Molecular profiling of primary endometrioid endometrial cancer and matched lung metastases: CTNNB1 mutation as a potential driver.

•Both primary endometrial cancers (ECs) and matched lung metastases shared a common ancestor with independent evolution at each site.•The two endometrioid ECs studied acquired additional mutations during the distant metastatic process.•Subclonal CTNNB1 hotspot mutations in the two primary ECs studied became clonal in the distant metastases.

Genomic and epigenomic basis of breast invasive lobular carcinomas lacking CDH1 genetic alterations.

CDH1 (E-cadherin) bi-allelic inactivation is the hallmark alteration of breast invasive lobular carcinoma (ILC), resulting in its discohesive phenotype. A subset of ILCs, however, lack CDH1 genetic/epigenetic inactivation, and their genetic underpinning is unknown. Through clinical targeted sequencing data reanalysis of 364 primary ILCs, we identified 25 ILCs lacking CDH1 bi-allelic genetic alterations. CDH1 promoter methylation was frequent (63%) in these cases. Targeted sequencing reanalysis revealed 3 ILCs harboring AXIN2 deleterious fusions (n = 2) or loss-of-function mutation (n = 1). Whole-genome sequencing of 3 cases lacking bi-allelic CDH1 genetic/epigenetic inactivation confirmed the AXIN2 mutation and no other cell-cell adhesion genetic alterations but revealed a new CTNND1 (p120) deleterious fusion. AXIN2 knock-out in MCF7 cells resulted in lobular-like features, including increased cellular migration and resistance to anoikis. Taken together, ILCs lacking CDH1 genetic/epigenetic alterations are driven by inactivating alterations in other cell adhesion genes (CTNND1 or AXIN2), endorsing a convergent phenotype in ILC.

Psychometric Evaluation of a Persian Version of Beliefs about Emotions Scale in Community and Clinical Samples.

Objective: This research is primarily conducted to determine the psychometric properties of the Beliefs about Emotions Scale (BES) in community and clinical samples. The BES is a scale measure used for evaluating individuals' beliefs in terms of how acceptable it is for them to experience and express their emotions. Method : This study was conducted on two separate samples. In the first part, 300 individuals were selected from a general sample in Tehran using the quota sampling method. For the second part of the study, we used purposive sampling to gather data from 119 patients suffering from Major Depressive Disorder (MDD) and 121 patients from Somatic Symptoms Disorder (SSD), whose disorders were diagnosed based on the DSM-5 diagnostic criteria. The BES structural validity was examined through Confirmatory Factor Analysis (CFA). Additionally, test-retest composite and internal consistency indices were explored to investigate the reliability of the BES score. Finally, the associations of the BES score with the Hospital Anxiety and Depression Scale (HADS), Young Schema Questionnaire (YSQ), Multidimensional Perfectionism Scale (MPS), Difficulties in Emotion Regulation Scale (DERS), and Emotion Regulation Questionnaire (ERQ) scores were highlighted to investigate the discriminant and convergent validity of the BES score. Results: According to CFAs, the one-factor model for the BES demonstrated a good fit with the data collected from both the clinical and community samples. The internal consistency (Cronbach's alpha) was satisfactory in the community sample (α = 0.84) and the clinical samples of SSD (α = 0.86) and MDD (α = 0.83). The community sample demonstrated high overall test-retest reliability (ICC = 0.93, P < 0.001; 95% CI: 0.89 - 0.95). In terms of convergent validity, the findings confirmed that in the MMD sample, there was a significant relationship between the BES and almost all measures (including Depression (r = 0.39, P < 0.01), Anxiety (r = 0.21, P < 0.05), Self-Sacrifice (r = 0.27, P < 0.01), MPS-total score (r = 0.22, P < 0.05), DERS total score (r = 0.50, P < 0.01), and Suppression (r = 0.38, P < 0.01). However, in the SSD group, this finding was not found. Conclusion: The results demonstrated that the Persian BES is a reliable and valid scale of maladaptive beliefs about emotions which could be implemented for both clinical and research aims.

