Variant classification changes over time in the clinical molecular diagnostic laboratory setting.

BACKGROUND: Variant classification in the setting of germline genetic testing is necessary for patients and their families to receive proper care. Variants are classified as pathogenic (P), likely pathogenic (LP), uncertain significance (VUS), likely benign (LB) and benign (B) using the standards and guidelines recommended by the American College of Medical Genetics and the Association for Molecular Pathology, with modifications for specific genes. As the literature continues to rapidly expand, and evidence continues to accumulate, prior classifications can be updated accordingly. In this study, we aim to characterise variant reclassifications in Ontario. METHODS: DNA samples from patients seen at hereditary cancer clinics in Ontario from January 2012 to April 2022 were submitted for testing. Patients met provincial eligibility criteria for testing for hereditary cancer syndromes or polycystic kidney disease. Reclassification events were determined to be within their broader category of significance (B to LB or vice versa, or P to LP or vice versa) or outside of their broader category as significance (ie, significant reclassifications from B/LB or VUS or P/LP, from P/LP to VUS or B/LB, or from VUS to any other category). RESULTS: Of the 8075 unique variants included in this study, 23.7% (1912) of variants were reassessed, and 7.2% (578) of variants were reclassified. Of these, 351 (60.7%) variants were reclassified outside of their broader category of significance. Overall, the final classification was significantly different for 336 (58.1%) variants. Importantly, most reclassified variants were downgraded to a more benign classification (n=245; 72.9%). Of note, most reclassified VUS was downgraded to B/LB (n=233; 84.7%). CONCLUSIONS: The likelihood for reclassification of variants on reassessment is high. Most reclassified variants were downgraded to a more benign classification. Our findings highlight the importance of periodic variant reassessment to ensure timely and appropriate care for patients and their families.

Expanding the spectrum of tyrosine kinase fusions in calcified chondroid mesenchymal neoplasms: Identification of a novel PDGFRA::USP8 gene fusion.

Calcified chondroid mesenchymal neoplasms represent a distinct, and recently recognized, spectrum of tumors. To date most cases have been reported to be characterized by FN1 gene fusions involving multiple potential tyrosine kinase partners. Following incidental identification of a tumor morphologically corresponding to calcified chondroid mesenchymal neoplasm, but with a PDGFRA::USP8 gene fusion, we undertook a retrospective review to identify and characterize additional such cases. A total of four tumors were identified. Each was multilobulated and composed of polygonal-epithelioid-stellate cells with a background of chondroid matrix containing distinctive patterns of calcification. Targeted RNA sequencing revealed an identical PDGFRA (exon 22)::USP8 (exon 5) gene fusion in each case. Subsequent immunohistochemical staining confirmed the presence of PDGFRα overexpression. In summary, we report a series of four tumors within the morphologic spectrum of calcified chondroid mesenchymal neoplasms. In contrast to prior reports, these tumors harbored a novel PDGFRA::USP8 gene fusion, rather than FN1 rearrangement. Our findings expand the molecular diversity of these neoplasms, and suggest they are united through activation of protein kinases.

Associations and outcomes of prenatally detected rhombencephalosynapsis.

