Routine noninvasive prenatal screening for fetal Rh D in maternal plasma-A 2-year experience from a single center in Belgium.

BACKGROUND: Hemolytic disease of the fetus and newborn (HDFN) due to rhesus D (RhD) immunization is a potentially life-threatening situation for which use of Rh Immunoglobulin (RhIg) has decreased risk drastically. Determination of fetal RHD on maternal plasma can be used to restrict prenatal RhIg administration to women carrying an RhD-positive child, avoiding unnecessary administration of blood-derived products. STUDY DESIGN AND METHODS: The aim of this study is to determine the performance of fetal RHD typing in our center. We prospectively collected 205 fetal RHD and 127 serological cord blood RhD data from RhD-negative women starting at 11 weeks of pregnancy (from October 2019 to October 2021). Real-time polymerase chain reaction targeting RHD exon 5 and 7 was used, similar to the screening program in The Netherlands, supplemented with an amplification control (beta-actin; ACTB) and a sex determination marker located on the Y-chromosome (SRY gene). RESULTS: Fetal RHD testing reached a sensitivity and specificity of 100%. No false-negative nor false-positive results were reported. Inconclusive results (6%, 13/205) were due to weak amplification in 10 cases, a maternal RHD variant in 2 cases (RHD*01N.71 and partial DVI), and a fetal RHD variant (partial DVI) in 1 case. Unnecessary administration of RhIg prophylaxis was avoided in 33% of cases and on the other hand was administered in one case (fetal partial DVI) which would have been missed with cord blood serology. DISCUSSION: This study demonstrates the high accuracy of routine prenatal fetal RHD gene screening after 11 weeks of pregnancy, encouraging routine clinical practice.

Mediastinal Myeloid Sarcoma with TP53 Mutation Preceding Acute Myeloid Leukemia with a PICALM-MLLT10 Fusion Gene.

INTRODUCTION: Myeloid sarcoma (MS), previously known as granulocytic sarcoma or chloroma, is a rare neoplastic condition defined as a tumor mass consisting of myeloblasts or immature myeloid cells occurring at an extramedullary site. Clinical presentation is diverse and determined by a tumor mass effect or local organ dysfunction. CASE REPORT: We report the case of a 25-year-old previously healthy male with rapidly progressive shortness of breath. A chest CT scan demonstrated a heterogenous anterosuperior mediastinal mass with pleural and pericardial invasion. A diagnosis of MS with both myeloid and lymphoid characteristics was made by pathologic, morphologic, and immunophenotypic investigation. Next generation analysis revealed a pathogenic TP53 mutation (c.1035_1036insCT, p.Glu346Leufs*25). After 4 cycles of chemotherapy only a partial metabolic response and tumor size reduction was obtained. A pretransplant bone marrow biopsy revealed the progression of disease to acute myeloid leukemia. Cytogenetic analysis demonstrated a t(10; 11)(p12;q21). Fluorescence in situ hybridization confirmed the presence of a PICALM-MLLT10 fusion gene. CONCLUSION: MS with a mediastinal localization is rare and often misdiagnosed as malignant lymphoma. Acute leukemia harboring a PICALM-MLLT10 fusion gene is characterized by a mixed T cell and myeloid phenotype. The rearrangement is a rare recurrent translocation associated with specific clinical features, as illustrated in this case report.

Bias reduction improves accuracy and informativity of high-throughput sequencing chimerism assays.

