Comprehensive Validation of Diagnostic Next-Generation Sequencing Panels for Acute Myeloid Leukemia Patients.

Next-generation sequencing has greatly advanced the molecular diagnostics of malignant hematological diseases and provides useful information for clinical decision making. Studies have shown that certain mutations are associated with prognosis and have a direct impact on treatment of affected patients. Therefore, reliable detection of pathogenic variants is critically important. Here, we compared four sequencing panels with different characteristics, from number of genes covered to technical aspects of library preparation and data analysis workflows, to find the panel with the best clinical utility for myeloid neoplasms with a special focus on acute myeloid leukemia. Using the Acrometrix Oncology Hotspot Control DNA and DNA from acute myeloid leukemia patients, panel performance was evaluated in terms of coverage, precision, recall, and reproducibility and different bioinformatics tools that can be used for the evaluation of any next-generation sequencing panel were tested. Taken together, our results support the reliability of the Acrometrix Oncology Hotspot Control to validate and compare sequencing panels for hematological diseases and show which panel-software combination (platform) has the best performance.

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.

Novel morphological and genetic features of fumarate hydratase deficient renal cell carcinoma in HLRCC syndrome patients with a tailored therapeutic approach.

The hereditary leiomyomatosis and renal cell carcinoma syndrome (HLRCC) is defined by germline mutations in the fumarate hydratase (FH) gene and associated with leiomyomas and aggressive renal cell carcinomas with FH deficiency. Here, we comprehensively characterize two new patients with HLRCC syndrome on a morphological, immunohistochemical and genetic level. The patients developed aggressive HLRCC syndrome-associated RCCs, uterine leiomyomas and dermal leiomyomas. One HLRCC syndrome-associated RCC exhibited an unusual morphology with accumulation of "colloid-like" cytoplasmic inclusions, which might serve as a novel sentinel feature to trigger further testing. This case showed partially retained FH expression, initially hampering correct diagnosis. Comprehensive next-generation sequencing analyses of HLRCC syndrome-associated RCC and leiomyomas in our patients revealed divergent genetic changes in the FH gene in different tumors from the same patient. While all leiomyomas (uterine and cutaneous) showed a FH loss of heterozygosity (LOH) as a wildtype allele inactivating event, one HLRCC-RCC showed a second, undescribed NM_000143.3; c.947C>T; p.Ala316Val FH mutation accompanying the preexisting splice site mutation c.378+2T>C. In the other HLRCC syndrome-associated RCC, the FH mutation (NM_000143.3; c.462T>G; p.Asn154Lys with a somatic LOH) represents another variant of unknown significance that we link to HLRCC - and thus classify as likely pathogenic. Due to the specific diagnosis of metastatic HLRCC syndrome-associated RCC, both cases were treated in first line with bevacizumab/erlotinib and showed remarkable and long lasting responses. These findings allow new morphological and molecular insights into the biology of the HLRCC syndrome, corroborate the "second hit" hypothesis of tumor formation in HLRCC patients and may promote a distinct therapeutic approach.

Combined genetic and epigenetic alterations of the TERT promoter affect clinical and biological behavior of bladder cancer.

In urothelial bladder cancer (UBC), risk stratification remains an important unmet need. Limitless self-renewal, governed by TERT expression and telomerase activation, is crucial for cancer progression. Thus, telomerase activation through the interplay of mutations (TERTpMut ) and epigenetic alterations in the TERT promoter may provide further insight into UBC behavior. Here, we investigated the combined effect of TERTpMut and the TERT Hypermethylated Oncological Region (THOR) status on telomerase activation and patient outcome in a UBC international cohort (n = 237). We verified that TERTpMut were frequent (76.8%) and present in all stages and grades of UBC. Hypermethylation of THOR was associated with higher TERT expression and higher-risk disease in nonmuscle invasive bladder cancers (NMIBC). TERTpMut alone predicted disease recurrence (HR: 3.18, 95%CI 1.84 to 5.51, p < 0.0001) but not progression in NMIBC. Combined THORhigh /TERTpMut increased the risk of disease recurrence (HR 5.12, p < 0.0001) and progression (HR 3.92, p = 0.025). Increased THOR hypermethylation doubled the risk of stage progression of both TERTpwt and TERTpMut NMIBC. These results highlight that both mechanisms are common and coexist in bladder cancer and while TERTpMut is an early event in bladder carcinogenesis THOR hypermethylation is a dynamic process that contributes to disease progression. While the absence of alterations comprises an extremely indolent phenotype, the combined genetic and epigenetic alterations of TERT bring additional prognostic value in NMIBC and provide a novel insight into telomere biology in cancer.

