Friedel Wenzel

Reliable detection of trisomy 21 using MALDI-TOF mass spectrometry

Purpose: Current diagnostic methods for chromosomal abnormalities rely mainly on karyotyping and occasionally fluorescent in situ hybridization or quantitative polymerase chain reaction. We describe an alternative molecular method for the detection of trisomy 21 involving mass spectrometric analysis of single nucleotide polymorphisms.Methods: In collaboration with Sequenom, Inc., 350 blinded amniotic fluid, amniocyte culture, chorionic villus, or amniotic fluid supernatant samples were analyzed for trisomy 21 using SNP analysis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Peak ratios were calculated for heterozygous genotypes and compared to control values generated from known euploid samples. An analytical algorithm using standard deviations from control values was used to determine the probability of a sample being affected or unaffected.Results: Seventy-three trisomy 21 samples from among the 350 blinded samples were correctly identified. There were no false-positive or false-negative results among the complete trisomy 21 samples. One sample exhibiting mosaicism for trisomy 21 was identified as being unaffected.Conclusions: MALDI-TOF mass spectrometry is a robust and reproducible method for the detection of trisomy 21. Its amenability to high-throughput analysis and high degree of multiplexing make it a potential future diagnostic tool for the detection of other aneuploidies as well.

Cell-free foetal DNA in maternal plasma does not appear to be derived from the rich pool of cell-free foetal DNA in amniotic fluid

Background: Large quantities of cell-free foetal DNA have been detected in amniotic fluid, and it has been proposed that this material may contribute to the pool of cell-free foetal DNA in maternal plasma. Methods: Twelve maternal blood samples were obtained from pregnant women about to undergo an amniocentesis. Cell-free DNA was extracted from the maternal plasma samples and the matched amniotic fluid samples. The amount of cell-free foetal DNA was quantified by real-time PCR assays for the SRY and RHD genes. Results: Amniotic fluid was found to contain vast quantities of cell-free DNA (median concentration = 3,978 copies/ml amniotic fluid). The concentration of cell-free foetal DNA in maternal plasma was much lower (median concentration = 96.6 copies/ml maternal plasma). No significant correlation could, however, be determined between these two pools of cell-free foetal DNA. Conclusions: Our data confirm that amniotic fluid contains prodigious quantities of cell-free foetal DNA, but as no relationship exists between this material and that in the maternal circulation, it is unlikely that the amnion contributes to the presence of cell-free foetal DNA in maternal plasma.

Detection of SNPs in the Plasma of Pregnant Women and in the Urine of Kidney Transplant Recipients by Mass Spectrometry

Abstract:  Recently, it has been discovered that cell‐free fetal DNA is smaller than corresponding maternal DNA. Therefore, circulating fetal DNA can be enriched by size‐fractionation. Such a selection improves the non‐invasive prenatal diagnosis of paternally inherited single gene mutations. Recent studies showed that MALDI‐TOF mass spectrometry (MS) can be used to reliably detect fetal‐specific single‐nucleotide polymorphisms (SNPs) in maternal plasma. In this study, we looked at whether the size‐fractionation approach could improve the detection of paternally inherited SNPs by MS assay. Our results indicated that the size‐fractionation approach improved the analysis of paternally inherited SNP alleles. Our previous studies showed that donor‐derived STR sequences could be detected in the urine of kidney transplant recipients. Here, we also examined whether donor‐specific SNPs could be detected in recipients urine by MS.

First successful pregnancy in Switzerland after prospective sex determination of the embryo through the separation of X-chromosome bearing spermatozoa

QUESTION UNDER STUDY The feasibility and the potential advantages of separating X-chromosome bearing spermatozoa for the prevention of a severe X-chromosome linked disorder with the use of intracytoplasmic sperm injection are presented. METHOD A carrier of muscular dystrophy type Becker was treated with intracytoplasmic sperm injection, using spermatozoa previously stained with the Hoechst dye 33342 and sorted with flow cytometry. RESULTS After transfer of one single blastocyst, an intrauterine pregnancy arose. In the ninth week of gestation, the female sex of the embryo was confirmed with proof of absence of the SRY gene of the Y-chromosome. After normal pregnancy, the patient delivered a healthy daughter. CONCLUSIONS The staining of spermatozoa with specific markers and sorting with flow cytometry provides a means of preventing significant disease in the offspring and may help in reducing the number of surplus embryos needed for preimplantation genetic diagnosis.

Chromosomal Microarrays in Prenatal Diagnosis: Time for a Change of Policy?

Microarrays have replaced conventional karyotyping as a first-tier test for unbalanced chromosome anomalies in postnatal cytogenetics mainly due to their unprecedented resolution facilitating the detection of submicroscopic copy number changes at a rate of 10–20% depending on indication for testing. A number of studies have addressed the performance of microarrays for chromosome analyses in high risk pregnancies due to abnormal ultrasound findings and reported an excess detection rate between 5% and 10%. In low risk pregnancies, clear pathogenic copy number changes at the submicroscopic level were encountered in 1% or less. Variants of unclear clinical significance, unsolicited findings, and copy number changes with variable phenotypic consequences are the main issues of concern in the prenatal setting posing difficult management questions. The benefit of microarray testing may be limited in pregnancies with only moderately increased risks (advanced maternal age, positive first trimester test). It is suggested to not change the current policy of microarray application in prenatal diagnosis until more data on the clinical significance of copy number changes are available.

