Publications by authors named "Ulrich Lehmann"

170 Publications

Activating human epidermal growth factor receptor 2 (HER2) gene mutation in bone metastases from breast cancer.

Virchows Arch 2018 Nov 9;473(5):577-582. Epub 2018 Aug 9.

Institute of Pathology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.

In addition to amplification, point mutations of the human epidermal growth factor receptor 2 (HER2) gene (ERBB2) have been shown to activate the corresponding signaling pathway in breast cancer. The prevalence of ERBB2/HER2 mutation in bone metastasis of breast cancer and the associated phenotype are not known. In this study, bone metastases from breast cancer patients (n = 231) were analyzed for ERBB2/HER2 mutation. In 7 patients (3%; median age 70 years, range 50-83 years), gain-of-function mutations of ERBB2/HER2 were detected. The most frequent mutation was p.L755S (71%). In 29% of mutated cases, p.V777L was found. Lobular breast cancer was present in 71% of mutated cases (n = 5) and in 49% of all samples (n = 231; p = 0.275). Mutation frequency was 4.4% in the lobular subgroup and 17.4% in the pleomorphic subtype of lobular cancer (n = 23), respectively. All but one mutated lobular cancers were of the pleomorphic subtype (p = 0.006). Mutated cancers belonged either to the luminal (n = 4) or to the triple-negative types (n = 3). With regard to protein expression and gene amplification, HER2 was negative in all mutated cases. Among the 14% of metastatic luminal cancers with estrogen receptor gene (ESR1) mutation, conveying resistance against aromatase inhibitors, no concomitant ERBB2/HER2 mutation occurred. We conclude that activating HER2 mutation is present in about 3% of bone metastases from breast cancers, with significantly higher rates in the pleomorphic subtype of lobular cancer. Since mutated cases appear to be HER2-negative by conventional testing, the opportunity for specific anti-HER2 therapy may be missed.
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http://dx.doi.org/10.1007/s00428-018-2414-1DOI Listing
November 2018

Hotspot mutations in cancer genes may be missed in routine diagnostics due to neighbouring sequence variants.

Exp Mol Pathol 2018 08 27;105(1):37-40. Epub 2018 May 27.

Institute of Pathology, Medical School Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. Electronic address:

The detection of hotspot mutations in key cancer genes is now an essential part of the diagnostic work-up in molecular pathology. Nearly all assays for mutation detection involve an amplification step. A second single nucleotide variant (SNV) on the same allele adjacent to a mutational hotspot can interfere with primer binding, leading to unnoticed allele-specific amplification of the wild type allele and thereby false-negative mutation testing. We present two diagnostic cases with false negative sequence results for JAK2 and SRSF2. In both cases mutations would have escaped detection if only one strand of DNA had been analysed. Because many commercially available diagnostic kits rely on the analysis of only one DNA strand they are prone to fail in cases like these. Detailed protocols and quality control measures to prevent corresponding pitfalls are presented.
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http://dx.doi.org/10.1016/j.yexmp.2018.05.010DOI Listing
August 2018

CDKN2A loss and PIK3CA mutation in myoepithelial-like metaplastic breast cancer.

J Pathol 2018 07 28;245(3):373-383. Epub 2018 May 28.

Institute of Pathology, Hannover Medical School, Hannover, Germany.

Metaplastic breast carcinoma comprises a heterogeneous group of tumours with poorly understood pathogenesis. A subset of metaplastic breast cancers show myoepithelial differentiation and constitute a morphological spectrum with ill-defined borders from fibromatosis-like spindle cell carcinoma to myoepithelial carcinoma. In a series of 34 metaplastic breast cancers with spindle cell and myoepithelial differentiation, we found recurrent genetic aberrations, which set them apart from other metaplastic breast cancers and suggest a unique pathogenesis. The majority of cases (28 of 34 patients; 82.4%) showed distinct chromosomal loss in the 9p21.3 region, including CDKN2A and CDKN2B. Biallelic loss of the CDKN2A/B region was found in 50% of deleted cases. Expression of the cyclin-dependent kinase inhibitor CDKN2A (p16) was missing in all samples affected by 9p21.3 loss. Other genomic alterations frequently occurring in triple-negative and metaplastic breast cancer were absent or found in only a minority of cases. Gains of whole chromosome 5 and chromosomal region 5p were observed in nine cases, and were associated with recurrences (p < 0.001). In 64.3% of cases, 9p21.3 loss was accompanied by concurrent PIK3CA mutation. Both genomic abnormalities were also detectable in adenomyoepitheliomas (4/12), which are considered to represent the precursor lesion of myoepithelial metaplastic breast cancer. In adenomyoepithelioma, PIK3CA mutation was present in both luminal epithelial and myoepithelial cells, whereas p16 loss was found only in the latter. We conclude that 9p21.3 (CDKN2A) loss and PIK3CA mutation characterize a subgroup of metaplastic breast cancers with myoepithelial and spindle cell differentiation. Myoepithelial cells in adenomyoepithelioma may show identical aberrations. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5091DOI Listing
July 2018

Molecular defects in BRAF wild-type ameloblastomas and craniopharyngiomas-differences in mutation profiles in epithelial-derived oropharyngeal neoplasms.

Virchows Arch 2018 Jun 15;472(6):1055-1059. Epub 2018 Mar 15.

Institute of Pathology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.