OBJECTIVE: To describe the association between prenatal imaging and neurodevelopmental outcomes of fetuses with rhombencephalosynapsis (RES). STUDY DESIGN: Thirty-four pregnancies complicated by RES were identified from our institutional databases based on US and/or MRI findings. Genetic testing results were gathered. In cases of termination of pregnancy, we studied the association between prenatal imaging and neuropathologic findings. For those who opted for expectant management, comprehensive developmental assessments and postnatal MRI imaging were evaluated. RESULTS: Over one third of fetuses in our cohort had complete RES. Common intracranial anomalies identified were mesencephalosynapsis, aqueduct stenosis and diencephalosynapsis. The degree of RES was not associated with the frequency of additional central nervous system anomalies. MRI had a good correlation with neuropathologic findings with regard to the degree of RES, aqueduct stenosis and mesencephalosynapsis. Postmortem autopsy showed that one third of our cases had VACTERL-H and almost all of those had complete RES. All liveborn neonates(n = 6) had aqueduct stenosis requiring ventriculoperitoneal shunting within days of delivery (median 5 days). While a large proportion of prenatally suspected complete RES were found to have partial RES on postnatal imaging, prenatal diagnosis of aqueduct stenosis remained unchanged. All children that were at least 2 years old (n = 3) had global developmental delay. CONCLUSION: Prenatal assessment of the RES severity is challenging and may be unreliable. Nevertheless, postnatal prognosis is poor for both complete and partial RES. Associated aqueductal stenosis, can be reliably assessed prenatally and this may contribute to worse postnatal prognosis than the degree of RES.

White matter abnormalities and iron deposition in prenatal mucolipidosis IV- fetal imaging and pathology.

Mucolipidosis type IV (MLIV; OMIM 252,650) is an autosomal recessive lysosomal disorder caused by mutations in MCOLN1. MLIV causes psychomotor impairment and progressive vision loss. The major hallmarks of postnatal brain MRI are hypomyelination and thin corpus callosum. Human brain pathology data is scarce and demonstrates storage of various inclusion bodies in all neuronal cell types. The current study describes novel fetal brain MRI and neuropathology findings in a fetus with MLIV. Fetal MRI was performed at 32 and 35 weeks of gestation due to an older sibling with spastic quadriparesis, visual impairment and hypomyelination. Following abnormal fetal MRI results, the parents requested termination of pregnancy according to Israeli regulations. Fetal autopsy was performed after approval of the high committee for pregnancy termination. A genetic diagnosis of MLIV was established in the fetus and sibling. Sequential fetal brain MRI showed progressive curvilinear hypointensities on T2-weighted images in the frontal deep white matter and a thin corpus callosum. Fetal brain pathology exhibited a thin corpus callosum and hypercellular white matter composed of reactive astrocytes and microglia, multifocal white matter abnormalities with mineralized deposits, and numerous aggregates of microglia with focal intracellular iron accumulation most prominent in the frontal lobes. This is the first description in the literature of brain MRI and neuropathology in a fetus with MLIV. The findings demonstrate prenatal white matter involvement with significant activation of microglia and astrocytes and impaired iron metabolism.

Syncytial variant of nodular sclerosing Hodgkin lymphoma in children: A prognostic factor?

Nodular sclerosing Hodgkin lymphoma (HL) has an excellent prognosis in children. The syncytial variant (SV) of HL in adults represents a clinic pathologic entity with a worse outcome. We report the clinical features and the course of the disease of three children with refractory HL. The three patients with SV were analyzed in a retrospective multi-institutional study conducted in Israel in 51 children diagnosed with refractory or recurrent HL between 1997 and 2014. All the three children developed multiple recurrences soon after diagnosis. All three received at least three different chemotherapy combinations with autologous bone marrow transplantation for two patients, allogenic bone marrow transplantation in one, and immunotherapy in one. One patient died of disease, one is in complete response of the disease but developed a second metastatic malignancy, and one is alive without disease. This retrospective study shows that SV histology may be a prognostic factor for poor outcome in children diagnosed with HL.

Bifocal primary pancreatic Burkitt's lymphoma in a 4-year-old child.

Pancreatic tumors are uncommon in children and rarely result in biliary obstruction. Primary pancreatic Burkitt's lymphoma is an exceptionally rare entity with only a few cases described in the literature. We present a case of a bifocal primary pancreatic Burkitt's lymphoma in a 4-year-old child presenting with jaundice. © 2016 Wiley Periodicals, Inc. J Clin Ultrasound 45:171-174, 2017.

DEPAS: De-novo Pathology Semantic Masks using a Generative Model.