BACKGROUND AND AIMS: Chimerism monitoring by means of high-throughput sequencing of biallelic polymorphisms has shown promising advantages for patient follow-up after hematopoietic stem cell transplantation. Yet, the presence of method bias precludes achievement of an assay's theoretically attainable informativity rate, as method bias necessitates the exclusion of some markers. This method bias arises because of preferential observation of one allele over the other, and for some allelic constellations because of stochasticity. RESULTS: This paper suggests how preferential allelic observation may lead to method bias, and when and why such bias necessitates the exclusion of markers. It is shown that also markers that remain informative suffer a reduction in trueness and precision due to method bias. A bias reduction approach in the data analysis phase is introduced and shown to improve trueness and precision under all circumstances, meriting its universal adoption. This bias reduction furthermore allows to achieve an assay's theoretically achievable informativity rate, though at the cost of reduced sensitivity. Several strategies to consider in the assay design phase that may lower biases are proposed. CONCLUSION: Improved design and data analysis of chimerism assays increase the accuracy, applicability, and cost-effectiveness of high-throughput sequencing chimerism assays.

Chimerism monitoring using biallelic single nucleotide or insertion/deletion polymorphisms: How many markers to screen?

BACKGROUND/AIMS: Chimerism monitoring by means of high-throughput sequencing or quantitative PCR of biallelic single nucleotide and insertion/deletion polymorphisms has shown potential for improved patient care when compared to the gold standard capillary electrophoresis assays. When designing chimerism assays the number of markers to screen needs consideration: it determines the informativity rate and accuracy of the assay, but screening too many markers increases the assay's cost and complexity. The minimal number of biallelic markers to screen is currently unstudied. MATERIALS/METHODS: A simulation framework accounting for marker minor allele frequencies, the number of markers screened, marker allelic constellations and donor-recipient relatedness was constructed. The framework was validated through analysis of 324 clinical samples. RESULTS: Empirical clinical data confirm the validity of the simulation framework. With guidelines suggesting to monitor at least three informative markers, we demonstrate that, for optimized assays, at least 40 biallelic markers need to be screened to achieve enough informative markers in over 99% of cases. We propose and discuss several assay optimization strategies. CONCLUSION: Currently used chimerism assays often screen too little or too many markers, leaving room for optimization. Through support of the simulation framework here introduced and validated, more informative, cost-effective chimerism assays can be designed.

Reliable and Scalable SARS-CoV-2 qPCR Testing at a High Sample Throughput: Lessons Learned from the Belgian Initiative.

We present our approach to rapidly establishing a standardized, multi-site, nation-wide COVID-19 screening program in Belgium. Under auspices of a federal government Task Force responsible for upscaling the country's testing capacity, we were able to set up a national testing initiative with readily available resources, putting in place a robust, validated, high-throughput, and decentralized qPCR molecular testing platform with embedded proficiency testing. We demonstrate how during an acute scarcity of equipment, kits, reagents, personnel, protective equipment, and sterile plastic supplies, we introduced an approach to rapidly build a reliable, validated, high-volume, high-confidence workflow based on heterogeneous instrumentation and diverse assays, assay components, and protocols. The workflow was set up with continuous quality control monitoring, tied together through a clinical-grade information management platform for automated data analysis, real-time result reporting across different participating sites, qc monitoring, and making result data available to the requesting physician and the patient. In this overview, we address challenges in optimizing high-throughput cross-laboratory workflows with minimal manual intervention through software, instrument and assay validation and standardization, and a process for harmonized result reporting and nation-level infection statistics monitoring across the disparate testing methodologies and workflows, necessitated by a rapid scale-up as a response to the pandemic.

Nationwide Harmonization Effort for Semi-Quantitative Reporting of SARS-CoV-2 PCR Test Results in Belgium.

From early 2020, a high demand for SARS-CoV-2 tests was driven by several testing indications, including asymptomatic cases, resulting in the massive roll-out of PCR assays to combat the pandemic. Considering the dynamic of viral shedding during the course of infection, the demand to report cycle threshold (Ct) values rapidly emerged. As Ct values can be affected by a number of factors, we considered that harmonization of semi-quantitative PCR results across laboratories would avoid potential divergent interpretations, particularly in the absence of clinical or serological information. A proposal to harmonize reporting of test results was drafted by the National Reference Centre (NRC) UZ/KU Leuven, distinguishing four categories of positivity based on RNA copies/mL. Pre-quantified control material was shipped to 124 laboratories with instructions to setup a standard curve to define thresholds per assay. For each assay, the mean Ct value and corresponding standard deviation was calculated per target gene, for the three concentrations (107, 105 and 103 copies/mL) that determine the classification. The results of 17 assays are summarized. This harmonization effort allowed to ensure that all Belgian laboratories would report positive PCR results in the same semi-quantitative manner to clinicians and to the national database which feeds contact tracing interventions.