Fetal Polydactyly A Study of 24 Cases Ascertained by Prenatal Sonography

Records of 24 pregnancies with fetal polydactyly were reviewed for the type of polydactyly, family history, associated sonographic findings, genetic testing, and postnatal/postmortem examination findings. The importance of fetal polydactyly can be mainly elucidated by the family history and absent or associated anomalies on a specialized malformation scan. Fetal karyotyping diagnoses frequent chromosomal anomalies in about half of cases with additional malformations, and array comparative genomic hybridization may be a future means of detecting cryptic chromosomal aberrations. Syndromic disorders of monogenic origin demand a careful interdisciplinary clinical assessment for establishing a clinical diagnosis and prognosis for the outcome of the child.

Sharing of a PTPN11 mutation by myelodysplastic bone marrow and a mature plasmacytoid dendritic cell proliferation provides evidence for their common myelomonocytic origin

Plasmacytoid dendritic cells (PDC) represent a subset of dendritic cells with unique morphology, function and cell surface phenotype [1]. They originate in the bone marrow from myeloid cell progenitors. PDC home in lymph nodes and are typically located close to high endothelial venules in small clusters. The presence of a significant number of PDC in lymph nodes indicates an ongoing immune reaction [2]. In other lymphoid tissues and in the bone marrow, PDC usually do not form aggregates but are scattered. PDC have been reported to give rise to two types of neoplasm [3]: a proliferation of mature PDC associated with a myeloid neoplasm, which most likely constitute a divergent form of differentiation of the malignant myelo-monocytic clone, and a blastic PDCneoplasm arising fromTdT+CD4+CD56+PDCprecursor cells.Mature PDC proliferations associatedwith amyeloid neoplasm aremost often encountered in chronicmyelomonocytic leukemia or, less often, in myelodysplastic syndromes (MDS) or acute leukemia with monocytic differentiation as to be expected from their putative divergent evolution form a putative malignant myelo-monocytic clone [4]. They are rare and occur predominantly (75%) in male patients with a median age of 69 years. Clinically, patients present with lymphadenopathy and skin lesions and, rarely, splenomegaly. Histopathologically, such proliferations are characterized by nodular, sometimes confluent, aggregates of clonally expanded mature PDC. Treatment of the underlying myeloid neoplasm may result in regression of the PDC component [5, 6]. Prognosis is usually poor, mainly due to the evolution of the myeloid neoplasm rather than expansion of PDC. The genomic landscape of such mature PDC proliferations is poorly understood. Several studies suggest a clonal relationship between the PDC and the associated myeloid neoplasm [7]. A unifying molecular pathway in terms of recurrent genomic aberrations has not yet been identified. We report a case of mature PDC proliferation associated with MDS with multilineage dysplasia (MDS-MLD according to the updated fourth edition of the WHO classification; refractory cytopenia with multilineage dysplasia (RCMD) according to the WHO 2008 classification). A clonal relationship of the PDC and the myeloid components was proven by a shared PTPN11 (protein tyrosine phosphatase, non-receptor type 11) mutation. Furthermore, using massive parallel sequencing (NGS) and array comparative genomic hybridization (aCGH), we document the genomic evolution in time of the myeloid component and the genomic stability of the PDC component.

Clonogenic versus morphogenic mutations in myeloid neoplasms: chronologic observations in a U2AF1, TET2, CSF3R and JAK2 ‘co-mutated’ myeloproliferative neoplasm suggest a hierarchical order of mutations and potential predictive value for kinase inhibitor treatment response

