Mutations in idiopathic cytopenia of undetermined significance assist diagnostics and correlate to dysplastic changes

Cytopenia is common in the elderly population and etiology may be difficult to assess. Here, we investigated the occurrence of mutations in patients with idiopathic cytopenia of undetermined significance and the usefulness in improving diagnostics. We included 60 patients with persistent cytopenia > 6 months without definite diagnosis of hematological neoplasm after routine assessment. Bone marrow material underwent a blinded morphology review and DNA was sequenced with a targeted 20 gene panel representing the most commonly mutated genes in myelodysplastic syndrome. Thirty seven (62%) patients carried at least one mutation at inclusion, and of these 95% carried a mutation in TET2, ASXL1, SRSF2, or DNMT3A. The most commonly mutated gene was TET2 observed in 43% of all patients. During one to eight years follow‐up seven patients progressed to a myeloid neoplasm and six of these had a detectable mutation at study entry. Median time to progression was 53 months (range 10–78), and at time of progression each patient had at least two mutations detected. Mutations in TP53 and NRAS were not present in patients at inclusion, but identified as secondary hits triggering progression. The morphology review was concordant in 68% of all cases, and 93% of the cases reclassified into the group “highly suspicious for MDS” had a mutation. All patients who had a concordant review “highly suspicious for MDS” had at least two mutations detected. Overall, we show that morphology examination is challenging in this heterogeneous group and targeted sequencing helps identify patients at risk of progression. Am. J. Hematol. 91:1234–1238, 2016. © 2016 Wiley Periodicals, Inc.     

NHE4 is critical for the renal handling of ammonia in rodents

Ammonia absorption by the medullary thick ascending limb of Henle’s loop (MTALH) is thought to be a critical step in renal ammonia handling and excretion in urine, in which it is the main acid component. Basolateral Na⁺/H⁺ exchangers have been proposed to play a role in ammonia efflux out of MTALH cells, which express 2 exchanger isoforms: Na⁺/H⁺ exchanger 1 (NHE1) and NHE4. Here, we investigated the role of NHE4 in urinary acid excretion and found that NHE4^–/– mice exhibited compensated hyperchloremic metabolic acidosis, together with inappropriate urinary net acid excretion. When challenged with a 7-day HCl load, NHE4^–/– mice were unable to increase their urinary ammonium and net acid excretion and displayed reduced ammonium medulla content compared with wild-type littermates. Both pharmacologic inhibition and genetic disruption of NHE4 caused a marked decrease in ammonia absorption by the MTALH. Finally, dietary induction of metabolic acidosis increased NHE4 mRNA expression in mouse MTALH cells and enhanced renal NHE4 activity in rats, as measured by in vitro microperfusion of MTALH. We therefore conclude that ammonia absorption by the MTALH requires the presence of NHE4 and that lack of NHE4 reduces the ability of MTALH epithelial cells to create the cortico-papillary gradient of NH₃/NH₄⁺ needed to excrete an acid load, contributing to systemic metabolic acidosis.     

Predicting acute uncomplicated urinary tract infection in women: a systematic review of the diagnostic accuracy of symptoms and signs

Background

Acute urinary tract infections (UTI) are one of the most common bacterial infections among women presenting to primary care. However, there is a lack of consensus regarding the optimal reference standard threshold for diagnosing UTI. The objective of this systematic review is to determine the diagnostic accuracy of symptoms and signs in women presenting with suspected UTI, across three different reference standards (10² or 10³ or 10⁵ CFU/ml). We also examine the diagnostic value of individual symptoms and signs combined with dipstick test results in terms of clinical decision making.

Methods

Searches were performed through PubMed (1966 to April 2010), EMBASE (1973 to April 2010), Cochrane library (1973 to April 2010), Google scholar and reference checking. Studies that assessed the diagnostic accuracy of symptoms and signs of an uncomplicated UTI using a urine culture from a clean-catch or catherised urine specimen as the reference standard, with a reference standard of at least ≥ 10² CFU/ml were included. Synthesised data from a high quality systematic review were used regarding dipstick results. Studies were combined using a bivariate random effects model.