Performance Assessment of the Devyser High-Throughput Sequencing-Based Assay for Chimerism Monitoring in Patients after Allogeneic Hematopoietic Stem Cell Transplantation.

Chimerism analysis is widely used to aid in the clinical management of patients after allogeneic hematopoietic stem cell transplantation. Many laboratories currently use assays based on PCR followed by capillary electrophoresis, with a limit of quantification of 1% to 5%. Assays with a lower limit of quantification could allow for earlier relapse detection, resulting in improved patient care. This study investigated the analytical, clinical, technical, and practical performance of the Devyser next-generation sequencing chimerism assay, a commercial high-throughput sequencing-based assay for chimerism analysis. Performance of this assay was compared with that of the Promega PowerPlex 16 HS assay, a commercial capillary electrophoresis-based assay. A limit of quantification of 0.1% was achievable with the Devyser assay. The repeatability, reproducibility, trueness, and linearity of the Devyser assay were acceptable. The Devyser assay showed potential for earlier relapse detection compared with the Promega assay. Conclusive analysis was not possible for 3% of donor-recipient pairs with the Devyser assay due to an insufficient number of informative markers; this factor was not an issue for the Promega assay. Further improvements in assay design or data analysis may allow the assay's applicability to be extended to all donor-recipient pairs studied. Technical performance criteria for chimerism analysis by high-throughput sequencing were suggested and evaluated. Both assays were found to be practical for use in a clinical diagnostics laboratory.

Evaluation of next-generation sequencing-based clonality analysis of T-cell receptor gamma gene rearrangements based on a new interpretation algorithm.

INTRODUCTION: T-cell receptor gene (TRG) rearrangement profiling is an essential component of the workup at diagnosis of T-cell malignancies. TRG amplification by polymerase chain reaction (PCR) and analysis by capillary electrophoresis (PCR-CE) is mostly widely used but is hampered by a subjective interpretation of its results and possible false-positive interpretation of clonality. Several studies evaluated the advantage of TRG rearrangement analysis by Next Generation Sequencing (TRG-NGS), however few have proposed an adequate data interpretation algorithm. METHODS: Eighty five fresh and 36 formalin-fixed paraffin embedded (FFPE) diagnostic samples suspected for a lymphoproliferative disorder were analyzed by PCR-CE and TRG NGS. Final clinical diagnosis was available for all fresh samples. Reproducibility, analytical specificity and sensitivity of the TRG NGS analysis was evaluated. RESULTS: We propose a new interpretation algorithm for TRG NGS data analysis. PCR-CE and TRG NGS showed identical results in 66/85 (78%) of fresh samples. Sensitivities to detect T-cell malignancies were comparable (96% versus 92%, respectively). The analysis of FFPE material was significantly more successful by TRG NGS (34/36 cases) in respect to PCR-CE (16/36 cases), most likely due to the small size of the amplicons. CONCLUSION: Assessment of T-cell clonality by TRG NGS has a significant added value in the diagnosis of T-cell disorders as an adjunct to PCR-CE, particularly in difficult to interpret cases or when analyzing FFPE samples.

Standardization of Somatic Variant Classifications in Solid and Haematological Tumours by a Two-Level Approach of Biological and Clinical Classes: An Initiative of the Belgian ComPerMed Expert Panel.

