Clinical Value of EGFR Copy Number Gain Determined by Amplicon-Based Targeted Next Generation Sequencing in Patients with EGFR-Mutated NSCLC

Background The clinical relevance of epidermal growth factor receptor (EGFR) copy number gain in patients with EGFR mutated advanced non-small cell lung cancer on first-line tyrosine kinase inhibitor treatment has not been fully elucidated. Objective We aimed to estimate EGFR copy number gain using amplicon-based next generation sequencing data and explored its prognostic value. Patients and Methods Next generation sequencing data were obtained for 1566 patients with non-small cell lung cancer. EGFR copy number gain was defined based on an increase in EGFR read counts relative to internal reference amplicons and normal controls in combination with a modified z-score >= 3.5. Clinical follow-up data were available for 60 patients treated with first-line EGFR-tyrosine kinase inhibitors. Results Specificity and sensitivity of next generation sequencing-based EGFR copy number estimations were above 90%. EGFR copy number gain was observed in 27.9% of EGFR mutant cases and in 7.4% of EGFR wild-type cases. EGFR gain was not associated with progression-free survival but showed a significant effect on overall survival with an adjusted hazard ratio of 3.14 (95% confidence interval 1.46-6.78, p = 0.003). Besides EGFR copy number gain, osimertinib in second or subsequent lines of treatment and the presence of T790M at relapse revealed significant effects in a multivariate analysis with adjusted hazard ratio of 0.43 (95% confidence interval 0.20-0.91, p = 0.028) and 0.24 (95% confidence interval 0.1-0.59, p = 0.001), respectively. Conclusions Pre-treatment EGFR copy number gain determined by amplicon-based next generation sequencing data predicts worse overall survival in EGFR-mutated patients treated with first-line EGFR-tyrosine kinase inhibitors. T790M at relapse and subsequent treatment with osimertinib predict longer overall survival

Translocation detection in lymphoma diagnosis by split-signal FISH: a standardised approach

Lymphomas originating from the lymphatic system comprise about 30 entities classified according to the World Health Organization (WHO). The histopathological diagnosis is generally considered difficult and prone to mistakes. Since non-random chromosomal translocations are specifically involved in different lymphoma entities, their detection will be increasingly important. Hence, a split-signal fluorescence in situ hybridisation (FISH) procedure would be helpful in discriminating the most difficult classifications. The Euro-FISH programme, a concerted action of nine European laboratories, has validated a robust, standardised protocol to improve the diagnostic approach on lymphoma entities. Therefore, 16 fluorescent probes and 10 WHO entities, supplemented with reactive cases, were selected. The results of the Euro-FISH programme show that all probes were correctly cytogenetically located, that the standardised protocol is robust, resulting in reliable results in approximately 90% of cases, and that the procedure could be implemented in every laboratory, bringing the relatively easy interpretation of split-signal probes within the reach of many pathology laboratories

Exon shuffling mimicked in cell culture

Undesired side products of DNA transfections are usually discarded. However, here, we show that such products may provide insight into mutational events that are also a major driving force in protein evolution. While studying the small heat-shock protein αA-crystallin, we transfected the hamster αA-crystallin gene into a mouse muscle cell line. One of the stable transfected cell lines expressed, in addition to the expected normal αA- and alternatively spliced αA(ins)-crystallins, two slightly larger, immunologically cross-reacting proteins. These proteins were found to be encoded by a mutant αA-crystallin gene with a large intragenic duplication, arisen by illegitimate recombination at two CCCAT homologies, ≈1.8 kilobases apart in the normal hamster αA-crystallin gene. As a consequence, a tandem-duplicated exon 3 sequence is present in the mature mRNA of this gene, resulting in a 41-residue repeat in the translated proteins. Cells expressing the elongated αA-crystallins have normal growth characteristics and the usual diffuse cytoplasmic distribution of immunoreactive αA-crystallin. Size-exclusion chromatography of cell extracts indicated that the mutant proteins are readily incorporated into the normal large water-soluble αA-crystallin complexes, showing that the insert does not disturb the integrity of these complexes. This viable αA-crystallin mutant thus mimics the origins and effects of exon duplication, which is a common consequence of exon shuffling in mammalian genome evolution

Double-staining chromogenic in situ hybridization as a useful alternative to split-signal fluorescence in situ hybridization in lymphoma diagnostics