Results

Sixteen studies incorporating 3,711 patients are included. The weighted prior probability of UTI varies across diagnostic threshold, 65.1% at ≥ 10² CFU/ml; 55.4% at ≥ 10³ CFU/ml and 44.8% at ≥ 10² CFU/ml ≥ 10⁵ CFU/ml. Six symptoms are identified as useful diagnostic symptoms when a threshold of ≥ 10² CFU/ml is the reference standard. Presence of dysuria (+LR 1.30 95% CI 1.20-1.41), frequency (+LR 1.10 95% CI 1.04-1.16), hematuria (+LR 1.72 95%CI 1.30-2.27), nocturia (+LR 1.30 95% CI 1.08-1.56) and urgency (+LR 1.22 95% CI 1.11-1.34) all increase the probability of UTI. The presence of vaginal discharge (+LR 0.65 95% CI 0.51-0.83) decreases the probability of UTI. Presence of hematuria has the highest diagnostic utility, raising the post-test probability of UTI to 75.8% at ≥ 10² CFU/ml and 67.4% at ≥ 10³ CFU/ml. Probability of UTI increases to 93.3% and 90.1% at ≥ 10² CFU/ml and ≥ 10³ CFU/ml respectively when presence of hematuria is combined with a positive dipstick result for nitrites. Subgroup analysis shows improved diagnostic accuracy using lower reference standards ≥ 10² CFU/ml and ≥ 10³ CFU/ml.

Conclusions

Individual symptoms and signs have a modest ability to raise the pretest-risk of UTI. Diagnostic accuracy improves considerably when combined with dipstick tests particularly tests for nitrites.     

The migration and invasion of human prostate cancer cell lines involves CD151 expression

The molecular mechanisms underlying prostate cancer metastasis remain poorly understood. The tetraspanin family member CD151 has been reported as an 'adaptor' between integrins and signal pathways. The role of CD151 in prostate cancer metastasis in vitro was investigated in this study. LNCap cells were transfected with wild-type CD151 cDNA, mutated CD151 cDNA and vector cDNA. The mutant (QRD194-196 to INF) CD151 cDNA was created using QuickChange 2 site directed Mutagenesis kit (Stratagene). siRNAs were also used to knock down the CD151 expression in the prostate cancer cell line PC3. Proliferation, migration and invasion properties were measured after gene transfection and gene knock-down. There was no difference in proliferation of untransfected or control transfected LNCap cells vs. CD151 transfected LNCap cells (P>0.05). There was greater motility of CD151-transfected vs. control cells, when transferring through migration chambers with or without matrigel-coated membranes (P<0.01, P<0.01). Fewer numbers of mutant-transfected cells were found on the membranes for both migration and invasion studies (P<0.01, P<0.01). CD151 knock-down PC3 cells showed decreased motility (P<0.01), but no change in proliferation (P>0.05). Our data show that CD151 does not change the proliferative properties of prostate cancer cells, but does promote migration and invasion, and suggest that CD151 plays a specific role in promoting prostate cancer cell motility.     

Comparison of gene expression profiles predicting progression in breast cancer patients treated with tamoxifen

Background Molecular signatures that predict outcome in tamoxifen treated breast cancer patients have been identified. For the first time, we compared these response profiles in an independent cohort of (neo)adjuvant systemic treatment naïve breast cancer patients treated with first-line tamoxifen for metastatic disease. Methods From a consecutive series of 246 estrogen receptor (ER) positive primary tumors, gene expression profiling was performed on available frozen tumors using 44K oligoarrays (n = 69). A 78-gene tamoxifen response profile (formerly consisting of 81 cDNA-clones), a 21-gene set (microarray-based Recurrence Score), as well as the HOXB13-IL17BR ratio (Two-Gene-Index, RT-PCR) were analyzed. Performance of signatures in relation to time to progression (TTP) was compared with standard immunohistochemical (IHC) markers: ER, progesterone receptor (PgR) and HER2. Results In univariate analyses, the 78-gene tamoxifen response profile, 21-gene set and HOXB13-IL17BR ratio were all significantly associated with TTP with hazard ratios of 2.2 (95% CI 1.3–3.7, P = 0.005), 2.3 (95% CI 1.3–4.0, P = 0.003) and 4.2 (95% CI 1.4–12.3, P = 0.009), respectively. The concordance among the three classifiers was relatively low, they classified only 45–61% of patients in the same category. In multivariate analyses, the association remained significant for the 78-gene profile and the 21-gene set after adjusting for ER and PgR. Conclusion The 78-gene tamoxifen response profile, the 21-gene set and the HOXB13-IL17BR ratio were all significantly associated with TTP in an independent patient series treated with tamoxifen. The addition of multigene assays to ER (IHC) improves the prediction of outcome in tamoxifen treated patients and deserves incorporation in future clinical studies.     