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.

Tracking the origin of simultaneous endometrial and ovarian cancer by next-generation sequencing - a case report.

BACKGROUND: Endometrioid adenocarcinoma of the uterus and ovarian endometrioid carcinoma share many morphological and molecular features. Differentiation between simultaneous primary carcinomas and ovarian metastases of an endometrial cancer may be very challenging but is essential for prognostic and therapeutic considerations. CASE PRESENTATION: In the present case study of a 33 year-old patient we used targeted amplicon next-generation re-sequencing for clarifying the origin of synchronous endometrioid cancer of the corpus uteri and the left ovary. The patient developed a metachronous lung metastasis of an endometrioid adenocarcinoma four years after hyster- and adnexectomy, vaginal brachytherapy and treatment with the synthetic steroid tibolone. Removal of the metastasis and megestrol treatment for seven years led to a complete remission. A total of 409 genes from the Ampliseq Comprehensive Cancer Panel (Ion Torrent, Thermo Fisher) were analysed by next generation sequencing and mutations in 10 genes, including ARID1A, CTNNB1, PIK3CA and PTEN were identified and confirmed by Sanger sequencing. Primary endometrial as well as ovarian cancer showed an identical mutational profile, suggesting the presence of an ovarian metastasis of the endometrial cancer, rather than a simultaneous endometrial and ovarian cancer. The metachronous lung metastasis showed a different mutational profile compared to the primary cancer. Immunohistochemical staining of the corresponding proteins suggested that the tumour development was driven by alterations in the protein function rather than by changes of the protein abundance in the cell. CONCLUSIONS: Our results have demonstrated next generation sequencing as a valuable tool in the differentiation of synchronous primary tumours and metastases, which has an important impact on the clinical decision making process. Similar to breast cancer, targeted therapies based on mutational tumour profiling will become increasingly important in endometrial and ovarian cancer. In summary, our results support the usage of next generation sequencing as a supplementary diagnostic tool, assisting in personalized precision medicine.

Detection of CCNE1/URI (19q12) amplification by in situ hybridisation is common in high grade and type II endometrial cancer.

One TCGA subgroup of endometrial cancer (EC) is characterised by extensive genomic DNA copy number alterations. CCNE1 located at 19q12 is frequently amplified in EC and a target for anti-cancer therapy. The relevance of URI, also located at 19q12, is unknown. To evaluate the prevalence of 19q12 (CCNE1/URI) in EC, we investigated different histologic types by in situ hybridisation (ISH) and copy number assay. We applied a previously established 19q12 ISH for the detection of CCNE1/URI copy numbers in EC (n = 270) using conventional bright field microscopy. In a subset (n = 21), 19q12 amplification status was validated by OncoScan assay. Manual ISH was controlled by a recently developed computational ISHProfiler algorithm. Associations of 19q12 status with Cyclin E1, URI and p53 expression, and clinico-pathological parameters were tested.Amplification of 19q12 (CCNE1/URI) was found in 10.4% (28/270) and was significantly associated with type II EC (high grade and non-endometrioid; p < 0.0001), advanced FIGO stage (p = 0.001), high Cyclin E1 expression (p = 0.008) and aberrant p53 expression (p = 0.04). 19q12 ISH data were confirmed by OncoScan and computational ISHProfiler techniques. The 19q12 in situ hybridisation is a feasible and robust biomarker assay in molecular pathology. Amplification of CCNE1/URI predominantly occurred in type II endometrial cancer. Prospective clinical trials are warranted to assess the utility of combined 19q12 amplification and Cyclin E1/URI protein expression analysis for the prediction of therapeutic response to chemotherapy and/or cyclin-dependent kinase inhibitors in patients with endometrial cancer.

Prognostic significance of POLE proofreading mutations in endometrial cancer.