Background: Malignant lymphomas are classified based on morphology, immunophenotype, genetics and clinical features. The pathological diagnosis is generally considered difficult and prone to mistakes. Since non-random chromosomal translocations are specifically involved in specific entities, their detection is an important adjunct for increasing the reliability of the diagnosis. Recently, split-signal fluorescence in situ hybridization has become available as a robust method to detect chromosomal breaks in paraffin-embedded formalin-fixed tissues. A bright field approach would bring this technology within the reach of every pathology laboratory. Design and Methods: Our study was initiated to determine the consistency between chromogenic in situ hybridization and fluorescence in situ hybridization, both using split-signal probes developed for the detection of chromosomal breaks. Five hundred and forty cases of 11 lymphoma entities and reactive, benign lymphoid tissues, collected from eight different pathology laboratories, placed on 15 fluorescence in situ hybridization pre-stained tissue microarray slides, were double stained for the chromogenic hybridization. For each core morphology and actual signal were compared to the original fluorescence hybridization results. In addition, hematoxylin background staining intensity and signal intensity of the double-staining chromogenic in situ hybridization procedure were analyzed. Results: With respect to the presence or absence of chromosomal breaks, 97% concordance was found between the results of the two techniques. Hematoxylin background staining intensity and signal intensity were found to correspond. The overall morphology after doublestaining chromogenic in situ hybridization had decreased compared to the initial morphology scored after split-signal fluorescence in situ hybridization staining. Conclusions: We conclude that double-staining chromogenic in situ hybridization is equally reliable as fluorescence in situ hybridization in detecting chromosomal breaks in lymphoid tissue. Although differences in morphology, hematoxylin staining and chromogenic signal intensity vary between the tumor entities none of the entities appeared more easy or difficult to score

Decision-making on maternal pertussis vaccination among women in a vaccine-hesitant religious group: Stages and needs

Introduction: As of December 2019, pregnant women in the Netherlands are offered pertussis vaccination to protect their newborn infant against pertussis infection. However, the manner in which pregnant women decide about this maternal pertussis vaccination is largely unknown. The aim of this study is to gain insight into the decision-making process regarding maternal pertussis vaccination, and to explore the related needs among the vaccine-hesitant subgroup of orthodox Protestant women. Methods: Charmaz’s grounded theory approach was used to develop a decision-making framework. To construct this framework we used an explorative multimethod approach in which in-depth interviews and online focus groups were supplemented by a literature search and research group meetings. This study was carried out in a hypothetical situation since the maternal pertussis vaccination had yet to be implemented in the Dutch immunisation programme at the time of the study. Results: Twenty-five orthodox Protestant women participated in an interview, an online focus group, or in both. The findings of this study resulted in a decision-making framework that included three stages of decision-making; an Orientation stage, a value-based Deliberation stage, and Final decision stage. The Orientation stage included the needs for decision-making categorised into Information needs and Conversation needs. Women indicated that -if they were to receive sufficient time for Orientation and Deliberation- they would be able to reach the stage of Final decision. Conclusion: The decision-making framework resulting from our findings can be used by health care professionals to provide women with information and consultation in the decision-making process. Future studies should investigate whether the stages of and needs for decision-making can be found across other vaccine-hesitant subgroups and vaccinations

The protein phosphatase 2A regulatory subunit PR70 is a gonosomal melanoma tumor suppressor gene

Male gender is independently and significantly associated with poor prognosis in melanoma of all clinical stages. The biological underpinnings of this sex difference remain largely unknown, but we hypothesized that gene expression from gonosomes (sex chromosomes) might play an important role. We demonstrate that loss of the inactivated X chromosome in melanomas arising in females is strongly associated with poor distant metastasis-free survival, suggesting a dosage benefit from two X chromosomes. The gonosomal protein phosphatase 2 regulatory subunit B, beta (PPP2R3B) gene is located on the pseudoautosomal region (PAR) of the X chromosome in females and the Y chromosome in males. We observed that, despite its location on the PAR that predicts equal dosage across genders, PPP2R3B expression was lower in males than in females and was independently correlated with poor clinical outcome. PPP2R3B codes for the PR70 protein, a regulatory substrate-recognizing subunit of protein phosphatase 2A. PR70 decreased melanoma growth by negatively interferingwith DNA replication and cell cycle progression through its role in stabilizing the cell division cycle 6 (CDC6)-chromatin licensing and DNA replication factor 1 (CDT1) interaction, which delays the firing of origins of DNA replication. Hence, PR70 functionally behaves as an X-linked tumor suppressor gene.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Array-Based Comparative Genomic Hybridization for the Genomewide Detection of Submicroscopic Chromosomal Abnormalities

Microdeletions and microduplications, not visible by routine chromosome analysis, are a major cause of human malformation and mental retardation. Novel high-resolution, whole-genome technologies can improve the diagnostic detection rate of these small chromosomal abnormalities. Array-based comparative genomic hybridization allows such a high-resolution screening by hybridizing differentially labeled test and reference DNAs to arrays consisting of thousands of genomic clones. In this study, we tested the diagnostic capacity of this technology using ∼3,500 flourescent in situ hybridization–verified clones selected to cover the genome with an average of 1 clone per megabase (Mb). The sensitivity and specificity of the technology were tested in normal-versus-normal control experiments and through the screening of patients with known microdeletion syndromes. Subsequently, a series of 20 cytogenetically normal patients with mental retardation and dysmorphisms suggestive of a chromosomal abnormality were analyzed. In this series, three microdeletions and two microduplications were identified and validated. Two of these genomic changes were identified also in one of the parents, indicating that these are large-scale genomic polymorphisms. Deletions and duplications as small as 1 Mb could be reliably detected by our approach. The percentage of false-positive results was reduced to a minimum by use of a dye-swap-replicate analysis, all but eliminating the need for laborious validation experiments and facilitating implementation in a routine diagnostic setting. This high-resolution assay will facilitate the identification of novel genes involved in human mental retardation and/or malformation syndromes and will provide insight into the flexibility and plasticity of the human genome