The integration of artificial intelligence (AI) into digital pathology has the potential to automate and improve various tasks, such as image analysis and diagnostic decision-making. Yet, the inherent variability of tissues, together with the need for image labeling, lead to biased datasets that limit the generalizability of algorithms trained on them. One of the emerging solutions for this challenge is synthetic histological images. Debiasing real datasets require not only generating photorealistic images but also the ability to control the cellular features within them. A common approach is to use generative methods that perform image translation between semantic masks that reflect prior knowledge of the tissue and a histological image. However, unlike other image domains, the complex structure of the tissue prevents a simple creation of histology semantic masks that are required as input to the image translation model, while semantic masks extracted from real images reduce the process's scalability. In this work, we introduce a scalable generative model, coined as DEPAS (De-novo Pathology Semantic Masks), that captures tissue structure and generates high-resolution semantic masks with state-of-the-art quality. We demonstrate the ability of DEPAS to generate realistic semantic maps of tissue for three types of organs: skin, prostate, and lung. Moreover, we show that these masks can be processed using a generative image translation model to produce photorealistic histology images of two types of cancer with two different types of staining techniques. Finally, we harness DEPAS to generate multi-label semantic masks that capture different cell types distributions and use them to produce histological images with on-demand cellular features. Overall, our work provides a state-of-the-art solution for the challenging task of generating synthetic histological images while controlling their semantic information in a scalable way.

Between Generating Noise and Generating Images: Noise in the Correct Frequency Improves the Quality of Synthetic Histopathology Images for Digital Pathology.

Artificial intelligence and machine learning techniques have the promise to revolutionize the field of digital pathology. However, these models demand considerable amounts of data, while the availability of unbiased training data is limited. Synthetic images can augment existing datasets, to improve and validate AI algorithms. Yet, controlling the exact distribution of cellular features within them is still challenging. One of the solutions is harnessing conditional generative adversarial networks that take a semantic mask as an input rather than a random noise. Unlike other domains, outlining the exact cellular structure of tissues is hard, and most of the input masks depict regions of cell types. This is also the case for non-small cell lung cancer, the most common type of lung cancer. Deciding whether a patient would receive immunotherapy depends on quantifying regions of stained cells. However, using polygon-based masks introduce inherent artifacts within the synthetic images - due to the mismatch between the polygon size and the single-cell size. In this work, we show that introducing random single-pixel noise with the appropriate spatial frequency into a polygon semantic mask can dramatically improve the quality of the synthetic images. We used our platform to generate synthetic images of immunohistochemistry-treated lung biopsies. We test the quality of the images using a three-fold validation procedure. First, we show that adding the appropriate noise frequency yields 87% of the similarity metrics improvement that is obtained by adding the actual single-cell features. Second, we show that the synthetic images pass the Turing test. Finally, we show that adding these synthetic images to the train set improves AI performance in terms of PD-L1 semantic segmentation performances. Our work suggests a simple and powerful approach for generating synthetic data on demand to unbias limited datasets to improve the algorithms' accuracy and validate their robustness.

Parents' Perceptions of Teachers' Authority and Parental Involvement: The Impact of Communality.

This study aimed to examine how the level of communality (communal affiliation) affects parents' perception of children attending public elementary schools, the concept of teacher authority, and the concept of parental involvement. The study population consisted of 300 parents living in various parts of Israel who agreed to complete a self-reporting anonymous questionnaire. The questionnaire comprised three subsections, two of which were based on previous studies: Scale of parents' perception of "parental involvement," which included 44 items, Cronbach's alpha value was 0.90.; The Scale of parents' perceptions of the concept of "Teacher's Authority," which included 25 items, Cronbach's alpha value was 0.79; and one was composed primarily for the current study, the Scale of parents' perception of "Communality Level" which included 19 items, Cronbach's alpha value was 0.88. The findings were analyzed using structural equation models (SEM). Applying these measures to the current study rendered the following results: RMSEA = 0.007, TLI = 0.995, CFI = 0.99, NFI = 0.904, df = 16, χ2 = 16.266, p = 0.435. Hence, the value of 1.01 ( x 2 d ⁢ f ) < 3, the TLI and CFI > 0.95. The research findings indicated that a high level of communality (communal affiliation) among parents predicted high levels of perceived teachers' authority (ß = 0.27) and parental involvement (ß = 0.30). By contrast, it was also found that living in the same residential characteristics as the teachers predicted low levels of both perceived teacher authority (ß = -0.18) and parental involvement (ß = -0.20). As regards the theoretical aspects, it adds a new layer to educational research about the variables that affect perceptions of teacher authority, an issue that has received little attention in the research literature. In terms of its practical applications, the model can help education systems in general and schools, in particular, to formulate policies and take steps to improve the ever-important relationship between the school and the parents. Furthermore, the model clarifies our understanding of and ways to strengthen the teacher's authority.

