increased genomic complexity. High proliferation signature scores correlated with increased number of large (>15 Mbp) CNAs (P = 0.03) as well as copy number gains at 7p (P = 0.02) and losses at 9q (P = 0.04). Furthermore, large/complex 13q losses were associated with improved survival (P < 0.05) as were losses/copy number neutral LOH at 19p13 (P = 0.01). In summary, this high-resolution genomic analysis identified novel aberrations and revealed that several CNAs correlated with genomic complexity, the proliferation status and survival.

Chronic lymphocytic leukemia (CLL) can be divided into prognostic subgroups based on the IGHV gene mutational status, and is further characterized by multiple subsets of cases with quasi-identical or stereotyped B cell receptors that also share clinical and biological features. We recently reported differential DNA methylation profiles in IGHV-mutated and IGHV-unmutated CLL subgroups. For the first time, we here explore the global methylation profiles of stereotyped subsets with different prognosis, by applying high-resolution methylation arrays on CLL samples from three major stereotyped subsets: the poor-prognostic subsets #1 (n = 15) and #2 (n = 9) and the favorable-prognostic subset #4 (n = 15). Overall, the three subsets exhibited significantly different methylation profiles, which only partially overlapped with those observed in our previous study according to IGHV gene mutational status. Specifically, gene ontology analysis of the differentially methylated genes revealed a clear enrichment of genes involved in immune response, such as B cell activation (e.g., CD80, CD86 and IL10), with higher methylation levels in subset #1 than subsets #2 and #4. Accordingly, higher expression of the co-stimulatory molecules CD80 and CD86 was demonstrated in subset #4 vs. subset #1, pointing to a key role for these molecules in the crosstalk of CLL subset #4 cells with the microenvironment. In summary, investigation of three prototypic, stereotyped CLL subsets revealed distinct DNA methylation profiles for each subset, which suggests subset-biased patterns of transcriptional control and highlights a key role for epigenetics during leukemogenesis.

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are mature CD5(+) B-cell malignancies with different biological/clinical characteristics. We recently reported an association between different prognostic subgroups of CLL (i.e., IGHV mutated and unmutated) and genomic methylation pattern. However, the relationship between DNA methylation and prognostic markers, such as the proliferation gene expression signature, has not been investigated in MCL. We applied high-resolution methylation microarrays (27,578 CpG sites) to assess the global DNA methylation profiles in 20 MCL (10 each with high/low proliferation signature) and 30 CLL (15 poor-prognostic IGHV unmutated subset #1 and 15 good-prognostic IGHV mutated subset #4) samples. Notably, MCL and each CLL subset displayed distinct genomic methylation profiles. After unsupervised hierarchical clustering, 17/20 MCL cases formed a cluster separate from CLL, while CLL subsets #1 and #4 formed subclusters. Surprisingly, few differentially methylated genes (n = 6) were identified between high vs. low proliferation MCL. In contrast, distinct methylation profiles were demonstrated for MCL and CLL. Importantly, certain functional classes of genes were preferentially methylated in either disease. For instance, developmental genes, in particular homeobox transcription factor genes (e.g., HLXB9, HOXA13), were more highly methylated in MCL, whereas apoptosis-related genes were enriched among targets methylated in CLL (e.g., CYFIP2, NR4A1). Results were validated using pyrosequencing, RQ-PCR and reexpression of specific genes. In summary, the methylation profile of MCL was homogeneous and no correlation with the proliferation signature was observed. Compared to CLL, however, marked differences were discovered such as the preferential methylation of homeobox genes in MCL.

Background The purpose of this study was to investigate the degree of ΔOD450 method variability and its effect on ΔOD450 measurements between selected clinical laboratories in the U.S. Method Four amniotic fluid specimens were sent to 7 clinical laboratories in the U.S. for ΔOD450 analysis. In addition, scanning spectrophotometric data from 152 amniotic fluid samples were used to compare ΔOD450 values calculated using both log and linear OD scales. Results We found that no 2 laboratories used exactly the same method and no laboratory used precisely the same method as originally described by Liley. Despite the varied methods, the ΔOD450 measurements were remarkably similar. The exception was 1 sample that was subjected to chloroform extraction. The ΔOD450 measurement on this sample was 82% lower than the mean. On average, ΔOD450 results determined from a linear OD scale were 37% lower than those determined from a log scale. Conclusion Although there is no standard method for performing the ΔOD450, inter-laboratory variation of ΔOD450 results is remarkably small. As our and other previous studies have reported, these data suggest that both chloroform extraction and use of a linear scale have the potential to result in lower ΔOD450 results.