Nonlobular Invasive Breast Carcinomas with Biallelic Pathogenic CDH1 Somatic Alterations: A Histologic, Immunophenotypic, and Genomic Characterization.

CDH1 encodes for E-cadherin, and its loss of function is the hallmark of invasive lobular carcinoma (ILC). Albeit vanishingly rare, biallelic CDH1 alterations may be found in nonlobular breast carcinomas (NL-BCs). We sought to determine the clinicopathologic characteristics and repertoire of genetic alterations of NL-BCs harboring CDH1 biallelic genetic alterations. Analysis of 5842 breast cancers (BCs) subjected to clinical tumor-normal sequencing with an FDA-cleared multigene panel was conducted to identify BCs with biallelic CDH1 pathogenic/likely pathogenic somatic mutations lacking lobular features. The genomic profiles of NL-BCs with CDH1 biallelic genetic alterations were compared with those of ILCs and invasive ductal carcinomas (IDCs), matched by clinicopathologic characteristics. Of the 896 CDH1-altered BCs, 889 samples were excluded based on the diagnosis of invasive mixed ductal/lobular carcinoma or ILC or the detection of monoallelic CDH1 alterations. Only 7 of the 5842 (0.11%) BCs harbored biallelic CDH1 alterations and lacked lobular features. Of these, 4/7 (57%) cases were ER-positive/HER2-negative, 1/7 (14%) was ER-positive/HER2-positive, and 2/7 (29%) were ER-negative/HER2-negative. In total, 5/7 (71%) were of Nottingham grade 2, and 2/7 (29%) were of grade 3. The NL-BCs with CDH1 biallelic genetic alterations included a mucinous carcinoma (n = 1), IDCs with focal nested growth (n = 2), IDC with solid papillary (n = 1) or apocrine (n = 2) features, and an IDC of no special type (NST; n = 1). E-cadherin expression, as detected by immunohistochemistry, was absent (3/5) or aberrant (discontinuous membranous/cytoplasmic/granular; 2/5). However, NL-BCs with CDH1 biallelic genetic alterations displayed recurrent genetic alterations, including TP53, PIK3CA (57%, 4/7; each), FGFR1, and NCOR1 (28%, 2/7, each) alterations. Compared with CDH1 wild-type IDC-NSTs, NL-BCs less frequently harbored GATA3 mutations (0% vs 47%, P = .03), but no significant differences were detected when compared with matched ILCs. Therefore, NL-BCs with CDH1 biallelic genetic alterations are vanishingly rare, predominantly comprise IDCs with special histologic features, and have genomic features akin to luminal B ER-positive BCs.

Genotype Distribution of Enterobius vermicularis Isolates from Northwest Provinces of Iran.

Background: Human infection with Enterobius vermicularis occurs worldwide. The most common clinical manifestation of a pinworm infection is an itchy anal region. This parasite is incidentally found in appendicitis. This study aims to characterize and genotype this parasite from different samples inferred by mt-DNA. Methods: Forty appendectomies for acute clinical appendicitis, 40 positive scotch-tape samples, and 10 adult females worm isolated from patients. Genetic differentiation, haplotype differences, and isolates population structure were analyzed based on the cytochrome c oxidase subunit I (cox1) gene. Results: It has been demonstrated that all isolations in the appendectomies specimens are similar, and the genetic difference divergence is seen in adult worm specimens. The neutral indices of the samples did not show a significant difference and show that there is no intra-specific and population distribution diversity. Conclusion: Our results show different haplotypes in the B type of E. vermicularis population and add new information about genotyping of these parasites in Iran. In comparison with other studies, intra-specific variation of this parasite from Iran was observed.

KIT genetic alterations in breast cancer.