BACKGROUND: Current risk stratification in endometrial cancer (EC) results in frequent over- and underuse of adjuvant therapy, and may be improved by novel biomarkers. We examined whether POLE proofreading mutations, recently reported in about 7% of ECs, predict prognosis. METHODS: We performed targeted POLE sequencing in ECs from the PORTEC-1 and -2 trials (n = 788), and analyzed clinical outcome according to POLE status. We combined these results with those from three additional series (n = 628) by meta-analysis to generate multivariable-adjusted, pooled hazard ratios (HRs) for recurrence-free survival (RFS) and cancer-specific survival (CSS) of POLE-mutant ECs. All statistical tests were two-sided. RESULTS: POLE mutations were detected in 48 of 788 (6.1%) ECs from PORTEC-1 and-2 and were associated with high tumor grade (P < .001). Women with POLE-mutant ECs had fewer recurrences (6.2% vs 14.1%) and EC deaths (2.3% vs 9.7%), though, in the total PORTEC cohort, differences in RFS and CSS were not statistically significant (multivariable-adjusted HR = 0.43, 95% CI = 0.13 to 1.37, P = .15; HR = 0.19, 95% CI = 0.03 to 1.44, P = .11 respectively). However, of 109 grade 3 tumors, 0 of 15 POLE-mutant ECs recurred, compared with 29 of 94 (30.9%) POLE wild-type cancers; reflected in statistically significantly greater RFS (multivariable-adjusted HR = 0.11, 95% CI = 0.001 to 0.84, P = .03). In the additional series, there were no EC-related events in any of 33 POLE-mutant ECs, resulting in a multivariable-adjusted, pooled HR of 0.33 for RFS (95% CI = 0.12 to 0.91, P = .03) and 0.26 for CSS (95% CI = 0.06 to 1.08, P = .06). CONCLUSION: POLE proofreading mutations predict favorable EC prognosis, independently of other clinicopathological variables, with the greatest effect seen in high-grade tumors. This novel biomarker may help to reduce overtreatment in EC.

KPNA2 is overexpressed in human and mouse endometrial cancers and promotes cellular proliferation.

Endometrial cancer is the most frequently occurring malignancy of the female genital tract in Western countries. Although in many cases surgically curable, about 30% of the tumours represent an aggressive and untreatable disease. In an attempt to establish a reliable prognostic marker for endometrial carcinomas disregarding their histological diversity, we investigated the expression of KPNA2, a mediator of nucleocytoplasmic transport, and other cell proliferation-associated proteins and their correlation with cancer progression. We analysed patient tissue microarrays (TMAs) assembled from 527 endometrial cancer tissue specimens and uterus samples from a Trp53 knockout mouse model of endometrial cancer. Our data show that KPNA2 expression was significantly up-regulated in human endometrial carcinomas and associated with higher tumour grade (p = 0.026), higher FIGO stage (p = 0.027), p53 overexpression (p < 0.001), activation of the PI3K/AKT pathway, and epithelial-mesenchymal transition. Increased nuclear KPNA2 immunoreactivity was identified as a novel predictor of overall survival, independent of well-established prognostic factors in Cox regression analyses (hazard ratio 1.7, 95% CI 1.13-2.56, p = 0.01). No significant association between KPNA2 expression and endometrial cancer subtype was detected. In the mouse model, KPNA2 showed increased expression levels from precancerous (EmgD, EIC) to far-advanced invasive lesions. We further investigated the cell proliferation capacity after siRNA-mediated KPNA2 knockdown in the human endometrial cancer cell line MFE-296. KPNA2 silencing led to decreased proliferation of the cancer cells, suggesting interplay of the protein with the cell cycle. Taken together, increased expression of KPNA2 is an independent prognostic marker for poor survival. The mechanism of enhanced nucleocytoplasmic transport by KPNA2 overexpression seems a common event in aggressive cancers since we have shown a significant correlation of KPNA2 expression and tumour aggressiveness in a large variety of other solid tumour entities. Introducing KPNA2 immunohistochemistry in routine diagnostics may allow for the identification of patients who need more aggressive treatment regimens.

The orphan adhesion G protein-coupled receptor GPR97 regulates migration of lymphatic endothelial cells via the small GTPases RhoA and Cdc42.