Dendritic cells are crucial for maintenance of tertiary lymphoid structures in the lung of influenza virus–infected mice

Tertiary lymphoid organs (TLOs) are organized aggregates of B and T cells formed in postembryonic life in response to chronic immune responses to infectious agents or self-antigens. Although CD11c⁺ dendritic cells (DCs) are consistently found in regions of TLO, their contribution to TLO organization has not been studied in detail. We found that CD11c^hi DCs are essential for the maintenance of inducible bronchus-associated lymphoid tissue (iBALT), a form of TLO induced in the lungs after influenza virus infection. Elimination of DCs after the virus had been cleared from the lung resulted in iBALT disintegration and reduction in germinal center (GC) reactions, which led to significantly reduced numbers of class-switched plasma cells in the lung and bone marrow and reduction in protective antiviral serum immunoglobulins. Mechanistically, DCs isolated from the lungs of mice with iBALT no longer presented viral antigens to T cells but were a source of lymphotoxin (LT) β and homeostatic chemokines (CXCL-12 and -13 and CCL-19 and -21) known to contribute to TLO organization. Like depletion of DCs, blockade of LTβ receptor signaling after virus clearance led to disintegration of iBALT and GC reactions. Together, our data reveal a previously unappreciated function of lung DCs in iBALT homeostasis and humoral immunity to influenza virus.The organized accumulation of lymphocytes in lymphoid organs serves to optimize both homeostatic immune surveillance and chronic responses to pathogenic stimuli (Cupedo and Mebius, 2005). During embryonic development, circulating hemopoietic cells gather at predestined sites throughout the body, where they are subsequently arranged in T and B cell–specific areas, which is characteristic of secondary lymphoid organs (SLOs). In contrast, the body seems to harbor a limited second set of selected sites that support neoformation of organized lymphoid aggregates in adult life. However, these are only revealed at times of local chronic inflammation when so-called tertiary lymphoid organs (TLOs) appear. As such, TLO was found in the pancreas of autoimmune diabetic mice (Kendall et al., 2007), around blood vessels in chronic allograft rejection (Nasr et al., 2007) and atherosclerosis (Gräbner et al., 2009), and in the brain in experimental allergic encephalitis (Magliozzi et al., 2004). In humans, TLO has been observed in the joint and lung of rheumatoid arthritis (Rangel-Moreno et al., 2006), around the airways of COPD patients (Hogg et al., 2004), and in the thyroid (Marinkovic et al., 2006). Certain infectious diseases are also accompanied by formation of TLO. Influenza virus infection of the respiratory tract leads to formation of inducible bronchus-associated lymphoid tissue (iBALT) that supports T and B cell proliferation and productive immunoglobulin class switching in germinal centers (GCs; Moyron-Quiroz et al., 2004, 2006).Although the embryonic development of SLO requires CD3⁻CD4⁺ lymphoid tissue–inducer cells, these are not a prerequisite for TLO induction (Marinkovic et al., 2006; Rangel-Moreno et al., 2007). Like SLOs, TLOs are formed in a highly regulated manner via production of homeostatic chemokines (CXCL13 and CCL19/CCL21), partially in response to signaling from the heterotrimer lymphotoxin (LT) α₁β₂ acting on the LTβ receptor on stromal lymphoid tissue organizer cells (Drayton et al., 2006). The instruction of stromal cells leads to formation of specialized high endothelial venules, and the organized production of chemokines leads to cellular organization of T cells and B cells in discrete areas. In all instances where TLOs have been described, antigen-presenting DCs have been found interspersed with T and B cell area, just as they are in SLO (Kratz et al., 1996; Cupedo et al., 2004; Moyron-Quiroz et al., 2004; Marinkovic et al., 2006; Tsuji et al., 2008). So far, the precise role of DCs in the functional organization of TLO has not been studied in great detail. Although DCs are mainly known for their function as antigen-presenting cells (Banchereau and Steinman, 1998), they are also a prominent source of homeostatic and inflammatory chemokines that can attract T and B cells and, thus, may contribute to TLO homeostasis (Beaty et al., 2007; GeurtsvanKessel and Lambrecht, 2008). In this paper, we have studied the precise contribution of DCs in the functional organization of iBALT, a specific form of TLO found in the lung after influenza virus infection (Moyron-Quiroz et al., 2004; Kocks et al., 2007).