AIMS: Activating somatic mutations or gene amplification of KIT result in constitutive activation of its receptor tyrosine kinase, which is targetable in various solid tumours. Here, we sought to investigate the presence of KIT genetic alterations in breast cancer (BC) and characterise the histological and genomic features of these tumours. METHODS: A retrospective analysis of 5,575 BCs previously subjected to targeted sequencing using the FDA-authorised Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Targets (MSK-IMPACT) assay was performed to identify BCs with KIT alterations. A histological assessment of KIT-altered BCs was conducted, and their repertoire of genetic alterations was compared with that of BCs lacking KIT genetic alterations, matched for age, histological type, oestrogen receptor/HER2 status and sample type. RESULTS: We identified 18 BCs (0.32%), including 9 primary and 9 metastatic BCs, with oncogenic/likely oncogenic genetic alterations affecting KIT, including activating somatic mutations (n=4) or gene amplification (n=14). All KIT-altered BCs were of high histological grade, although no distinctive histological features were observed. When compared with BCs lacking KIT genetic alterations, no distinctive genetic features were identified. In two metastatic KIT-altered BCs in which the matched primary BC had also been analysed by MSK-IMPACT, the KIT mutations were found to be restricted to the metastatic samples, suggesting that they were late events in the evolution of these cancers. CONCLUSIONS: KIT genetic alterations are vanishingly rare in BC. KIT-altered BCs are of high grade but lack distinctive histological features. Genetic alterations in KIT might be late events in the evolution and/or progression of BC.

Ovarian cancer mutational processes drive site-specific immune evasion.

High-grade serous ovarian cancer (HGSOC) is an archetypal cancer of genomic instability1-4 patterned by distinct mutational processes5,6, tumour heterogeneity7-9 and intraperitoneal spread7,8,10. Immunotherapies have had limited efficacy in HGSOC11-13, highlighting an unmet need to assess how mutational processes and the anatomical sites of tumour foci determine the immunological states of the tumour microenvironment. Here we carried out an integrative analysis of whole-genome sequencing, single-cell RNA sequencing, digital histopathology and multiplexed immunofluorescence of 160 tumour sites from 42 treatment-naive patients with HGSOC. Homologous recombination-deficient HRD-Dup (BRCA1 mutant-like) and HRD-Del (BRCA2 mutant-like) tumours harboured inflammatory signalling and ongoing immunoediting, reflected in loss of HLA diversity and tumour infiltration with highly differentiated dysfunctional CD8+ T cells. By contrast, foldback-inversion-bearing tumours exhibited elevated immunosuppressive TGFß signalling and immune exclusion, with predominantly naive/stem-like and memory T cells. Phenotypic state associations were specific to anatomical sites, highlighting compositional, topological and functional differences between adnexal tumours and distal peritoneal foci. Our findings implicate anatomical sites and mutational processes as determinants of evolutionary phenotypic divergence and immune resistance mechanisms in HGSOC. Our study provides a multi-omic cellular phenotype data substrate from which to develop and interpret future personalized immunotherapeutic approaches and early detection research.

Psychometric properties of the Persian version of the somatic symptom disorder-B Criteria Scale (SSD-12) in community and clinical samples.

OBJECTIVE: In the DSM-5's diagnostic criteria of somatic symptom disorders (SSD), the presence of psychological problems (i.e., excessive thoughts, feelings, or behaviors) is emphasized more than the absence of the medical causes of patients' bothersome symptoms. In this regard, the Somatic Symptom Disorder-B Criteria Scale (SSD-12) is a screening tool for assessing these psychological features in somatic symptom disorder. This study aimed to validate the Persian version of SSD-12 in the Iranian community (non-clinical) and clinical samples. METHODS: Data was gathered from 291 individuals in a community sample (aged 18 to 54, M-age = 36.62, SD = 10.56, 79.7% females) and from clinical setting, including 118 patients diagnosed with major depressive disorder (MDD, aged 18 to 60, M-age = 36.52, SD = 11.39, 75.8% females) and 120 patients diagnosed with somatic symptom disorders (aged 18 to 60, M-age = 35.17, SD = 8.77, 73.7% females). To assess the convergent validity of SSD-12 in the clinical samples, participants were asked to complete measures assessing anxiety, depression, somatic symptoms, health anxiety, and emotional regulation. RESULTS: Confirmatory Factor Analyses (CFAs) showed that the three-factor model of the SSD-12 reached acceptable fit in the community and clinical samples and yielded excellent internal consistency across the samples. Also, test-retest reliability analysis results were good in the community sample. Convergent validity could be shown in the clinical samples. A cut-off score greater than 14 was in the optimal state with a sensitivity of 70.83 and a specificity of 70.07. CONCLUSION: The current study provides evidence on the factor structure, reliability, and validity of the Persian SSD-12 in the Iranian community and clinical samples. A sum score of 14 can be recommended as the cut-off point. Further studies are needed to assess SSD-12 in different clinical populations and larger samples.