The important role of the lymphatic vascular system in pathological conditions such as inflammation and cancer has been increasingly recognized, but its potential as a pharmacological target is poorly exploited. Our study aimed at the identification and molecular characterization of lymphatic-specific G protein-coupled receptors (GPCRs) to assess new targets for pharmacological manipulation of the lymphatic vascular system. We used a TaqMan quantitative RT-PCR-based low density array to determine the GPCR expression profiles of ex vivo isolated intestinal mouse lymphatic (LECs) and blood vascular endothelial cells (BECs). GPR97, an orphan adhesion GPCR of unknown function, was the most highly and specifically expressed GPCR in mouse lymphatic endothelium. Using siRNA silencing, we found that GPR97-deficient primary human LECs displayed increased adhesion and collective cell migration, whereas single cell migration was decreased as compared with nontargeting siRNA-transfected control LECs. Loss of GPR97 shifted the ratio of active Cdc42 and RhoA and initiated cytoskeletal rearrangements, including F-actin redistribution, paxillin and PAK4 phosphorylation, and ß1-integrin activation. Our data suggest a possible role of GPR97 in lymphatic remodeling and furthermore provide the first insights into the biological functions of GPR97.

ROS-induced ATF3 causes susceptibility to secondary infections during sepsis-associated immunosuppression.

Sepsis, sepsis-induced hyperinflammation and subsequent sepsis-associated immunosuppression (SAIS) are important causes of death. Here we show in humans that the loss of the major reactive oxygen species (ROS) scavenger, glutathione (GSH), during SAIS directly correlates with an increase in the expression of activating transcription factor 3 (ATF3). In endotoxin-stimulated monocytes, ROS stress strongly superinduced NF-E2-related factor 2 (NRF2)-dependent ATF3. In vivo, this ROS-mediated superinduction of ATF3 protected against endotoxic shock by inhibiting innate cytokines, as Atf3(-/-) mice remained susceptible to endotoxic shock even under conditions of ROS stress. Although it protected against endotoxic shock, this ROS-mediated superinduction of ATF3 caused high susceptibility to bacterial and fungal infections through the suppression of interleukin 6 (IL-6). As a result, Atf3(-/-) mice were protected against bacterial and fungal infections, even under conditions of ROS stress, whereas Atf3(-/-)Il6(-/-) mice were highly susceptible to these infections. Moreover, in a model of SAIS, secondary infections caused considerably less mortality in Atf3(-/-) mice than in wild-type mice, indicating that ROS-induced ATF3 crucially determines susceptibility to secondary infections during SAIS.

Fumarates improve psoriasis and multiple sclerosis by inducing type II dendritic cells.

Fumarates improve multiple sclerosis (MS) and psoriasis, two diseases in which both IL-12 and IL-23 promote pathogenic T helper (Th) cell differentiation. However, both diseases show opposing responses to most established therapies. First, we show in humans that fumarate treatment induces IL-4-producing Th2 cells in vivo and generates type II dendritic cells (DCs) that produce IL-10 instead of IL-12 and IL-23. In mice, fumarates also generate type II DCs that induce IL-4-producing Th2 cells in vitro and in vivo and protect mice from experimental autoimmune encephalomyelitis. Type II DCs result from fumarate-induced glutathione (GSH) depletion, followed by increased hemoxygenase-1 (HO-1) expression and impaired STAT1 phosphorylation. Induced HO-1 is cleaved, whereupon the N-terminal fragment of HO-1 translocates into the nucleus and interacts with AP-1 and NF-κB sites of the IL-23p19 promoter. This interaction prevents IL-23p19 transcription without affecting IL-12p35, whereas STAT1 inactivation prevents IL-12p35 transcription without affecting IL-23p19. As a consequence, GSH depletion by small molecules such as fumarates induces type II DCs in mice and in humans that ameliorate inflammatory autoimmune diseases. This therapeutic approach improves Th1- and Th17-mediated autoimmune diseases such as psoriasis and MS by interfering with IL-12 and IL-23 production.

Inducible Cre mice.

The Cre/lox site-specific recombination system has emerged as an important tool for the generation of conditional somatic mouse mutants. This method allows one to control gene activity in space and time in almost any tissue of the mouse, thus opening new avenues for studying gene function and for establishing sophisticated animal models of human diseases. A major technical advance in terms of in vivo inducibility was the development of ligand-dependent Cre recombinases that can be activated by administration of tamoxifen to the animal. Here we describe how tamoxifen-dependent Cre recombinases, so-called CreER recombinases, work and how they can be used to generate time- and tissue-specific mouse mutants. The focus will be on the CreER(T2) recombinase, which is currently the most successful CreER version. We will give an overview of available CreER(T2) transgenic mouse lines and present protocols that detail the generation of experimental mice for inducible gene knockout studies, the induction of recombination by tamoxifen treatment, and the analysis of the quality and quantity of recombination by reporter gene and target gene studies. Most of the protocols can also be used as general guidelines for the generation and characterization of Cre/lox-mediated genome modifications in mice.