Somatic hypermutation (SHM) aberrantly targets proto-oncogenes in various non-Hodgkin's lymphoma. To test the association of SHM with transformation of follicular lymphoma (FL), we sequenced mutational hot spots in five proto-oncogenes (BCL6, PAX5, RHOH, MYC and PIM1) in 32 low-grade FL (lgFL) with follicular histology and 26 transformed FL (tFL) with diffuse large cell histology. No difference was detected in the fraction of specimens mutated (75% of lgFL and 77% of tFL) or in the mutation load (0.08 for lgFL vs. 0.06 mutations/100 bp/allele for tFL). Serial specimens were examined from 25 patients showing stable low-grade FL (slgFL; n=6) or a low-grade FL that later transformed into diffuse large cell lymphoma (tFL; n=19). slgFL and tFL patients accumulated similar numbers of mutations in the interval between biopsies. These data indicate that mutations attributable to aberrant SHM occur with similar frequency in low-grade and transformed FL; transformation is not associated with a higher rate of aberrant SHM. Moreover, the frequency of mutations attributable to aberrant SHM in tFL was significantly lower than that reported for de novo diffuse large B cell lymphoma, suggesting differing oncogenic mechanisms in transformed follicular lymphoma and de novo diffuse large B cell lymphoma.

The BIOMED-2 PCR-based immunoglobulin gene rearrangement assays have quickly become the most commonly used laboratory method for detection of B-cell clonality. Therefore, the reliability of these assays under various conditions has become increasingly important. When studying paired cases of follicular lymphoma (FL) from individual patients, we used these assays to assess clonality in 40 formalin-fixed paraffin-embedded (FFPE) specimens from 19 patients diagnosed with FL. The assays of IGH rearrangement failed to give a clonal result in 26/40 (65%) specimens, while the IGK assays failed in only 3/40 (8%) specimens. The high failure rate of the IGH assays for this set of FFPE lymphomas cannot be explained by systematic problems with DNA extraction or amplification because the same IGH assays resulted in a low failure rate (3/32, 9%) for FFPE small lymphocytic lymphoma/chronic lymphocytic leukemia specimens and for fresh frozen FL specimens (1/6, 17%). Furthermore, in a second validation set of 13 FFPE follicular lymphoma the failure rate was 9/13 (69%). Therefore, the BIOMED-2 IGH assay did not perform well on FFPE follicular lymphoma specimens, and the IGK assay may be superior for assessing clonality when no fresh/frozen tissue is available.

Dermatan sulfate (DS) accelerates the inhibition of thrombin by heparin cofactor II (HCII). A hexasaccharide consisting of three L-iduronic acid 2-O-sulfate (IdoA2SO₃)[rightward arrow]N-acetyl-D-galactosamine 4-O-sulfate (GalNAc4SO₃) subunits was previously isolated from porcine skin DS and shown to bind HCII with high affinity. DS from porcine intestinal mucosa has a much lower content of this disaccharide but activates HCII with potency similar to that of porcine skin DS. Therefore, we sought to characterize oligosaccharides from porcine mucosal DS that interact with HCII. DS was partially depolymerized with chondroitinase ABC, and oligosaccharides containing 2-12 monosaccharide units were isolated. The oligosaccharides were then fractionated by anion-exchange and affinity chromatography on HCII-Sepharose, and the disaccharide compositions of selected fractions were determined. We found that the smallest oligosaccharides able to bind HCII were hexasaccharides. Oligosaccharides 6-12 units long that lacked uronic acid (UA)2SO₃ but contained one or two GalNAc4,6SO₃ residues bound, and binding was proportional to both oligosaccharide size and number of GalNAc4,6SO₃ residues. Intact DS and bound dodecasaccharides contained predominantly IdoA but little D-glucuronic acid. Decasaccharides and dodecasaccharides containing one or two GalNAc4,6SO₃ residues stimulated thrombin inhibition by HCII and prolonged the clotting time of normal but not HCII-depleted human plasma. These data support the hypothesis that modification of IdoA[rightward arrow]GalNAc4SO₃ subunits in the DS polymer by either 2-O-sulfation of IdoA or 6-O-sulfation of GalNAc can generate molecules with HCII-binding sites and anticoagulant activity.