The aim of this study was to evaluate the mutation profile of BRAF wild-type craniopharyngiomas and ameloblastomas. Pre-screening by immunohistochemistry and pyrosequencing for identifying BRAF wild-type tumors was performed on archived specimens of ameloblastic tumors (n = 20) and craniopharyngiomas (n = 62). Subsequently, 19 BRAF wild-type tumors (nine ameloblastic tumors and ten craniopharyngiomas) were analyzed further using next-generation sequencing (NGS) targeting hot spot mutations of 22 cancer-related genes. Thereby, we found craniopharyngiomas mainly CTNNB1 mutated (8/10), including two FGFR3/CTNNB1-double mutated tumors. Ameloblastic tumors were often FGFR2 mutated (4/9; including one FGFR2/TP53/PTEN-triple mutated case) and rarely CTNNB1/TP53-double mutated (1/9) and KRAS-mutated (1/9). In the remaining samples, no mutation could be detected in the 22 genes under investigation. In conclusion, mutation profiles of BRAF wild-type craniopharyngiomas and ameloblastomas share mutations of FGFR genes and have additional mutations with potential for targeted therapy.
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http://dx.doi.org/10.1007/s00428-018-2323-3DOI Listing
June 2018

Detection of Aberrant DNA Methylation Patterns in the RB1 Gene.

Methods Mol Biol 2018 ;1726:35-47

Institute of Pathology, Medizinische Hochschule Hannover, Hannover, Germany.

The retinoblastoma protein (pRb) plays a central role in the regulation of cell cycle by interaction with members of the E2F transcription factor family. As a tumor suppressor protein, pRb is frequently dysregulated in several major cancers. In addition to mutations, inactivation of pRb is also caused by epigenetic mechanisms including alterations of DNA methylation. There are three CpG islands located within the RB1 gene that encodes pRb that are closely associated with the regulation of pRb expression. Aberrant DNA methylation at the RB1 gene has been reported in sporadic retinoblastoma as well as other cancers including glioblastoma, hepatocellular carcinoma, and breast cancer. Recent studies have revealed that the RB1 gene is imprinted. Therefore, quantitative analysis is required to detect aberrations in DNA methylation associated with imprint deregulation. Pyrosequencing is considered as the method of choice for quantitative and reproducible analysis of DNA methylation with single base resolution. In this chapter, we provide a detailed protocol for the quantitative analysis of RB1 gene methylation using bisulfite Pyrosequencing.
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http://dx.doi.org/10.1007/978-1-4939-7565-5_5DOI Listing
January 2019

Next-Generation Sequencing Analysis of Laser-Microdissected Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens.

Methods Mol Biol 2018 ;1723:111-118

Institute of Pathology, Medizinische Hochschule Hannover, Hannover, Germany.

In recent years, next-generation sequencing (NGS) became widely used in molecular pathology. Comprehensive mutational profiling improved diagnosis and prognosis, as well as the identification of therapeutically relevant genetic alterations. However, the vast majority of studies analyzing tissue samples use DNA extracted from bulk tissue or only manually microdissected specimens. Laser-assisted microdissection offers the possibility of isolating morphologically defined small tissue compartments (like individual glands) or even of single cells for further molecular analysis. Even formalin-fixed paraffin-embedded (FFPE) tissue specimens can be used for laser-assisted microdissection. Combining these two innovative powerful methodological approaches provides invaluable insights into the genetic profile of any cell type and tissue compartment of interest, contributing to a better understanding of fundamental biological processes and disease-specific mechanisms.In this chapter, a detailed protocol is provided for microdissection of human mammary adenomyoepithelioma tissue specimens and subsequent targeted resequencing of a panel of cancer-related genes using IonTorrent/PGM technology.
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http://dx.doi.org/10.1007/978-1-4939-7558-7_5DOI Listing
January 2019

Absence of MGMT promoter methylation in diffuse midline glioma, H3 K27M-mutant.

Acta Neuropathol Commun 2017 12 15;5(1):98. Epub 2017 Dec 15.

Institute of Pathology, Department of Neuropathology, Hannover Medical School (MHH), Carl-Neuberg-Str. 1, D-30625, Hannover, Germany.

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http://dx.doi.org/10.1186/s40478-017-0500-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5732448PMC
December 2017

Lobular carcinoma in situ and invasive lobular breast cancer are characterized by enhanced expression of transcription factor AP-2β.

Lab Invest 2018 01 16;98(1):117-129. Epub 2017 Oct 16.

Institute of Pathology, Hannover Medical School, Hannover, Germany.

Transcription factor AP-2β (TFAP2B) regulates embryonic organ development and is overexpressed in alveolar rhabdomyosarcoma, a rare childhood malignancy. Gene expression profiling has implicated AP-2β in breast cancer (BC). This study characterizes AP-2β expression in the mammary gland and in BC. AP-2β protein expression was assessed in the normal mammary gland epithelium, in various reactive, metaplastic and pre-invasive neoplastic lesions and in two clinical BC cohorts comprising >2000 patients. BCs from various genetically engineered mouse (GEM) models were also evaluated. Human BC cell lines served as functional models to study siRNA-mediated inhibition of AP-2β. The normal mammary gland epithelium showed scattered AP-2β-positive cells in the luminal cell layer. Various reactive and pre-invasive neoplastic lesions, including apocrine metaplasia, usual ductal hyperplasia and lobular carcinoma in situ (LCIS) showed enhanced AP-2β expression. Cases of ductal carcinoma in situ (DCIS) were more often AP-2β-negative (P<0.001). In invasive BC cohorts, AP-2β-positivity was associated with the lobular BC subtype (P<0.001), loss of E-cadherin (P<0.001), a positive estrogen receptor (ER) status (P<0.001), low Ki67 (P<0.001), low/intermediate Oncotype DX recurrence scores (P<0.001), and prolonged event-free survival (P=0.003). BCs from GEM models were all AP-2β-negative. In human BC cell lines, AP-2β expression was independent from ER-signaling. SiRNA-mediated inhibition of AP-2β diminished proliferation of lobular BC cell lines in vitro. In summary, AP-2β is a new mammary epithelial differentiation marker. Its expression is preferentially retained and enhanced in LCIS and invasive lobular BC and has prognostic implications. Our findings indicate that AP-2β controls tumor cell proliferation in this slow-growing BC subtype.
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http://dx.doi.org/10.1038/labinvest.2017.106DOI Listing
January 2018

Rare ADAR and RNASEH2B variants and a type I interferon signature in glioma and prostate carcinoma risk and tumorigenesis.