Myeloproliferative neoplasms (MPN) are characterized by abnormal growth of functional and differentiated blood cells in the bone marrow. The generic term MPN includes, among others, polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). They arise from clonal outgrowth of a single hematopoietic stem cell bearing somatic mutations that lead to or at least support expansion. Mutations in JAK2, MPL and CALR are thought as drivers in MPN [1–4]. They induce the MPN-phenotype (‘morphogenic or phenotypic mutations’), confer a relevant growth advantage and support positive selection of the mutant clone [1]. Furthermore and analogous to JAK2, MPL, and CALR, activating mutations in the colonystimulating factor 3 receptor (CSF3R) leading to hypersensitivity towards granulocyte colony-stimulating factor (G-CSF) have been identified as causative/morphogenic (and disease-defining) for chronic neutrophilic leukemia (CNL) [5]. Importantly, CSF3R and CSF3R-mutants signal through JAK2 [6]. Suitably, JAK2 inhibitors show a distinct inhibition of G-CSF-induced activation in cells with membrane-proximal mutations of CSF3R [6]. In most cases of ET and PV usually only the driver mutation is found, whereas PMF patients frequently show additional somatic mutations, the cumulative number of which seems to determine a worse prognosis [7]. With the help of massive parallel sequencing (MPS) techniques like NGS, a growing number of concurrent mutations e.g. of spliceosome genes like U2AF1 or epigenetic regulatory genes like TET2 have been identified in MPN, helping to explain MPN’s heterogeneity as well as the specific clinical course of some patients [7–9]. Although these mutations alone cannot cause a MPN-phenotype and are frequently found in other hematologic malignancies, they seem to generally increase the genetic instability of the affected cells, thereby potentially giving them additional pro-oncogenic advantages and are probably able to modify disease progression [1]. For instance, mutations of the splicing factor U2AF1 have been found in 8% of MPN in total and up to 16% of PMF and are regarded to be an independent risk factor of developing severe anemia, therefore influencing the overall survival significantly [9,10]. Here, we describe a patient with a four-fold co-mutated JAK2-positive MPN, who was initially treated by ruxolitinib and tracked chronologically by MPS. The case shows a hierarchical order of mutations potentially being of predictive value for kinase inhibitor treatment response. In November 2015 a 66-year-old man was admitted to our hospital because of progressive fatigue and arthralgias. Body examination revealed splenomegaly (16.8 cm) and laboratory investigations showed severe normochromic and normocytic anemia (hemoglobin 79 g/L) and thrombopenia (34 g/L) as well as LDH increase (302U/L), while the white blood cell count was only slightly increased (10.1 G/L) due to absolute neutrophilia of 7.61 G/L. The peripheral blood showed leukoerythroblastosis without any dysplasia. A bone marrow biopsy was performed. The aspiration was a dry tap. By histology, the bone marrow was hypocellular and displayed significant collagenous sclerosis and myelofibrosis (MF3) with edema and increased microvascularization with dilated sinuses. The megakaryopoiesis showed occasional clusters and cells with cloudy nuclei. The remaining linages were decreased, yet unremarkable. There was no increase of blasts (Figure 1(A–C)). With the help of MPS (applying the AmpliSeq Cancer Hotspot Panel v2 and a customized panel for ‘myeloid’ mutations in 30 genes [11]) the following mutations were identified: JAK2 p.V617F (allelic burden 16%), U2AF1 p.Q157P (allelic burden 49%), and CSF3R p.T618I (allelic burden 8%). The

Abstract 1910: 3′UTR poly(T/U) tract deletions and altered expression of EWSR1 are a hallmark of mismatch repair-deficient cancers.

Background: Microsatellite instability (MSI), the genome-wide accumulation of DNA replication errors, is the hallmark lesion of DNA mismatch repair (MMR) deficient cancers, present in Lynch syndrome (LS)-related and 10-20% of sporadic colorectal (CRC), gastric and endometrial cancers. MSI testing is widely used to guide clinical management but the functional significance of MSI at distinct genic loci remains largely elusive. Here, we characterize a novel MSI target locus consisting of a mononucleotide (T/U)16 tract located in the 3’untranslated region (UTR) of the Ewing sarcoma break point region 1 (EWSR1) gene (EWS16T). Methods: The diagnostic accuracy of EWS16T to identify MMR-deficient cancers was determined by analyzing 319 cancers (240 colorectal, 72 gastric, 7 endometrial) from two different populations. The functional consequences of EWS16T contractions were assessed in vitro (siRNA-mediated poly(A) site selection and pull-down assays) and in vivo (mRNA and protein expression by qPCR and tissue microarray analysis). Results: The EWS16T locus discriminates MMR-proficient (n=128) from deficient (n=191) cancers with perfect diagnostic sensitivity (100%) and specificity (100%). Biochemical analyses indicate that EWS16T contractions alter poly(A) site selection by promoting SFPQ-mediated distal poly(A) site usage in EWSR1 pre-mRNAs and result in decreased mRNA as well as EWS protein expression. In contrast to their MMR-proficient counterparts (n=64), MMR-deficient, LS-related CRC (n=94) display altered subcellular localization of EWS (p Conclusions: The EWS16T locus represents a novel, quasi-monomorphic MSI target locus to accurately identify both hereditary and sporadic MMR-deficient cancers. Contractions therein affect multiple regulatory mechanisms implicating the RNA-/DNA-binding Ewing sarcoma protein in MSI-associated colorectal tumorigenesis. Citation Format: Salvatore Piscuoglio, Shivendra Kishore, Michal Kovac, Annette Gylling, Friedel Wenzel, Francesca Trapani, Hans Joerg Altermatt, Valentina Mele, Giancarlo Marra, Paivi Peltomaki, Luigi Terracciano, Mihaela Zavolan, Karl Heinimann. 3′UTR poly(T/U) tract deletions and altered expression of EWSR1 are a hallmark of mismatch repair-deficient cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1910. doi:10.1158/1538-7445.AM2013-1910