In most diagnostic laboratories, targeted next-generation sequencing (NGS) is currently the default assay for the detection of somatic variants in solid as well as haematological tumours. Independent of the method, the final outcome is a list of variants that differ from the human genome reference sequence of which some may relate to the establishment of the tumour in the patient. A critical point towards a uniform patient management is the assignment of the biological contribution of each variant to the malignancy and its subsequent clinical impact in a specific malignancy. These so-called biological and clinical classifications of somatic variants are currently not standardized and are vastly dependent on the subjective analysis of each laboratory. This subjectivity can thus result in a different classification and subsequent clinical interpretation of the same variant. Therefore, the ComPerMed panel of Belgian experts in cancer diagnostics set up a working group with the goal to harmonize the biological classification and clinical interpretation of somatic variants detected by NGS. This effort resulted in the establishment of a uniform, two-level classification workflow system that should enable high consistency in diagnosis, prognosis, treatment and follow-up of cancer patients. Variants are first classified into a tumour-independent biological five class system and subsequently in a four tier ACMG clinical classification. Here, we describe the ComPerMed workflow in detail including examples for each step of the pipeline. Moreover, this workflow can be implemented in variant classification software tools enabling automatic reporting of NGS data, independent of panel, method or analysis software.

The human alphaE-catenin gene CTNNA1: mutational analysis and rare occurrence of a truncated splice variant.

Abnormal expression of the alphaE-catenin protein, a component of the E-cadherin/catenin cell adhesion complex, is frequently observed in human cancer cells. An inverse correlation between alphaE-catenin expression and tumor malignancy can be of prognostic value. Mutations of the alphaE-catenin gene, CTNNA1, were described in several human cancer cell lines and were found to result in aberrant cell adhesion. We have developed a polymerase chain reaction/single-strand conformation polymorphism-based method for mutation analysis of this gene in human tumor DNA. This approach enabled us to identify several polymorphisms in a set of desmoid tumors, demonstrating that this method is suitable for alphaE-catenin mutational analysis. On the basis of our genomic characterization data, we found that the previously reported alternative splicing of the alphaE-catenin gene actually generates a frame-shift, resulting in a truncated alphaE-catenin protein. This finding is unlike the other alpha-catenin family members alphaN-catenin and vinculin, which show in-frame alternative inserts. Furthermore, real-time quantitative reverse transcriptase-PCR analysis did not reveal relevant expression levels of this alternatively spliced alphaE-catenin variant neither in any human tissue or cell line tested, nor at any mouse developmental stage tested. Thus, contrary to previous notions, alternative splicing with in-frame insertion nearby the C-terminal end of the protein is not a general feature for all members of the alpha-catenin/vinculin family.

A real-time polymerase chain reaction assay for rapid, sensitive, and specific quantification of the JAK2V617F mutation using a locked nucleic acid-modified oligonucleotide.

The JAK2V617F mutation has emerged as an essential molecular determinant of myeloproliferative neoplasms (MPNs). The aim of this study was to evaluate the analytical and clinical performances of a real-time PCR (qPCR) assay using a combination of hydrolysis probes and a wild-type blocking oligonucleotide, all containing locked nucleic acid (LNA) bases. Moreover, we validated a procedure for precise quantification of the JAK2V617F allele burden. We used DNA samples from patients suspected to suffer from MPN and dilutions of HEL cells, carrying the mutation, to compare the LNA-qPCR assay to two previously published methods. All assays detected the same 36 JAK2V617F positive patients of 116 suspected MPN diagnostic samples. No amplification of normal donor DNA was observed in the LNA-qPCR, and the assay was able to detect and reproducibly quantify as few as 0.4% of the JAK2V617F allele in wild-type alleles. Quantification of the JAK2V617F allele burden showed similar proportion levels among the different MPN entities as described by other groups. In conclusion, the LNA-qPCR is a rapid, robust, sensitive, and highly specific assay for quantitative JAK2V617F determination that can be easily implemented in clinical molecular diagnostic laboratories. Moreover, precise quantification allows determination of JAK2V617F burden at diagnosis as well as the evaluation of response to JAK2 inhibitors.