Acta Neuropathol 2017 12 13;134(6):905-922. Epub 2017 Oct 13.

Department of Human Genetics OE 6300, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.

In search of novel germline alterations predisposing to tumors, in particular to gliomas, we studied a family with two brothers affected by anaplastic gliomas, and their father and paternal great-uncle diagnosed with prostate carcinoma. In this family, whole-exome sequencing yielded rare, simultaneously heterozygous variants in the Aicardi-Goutières syndrome (AGS) genes ADAR and RNASEH2B co-segregating with the tumor phenotype. AGS is a genetically induced inflammatory disease particularly of the brain, which has not been associated with a consistently increased cancer risk to date. By targeted sequencing, we identified novel ADAR and RNASEH2B variants, and a 3- to 17-fold frequency increase of the AGS mutations ADAR,c.577C>G;p.(P193A) and RNASEH2B,c.529G>A;p.(A177T) in the germline of familial glioma patients as well as in test and validation cohorts of glioblastomas and prostate carcinomas versus ethnicity-matched controls, whereby rare RNASEH2B variants were significantly more frequent in familial glioma patients. Tumors with ADAR or RNASEH2B variants recapitulated features of AGS, such as calcification and increased type I interferon expression. Patients carrying ADAR or RNASEH2B variants showed upregulation of interferon-stimulated gene (ISG) transcripts in peripheral blood as seen in AGS. An increased ISG expression was also induced by ADAR and RNASEH2B variants in tumor cells and was blocked by the JAK inhibitor Ruxolitinib. Our data implicate rare variants in the AGS genes ADAR and RNASEH2B and a type I interferon signature in glioma and prostate carcinoma risk and tumorigenesis, consistent with a genetic basis underlying inflammation-driven malignant transformation in glioma and prostate carcinoma development.
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http://dx.doi.org/10.1007/s00401-017-1774-yDOI Listing
December 2017

EGFR T790M mutation testing of non-small cell lung cancer tissue and blood samples artificially spiked with circulating cell-free tumor DNA: results of a round robin trial.

Virchows Arch 2017 Oct 8;471(4):509-520. Epub 2017 Sep 8.

Institue of Pathology, University Hospital Cologne, Kerpener Strasse 62, 50924, Cologne, Germany.

The European Commision (EC) recently approved osimertinib for the treatment of adult patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC) harboring EGFR T790M mutations. Besides tissue-based testing, blood samples containing cell-free circulating tumor DNA (ctDNA) can be used to interrogate T790M status. Herein, we describe the conditions and results of a round robin trial (RRT) for T790M mutation testing in NSCLC tissue specimens and peripheral blood samples spiked with cell line DNA mimicking tumor-derived ctDNA. The underlying objectives of this two-staged external quality assessment (EQA) approach were (a) to evaluate the accuracy of T790M mutations testing across multiple centers and (b) to investigate if a liquid biopsy-based testing for T790M mutations in spiked blood samples is feasible in routine diagnostic. Based on a successfully completed internal phase I RRT, an open RRT for EGFR T790M mutation testing in tumor tissue and blood samples was initiated. In total, 48 pathology centers participated in the EQA. Of these, 47 (97.9%) centers submitted their analyses within the pre-defined time frame and 44 (tissue), respectively, 40 (plasma) successfully passed the test. The overall success rates in the RRT phase II were 91.7% (tissue) and 83.3% (blood), respectively. Thirty-eight out of 48 participants (79.2%) successfully passed both parts of the RRT. The RRT for blood-based EGFR testing initiated in Germany is, to the best of our knowledge, the first of his kind in Europe. In summary, our results demonstrate that blood-based genotyping for EGFR resistance mutations can be successfully integrated in routine molecular diagnostics complementing the array of molecular methods already available at pathology centers in Germany.
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http://dx.doi.org/10.1007/s00428-017-2226-8DOI Listing
October 2017

Estrogen receptor (ESR1) mutation in bone metastases from breast cancer.

Mod Pathol 2018 01 11;31(1):56-61. Epub 2017 Aug 11.

Institute of Pathology, Hannover Medical School, Hannover, Germany.

Activating mutations of estrogen receptor α gene (ESR1) in breast cancer can cause endocrine resistance of metastatic tumor cells. The skeleton belongs to the metastatic sides frequently affected by breast cancer. The prevalence of ESR1 mutation in bone metastasis and the corresponding phenotype are not known. In this study bone metastases from breast cancer (n=231) were analyzed for ESR1 mutation. In 27 patients (12%) (median age 73 years, range: 55-82 years) activating mutations of ESR1 were detected. The most frequent mutation was p.D538G (53%), no mutations in exon 4 (K303) or 7 (S463) were found. Lobular breast cancer was present in 52% of mutated cases (n=14) and in 49% of all samples (n=231), respectively. Mutated cancers constantly displayed strong estrogen receptor expression. Progesterone receptor was positive in 78% of the mutated cases (n=21). From 194 estrogen receptor-positive samples, 14% had ESR1 mutated. Except for one mutated case, no concurrent HER2 overexpression was noted. Metastatic breast cancer with activating mutations of ESR1 had a higher Ki67 labeling index than primary luminal cancers (median 30%, ranging from 5 to 60% with 85% of cases revealing ≥20% Ki67-positive cells). From those patients from whom information on endocrine therapy was available (n=7), two had received tamoxifen only, 4 tamoxifen followed by aromatase inhibitors and one patient had been treated with aromatase inhibitors only. We conclude that ESR1 mutation is associated with estrogen receptor expression and high proliferative activity and affects about 14% of estrogen receptor-positive bone metastases from breast cancer.
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http://dx.doi.org/10.1038/modpathol.2017.95DOI Listing
January 2018

Subclonal analysis in a lobular breast cancer with classical and solid growth pattern mimicking a solid-papillary carcinoma.

