Citations

Abstract Adenosine signaling has been implicated in chronic lung diseases such as asthma and chronic obstructive pulmonary disease; however, the specific roles of the various adenosine receptors in processes central to these disorders are not well understood. In this study, we have investigated the role(s) of the A3 adenosine receptor in adenosine-dependent pulmonary inflammation observed in adenosine deaminase (ADA)-deficient mice. The A3 receptor (A3R) was found to be expressed in eosinophils and mucus-producing cells in the airways of ADA-deficient mice. Treatment of ADA-deficient mice with MRS 1523, a selective A3R antagonist, prevented airway eosinophilia and mucus production. Similar findings were seen in the lungs of ADA/A3 double knockout mice. Although eosinophils were decreased in the airways of ADA-deficient mice following antagonism or removal of the A3R, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3R is needed for the migration of eosinophils into the airways. These findings identify an important role for the A3R in regulating lung eosinophilia and mucus production in an environment of elevated adenosine.

Abstract Activation-induced cytidine deaminase (AID) initiates Ab class-switch recombination (CSR) in activated B cells resulting in exchanging the IgH C region and improved Ab effector function. During CSR, AID instigates DNA double-strand break (DSB) formation in switch (S) regions located upstream of C region genes. DSBs are necessary for CSR, but improper regulation of DSBs can lead to chromosomal translocations that can result in B cell lymphoma. The protein kinase ataxia telangiectasia mutated (ATM) is an important proximal regulator of the DNA damage response (DDR), and translocations involving S regions are increased in its absence. ATM phosphorylates H2AX, which recruits other DNA damage response (DDR) proteins, including mediator of DNA damage checkpoint 1 (Mdc1) and p53 binding protein 1 (53BP1), to sites of DNA damage. As these DDR proteins all function to promote repair and recombination of DSBs during CSR, we examined whether mouse splenic B cells deficient in these proteins would show alterations in S region DSBs when undergoing CSR. We find that in atm−/− cells Sμ DSBs are increased, whereas DSBs in downstream Sγ regions are decreased. We also find that mutations in the unrearranged Sγ3 segment are reduced in atm−/− cells. Our data suggest that ATM increases AID targeting and activity at downstream acceptor S regions during CSR and that in atm−/− cells Sμ DSBs accumulate as they lack a recombination partner.

Background The GGGGCC-repeat expansion in C9orf72 is the most frequent mutation found in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Most of the studies on C9orf72 have relied on repeat-primed PCR (RP-PCR) methods for detection of the expansions. To investigate the inherent limitations of this technique, we compared methods and results of 14 laboratories. Methods The 14 laboratories genotyped DNA from 78 individuals (diagnosed with ALS or FTD) in a blinded fashion. Eleven laboratories used a combination of amplicon-length analysis and RP-PCR, whereas three laboratories used RP-PCR alone; Southern blotting techniques were used as a reference. Results Using PCR-based techniques, 5 of the 14 laboratories got results in full accordance with the Southern blotting results. Only 50 of the 78 DNA samples got the same genotype result in all 14 laboratories. There was a high degree of false positive and false negative results, and at least one sample could not be genotyped at all in 9 of the 14 laboratories. The mean sensitivity of a combination of amplicon-length analysis and RP-PCR was 95.0% (73.9–100%), and the mean specificity was 98.0% (87.5–100%). Overall, a sensitivity and specificity of more than 95% was observed in only seven laboratories. Conclusions Because of the wide range seen in genotyping results, we recommend using a combination of amplicon-length analysis and RP-PCR as a minimum in a research setting. We propose that Southern blotting techniques should be the gold standard, and be made obligatory in a clinical diagnostic setting.

SummaryPrimary breast diffuse large B‐cell lymphoma (DLBCL) is a rare subtype of non‐Hodgkin lymphoma (NHL) with limited data on pathology and outcome. A multicentre retrospective study was undertaken to determine prognostic factors and the incidence of central nervous system (CNS) relapses. Data was retrospectively collected on patients from 8 US academic centres. Only patients with stage I/II disease (involvement of breast and localized lymph nodes) were included. Histologies apart from primary DLBCL were excluded. Between 1992 and 2012, 76 patients met the eligibility criteria. Most patients (86%) received chemotherapy, and 69% received immunochemotherapy with rituximab; 65% received radiation therapy and 9% received prophylactic CNS chemotherapy. After a median follow‐up of 4·5 years (range 0·6–20·6 years), the Kaplan–Meier estimated median progression‐free survival was 10·4 years (95% confidence interval [CI] 5·8–14·9 years), and the median overall survival was 14·6 years (95% CI 10·2–19 years). Twelve patients (16%) had CNS relapse. A low stage‐modified International Prognostic Index (IPI) was associated with longer overall survival. Rituximab use was not associated with a survival advantage. Primary breast DLBCL has a high rate of CNS relapse. The stage‐modified IPI score is associated with survival.

Aims To explore the immunohistochemical utility of proliferating cell nuclear antigen (PCNA), insulin-like growth factor 2 (IGF2) and clusterin in the distinction between malignant and benign liver nodular lesions. Methods Immunohistochemical stains for PCNA, IGF2 and clusterin were performed on 284 liver nodular lesions, including 33 hepatocellular adenomas (HCA), 40 focal nodular hyperplasias (FNH), 77 large regenerative nodules (LRN) and 134 hepatocellular carcinomas (HCC). Results Strong and diffuse nuclear PCNA immunoreactivity was observed in 103 (77%) HCCs but in only 2 (6%) HCAs. None of the FNH and LRN cases showed a strong and diffuse staining pattern. All HCAs, 95% of FNHs and 92% of LRNs showed cytoplasmic IGF2 expression, with a strong staining observed in 70% of HCAs, 20% of FNHs and 30% of LRNs. This was in marked contrast to that observed in HCCs, where 66% of HCCs demonstrated a weak and focal/patchy immunostaining pattern and another 25% showed no detectable IGF2 immunoreactivity. In comparison with their adjacent non-lesional hepatocytes, 75% of HCCs showed decreased IGF2 expression. However, decreased IGF2 expression was not evident in HCAs, FNHs and LRNs. Cytoplasmic staining for clusterin was seen in both benign and malignant nodular lesions. However, an enhanced and exaggerated pericanalicular staining pattern was observed in 75% of HCCs, which was not demonstrated in HCAs, FNHs and LRNs. Conclusions PCNA, IGF2 and clusterin show unique immunostaining characteristics in HCCs, which can be useful adjuncts to other currently available markers to aid in the distinction of HCC from benign liver nodular lesions.

Abstract BACKGROUND Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn relies on appropriate assay design, interpretation, and reporting. METHODS We conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501+3A>T). Differences in protocols were then assessed to determine which elements were critical in reliable assay design. RESULTS PCR primer design strategies, PCR conditions, and product detection methods, combined with a prior knowledge of expected alternative transcripts, were the key factors for accurate splicing assay results. For example, because of the position of primers and PCR extension times, several isoforms associated with BRCA1, c.594-2A>C and c.671-2A>G, were not detected by many sites. Variation was most evident for the detection of low-abundance transcripts (e.g., BRCA2 c.8632+1G>A Δ19,20 and BRCA1 c.135-1G>T Δ5q and Δ3). Detection of low-abundance transcripts was sometimes addressed by using more analytically sensitive detection methods (e.g., BRCA2 c.426-12_8delGTTTT ins18bp). CONCLUSIONS We provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.