Recurrent WWTR1 S89W mutations and Hippo pathway deregulation in clear cell carcinomas of the cervix.

Clear cell carcinoma (CCC) of the cervix (cCCC) is a rare and aggressive type of human papillomavirus (HPV)-negative cervical cancer with limited effective treatment options for recurrent or metastatic disease. Historically, CCCs of the lower genital tract were associated with in utero diethylstilbestrol exposure; however, the genetic landscape of sporadic cCCCs remains unknown. Here we sought to define the molecular underpinning of cCCCs. Using a combination of whole-exome, targeted capture, and RNA-sequencing, we identified pathogenic genetic alterations in the Hippo signaling pathway in 50% (10/20) of cCCCs, including recurrent WWTR1 S89W somatic mutations in 40% (4/10) of the cases harboring mutations in the Hippo pathway. Irrespective of the presence or absence of Hippo pathway genetic alterations, however, all primary cCCCs analyzed in this study (n = 20) harbored features of Hippo pathway deregulation at the transcriptomic and protein levels. In vitro functional analysis revealed that expression of the WWTR1 S89W mutation leads to reduced binding of TAZ to 14-3-3, promoting constitutive nuclear translocation of TAZ and Hippo pathway repression. WWTR1 S89W expression was found to lead to acquisition of oncogenic behavior, including increased proliferation, migration, and colony formation in vitro as well as increased tumorigenesis in vivo, which could be reversed by targeted inhibition of the TAZ/YAP1 complex with verteporfin. Finally, xenografts expressing WWTR1 S89W displayed a shift in tumor phenotype, becoming more infiltrative as well as less differentiated, and were found to be composed of cells with conspicuous cytoplasmic clearing as compared to controls. Our results demonstrate that Hippo pathway alterations are likely drivers of cCCCs and likely contribute to the clear cell phenotype. Therapies targeting this pathway may constitute a new class of treatment for these rare, aggressive tumors. © 2022 The Pathological Society of Great Britain and Ireland.

Novel oxygen sensing mechanism in the spinal cord involved in cardiorespiratory responses to hypoxia.

As blood oxygenation decreases (hypoxemia), mammals mount cardiorespiratory responses, increasing oxygen to vital organs. The carotid bodies are the primary oxygen chemoreceptors for breathing, but sympathetic-mediated cardiovascular responses to hypoxia persist in their absence, suggesting additional high-fidelity oxygen sensors. We show that spinal thoracic sympathetic preganglionic neurons are excited by hypoxia and silenced by hyperoxia, independent of surrounding astrocytes. These spinal oxygen sensors (SOS) enhance sympatho-respiratory activity induced by CNS asphyxia-like stimuli, suggesting they bestow a life-or-death advantage. Our data suggest the SOS use a mechanism involving neuronal nitric oxide synthase 1 (NOS1) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). We propose NOS1 serves as an oxygen-dependent sink for NADPH in hyperoxia. In hypoxia, NADPH catabolism by NOS1 decreases, increasing availability of NADPH to NOX and launching reactive oxygen species-dependent processes, including transient receptor potential channel activation. Equipped with this mechanism, SOS are likely broadly important for physiological regulation in chronic disease, spinal cord injury, and cardiorespiratory crisis.