J Pathol Clin Res 2017 Jul 19;3(3):191-202. Epub 2017 Jul 19.

Institute of Pathology, Hannover Medical SchoolHannoverGermany.

Recently, a new variant of invasive lobular breast cancer (ILBC) with solid-papillary-like growth pattern has been described. We present a case of ILBC with solid-papillary-like growth pattern in the main tumour mass and classical invasive lobular growth pattern in adjacent satellite foci. The two tumour components were subjected to comprehensive molecular analyses. Both components were ER/PR-positive, HER2-negative, and showed a complete loss of E-cadherin and beta-catenin protein expression, as determined by immunohistochemistry. Gene expression profiling classified the main tumour and a satellite focus as luminal-B and luminal-A subtypes, respectively. Whole-genome copy number profiles were highly similar in both tumour components. Shared copy number alterations (CNAs) included gains of chromosome 1q21.1-q43 and losses of chromosome 16q11.2-q24.3, the locus of the /E-cadherin tumour suppressor gene. CNAs detected only in the main tumour included a gain of chromosome 20q12-q13.33 and a loss of chromosome 1p36.33-p34.3, which has recently been associated with the solid variant of ILBC. Next generation sequencing revealed an identical, truncating mutation (p.G169fs*5) in both tumour components confirming a common clonal ancestry. In conclusion, we confirm the recently described variant of ILBC with solid-papillary-like growth pattern and provide evidence that it evolves from classical ILBC by subclonal evolution.
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http://dx.doi.org/10.1002/cjp2.76DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527319PMC
July 2017

Molecular Analysis of Circulating Cell-Free DNA from Lung Cancer Patients in Routine Laboratory Practice: A Cross-Platform Comparison of Three Different Molecular Methods for Mutation Detection.

J Mol Diagn 2017 09 16;19(5):722-732. Epub 2017 Jul 16.

Institute of Pathology, Hannover Medical School, Hannover, Germany.

Circulating cell-free DNA (cfDNA), which is isolated from blood plasma, represents a noninvasive source for the detection of mutations conferring resistance against epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small-cell lung cancer patients. In advanced disease stages, performing regular biopsies is often not possible because of the general health condition of the patients. Furthermore, a biopsy of a single tumor lesion or metastasis may not reflect the heterogeneous genotype of the tumor and its metastases. Plasma cfDNA represents an alternative material for molecular monitoring of patients under therapy. Herein, we present a cross-platform comparison of three different molecular methods [digital PCR, next-generation sequencing (NGS), and quantitative PCR] to detect clinically relevant mutations in cfDNA. We validated our workflow with commercially available cfDNA reference material (5.0%, 1.0%, and 0.1% mutation frequency, respectively). Digital PCR and NGS detect reliably 0.1% allele frequency of the EGFR p.T790M mutation. Furthermore, we analyzed 55 cfDNA preparations from patients with lung cancer to compare reliability and sensitivity of the three methods under routine conditions and achieved 96.0% concordance of p.T790M results. A limit of detection for mutation calling using digital PCR (>0.1%) and NGS (>0.2%) was established. In total, 62.5% of known primary EGFR mutations were successfully detected in cfDNA. In 56.0% of the patients with detectable EGFR primary mutations, we identified a resistance conferring the EGFR p.T790M mutation.
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http://dx.doi.org/10.1016/j.jmoldx.2017.05.008DOI Listing
September 2017

Transposable Elements in Human Cancer: Causes and Consequences of Deregulation.

Int J Mol Sci 2017 May 4;18(5). Epub 2017 May 4.

Institute of Pathology, Medizinische Hochschule Hannover, Hannover 30625, Germany.

Transposable elements (TEs) comprise nearly half of the human genome and play an essential role in the maintenance of genomic stability, chromosomal architecture, and transcriptional regulation. TEs are repetitive sequences consisting of RNA transposons, DNA transposons, and endogenous retroviruses that can invade the human genome with a substantial contribution in human evolution and genomic diversity. TEs are therefore firmly regulated from early embryonic development and during the entire course of human life by epigenetic mechanisms, in particular DNA methylation and histone modifications. The deregulation of TEs has been reported in some developmental diseases, as well as for different types of human cancers. To date, the role of TEs, the mechanisms underlying TE reactivation, and the interplay with DNA methylation in human cancers remain largely unexplained. We reviewed the loss of epigenetic regulation and subsequent genomic instability, chromosomal aberrations, transcriptional deregulation, oncogenic activation, and aberrations of non-coding RNAs as the potential mechanisms underlying TE deregulation in human cancers.
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http://dx.doi.org/10.3390/ijms18050974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5454887PMC
May 2017

Impact of Molecular Genetics on Outcome in Myelofibrosis Patients after Allogeneic Stem Cell Transplantation.

Biol Blood Marrow Transplant 2017 Jul 4;23(7):1095-1101. Epub 2017 Apr 4.

Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.