The commonly used cGMP-dependent protein kinase type I (cGKI) inhibitor Rp-8-Br-PET-cGMPS can activate cGKI in vitro and in intact cells.

Small-molecule modulators of cGMP signaling are of interest to basic and clinical research. The cGMP-dependent protein kinase type I (cGKI) is presumably a major mediator of cGMP effects, and the cGMP analogue Rp-8-Br-PET-cGMPS (Rp-PET) (chemical name: beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer) is currently considered one of the most permeable, selective, and potent cGKI inhibitors available for intact cell studies. Here, we have evaluated the properties of Rp-PET using cGKI-expressing and cGKI-deficient primary vascular smooth muscle cells (VSMCs), purified cGKI isozymes, and an engineered cGMP sensor protein. cGKI activity in intact VSMCs was monitored by cGMP/cGKI-stimulated cell growth and phosphorylation of vasodilator-stimulated phosphoprotein. Unexpectedly, Rp-PET (100 microm) did not efficiently antagonize activation of cGKI by the agonist 8-Br-cGMP (100 microm) in intact VSMCs. Moreover, in the absence of 8-Br-cGMP, Rp-PET (100 microm) stimulated cell growth in a cGKIalpha-dependent manner. Kinase assays with purified cGKI isozymes confirmed the previously reported inhibition of the cGMP-stimulated enzyme by Rp-PET in vitro. However, in the absence of the agonist cGMP, Rp-PET partially activated the cGKIalpha isoform. Experiments with a fluorescence resonance energy transfer-based construct harboring the cGMP binding sites of cGKI suggested that binding of Rp-PET induces a conformational change similar to the agonist cGMP. Together, these findings indicate that Rp-PET is a partial cGKIalpha agonist that under certain conditions stimulates rather than inhibits cGKI activity in vitro and in intact cells. Data obtained with Rp-PET as cGKI inhibitor should be interpreted with caution and not be used as sole evidence to dissect the role of cGKI in signaling processes.

Rescue of cGMP kinase I knockout mice by smooth muscle specific expression of either isozyme.

Smooth muscle expresses the Ialpha and the Ibeta isoforms of cGMP-dependent protein kinase I (cGKI). Inactivation of the murine cGKI gene prkg1 leads to multiple phenotypes and premature death at approximately 6 weeks. We reconstituted mice with the cGKIalpha or -Ibeta isozyme to test which isozyme was needed to support basic smooth muscle functions. Mice were generated by gene targeting. The cGKIalpha or the -Ibeta coding sequences were placed under the control of the SM22alpha promoter to express either isoform selectively in smooth muscle cells (SM-Ialpha or SM-Ibeta transgene). To generate smooth muscle-specific cGKIalpha or cGKIbeta rescue mice, the SM-Ialpha or SM-Ibeta transgenes were crossed on a cGKI-/- genetic background. The levels of cGKIalpha or -Ibeta expression were comparable to endogenous cGKI expression in wild-type aortic and intestinal smooth muscles. In cGKIalpha or -Ibeta rescue mice, expression of the isozymes was not detectable in non-smooth muscle tissues and cells. Median survival time of the Ialpha and Ibeta rescue mice was 52 weeks. Both isozymes mediated the 8-bromo-cGMP-induced relaxation of precontracted jejunum and aorta muscle strips. Activation of both isozymes reduced hormone- or K+-induced [Ca2+]i levels. The cGKIalpha and cGKIbeta rescue mice did not show a significant difference in intestinal passage time of BaSO4 in comparison with wild-type animals. Telemetric blood pressure measurements in conscious freely moving animals did not show differences between rescues and control mice in basal blood pressure and its regulation by DETA-NO, sodium nitroprusside, carbachol, or Y-27632. These results show that cGKI in smooth muscle is essential and that either cGKI isozyme alone can rescue basic vascular and intestinal smooth muscle functions.