Pathogenesis of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) encompasses a heterogeneous group of fundamentally different diseases with different histologic, genomic, and immunologic profiles, which are aggregated under this term because of their lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression. Massively parallel sequencing and other omics technologies have demonstrated the level of heterogeneity in TNBCs and shed light into the pathogenesis of this therapeutically challenging entity in breast cancer. In this review, we discuss the histologic and molecular classifications of TNBC, the genomic alterations these different tumor types harbor, and the potential impact of these alterations on the pathogenesis of these tumors. We also explore the role of the tumor microenvironment in the biology of TNBCs and its potential impact on therapeutic response. Dissecting the biology and understanding the therapeutic dependencies of each TNBC subtype will be essential to delivering on the promise of precision medicine for patients with triple-negative disease.

ATM Germline-Mutated Gastroesophageal Junction Adenocarcinomas: Clinical Descriptors, Molecular Characteristics, and Potential Therapeutic Implications.

BACKGROUND: Gastroesophageal junction (GEJ) adenocarcinoma is a rare cancer associated with poor prognosis. The genetic factors conferring predisposition to GEJ adenocarcinoma have yet to be identified. METHODS: We analyzed germline testing results from 23 381 cancer patients undergoing tumor-normal sequencing, of which 312 individuals had GEJ adenocarcinoma. Genomic profiles and clinico-pathologic features were analyzed for the GEJ adenocarcinomas. Silencing of ATM and ATR was performed using validated short-interfering RNA species in GEJ, esophageal, and gastric adenocarcinoma cell lines. All statistical tests were 2-sided. RESULTS: Pathogenic or likely pathogenic ATM variants were identified in 18 of 312 patients (5.8%), and bi-allelic inactivation of ATM through loss of heterozygosity of the wild-type allele was detected in all (16 of 16) samples with sufficient tumor content. Germline ATM-mutated GEJ adenocarcinomas largely lacked somatic mutations in TP53, were more likely to harbor MDM2 amplification, and harbored statistically significantly fewer somatic single nucleotide variants (2.0 mutations/Mb vs 7.9 mutations/Mb; P < .001). A statistically significantly higher proportion of germline ATM-mutated than ATM-wild-type GEJ adenocarcinoma patients underwent a curative resection (10 [100%] vs 92 [86.8%], P = .04; Fisher's exact test.), A synthetic lethal interaction between short-interfering RNA silencing of ATM and ATR was observed in the models analyzed. CONCLUSIONS: Our results indicate that germline pathogenic variants in ATM drive oncogenesis in GEJ adenocarcinoma and might result in a distinct clinical phenotype. Given the high prevalence of germline ATM-mutated GEJ adenocarcinomas, genetic testing for individuals with GEJ adenocarcinomas may be considered to better inform prognostication, treatment decisions, and future cancer risk.

Cancer-Causative Mutations Occurring in Early Embryogenesis.

Mosaic mutations in normal tissues can occur early in embryogenesis and be associated with hereditary cancer syndromes when affecting cancer susceptibility genes (CSG). Their contribution to apparently sporadic cancers is currently unknown. Analysis of paired tumor/blood sequencing data of 35,310 patients with cancer revealed 36 pathogenic mosaic variants affecting CSGs, most of which were not detected by prior clinical genetic testing. These CSG mosaic variants were consistently detected at varying variant allelic fractions in microdissected normal tissues (n = 48) from distinct embryonic lineages in all individuals tested, indicating their early embryonic origin, likely prior to gastrulation, and likely asymmetrical propagation. Tumor-specific biallelic inactivation of the CSG affected by a mosaic variant was observed in 91.7% (33/36) of cases, and tumors displayed the hallmark pathologic and/or genomic features of inactivation of the respective CSGs, establishing a causal link between CSG mosaic variants arising in early embryogenesis and the development of apparently sporadic cancers. SIGNIFICANCE: Here, we demonstrate that mosaic variants in CSGs arising in early embryogenesis contribute to the oncogenesis of seemingly sporadic cancers. These variants can be systematically detected through the analysis of tumor/normal sequencing data, and their detection may affect therapeutic decisions as well as prophylactic measures for patients and their offspring. See related commentary by Liggett and Sankaran, p. 889. This article is highlighted in the In This Issue feature, p. 873.