Molecular genetics may influence outcome for patients with myelofibrosis. To determine the impact of molecular genetics on outcome after allogeneic stem cell transplantation, we screened 169 patients with primary myelofibrosis (n = 110), post-essential thrombocythemia/polycythemia vera myelofibrosis (n = 46), and myelofibrosis in transformation (n = 13) for mutations in 16 frequently mutated genes. The most frequent mutation was JAK2V617F (n = 101), followed by ASXL1 (n = 49), calreticulin (n = 34), SRSF2 (n = 16), TET2 (n = 10), U2AF1 (n = 11), EZH2 (n = 7), MPL (n = 6), IDH2 (n = 5), IDH1 (n = 4), and CBL (n = 1). The cumulative incidence of nonrelapse mortality (NRM) at 1 year was 21% and of relapse at 5 years 25%. The 5-year rates progression-free (PFS) and overall survival (OS) were and 56%, respectively. In a multivariate analysis CALR mutation was an independent factor for lower NRM (HR, .415; P = .05), improved PFS (HR, .393; P = .01), and OS (HR, .448; P = .03). ASXL1 and IDH2 mutations were independent risk factors for lower PFS (HR, 1.53 [P = .008], and HR, 5.451 [P = .002], respectively), whereas no impact was observed for "triple negative" patients. Molecular genetics, especially CALR, IDH2, and ASXL1 mutations, may thus be useful to predict outcome independently from known clinical risk factors after allogeneic stem cell transplantation for myelofibrosis.
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http://dx.doi.org/10.1016/j.bbmt.2017.03.034DOI Listing
July 2017

is frequently methylated and related to poor survival in human hepatocellular carcinoma.

World J Gastroenterol 2017 Mar;23(9):1568-1575

Sumadi Lukman Anwar, Till Krech, Britta Hasemeier, Elisa Schipper, Hans Kreipe, Ulrich Lehmann, Institute of Pathology, Medizinische Hochschule Hannover, D30625 Hannover, Germany.

Aim: To screen clinically relevant microRNAs (miRNAs) silenced by DNA methylation in human hepatocellular carcinoma (HCC).

Methods: Knockdown of DNA methyltransferases (DNMTs) using siRNAs and miRNA profiling in HCC cell lines were performed to identify DNA hypermethylation-mediated miRNA downregulation. Confirmation using individual quantitative real-time PCR (qRT-PCR) assays was then performed followed by DNA methylation quantification at the promoter of the miRNA genes. Quantification of DNA methylation and miRNA expression was then performed in primary HCC tumor samples and related with clinicopathological variables.

Results: miRNA profiling after DNMT knockdown in HCC cell lines revealed upregulation of miR-23, miR-25 and miR-183. After qRT-PCR confirmation and CpG island methylation quantification of these miRNAs in cell lines, further analysis in primary HCC specimens showed that is hypermethylated in 30% of HCC ( = 40). Expression of mature miR-183 showed an inverse correlation with DNA methylation levels. In HCC cells, knockdown and 5-aza-2'-deoxycytidine treatment reduced methylation and stimulated expression of miR-183. In HCC patients, hypermethylation at promoter significantly correlates with poor survival (log-rank test = 0.03). DNA methylation analysis in healthy liver, benign liver tumors (hepatocellular adenoma and focal nodular hyperplasia) and their corresponding adjacent tissues showed absence of hypermethylation supporting the notion that aberrant methylation at is specific for the malignant transformation of hepatocytes.

Conclusion: Our data indicate that hypermethylation of is a frequent event in HCC and potentially useful as a novel surrogate diagnostic and prognostic marker.
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http://dx.doi.org/10.3748/wjg.v23.i9.1568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5340808PMC
March 2017

Precise ERBB2 copy number assessment in breast cancer by means of molecular inversion probe array analysis.

Oncotarget 2016 Dec;7(50):82733-82740

Institute of Human Genetics, Hannover Medical School, Hannover, Germany.

HER2/ERBB2 amplification/overexpression determines the eligibility of breast cancer patients to HER2-targeted therapy. This study evaluates the agreement between ERBB2 copy number assessment by fluorescence in situ hybridization, a standard method recommended by the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP), and newly available DNA extraction-based methods. A series of n=29 formalin-fixed paraffin-embedded breast cancers were subjected to ERBB2 copy number assessment by fluorescence in situ hybridization (FISH, Vysis, Abbott). Following macrodissection of invasive breast cancer tissue and DNA extraction, ERBB2 copy number was also determined by molecular inversion probe array analysis (MIP, OncoScan, Affymetrix) and next generation sequencing combined with normalized amplicon coverage analysis (NGS/NAC, AmpliSeq, Ion Torrent). ERBB2 copy number values obtained by MIP or NGS/NAC were tightly correlated with ERBB2 copy number values obtained by conventional FISH (rs = 0.940 and rs = 0.894, P < 0.001). Using ASCO/CAP guideline-conform thresholds for categorization of breast cancers as HER2-negative, equivocal or positive, nearly perfect concordance was observed for HER2 classification by FISH and MIP (93% concordant classifications, κ = 0.87). Substantial concordance was observed for FISH and NGS/NAC (83% concordant classifications, κ = 0.62). In conclusion, MIP facilitates precise ERBB2 copy number detection and should be considered as an ancillary method for clinical HER2 testing.
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http://dx.doi.org/10.18632/oncotarget.12421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347728PMC
December 2016

A liver nodule in a patient transplanted for primary sclerosing cholangitis: an interdisciplinary diagnostic approach.

Z Gastroenterol 2017 Jan 5;55(1):56-62. Epub 2016 Oct 5.

Department of Gastroenterology, Digestive Diseases and Intensive Care Medicine (Department of Medicine III), University Hospital RWTH Aachen, Aachen, Germany.

