Setting a diagnostic benchmark for tumor BRCA testing: detection of BRCA1 and BRCA2 large genomic rearrangements in FFPE tissue - A pilot study.

PARP inhibitors are used for treatment of tumors lacking function of the double-strand DNA break repair proteins BRCA1 or BRCA2 and are already approved for several cancer types. Thus, it is clinically crucial to determine germline as well as somatic BRCA1/2 mutations in those patients. The amplicon-based Oncomine BRCA1 and BRCA2 Assay is a test routinely used in diagnostics with FFPE specimens. The assay is validated for the detection of mutations, however, data on its performance in detecting large genomic rearrangements in FFPE tissue, is scarce. We cross-validated Oncomine BRCA1 and BRCA2 Assay in blood samples and/or FFPE tissue with multiplex ligation-dependent probe amplification (MLPA) for exon deletions and with OncoScan and an in-house hybridization-based target capture assay (MelArray) with a customized pipeline for the detection of loss of heterozygosity (LOH) and heterozygous versus complete gene loss. The Oncomine BRCA1 and BRCA2 Assay could detect both exon deletion and mono- and bi-allelic losses of the BRCA1/2 genes. We show that the therapeutically relevant large genomic rearrangements are reliably detected with the amplicon-based Oncomine BRCA1 and BRCA2 Assay in FFPE tumor tissue. Based on our data, we suggest tumor BRCA testing as standard diagnostic prescreening prior to germline BRCA testing.

Role of latent membrane protein 2 isoforms in Epstein-Barr virus latency.

The oncogenic Epstein-Barr virus (EBV) infects the majority of the human population without doing harm and establishes a latent infection in the memory B-cell compartment. To accomplish this, EBV hijacks B-cell differentiation pathways and uses its own viral genes to interfere with B-cell signalling to achieve life-long persistence. EBV latent membrane protein 2A (LMP2A) provides a surrogate B-cell receptor signal essential for cell survival and is believed to have a crucial role in the maintenance of latency by blocking B-cell activation which would otherwise lead to lytic EBV infection. These two functions demand tight control of LMP2A activity and expression levels. Based on recent insights in the function of LMP2B, an isoform of LMP2A, we propose a model for how LMP2B modulates the activity of LMP2A contributing to maintenance of EBV latency.

Latent membrane protein 2B regulates susceptibility to induction of lytic Epstein-Barr virus infection.

The B-lymphotropic Epstein-Barr virus (EBV) encodes two isoforms of latent membrane protein 2 (LMP2), LMP2A and LMP2B, which are expressed during latency in B cells. The function of LMP2B is largely unknown, whereas LMP2A blocks B-cell receptor (BCR) signaling transduction and induction of lytic EBV infection, thereby promoting B-cell survival. Transfection experiments on LMP2B in EBV-negative B cells and the silencing of LMP2B in EBV-harboring Burkitt's lymphoma-derived Akata cells suggest that LMP2B interferes with the function of LMP2A, but the role of LMP2B in the presence of functional EBV has not been established. Here, LMP2B, LMP2A, or both were overexpressed in EBV-harboring Akata cells to study the function of LMP2B. The overexpression of LMP2B increased the magnitude of EBV switching from its latent to its lytic form upon BCR cross-linking, as indicated by a more-enhanced upregulation and expression of EBV lytic genes and significantly increased production of transforming EBV compared to Akata vector control cells or LMP2A-overexpressing cells. Moreover, LMP2B lowered the degree of BCR cross-linking required to induce lytic EBV infection. Finally, LMP2B colocalized with LMP2A as demonstrated by immunoprecipitation and immunofluorescence and restored calcium mobilization upon BCR cross-linking, a signaling process inhibited by LMP2A. Thus, our findings suggest that LMP2B negatively regulates the function of LMP2A in preventing the switch from latent to lytic EBV replication.

Targeted next-generation-sequencing for reliable detection of targetable rearrangements in lung adenocarcinoma-a single center retrospective study.