Neuroendocrine tumours of the breast: a genomic comparison with mucinous breast cancers and neuroendocrine tumours of other anatomic sites.

AIMS: Breast neuroendocrine tumours (NETs) constitute a rare histologic subtype of oestrogen receptor (ER)-positive breast cancer, and their definition according to the WHO classification was revised in 2019. Breast NETs display histologic and transcriptomic similarities with mucinous breast carcinomas (MuBCs). Here, we sought to compare the repertoire of genetic alterations in breast NETs with MuBCs and NETs from other anatomic origins. METHODS: On histologic review applying the new WHO criteria, 18 breast tumours with neuroendocrine differentiation were reclassified as breast NETs (n=10) or other breast cancers with neuroendocrine differentiation (n=8). We reanalysed targeted sequencing or whole-exome sequencing data of breast NETs (n=10), MuBCs type A (n=12) and type B (n=11). RESULTS: Breast NETs and MuBCs were found to be genetically similar, harbouring a lower frequency of PIK3CA mutations, 1q gains and 16q losses than ER-positive/HER2-negative breast cancers. 3/10 breast NETs harboured the hallmark features of ER-positive disease (ie, PIK3CA mutations and concurrent 1q gains/16q losses). Breast NETs showed an enrichment of oncogenic/likely oncogenic mutations affecting transcription factors compared with common forms of ER-positive breast cancer and with pancreatic and pulmonary NETs. CONCLUSIONS: Breast NETs are heterogeneous and are characterised by an enrichment of mutations in transcription factors and likely constitute a spectrum of entities histologically and genomically related to MuBCs. While most breast NETs are distinct from ER-positive/HER2-negative IDC-NSTs, a subset of breast NETs appears to be genetically similar to common forms of ER-positive breast cancer, suggesting that some breast cancers may acquire neuroendocrine differentiation later in tumour evolution.

Comparability of laboratory-developed and commercial PD-L1 assays in non-small cell lung carcinoma.

PD-L1 expression in non-small cell lung cancer (NSCLC) is predictive of response to treatment with PD-1 and PD-L1 inhibitors. Different inhibitors have been developed with different PD-L1 assays, which use different PD-1 antibody clones on different immunohistochemistry platforms. Depending on instrument and reagent availability, laboratory-developed tests with cross-platform use of PD-L1 antibodies may have practical benefits over commercial assays. The 22C3 pharmDx Assay (referred to as 22C3 DAKO), the VENTANA PD-L1 SP263 Assay (referred to as SP263 VENTANA) and a lab-developed test using the 22C3 antibody on the VENTANA BenchMark ULTRA IHC/ISH system (referred to as 22C3 VENTANA) were performed on whole sections of 85 NSCLC surgical resections. All sections were independently scored by three pathologists using tumor proportion scores. Correlation coefficients for continuous scores in pairwise comparisons between assays ranged from 0.976 to 0.978. When using a 1% positivity threshold (dichotomous scores), the 22C3 DAKO assay and 22C3 VENTANA assays showed the greatest agreement (93% agreement, κ = 0.86, 95% CI 0.75-0.97), and the 22C3 DAKO and SP263 VENTANA assays tended to show slightly less agreement (84% agreement, κ = 0.66, 95% CI 0.50-0.82). When using a 50% positivity threshold (dichotomous scores), all pairwise comparisons showed similar agreement (96-99% agreement, κ = 0.89-0.97). Overall, there was no significant difference between assays at 1% or 50% thresholds (P = .77). These data are consistent with potential interchangeability of these assays, which may widen the scope of PD-L1 assays available to laboratories and reduce logistical barriers to testing.