We report the case of a 53-year-old female patient who was transplanted with the liver of a 71-year-old male donor for advanced primary sclerosing cholangitis (PSC) and who additionally was diagnosed with a histologically non-classifiable colitis shortly before transplantation. Upon follow-up abdominal ultrasound 4 months after transplantation, a liver lesion measuring 16 × 23 mm was detected in the transplanted liver. This lesion had not been noticed immediately after transplantation and showed a pattern suspicious for malignancy in contrast-enhanced ultrasound. In line, a biopsy revealed the presence of a metastasis of an adenocarcinoma of colorectal origin, suggesting that a colitis- and PSC-associated colorectal cancer of the recipient might have been overseen upon the initial diagnostic workup. Despite two negative follow-up colonoscopies, this hypothesis was further supported by a strong positive signal in projection to the cecum in a subsequently performed PET/CT-scan. However, surgical resection of the right colon that was performed simultaneously with the atypical resection of the liver metastasis only revealed an inflamed diverticulum but no malignancy in the resected colon segment. Moreover, cytogenetic and molecular genetic testing on the resected specimens clearly attributed the metastasis to the male donor. On the one hand, this case underlines the necessity of endoscopic surveillance of patients with PSC and/or inflammatory bowel disease as well as the challenges in diagnosis of colitis-associated cancer. On the other hand, it shows that the acceptance of organs from elderly donors in times of organ shortage might be linked to an increased risk of donor transmitted malignancies.
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http://dx.doi.org/10.1055/s-0042-111048DOI Listing
January 2017

A novel germline POLE mutation causes an early onset cancer prone syndrome mimicking constitutional mismatch repair deficiency.

Fam Cancer 2017 01;16(1):67-71

Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany.

In a 14-year-old boy with polyposis and rectosigmoid carcinoma, we identified a novel POLE germline mutation, p.(Val411Leu), previously found as recurrent somatic mutation in 'ultramutated' sporadic cancers. This is the youngest reported cancer patient with polymerase proofreading-associated polyposis indicating that POLE mutation p.(Val411Leu) may confer a more severe phenotype than previously reported POLE and POLD1 germline mutations. The patient had multiple café-au-lait macules and a pilomatricoma mimicking the clinical phenotype of constitutional mismatch repair deficiency. We hypothesize that these skin features may be common to different types of constitutional DNA repair defects associated with polyposis and early-onset cancer.
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http://dx.doi.org/10.1007/s10689-016-9925-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5243902PMC
January 2017

Prognostic factors in the myoepithelial-like spindle cell type of metaplastic breast cancer.

Virchows Arch 2016 Aug 25;469(2):191-201. Epub 2016 May 25.

Institute of Pathology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.

Metaplastic breast carcinoma (MBC) comprises a heterogeneous group of tumors with difficult to predict biological behavior. A subset of MBC, characterized by spindle-shaped tumor cells with a myoepithelial-like immunophenotype, was entered into a retrospective study (n = 42, median follow-up time 43 months). Molecular parameters (DNA sequences of mutation hot spots in AKT1, ALK, APC, BRAF, CDH1, CTNNB1, EGFR, ERBB2, FBXW7, FGFR2, FOXL2, GNAQ, GNAS, KIT, KRAS, MAP2K1, MET, MSH6, NRAS, PDGFRA, PIK3CA, PTEN, SF3B1, SMAD4, SRC, SRSF2, STK11, TP53, and U2AF1; copy numbers for EGFR, c-myc, FGFR, PLAG, c-met) were assessed. None of the patients had axillary lymph node involvement. In 13 cases, local recurrence developed after surgery (30.9 %). Distant metastasis occurred in seven patients (17 %; four after local recurrence). The most frequent genetic alteration was PIK3CA mutation (50 % of cases). None of the pathological parameters (size, grade, stage, Ki-67 labeling index) was significantly associated with disease-free survival (DFS) or overall survival (OS). PIK3CA mutation, especially the H1047R type, tended to adversely affect OS. Type of resection (mastectomy vs. breast-conserving therapy, width of margins) or adjuvant radiotherapy had no influence on DFS or OS, whereas in the group treated with radio-/chemotherapy, no local recurrence or metastasis and no death occurred. We conclude that the spindle cell type of MBC with myoepithelial features exhibits a higher frequency of PIK3CA mutation than other types of metaplastic or basal-like breast cancer and may benefit from combined radio-/chemotherapy. Classical pathological parameters are not helpful in identifying the high-risk tumors among this subgroup of MBC.
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http://dx.doi.org/10.1007/s00428-016-1950-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4978764PMC
August 2016

Routine clinical mutation profiling using next generation sequencing and a customized gene panel improves diagnostic precision in myeloid neoplasms.

Oncotarget 2016 May;7(21):30084-93

Institute of Pathology, Medizinische Hochschule Hannover, Hannover, Germany.

Microscopic examination of myelodysplastic syndromes (MDS) and myelodysplastic-myeloproliferative neoplasms (MDS/MPN) may be challenging because morphological features can overlap with those of reactive states. Demonstration of clonal hematopoiesis provides a diagnostic clue and has become possible by comprehensive mutation profiling of a number of frequently mutated genes, some of them with large coding regions.To emphasize the potential benefit of NGS in hematopathology we present sequencing results from routinely processed formalin-fixed and paraffin-embedded (FFPE) bone marrow trephines (n = 192). A customized amplicon-based gene panel including 23 genes frequently mutated in myeloid neoplasms was established and implemented. Thereby, 629,691 reads per sample (range 179,847-1,460,412) and a mean coverage of 2,702 (range 707-6,327) could be obtained, which are sufficient for comprehensive mutational profiling. Seven samples failed in sequencing (3.6%). In 185 samples we found in total 269 pathogenic variants (mean 1.4 variants per patient, range 0-5), 125 Patients exhibit at least one pathogenic mutation (67.6%). Variants show allele frequencies ranging from 6.7% up to 95.7%. Most frequently mutated genes were TET2 (28.7%), SRSF2 (19.5%), ASXL1 (8.6%) and U2AF1 (8.1%). The mutation profiling increases the diagnostic precision and adds prognostic information.
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http://dx.doi.org/10.18632/oncotarget.8310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5058665PMC
May 2016

NGS-based BRCA1/2 mutation testing of high-grade serous ovarian cancer tissue: results and conclusions of the first international round robin trial.

