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J. Mol. Pathol., Volume 3, Issue 1 (March 2022) – 6 articles

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15 pages, 3404 KiB  
Article
Evaluation of the TruSight Tumor 170 Assay and Its Value in Clinical Diagnostics
by Carina Heydt, Roberto Pappesch, Katrin Stecker, Martin März and Sabine Merkelbach-Bruse
J. Mol. Pathol. 2022, 3(1), 53-67; https://doi.org/10.3390/jmp3010006 - 05 Feb 2022
Viewed by 3291
Abstract
Background: Parallel sequencing technologies have become integrated into clinical practice. This study evaluated the TruSight Tumor 170 assay for the simultaneous detection of somatic gene mutations (SNPs and indels), gene fusions and CNVs, and its implementation into routine diagnostics. Methods: Forty-four formalin-fixed, paraffin-embedded [...] Read more.
Background: Parallel sequencing technologies have become integrated into clinical practice. This study evaluated the TruSight Tumor 170 assay for the simultaneous detection of somatic gene mutations (SNPs and indels), gene fusions and CNVs, and its implementation into routine diagnostics. Methods: Forty-four formalin-fixed, paraffin-embedded tissue samples analyzed previously with validated methods were evaluated with the TruSight Tumor 170 assay (Illumina). For data analysis the TruSight Tumor 170 app, the BaseSpace Variant Interpreter (Illumina), and the Molecular Health Guide Software (Molecular Health) were used. Results: All somatic gene mutations were identified when covered by the assay. Two high-level MET amplifications were detected by CNV analysis. Focal MET amplifications with a copy number below 10 were not reliably detected at the DNA-level. Twenty-one of 31 fusions and splice variants were confirmed with the assay on the RNA-level. The remaining eight aberrations were incorrect by previous methods. In two cases, no splicing was observed. Conclusions: The TruSight Tumor 170 gives reliable results even if low DNA and RNA concentrations are applied in comparison to other methods and can be used in a routine workflow to detect somatic gene mutations, gene fusions, and splice variants. However, we were not able to detect most focal gene amplifications/deletions. Full article
(This article belongs to the Collection Feature Papers in Journal of Molecular Pathology)
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1 pages, 154 KiB  
Editorial
Acknowledgment to Reviewers of Journal of Molecular Pathology in 2021
by Journal of Molecular Pathology Editorial Office
J. Mol. Pathol. 2022, 3(1), 52; https://doi.org/10.3390/jmp3010005 - 29 Jan 2022
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Abstract
Rigorous peer-reviews are the basis of high-quality academic publishing [...] Full article
14 pages, 3071 KiB  
Article
Heterogeneity of Fecal Calprotectin Reflecting Generation of Neutrophil Extracellular Traps (NETs) in the Gut: New Immunoassays Are Available
by Magne K. Fagerhol and Jarle Rugtveit
J. Mol. Pathol. 2022, 3(1), 38-51; https://doi.org/10.3390/jmp3010004 - 21 Jan 2022
Cited by 4 | Viewed by 2704
Abstract
Background: We aimed at obtaining more information on the structure of fecal calprotectin (CP) as a basis for establishing improved quantitative assays and detection of Neutrophil Extracellular Traps (NETs) in stools. Commercial fecal CP assays produce different results, probably due to differences in [...] Read more.