Oncogenic rearrangements leading to targetable gene fusions are well-established cancer driver events in lung adenocarcinoma. Accurate and reliable detection of these gene fusions is crucial to select the appropriate targeted therapy for each patient. We compared the targeted next-generation-sequencing Oncomine Focus Assay (OFA; Thermo Fisher Scientific) with conventional ALK FISH and anti-Alk immunohistochemistry in a cohort of 52 lung adenocarcinomas (10 ALK rearranged, 18 non-ALK rearranged, and 24 untested cases). We found a sensitivity and specificity of 100% for detection of ALK rearrangements using the OFA panel. In addition, targeted next generation sequencing allowed us to analyze a set of 23 driver genes in a single assay. Besides EML4-ALK (11/52 cases), we detected EZR-ROS1 (1/52 cases), KIF5B-RET (1/52 cases) and MET-MET (4/52 cases) fusions. All EML4-ALK, EZR-ROS1 and KIF5B-RET fusions were confirmed by multiplexed targeted next generation sequencing assay (Oncomine Solid Tumor Fusion Transcript Kit, Thermo Fisher Scientific). All cases with EML4-ALK rearrangement were confirmed by Alk immunohistochemistry and all but one by ALK FISH. In our experience, targeted next-generation sequencing is a reliable and timesaving tool for multiplexed detection of targetable rearrangements. Therefore, targeted next-generation sequencing represents an efficient alternative to time-consuming single target assays currently used in molecular pathology.

Zebularine reactivates silenced E-cadherin but unlike 5-Azacytidine does not induce switching from latent to lytic Epstein-Barr virus infection in Burkitt's lymphoma Akata cells.

Epigenetic silencing of regulatory genes by aberrant methylation contributes to tumorigenesis. DNA methyltransferase inhibitors (DNMTI) represent promising new drugs for anti-cancer therapies. The DNMTI 5-Azacytidine is effective against myelodysplastic syndrome, but induces switching of latent to lytic Epstein-Barr virus (EBV) in vitro and results in EBV DNA demethylation with the potential of induction of lytic EBV in vivo. This is of considerable concern given that recurrent lytic EBV has been linked with an increased incidence of EBV-associated lymphomas. Based on the distinct properties of action we hypothesized that the newer DNMTI Zebularine might differ from 5-Azacytidine in its potential to induce switching from latent to lytic EBV. Here we show that both 5-Azacytidine and Zebularine are able to induce expression of E-cadherin, a cellular gene frequently silenced by hypermethylation in cancers, and thus demonstrate that both DNMTI are active in our experimental setting consisting of EBV-harboring Burkitt's lymphoma Akata cells. Quantification of mRNA expression of EBV genes revealed that 5-Azacytidine induces switching from latent to lytic EBV and, in addition, that the immediate-early lytic infection progresses to early and late lytic infection. Furthermore, 5-Azacytidine induced upregulation of the latent EBV genes LMP2A, LMP2B, and EBNA2 in a similar fashion as observed following switching of latent to lytic EBV upon cross-linking of the B-cell receptor. In striking contrast, Zebularine did not exhibit any effect neither on lytic nor on latent EBV gene expression. Thus, Zebularine might be safer than 5-Azacytidine for the treatment of cancers in EBV carriers and could also be applied against EBV-harboring tumors, since it does not induce switching from latent to lytic EBV which may result in secondary EBV-associated malignancies.

Cytology smears as excellent starting material for next-generation sequencing-based molecular testing of patients with adenocarcinoma of the lung.