Virchows Arch 2016 Jun 22;468(6):697-705. Epub 2016 Mar 22.

Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld, 224, Heidelberg, Germany.

With the approval of olaparib as monotherapy treatment in platinum-sensitive, relapsed high-grade serous ovarian cancer by the European Medical Agency (EMA), comprehensive genotyping of BRCA1 and BRCA2 in tumor tissue has become a mandatory pre-therapeutic test. This requires significant advances in routine tumor test methodologies due to the large size of both genes and the lack of mutational hot spots. Classical focused screening approaches, like Sanger sequencing, do not allow for a sensitive, rapid, and economic analysis of tumor tissue. Next-generation sequencing (NGS) approaches employing targeted panels for BRCA1/2 to interrogate formalin-fixed and paraffin-embedded tumor samples from either surgical resection or biopsy specimens can overcome these limitations. Although focused NGS methods have been implemented by few centers in routine molecular diagnostics for the analysis of some druggable oncogenic mutations, the reliable diagnostic testing of the entire coding regions of BRCA1 and BRCA2 was a new challenge requiring extensive technological improvement and quality management. Here, we describe the implementation and results of the first round robin trial for BRCA1/2 mutation testing in tumor tissue that was conducted in central Europe on May 2015, shortly after the approval and prior to the official release of olaparib. The high success rate of 81 % (21/26 test centers) demonstrates that BRCA1/2 multicenter mutation testing is well feasible in FFPE tumor tissue, extending to other tumor entities beyond ovarian cancer. The high number of test centers passing the trial demonstrates the success of the concerted efforts by German, Swiss, and Austrian pathology centers to ensure quality-controlled NGS-based testing and proves the potential of this technology in routine molecular pathology. On the basis of our results, we provide recommendations for predictive testing of tumor tissue for BRCA1/2 to clinical decision making in ovarian cancer patients.
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http://dx.doi.org/10.1007/s00428-016-1919-8DOI Listing
June 2016

Loss of DNA methylation at imprinted loci is a frequent event in hepatocellular carcinoma and identifies patients with shortened survival.

Clin Epigenetics 2015 15;7:110. Epub 2015 Oct 15.

Institute of Pathology, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.

Background: Aberrant DNA methylation at imprinted loci is an important molecular mechanism contributing to several developmental and pathological disorders including cancer. However, knowledge about imprinting defects due to DNA methylation changes is relatively limited in hepatocellular carcinoma (HCC), the third leading cause of cancer death worldwide. Therefore, comprehensive quantitative DNA methylation analysis at imprinted loci showing ~50 % methylation in healthy liver tissues was performed in primary HCC specimens and the peritumoural liver tissues.

Results: We found frequent and extensive DNA methylation aberrations at many imprinted loci in HCC. Unsupervised cluster analysis of DNA methylation patterns at imprinted loci revealed subgroups of HCCs with moderate and severe loss of methylation. Hypomethylation at imprinted loci correlated significantly with poor overall survival (log-rank test, p = 0.02). Demethylation at imprinted loci was accompanied by loss of methylation at LINE-1, a commonly used marker for global DNA methylation levels (p < 0.001). In addition, we found that loss of methylation at imprinted loci correlated with the presence of a CTNNB1 mutation (Fisher's exact test p = 0.03). Re-analysis of publically available genome-wide methylation data sets confirmed our findings. The analysis of benign liver tumours (hepatocellular adenoma (HCA) and focal nodular hyperplasia (FNH)), the corresponding adjacent liver tissues, and healthy liver tissues showed that aberrant DNA methylation at imprinted loci is specific for HCC.

Conclusions: Our analyses demonstrate frequent and widespread DNA methylation aberrations at imprinted loci in human HCC and identified a hypomethylated subgroup of patients with shorter overall survival.
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http://dx.doi.org/10.1186/s13148-015-0145-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4606497PMC
October 2015

Breast Cancer Anti-Estrogen Resistance 4 (BCAR4) Drives Proliferation of IPH-926 lobular Carcinoma Cells.

PLoS One 2015 28;10(8):e0136845. Epub 2015 Aug 28.

Institute of Pathology, Hannover Medical School, Hannover, Germany.

Background: Most breast cancers depend on estrogenic growth stimulation. Functional genetic screenings in in vitro cell models have identified genes, which override growth suppression induced by anti-estrogenic drugs like tamoxifen. Using that approach, we have previously identified Breast Cancer Anti-Estrogen Resistance 4 (BCAR4) as a mediator of cell proliferation and tamoxifen-resistance. Here, we show high level of expression and function of BCAR4 in human breast cancer.

Methods: BCAR4 mRNA expression was evaluated by (q)RT-PCR in a panel of human normal tissues, primary breast cancers and cell lines. A new antibody raised against C78-I97 of the putative BCAR4 protein and used for western blot and immunoprecipitation assays. Furthermore, siRNA-mediated gene silencing was implemented to study the function of BCAR4 and its downstream targets ERBB2/3.

Results: Except for placenta, all human normal tissues tested were BCAR4-negative. In primary breast cancers, BCAR4 expression was comparatively rare (10%), but associated with enhanced proliferation. Relative high BCAR4 mRNA expression was identified in IPH-926, a cell line derived from an endocrine-resistant lobular breast cancer. Moderate BCAR4 expression was evident in MDA-MB-134 and MDA-MB-453 breast cancer cells. BCAR4 protein was detected in breast cancer cells with ectopic (ZR-75-1-BCAR4) and endogenous (IPH-926, MDA-MB-453) BCAR4 mRNA expression. Knockdown of BCAR4 inhibited cell proliferation. A similar effect was observed upon knockdown of ERBB2/3 and exposure to lapatinib, implying that BCAR4 acts in an ERBB2/3-dependent manner.