Intratumoral HLA-DR-/CD33+/CD11b+ Myeloid-Derived Suppressor Cells Predict Response to Neoadjuvant Chemoradiotherapy in Locally Advanced Rectal Cancer.

Capecitabine-based neoadjuvant chemoradiation therapy (nCRT) is currently the mainstay of treatment for locally advanced rectal cancer (LARC), prior to surgical tumor removal. While response to this treatment is partial, it carries significant risk of side effects. As of today, there is no accepted model to predict tumor response, and allow for patient stratification. The level of circulating Myeloid-derived suppressor cells (MDSCs), a subpopulation of early myeloid cells (EMCs), has been shown to correlate with prognosis and response to therapy in advanced colon cancer, but their role in LARC is not clear. We sought to study the effect of intratumoral and circulating levels of different EMCs subpopulations including MDSCs on response to nCRT. We analyzed tumor, normal mucosa, and peripheral blood samples from 25 LARC patients for their different EMCs subpopulation before and after nCRT, and correlated them with degree of pathologic response, as determined postoperatively. In addition, we compared LARC patient to 10 healthy donors and 6 metastatic patients. CD33+HLA-DR-CD16-CD11b+EMCs in the circulation of LARC patients were found to inhibit T-cell activation. Furthermore, elevated levels of CD33+HLA-DR- myeloid cells were found in the tumor relative to normal mucosa, but not in the circulation when compared to healthy subjects. Moreover, intratumoral, but not circulating levels of MDSCs correlated with clinical stage and response to therapy in patients treated with nCRT, with high levels of MDSCs significantly predicting poor response to nCRT. Importantly, therapy by itself, had significant differential effects on MDSC levels, leading to increased circulating MDSCs, concomitantly with decreasing intratumoral MDSCs. Our results suggest that high levels of intratumoral, but not circulating MDSCs may confer drug resistance due to immunomodulatory effects, and serve as a biomarker for patient stratification and decision-making prior to nCRT.

Monogenic Inflammatory Bowel Disease: It's Never Too Late to Make a Diagnosis.

Background: More than 50 different monogenic disorders have been identified as directly causing inflammatory bowel diseases, typically manifesting in the first years of life. We present the clinical course and immunological work-up of an adult patient who presented in adolescent years with an atypical gastrointestinal phenotype and was diagnosed more than two decades later with a monogenic disorder with important therapeutic implications. Methods: Whole exome sequencing was performed in a 37-years-old patient with a history of diarrhea since adolescence. Sanger sequencing was used to validate the suspected variant. Mass cytometry (CyTOF) and flow cytometry were conducted on peripheral blood mononuclear cells for deep immunophenotyping. Next-generation sequencing of the TCRB and IgH was performed for global immune repertoire analysis of circulating lymphocytes. Results: We identified a novel deleterious c.1455C>A (p.Y485X) mutation in LRBA. CyTOF studies demonstrated significant changes in immune landscape in the LRBA-deficient patient, including an increase in myeloid derived suppressor cells and double-negative T cells, decreased B cells, low ratio of naïve:memory T cells, and reduced capacity of T cells to secrete various cytokines following stimulation, including tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). In addition, this patient exhibited low frequency of regulatory T cells, with a reduction in their CTLA4 expression and interleukin (IL)-10 secretion. Finally, we show marked oligoclonal expansion of specific B- and T-cell clones in the peripheral blood of the LRBA-deficient patient. Conclusions: LRBA deficiency is characterized by marked immunological changes in innate and adaptive immune cells. This case highlights the importance of advanced genetic studies in patients with a unique phenotype, regardless of their age at presentation.