CD40 ligand (CD40L) is a member of the tumor necrosis factor superfamily and is produced in a variety of cells, including platelets. The soluble form (sCD40L) is a mediator of both inflammatory and hemostasis processes and has been implicated in the pathogenesis of atherosclerosis. Clinical studies have revealed increased sCD40L in patients with unstable angina (1) and identified an association between increased sCD40L and future risk for death or nonfatal myocardial infarction (2)(3). While prospectively measuring sCD40L in a cohort of persons at risk for cardiovascular complications, we identified both preanalytical and analytical sources of error. This report documents the effects of specimen type (serum and plasma), processing (time and temperature), and commercial reagent selection on sCD40L ELISA results. These findings raise concerns about the accuracy of sCD40L results reported in recent clinical studies. After obtaining informed consent, we enrolled 147 patients older than 60 years referred for diagnostic cardiac catheterization in an Institutional Review Board-approved study to evaluate the value of clinical, echocardiographic, and biomarker variables for prediction of future cardiovascular complications. When combined with clinical predictors, B-type natriuretic peptide and C-reactive protein, but not sCD40L, were independent predictors of death or cardiovascular hospitalization at 6 months (data not shown). The unexpectedly poor correlation between undiluted plasma sCD40L results and clinical outcomes in this study motivated us to perform the following investigations. Venous blood from the 147 study participants [mean (SD) age, 70.8 (6.9) years] was collected into plastic tubes containing tripotassium EDTA (BD Vacutainer; Becton Dickinson) before catheterization and placed on ice for 1–4 h before processing. Blood from 10 control individuals [mean (SD) age, 38.7 (8.4) years] was collected into both EDTA and plain glass tubes (Becton Dickinson) and maintained at room temperature for 30 min before processing. Study and control samples were centrifuged twice: …

We recently described the use of a quantitative serum human chorionic gonadotropin (hCG) assay with urine samples (1) . Quantitative urine hCG assays are important in validations of point-of-care qualitative hCG tests and in cases of suspected false-positive quantitative serum hCG results because of heterophilic antibodies (2)(3). Here, we report our study of the ADVIA Centaur® (Bayer Corporation) total-hCG (ThCG) serum assay with urine. Recovery of hCG (US Pharmaco-peia) added to urine was >100% at all concentrations (Table 1⇓ ). At concentrations ≤200 IU/L, recovery was >130%. Urine hCG measurements ≤200 IU/L were linear but were not parallel to expected amounts. For samples with …

Abstract 536 Introduction: Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are B-cell malignancies of different postulated origin, genetics, clinical presentation and prognosis. Several studies have reported that both MCL and CLL individually exhibit aberrant methylation in comparison to normal B-cells. However, a comprehensive comparison of the methylation profiles of these two B-cell disorders has not been performed yet. This strategy has the potential to identify cellular pathways and genes that are specifically targeted in each disease. Methods: We applied the genome-wide Illumina Infinium HumanMethylation27 BeadChip array (Illumina, San Diego, USA) which measures methylation levels at 27,578 CpG dinucleotides covering 14,495 genes, to compare the methylation profiles in: (i) 20 MCL cases; and, (ii) 30 CLL cases, 15 each with unmutated stereotyped subset #1 (IGHV1-5-7/IGKV1(D)-39) B cell receptors (BCRs) or mutated stereotyped subset #4 (IGHV4-34/IGKV2-30) BCRs, where these two subsets represent prototypes of unmutated and mutated CLL. The methylation status for each detected CpG site ranged between 0.1 (completely unmethylated) to 1 (completely methylated). Results: As expected, major differences in methylation patterns between MCL and CLL were observed. When the methylation profiles of the two entities were compared, 51 genes were identified as differentially methylated in all comparisons (MCL versus both CLL subsets combined and each subset separately). Among the 19 genes highly methylated in MCL were six (32%) homeobox or homeodomain-containing transcription factors (e.g. POU4F1, PITX3), whereas genes enhancing cell proliferation and tumor progression such as MERTK and CAMP were hypomethylated in MCL. Of the 32 genes hypermethylated in CLL were six pro-apoptotic genes, including DYRK2 and CYFIP2, the tumor suppressor PRDM2 and the cell cycle regulator CCND1. Conclusions: We report for the first time disease-biased methylation profiles for different functional classes of genes in MCL or CLL. Homeobox genes were highly methylated in MCL, whereas CLL was characterized by methylation of apoptosis-related genes. The identified differences in global methylation profiles between MCL and CLL may assist in unfolding distinct epigenetic silencing mechanisms involved in the pathogenesis of these B-cell malignancies. Disclosures: No relevant conflicts of interest to declare.