Conclusion: BCAR4 encodes a functional protein, which drives proliferation of endocrine-resistant breast cancer cells. Lapatinib, a clinically approved EGFR/ERBB2 inhibitor, counteracts BCAR4-driven tumor cell growth, a clinical relevant observation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0136845PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4552740PMC
May 2016

Pyrosequencing Technology. Preface.

Methods Mol Biol 2015 ;1315:v-vi

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March 2016

MicroRNAs: Emerging Novel Clinical Biomarkers for Hepatocellular Carcinomas.

J Clin Med 2015 Aug 18;4(8):1631-50. Epub 2015 Aug 18.

Institute of Pathology, Medizinische Hochschule Hannover, Hannover D30625, Germany.

The discovery of small non-coding RNAs known as microRNAs has refined our view of the complexity of gene expression regulation. In hepatocellular carcinoma (HCC), the fifth most frequent cancer and the third leading cause of cancer death worldwide, dysregulation of microRNAs has been implicated in all aspects of hepatocarcinogenesis. In addition, alterations of microRNA expression have also been reported in non-cancerous liver diseases including chronic hepatitis and liver cirrhosis. MicroRNAs have been proposed as clinically useful diagnostic biomarkers to differentiate HCC from different liver pathologies and healthy controls. Unique patterns of microRNA expression have also been implicated as biomarkers for prognosis as well as to predict and monitor therapeutic responses in HCC. Since dysregulation has been detected in various specimens including primary liver cancer tissues, serum, plasma, and urine, microRNAs represent novel non-invasive markers for HCC screening and predicting therapeutic responses. However, despite a significant number of studies, a consensus on which microRNA panels, sample types, and methodologies for microRNA expression analysis have to be used has not yet been established. This review focuses on potential values, benefits, and limitations of microRNAs as new clinical markers for diagnosis, prognosis, prediction, and therapeutic monitoring in HCC.
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http://dx.doi.org/10.3390/jcm4081631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4555081PMC
August 2015

Comprehensive Molecular Profiling of Archival Bone Marrow Trephines Using a Commercially Available Leukemia Panel and Semiconductor-Based Targeted Resequencing.

PLoS One 2015 29;10(7):e0133930. Epub 2015 Jul 29.

Institute of Pathology, Hannover Medical School, Hannover, Germany.

Comprehensive mutation profiling becomes more and more important in hematopathology complementing morphological and immunohistochemical evaluation of fixed, decalcified and embedded bone marrow biopsies for diagnostic, prognostic and also predictive purposes. However, the number and the size of relevant genes leave conventional Sanger sequencing impracticable in terms of costs, required input DNA, and turnaround time. Since most published protocols and commercially available reagents for targeted resequencing of gene panels are established and validated for the analysis of fresh bone marrow aspirate or peripheral blood it remains to be proven whether the available technology can be transferred to the analysis of archival trephines. Therefore, the performance of the recently available Ion AmpliSeq AML Research panel (LifeTechnologies) was evaluated for the analysis of fragmented DNA extracted from archival bone marrow trephines. Taking fresh aspirate as gold standard all clinically relevant mutations (n = 17) as well as 25 well-annotated SNPs could be identified reliably with high quality in the corresponding archival trephines of the training set (n = 10). Pre-treatment of the extracted DNA with Uracil-DNA-Glycosylase reduced the number of low level artificial sequence variants by more than 60%, vastly reducing time required for proper evaluation of the sequencing results. Subsequently, randomly picked FFPE samples (n = 41) were analyzed to evaluate sequencing performance under routine conditions. Thereby all known mutations (n = 43) could be verified and 36 additional mutations in genes not yet covered by the routine work-up (e.g., TET2, ASXL1, DNMT3A), demonstrating the feasibility of this approach and the gain of diagnostically relevant information. The dramatically reduced amount of input DNA, the increase in sensitivity as well as calculated cost-effectiveness, low hands on , and turn-around-time, necessary for the analysis of 237 amplicons strongly argue for replacing Sanger sequencing by this semiconductor-based targeted resequencing approach.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133930PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519100PMC
May 2016

Lobular breast cancer--the most common special subtype or a most special common subtype?

Authors:
Ulrich Lehmann

Breast Cancer Res 2015 Jul 28;17:99. Epub 2015 Jul 28.

Institute of Pathology, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625, Hannover, Germany.

Lobular breast cancer is not only the second most common breast cancer subtype, known for decades, but also a tumour entity that still poses many unresolved questions. These include questions about the targets and cooperation partners of E-cadherin, the best model systems for translational research, and the best tools for detection, surveillance and therapy. Leading experts review the molecular and cellular bases, the model systems, the histopathology and profiling approaches, risk factors, imaging tools and therapeutic options for lobular breast cancer.
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http://dx.doi.org/10.1186/s13058-015-0606-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4531830PMC
July 2015

Quantitative DNA Methylation Analysis by Pyrosequencing®.

Methods Mol Biol 2015 ;1315:175-88

Institute of Pathology, Medizinische Hochschule Hannover, Carl-Neuberg Strasse 1, Hannover, D-30625, Germany.

DNA methylation is an epigenetic mark playing an important role in development and disease. Aberrant DNA methylation was identified as an alternative mechanism for gene inactivation complementing deletions and mutations in cancer initiation and progression. However, to accurately compare differences in DNA methylation among various tissue types, adequate quantitative approaches are required. Pyrosequencing(®), as a sequencing-by-synthesis method, allows such quantification with single CpG resolution and the ability for threshold determination. This book chapter provides a detailed protocol for DNA methylation analysis by Pyrosequencing, including information on assay design and practical procedure. Additionally, emphasis is placed on the discussion of strengths and weaknesses of the methodology.
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http://dx.doi.org/10.1007/978-1-4939-2715-9_13DOI Listing
March 2016
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