Background: We aimed at obtaining more information on the structure of fecal calprotectin (CP) as a basis for establishing improved quantitative assays and detection of Neutrophil Extracellular Traps (NETs) in stools. Commercial fecal CP assays produce different results, probably due to differences in antibodies, extraction procedures, and standards used. In addition, the structure of fecal CP may be different from that in the standard so that rules for immunoassays are violated. We aimed at solving these problems by studying the structure of fecal CP and developing new antibodies and assay procedures including some for NETs in stools. Methods and Findings: Stool samples from children with abdominal symptoms were extracted by a conventional and a new procedure. Some extracts were run on anion exchange and size exclusion chromatography, and fractions were tested on ELISAs by use of ten new mouse monoclonal antibodies against the CP subunit S100A9. Hybrid ELISAs (named HELISA) were established using anti-DNA or anti-histones for coating of microwells, and enzyme labelled anti-CP was used for development. By ion exchange chromatography, five to ten fecal CP subfraction peaks differing in net electric charge were found, all of which contained the major chromatin components. The presence of DNA and histones followed calprotectin in the chromatographic fractions suggesting that NETs are generated in the gut lumen. The new CP monoclonals reacted very differently against the subfractions so that a mixture of them (called MiMo) must be used to obtain reliable assay values for fecal CP. A new method called FELISA was developed where standards and samples are applied directly in Nunc (Denmark) MaxiSorp plates, without any catching antibody. It takes advantage of the property of CP to bind strongly to the plastic in wells. This method has a higher sensitivity because it will detect CP molecules with only one antigenic epitope available. It will give more reliable estimates and more efficient selection of patients for complex diagnostic procedures. We also developed an alternative to the FELISA: a competitive ELISA where S100A9 coated in microwells will compete with CP in standards and samples for binding to a properly diluted HRP-anti-CP solution. In this method, the presence of other proteins in extraction or dilution buffers will not interfere. Using the HELISA, about 65% of the patients had detectable fecal NETs in concentrations between 150 and 1500 ng/mL; however, the values correlated poorly with CP values. Extraction of fecal samples with a simple buffer of TBS, and pH 5 with 5 mM EDTA, gave a yield of about 90%, while the yields of commercial kits are not specified or lie around 50%. A fecal CP standard will bring methods in accordance with the requirements for immunoassays that the structure of CP in the standard and sample must be the same. A mixture of fecal anion exchange fractions as a standard may be a solution to this problem. The principle worked in the first trial by giving the same values after storage of such a standard at 5C for four months. Conclusions: Fecal CP consists of at least five subfractions containing NETs or degradation products thereof. Commercial kits should not be accepted for clinical use unless it has been shown that they can detect all subfractions which may require the use of a mixture of monoclonals. The methods presented here can be used for such a quality control. The HELISA methods can be used for assays on NETs in stools and to study their possible pathogenic effects in the gut. Use of the FELISA and the S100A9 competitive method may give increased sensitivity, higher precision, and better selection of patients for more complex procedures. Full article
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14 pages, 642 KiB  
Review
Molecular Testing and Treatment Strategies in RET-Rearranged NSCLC Patients: Stay on Target to Look Forward
by Maria Lucia Reale, Valentina Bertaglia, Angela Listì, Silvia Novello and Francesco Passiglia
J. Mol. Pathol. 2022, 3(1), 24-37; https://doi.org/10.3390/jmp3010003 - 18 Jan 2022
Cited by 4 | Viewed by 3666
Abstract
RET alterations are recognized as key oncogenic drivers in different cancer types, including non-small cell lung cancer (NSCLC). Multikinase inhibitors (MKIs) with anti-RET activities resulted in variable efficacy with significant toxicities because of low target specificity. Selective RET kinase inhibitors, such as [...] Read more.
RET alterations are recognized as key oncogenic drivers in different cancer types, including non-small cell lung cancer (NSCLC). Multikinase inhibitors (MKIs) with anti-RET activities resulted in variable efficacy with significant toxicities because of low target specificity. Selective RET kinase inhibitors, such as pralsetinib and selepercatinib, demonstrated high efficacy and favorable tolerability in advanced RET-rearranged NSCLC patients, leading to their introduction in the clinical setting. Among the different approaches available for the identification of RET rearrangements, next-generation sequencing (NGS) assays present substantial advantages in terms of turnaround time and diagnostic accuracy, even if potentially limited by accessibility issues. The recent advent of novel effective targeted therapies raises several questions regarding the emergence of resistance mechanisms and the potential ways to prevent/overcome them. In this review, we discuss molecular testing and treatment strategies to manage RET fusion positive NSCLC patients with a focus on resistance mechanisms and future perspectives in this rapidly evolving scenario. Full article
(This article belongs to the Collection Feature Papers in Journal of Molecular Pathology)
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9 pages, 2814 KiB  
Brief Report
Tissue Microarray from Cell Block Material (cbTMA)—An Additional Shot for Cytology in the Predictive Pathology Era: The PD-L1 Experience
by Antonino Iaccarino, Gennaro Acanfora, Pasquale Pisapia, Umberto Malapelle, Claudio Bellevicine, Giancarlo Troncone and Elena Vigliar
J. Mol. Pathol. 2022, 3(1), 15-23; https://doi.org/10.3390/jmp3010002 - 12 Jan 2022
Viewed by 2729
Abstract
Generally, predictive biomarker tests are clinically validated on histological formalin-fixed, paraffin-embedded (FFPE) samples. In addition to FFPE samples, cytological samples have also emerged as a useful approach to detect predictive biomarkers. However, as of today, despite the promising results reported in the recent [...] Read more.