BACKGROUND: Molecular testing of lung adenocarcinomas (ADCs) is crucial for therapy stratification of patients. Because of the often limited diagnostic material, the authors aimed to explore the suitability of cytology smears for next-generation sequencing (NGS) and compared the results with concurrent histological specimens or cell blocks. METHODS: A total of 16 formalin-fixed paraffin-embedded (FFPE) ADCs with known genetic alterations were used as the first cohort for targeted DNA and RNA sequencing. In the second cohort of 8 cases, 8 cytological smears were compared with matching histological specimens or cell blocks for the study. For NGS library amplification, commercially available panels for DNA and RNA sequencing were applied. The Ion Torrent Personal Genome Machine and the Ion Reporter workflow (version 5.0) were used for sequencing. RESULTS: All DNA libraries derived from FFPE and non-formalin-fixed cytological smear samples produced acceptable quality metrics, thereby enabling successful targeted DNA sequencing (100% performance). Targeted RNA sequencing failed in 1 FFPE case and 1 cytology probe by not reaching enough mapped fusion reads (92% performance rate). All previously detected mutations and gene rearrangements could be confirmed (sensitivity of 100%), whereas specificity of the DNA-based NGS assay reached 96%. CONCLUSIONS: The results of the current study demonstrated the suitability of non-formalin cytology specimens for the simultaneous NGS testing of lung ADCs using amplicon resequencing panels. These assays allowed for the input of cytological smears equal to concurrent histology or cell blocks and proved to be accurate in the detection of therapeutically actionable somatic mutations and gene rearrangements. Cancer Cytopathol 2017;125:30-40. © 2016 American Cancer Society.

Value of immunohistochemistry in the detection of BRAF(V600E) mutations in fine-needle aspiration biopsies of papillary thyroid carcinoma.

BACKGROUND: Fine-needle aspiration biopsy (FNAB) is important in the diagnostic establishment of suspicious thyroid nodules. In thyroid neoplasms, mutation of the BRAF gene occurs rather exclusively in papillary thyroid carcinoma (PTC) and results in>98% of the cases in V600E amino acid substitution. In the current study, the authors investigated the diagnostic value of a recently described monoclonal antibody that detects this specific mutation on FNAB specimens from patients with PTC. METHODS: BRAF(V600E) status of FNAB cell blocks from 55 patients with PTC was analyzed by immunohistochemistry (IHC) with the new BRAF(V600E) antibody (clone VE1) and by Sanger sequencing (SaS). In discrepant cases, ultra-deep sequencing was also performed. Available corresponding histological specimens were investigated by IHC and, in selected cases, with SaS as well. RESULTS: All cases yielded evaluable IHC staining results of the cell block sections with good interobserver agreement (kappa value, 0.650). Ten tumors (18.2%) demonstrated no staining, 10 tumors (18.2%) demonstrated equivocal staining, 25 tumors (45.4%) demonstrated moderate staining, and 10 tumors (18.2%) demonstrated strong staining. SaS was able to be performed in 48 cases. Nineteen cases demonstrated wild-type BRAF and 29 cases were found to have the BRAF(V600E) mutation. After performing ultra-deep sequencing 1 false-positive and 2 false-negative VE1 IHC cases remained, resulting in a sensitivity of 93.8% and a specificity of 93.8%. CONCLUSIONS: BRAF(V600E) mutations in FNAB specimens from patients with PTC can be reliably detected in most cases by IHC with a new mutation-specific antibody. Interpretation of VE1 IHC staining results on cell block slides of PTC can be difficult in some cases.

Automated assessment of ß-cell area and density per islet and patient using TMEM27 and BACE2 immunofluorescence staining in human pancreatic ß-cells.

In this study we aimed to establish an unbiased automatic quantification pipeline to assess islet specific features such as ß-cell area and density per islet based on immunofluorescence stainings. To determine these parameters, the in vivo protein expression levels of TMEM27 and BACE2 in pancreatic islets of 32 patients with type 2 diabetes (T2D) and in 28 non-diabetic individuals (ND) were used as input for the automated pipeline. The output of the automated pipeline was first compared to a previously developed manual area scoring system which takes into account the intensity of the staining as well as the percentage of cells which are stained within an islet. The median TMEM27 and BACE2 area scores of all islets investigated per patient correlated significantly with the manual scoring and with the median area score of insulin. Furthermore, the median area scores of TMEM27, BACE2 and insulin calculated from all T2D were significantly lower compared to the one of all ND. TMEM27, BACE2, and insulin area scores correlated as well in each individual tissue specimen. Moreover, islet size determined by costaining of glucagon and either TMEM27 or BACE2 and ß-cell density based either on TMEM27 or BACE2 positive cells correlated significantly. Finally, the TMEM27 area score showed a positive correlation with BMI in ND and an inverse pattern in T2D. In summary, automated quantification outperforms manual scoring by reducing time and individual bias. The simultaneous changes of TMEM27, BACE2, and insulin in the majority of the ß-cells suggest that these proteins reflect the total number of functional insulin producing ß-cells. Additionally, ß-cell subpopulations may be identified which are positive for TMEM27, BACE2 or insulin only. Thus, the cumulative assessment of all three markers may provide further information about the real ß-cell number per islet.