Introduction: Follicular lymphoma (FL) is the second most prevalent non-Hodgkins lymphoma (NHL) in the United States. Although the clinical course is usually indolent, about 50% of low-grade FL transform to an aggressive lymphoma within 15 years of diagnosis. Somatic hypermutation (SHM) is a normal process in B cells which targets the immunoglobulin locus and introduces point mutations and small insertions/deletions. In diffuse large B cell lymphoma, the SHM misfires and aberrantly targets multiple loci, including some proto-oncogenes (PIM-1, PAX-5, RhoH/TTF and C-MYC). Hypothesis: We hypothesized that acquired mutations in the same proto-oncogenes could be responsible for the clinical transformation of follicular lymphoma. Materials and methods: The clinical database of the Department of Pathology, Washington University School of Medicine, St. Louis was searched to find patients with serial biopsies and the diagnosis of either stable (sFL) or transformed follicular lymphoma (tFL). Thirty-five (35) formalin-fixed paraffin-embedded (FFPE) tissue samples from 17 patients were identified. Eleven of seventeen patients had transformed to large cell lymphoma and six had stable low-grade follicular lymphoma. To exclude the possibility of a second unrelated lymphoma the common clonal origin of the original and follow-up lymphoma was confirmed by comparing immunoglobulin heavy and/or light chain variable gene sequences. Previously identified mutational hot spots in four genes, BCL-6, PAX-5, RhoH/TTF and C-MYC, were amplified and sequenced directly. Results: Fifty-five mutations were found in the four genes sequenced; all patients had at least one mutation. The number of mutations was similar in both the low grade and the transformed specimens overall, and most paired specimens had identical mutations or a single mutation change over time. Of the mutations identified, only mutations in Myc exon 2 correlated with the transformation. Acquired missense mutations were detected in the trans-activation domain of Myc in two of the eleven transformed FL specimens but none of the 24 low-grade follicular lymphoma specimens. The first case had two mutations: Pro57 to Ser57 and Glu39 to Asp39. The second case had a single Glu39 to Asp39 change. One of the mutations (Pro57 to Ser57) is close to a functionally critical phosphorylation site, Thr58, and has been reported in both Diffuse large B cell and Burkitt’s lymphomas. The other mutation (Glu39 to Asp39) has also been reported in both Burkitts and DLBCL cases, and is not reported as a normal variant. Conclusion: There is not an accumulation in the overall number of SHM with transformation as the frequency of SHM is similar in low-grade FL and transformed FL. However, mutations in the trans-activation domain in c-Myc, caused by an aberrant SHM, may play a role in the transformation of low-grade FL to a diffuse, large cell lymphoma in a subset of patients. | Gene Name | % Specimens with SHM | Mutation frequency per 100 bp | Number of unique single base pair substitutions | Number of unique deletions or insertions | Number of substitutions targeting G+C vs A+T | |:------------------- | -------------------- | ----------------------------- | ----------------------------------------------- | ---------------------------------------- | -------------------------------------------- | | NA = Not applicable | | BCL-6 | 63% | 0.14 | 25 | 3 | 16 vs 9 | | PAX-5 | 43% | 0.06 | 7 | 2 | 7 vs 0 | | Rho/TTF | 17% | 0.03 | 8 | 2 | 1 vs 7 | | C-MYC | 31% | 0.02 | 6 | 1 | 6 vs 0 | | All genes | 83% | NA | 47 | 8 | 31 vs 17 | Somatic Hypermutation Analysis of 17 serial Follicular Lymphoma cases (35 specimens)