Genetic diversity analysis of Blastocystis subtypes and their distribution among the domestic animals and pigeons in northwest of Iran.

Blastocystis is a unicellular, anaerobic, eukaryotic protist, a common parasite found in the intestinal tracts of animals and humans. During the last few years, the host fecal DNA analysis by nucleic acid-based method has led to significant advances in Blastocystis diagnostics and enabled subtypes (STs). The zoonotic transmission of Blastocystis to humans is not well understood, therefore the present study was conducted to identify Blastocystis subtypes in Iran from different animal hosts from northwest of Iran. A total of 427 fresh fecal specimens were collected from cattle, sheep, poultry and pigeon (40,150,132,105 respectively). To detect the Blastocystis sp., each fecal specimen was examined microscopically. Total DNA from the samples that were positive for Blastocystis sp. was isolated, and the barcoding region of the small subunit of ribosomal rRNA (18S rRNA) was amplified and sequenced. Subsequently, sequence analyses, genetic diversity indices and evolutionary relationships of Blastocystis subtype populations were carried out. In total, 14.98% of the analyzed samples were positive for Blastocystis sp. and the subtypes detected were ST3,7,10 and 14. Among these, the ST10 was the main subtype that was found only in the cattle, sheep and poultry and the zoonotic subtype ST3 was present only from cattle. Our study shows the presence of Blastocystis subtypes in the sheep in north west of Iran and also demonstrated that the genetic approaches are crucial to understand the host specify of subtypes and the mode of infection. The study suggests that the genetic approaches will help us to understand the host specificity of subtypes and their role in infection if they are obtained from human and animals from the same geographical locations. Therefore, it is important to study the zoonotic aspects of this parasite with large number of samples from different groups of animals and from different geographical locations.

Differential expression and prognostic relevance of autophagy-related markers ATG4B, GABARAP, and LC3B in breast cancer.

PURPOSE: Previous studies indicate that breast cancer molecular subtypes differ with respect to their dependency on autophagy, but our knowledge of the differential expression and prognostic significance of autophagy-related biomarkers in breast cancer is limited. METHODS: Immunohistochemistry (IHC) was performed on tissue microarrays from a large population of 3992 breast cancer patients divided into training and validation cohorts. Consensus staining scores were used to evaluate the expression levels of autophagy proteins LC3B, ATG4B, and GABARAP and determine the associations with clinicopathological variables and molecular biomarkers. Survival analyses were performed using the Kaplan-Meier function and Cox proportional hazards regression models. RESULTS: We found subtype-specific expression differences for ATG4B, with its expression lowest in basal-like breast cancer and highest in Luminal A, but there were no significant associations with patient prognosis. LC3B and GABARAP levels were highest in basal-like breast cancers, and high levels were associated with worse outcomes across all subtypes (DSS; GABARAP: HR 1.43, LC3B puncta: HR 1.43). High ATG4B levels were associated with ER, PR, and BCL2 positivity, while high LC3B and GABARAP levels were associated with ER, PR, and BCL2 negativity, as well as EGFR, HER2, HER3, CA-IX, PD-L1 positivity, and high Ki67 index (p < 0.05 for all associations). Exploratory multi-marker analysis indicated that the combination of ATG4B and GABARAP with LC3B could be useful for further stratifying patient outcomes. CONCLUSIONS: ATG4B levels varied across breast cancer subtypes but did not show prognostic significance. High LC3B expression and high GABARAP expression were both associated with poor prognosis and with clinicopathological characteristics of aggressive disease phenotypes in all breast cancer subtypes.

Immunohistochemical analysis of IDH2 R172 hotspot mutations in breast papillary neoplasms: applications in the diagnosis of tall cell carcinoma with reverse polarity.