Ultra-deep sequencing confirms immunohistochemistry as a highly sensitive and specific method for detecting BRAF V600E mutations in colorectal carcinoma.

The activating BRAF (V600) mutation is a well-established negative prognostic biomarker in metastatic colorectal carcinoma (CRC). A recently developed monoclonal mouse antibody (clone VE1) has been shown to detect reliably BRAF (V600E) mutated protein by immunohistochemistry (IHC). In this study, we aimed to compare the detection of BRAF (V600E) mutations by IHC, Sanger sequencing (SaS), and ultra-deep sequencing (UDS) in CRC. VE1-IHC was established in a cohort of 68 KRAS wild-type CRCs. The VE1-IHC was only positive in the three patients with a known BRAF (V600E) mutation as assessed by SaS and UDS. The test cohort consisted of 265 non-selected, consecutive CRC samples. Thirty-nine out of 265 cases (14.7%) were positive by VE1-IHC. SaS of 20 randomly selected IHC negative tumors showed BRAF wild-type (20/20). Twenty-four IHC-positive cases were confirmed by SaS (24/39; 61.5%) and 15 IHC-positive cases (15/39; 38.5%) showed a BRAF wild-type by SaS. UDS detected a BRAF (V600E) mutation in 13 of these 15 discordant cases. In one tumor, the mutation frequency was below our threshold for UDS positivity, while in another case, UDS could not be performed due to low DNA amount. Statistical analysis showed sensitivities of 100% and 63% and specificities of 95 and 100% for VE1-IHC and SaS, respectively, compared to combined results of SaS and UDS. Our data suggests that there is high concordance between UDS and IHC using the anti-BRAF(V600E) (VE1) antibody. Thus, VE1 immunohistochemistry is a highly sensitive and specific method in detecting BRAF (V600E) mutations in colorectal carcinoma.

VHL gene mutations and their effects on hypoxia inducible factor HIFα: identification of potential driver and passenger mutations.

Mutations of the von Hippel-Lindau (VHL) gene are frequent in clear cell renal cell carcinomas (ccRCC). Nonsense and frameshift mutations abrogate the function of the VHL protein (pVHL), whereas missense mutations can have different effects. To identify those missense mutations with functional consequences, we sequenced VHL in 256 sporadic ccRCC and identified 187 different VHL mutations of which 65 were missense mutations. Location and destabilizing effects of VHL missense mutations were determined in silico. The majority of the thermodynamically destabilizing missense mutations were located in exon 1 in the core of pVHL, whereas protein surface mutations in exon 3 affected the interaction domains of elongin B and C. Their impact on pVHL's functionality was further investigated in vitro by stably reintroducing VHL missense mutations into a VHL null cell line and by monitoring the green fluorescent protein (GFP) signals after the transfection of a hypoxia inducible factor (HIF)α-GFP expression vector. pVHL's functionality ranged from no effect to complete HIF stabilization. Interestingly, Asn78Ser, Asp121Tyr, and Val130Phe selectively influenced HIF1α and HIF2α degradation. In summary, we obtained three different groups of missense mutations: one with severe destabilization of pVHL; a second without destabilizing effects on pVHL but relevance for the interaction with HIFα, elongin B, and elongin C; and a third with pVHL functions comparable with wild type. We therefore conclude that the specific impact of missense mutations may help to distinguish between driver and passenger mutations and may explain responses of ccRCC patients to HIF-targeted therapies.

Bace2 is a ß cell-enriched protease that regulates pancreatic ß cell function and mass.