Concealed and Unconcealed Motives for Joining the Parent-Teacher Association: Mapping Sentence and Smallest Space Analysis.

Background and Subject: A Parent-Teacher Association (PTA) is an organization that enables parents to be involved in their children's schools. Participation in the PTA is one of many steps parents can take to ensure their involvement. Few if any studies have examined parents' concealed and unconcealed motives (UCM) for joining the PTA. Purpose/Hypotheses: The main purpose was to identify the structure of the concept Motives for Joining the PTA, so as to enhance our understanding of what motivates Israeli parents to join the PTA. The second purpose was to differentiate between parents' concealed and UCM. Method/Procedure: A self-report anonymous questionnaire, containing 40 items (30 items about aspects of being a member of a PTA and 10 items for reporting background variables), was administrated. Data were collected from a sample of 155 Israeli parents. The initial data processing stage involved EFA (Exploratory factor analysis) using SPSS software. Stage two was a Smallest Space Analysis (SSA), conducted using the Hebrew University Data Analysis Program (HUDAP). Results: EFA indicated three main factors. The internal consistency of the scores in the entire scale, measured using Cronbach's alpha coefficient, was 0.89. Data deployment on the SSA map exhibited both a polarized form (an angular form) and a radial form in a Radex configuration. The first layer (the polarized facet) was composed of three major motives related to self-serving altruistic ideological motives/ (SSAIM), self-serving altruistic pedagogical motives (SSAPM), and egoistic motives (EM). The second layer (the radial form) was composed of concealed motives, UCM, and politically correct-driven motives.

Case Report: Histoplasmosis: First Autochthonous Case from Israel.

We present an autochthonous Histoplasma infection in Israel. The patient presented with hoarseness and weight loss. Pathology findings and molecular tests confirmed the diagnosis, and the patient responded to antifungal therapy. Because the patient had an atypical presentation of histoplasmosis, it is likely that other more typical cases have gone unreported. To our knowledge, no other cases have been previously reported from Israel or the Middle East region.

Automated image analysis of placental villi and syncytial knots in histological sections.

INTRODUCTION: Delayed villous maturation and accelerated villous maturation diagnosed in histologic sections are morphologic manifestations of pathophysiological conditions. The inter-observer agreement among pathologists in assessing these conditions is moderate at best. We investigated whether automated image analysis of placental villi and syncytial knots could improve standardization in diagnosing these conditions. METHODS: Placentas of antepartum fetal death at or near term were diagnosed as normal, delayed or accelerated villous maturation. Histologic sections of 5 cases per group were photographed at ×10 magnification. Automated image analysis of villi and syncytial knots was performed, using ImageJ public domain software. Analysis of hundreds of histologic images was carried out within minutes on a personal computer, using macro commands. RESULTS: Compared to normal placentas, villi from delayed maturation were larger and fewer, with fewer and smaller syncytial knots. Villi from accelerated maturation were smaller. The data were further analyzed according to horizontal placental zones and groups of villous size. DISCUSSION: Normal placentas can be discriminated from placentas of delayed or accelerated villous maturation using automated image analysis. Automated image analysis of villi and syncytial knots is not equivalent to interpretation by the human eye. Each method has advantages and disadvantages in assessing the 2-dimensional histologic sections representing the complex, 3-dimensional villous tree. Image analysis of placentas provides quantitative data that might help in standardizing and grading of placentas for diagnostic and research purposes.