Generally, predictive biomarker tests are clinically validated on histological formalin-fixed, paraffin-embedded (FFPE) samples. In addition to FFPE samples, cytological samples have also emerged as a useful approach to detect predictive biomarkers. However, as of today, despite the promising results reported in the recent literature, their full implementation in routine clinical practice is still lagging owing to a lack of standardized preparatory protocols, challenging assessments of cyto-histological correlation, and variable inter-observer agreement. The aim of this report was to explore the possibility of implementing a large-scale validation of predictive biomarker testing on cytological material. To this aim, we evaluated the technical feasibility of PD-L1 assessment on a cell block (CB)-derived tissue microarray (cbTMA). Consecutive and unselected CBs prepared from metastatic lymph node fine-needle cytology (FNC) samples were retrospectively collected and used for TMA construction. PD-L1 immunohistochemistry (IHC) was carried out on cbTMA sections with the companion diagnostic kit SP263 assay. TMA contained 33 CB-derived cores. A total of 20 sections were hematoxylin and eosin (H&E) stained. Overall, 29 (88%) samples were visible at least in one H&E-stained slide. Four cases out of five sections stained with the SP263 assay (4/29, 13.8%) showed PD-L1 positivity in neoplastic and/or immune cells; remarkably, no unspecific background was observed. Although our study was based on a limited and non-selected series, our findings do provide proof of concept for the use of cbTMA in predictive biomarker testing on cytological material in large-scale post-clinical trial validation studies, multicenter studies, and quality control programs. Full article
(This article belongs to the Special Issue Molecular Cytopathology)
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14 pages, 1244 KiB  
Review
The Multifaceted Profile of Thyroid Disease in the Background of DICER1 Germline and Somatic Mutations: Then, Now and Future Perspectives
by Sule Canberk, Marcelo Correia, Ana Rita Lima, Massimo Bongiovanni, Manuel Sobrinho-Simões, Paula Soares and Valdemar Máximo
J. Mol. Pathol. 2022, 3(1), 1-14; https://doi.org/10.3390/jmp3010001 - 11 Jan 2022
Viewed by 3185
Abstract
DICER1 protein is a member of the ribonuclease (RNAse) III family with a key role in the biogenesis of microRNAs (miRNA) and in microRNA processing, potentially affecting gene regulation at the post-transcriptional level. The role of DICER1 and its relevance to thyroid cellular [...] Read more.
DICER1 protein is a member of the ribonuclease (RNAse) III family with a key role in the biogenesis of microRNAs (miRNA) and in microRNA processing, potentially affecting gene regulation at the post-transcriptional level. The role of DICER1 and its relevance to thyroid cellular processes and tumorigenesis have only recently been explored, following the acknowledgement that DICER1 germline and somatic changes can contribute not only to non-toxic multinodule goiter (MNG) lesions detected in individuals of affected families but also to a series of childhood tumours, including thyroid neoplasms, which can be identified from early infancy up until the decade of 40s. In a context of DICER1 germline gene mutation, thyroid lesions have recently been given importance, and they may represent either an index event within a syndromic context or the isolated event that may trigger a deeper and broader genomic analysis screening of individuals and their relatives, thereby preventing the consequences of a late diagnosis of malignancy. Within the syndromic context MNG is typically the most observed lesion. On the other hand, in a DICER1 somatic mutation context, malignant tumours are more common. In this review we describe the role of DICER protein, the genomic events that affect the DICER1 gene and their link to tumorigenesis as well as the frequency and pattern of benign and malignant thyroid lesions and the regulation of DICER1 within the thyroidal environment. Full article
(This article belongs to the Special Issue Molecular Pathology in Solid Tumors)
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