Decreased ß cell mass and function are hallmarks of type 2 diabetes. Here we identified, through a siRNA screen, beta site amyloid precursor protein cleaving enzyme 2 (Bace2) as the sheddase of the proproliferative plasma membrane protein Tmem27 in murine and human ß cells. Mice with functionally inactive Bace2 and insulin-resistant mice treated with a newly identified Bace2 inhibitor both display augmented ß cell mass and improved control of glucose homeostasis due to increased insulin levels. These results implicate Bace2 in the control of ß cell maintenance and provide a rational strategy to inhibit this protease for the expansion of functional pancreatic ß cell mass.

Graph-based pancreatic islet segmentation for early type 2 diabetes mellitus on histopathological tissue.

It is estimated that in 2010 more than 220 million people will be affected by type 2 diabetes mellitus (T2DM). Early evidence indicates that specific markers for alpha and beta cells in pancreatic islets of Langerhans can be used for early T2DM diagnosis. Currently, the analysis of such histological tissues is manually performed by trained pathologists using a light microscope. To objectify classification results and to reduce the processing time of histological tissues, an automated computational pathology framework for segmentation of pancreatic islets from histopathological fluorescence images is proposed. Due to high variability in the staining intensities for alpha and beta cells, classical medical imaging approaches fail in this scenario. The main contribution of this paper consists of a novel graph-based segmentation approach based on cell nuclei detection with randomized tree ensembles. The algorithm is trained via a cross validation scheme on a ground truth set of islet images manually segmented by 4 expert pathologists. Test errors obtained from the cross validation procedure demonstrate that the graph-based computational pathology analysis proposed is performing competitively to the expert pathologists while outperforming a baseline morphological approach.

Silencing of latent membrane protein 2B reduces susceptibility to activation of lytic Epstein-Barr virus in Burkitt's lymphoma Akata cells.

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) blocks B-cell receptor (BCR) signalling after BCR cross-linking to inhibit activation of lytic EBV, and ectopically expressed LMP2B negatively regulates LMP2A. Here, it is demonstrated that silencing of LMP2B in EBV-harbouring Burkitt's lymphoma Akata cells results in reduced expression of EBV immediate-early lytic BZLF1 gene mRNA and late lytic gp350/220 protein upon BCR cross-linking. Similarly, reduction of lytic EBV activation was observed in Akata cells overexpressing LMP2A. In contrast, silencing of LMP2A expression resulted in higher lytic EBV mRNA and protein expression in BCR cross-linked Akata cells. These observations indicate a role for LMP2B distinct from that of LMP2A in regulation of lytic EBV activation in the host cell and support the hypothesis that LMP2B exhibits a negative-regulatory effect on the ability of LMP2A to maintain EBV latency by preventing the switch to lytic replication.

Ligand binding properties of bacterial hemoglobins and flavohemoglobins.

Bacterial hemoglobins and flavohemoglobins share a common globin fold but differ otherwise in structural and functional aspects. The bases of these differences were investigated through kinetic studies on oxygen, carbon monoxide, and nitric oxide binding. The novel bacterial hemoglobins from Clostridium perfringens and Campylobacter jejuni and the flavohemoglobins from Bacillus subtilis and Salmonella enterica serovar Typhi have been analyzed. Examination of the biochemical and ligand binding properties of these proteins shows a clear distinction between the two groups. Flavohemoglobins show a much greater tendency to autoxidation compared to bacterial hemoglobins. The differences in affinity for oxygen, carbon monoxide, and nitric oxide between bacterial hemoglobins and flavohemoglobins are mainly due to differences in the association rate constants. The second-order rate constants for oxygen and carbon monoxide binding to bacterial hemoglobins are severalfold higher than those for flavohemoglobins. A similar trend is observed for NO association with the oxidized iron(III) form of the proteins. No major differences are observed among the values obtained for the dissociation rate constants for the two groups of bacterial proteins studied, and these constants are all rather similar to those for myoglobin. Taken together, our data suggest that differences exist between the mechanisms of ligand binding to bacterial hemoglobins and flavohemoglobins, suggesting different functions in the cell.