Tall cell carcinoma with reverse polarity is a rare subtype of breast carcinoma with solid and papillary growth and nuclear features reminiscent of those of the tall cell variant of papillary thyroid carcinoma. These tumors harbor recurrent IDH2 R172 hotspot mutations or TET2 mutations, co-occurring with mutations in PI3K pathway genes. Diagnosis of tall cell carcinomas with reverse polarity is challenging in view of their rarity and the range of differential diagnosis. We sought to determine the sensitivity and specificity of IDH2 R172 immunohistochemistry for the detection of IDH2 R172 hotspot mutations in this entity. We evaluated 14 tall cell carcinomas with reverse polarity (ten excision and five core needle biopsy specimens), 13 intraductal papillomas, 16 solid papillary carcinomas, and 5 encapsulated papillary carcinomas by Sanger sequencing of the IDH2 R172 hotspot locus and of exons 9 and 20 of PIK3CA, and by immunohistochemistry using monoclonal antibodies (11C8B1) to the IDH2 R172S mutation. The 14 tall cell carcinomas with reverse polarity studied harbored IDH2 R172 hotspot mutations, which co-occurred with PIK3CA hotspot mutations in 50% of cases. None of the other papillary neoplasms analyzed displayed IDH2 R172 mutations, however PIK3CA hotspot mutations were detected in 54% of intraductal papillomas, 6% of solid papillary carcinomas, and 20% of encapsulated papillary carcinomas tested. Immunohistochemical analysis with anti-IDH2 R172S antibodies (11C8B1) detected IDH2 R172 mutated protein in 93% (14/15) of tall cell carcinomas with reverse polarity samples including excision (n = 9/10) and core needle biopsy specimens (n = 5), whereas the remaining papillary neoplasms (n = 34) were negative. Our findings demonstrate that immunohistochemical analysis of IDH2 R172 is highly sensitive and specific for the detection of IDH2 R172 hotspot mutations, and likely suitable as a diagnostic tool in the evaluation of excision and core needle biopsy material of tall cell carcinomas with reverse polarity.

Use of Programmed Death Ligand-1 (PD-L1) Staining to Separate Sarcomatoid Malignant Mesotheliomas From Benign Mesothelial Reactions.

CONTEXT.­: The separation of benign from malignant mesothelial proliferations is a difficult morphologic problem. Some mesotheliomas stain for programmed death ligand-1 (PD-L1). OBJECTIVE.­: To determine whether PD-L1 staining can separate mesotheliomas from reactive mesothelial proliferations (RMPs). DESIGN.­: We stained 2 tissue microarrays containing in toto 62 malignant mesotheliomas and 88 RMPs, using anti-PD-L1 antibody 22C3. Staining was graded by using an immunoreactive score encompassing intensity/distribution and was divided into negative, weak, moderate, and strong. Because PD-L1 staining can be heterogeneous, we also stained 10 whole sections of sarcomatoid/desmoplastic mesotheliomas (SMMs) and 10 whole sections of spindled RMPs in the form of organizing pleuritis, the major morphologic differential of SMM. These 20 cases were not on the tissue microarrays. RESULTS.­: RMPs showed generally negative, or occasionally weak staining in either the epithelioid or spindle cell compartments, whereas moderate to strong staining was seen in 10 of 14 SMMs but only in 6 of 48 epithelioid mesotheliomas (EMMs). The difference between SMM and RMP staining was statistically significant (P < .001), but between EMM and RMP was not significant. Whole section cases of organizing pleuritis showed no staining (N = 8) or weak staining (N = 2), whereas moderate/strong staining was seen in 9 of 10 whole sections of SMMs, a statistically significant difference (P = .02). There were no "hot spots" of RMP staining, suggesting that heterogeneous staining of RMPs is not a confounder. CONCLUSIONS.­: Strong diffuse PD-L1 staining using antibody 22C3 supports a diagnosis of SMM when the differential diagnosis is RMPs, but PD-L1 staining is not useful for separating EMMs from RMPs.

PACAP-PAC1 Receptor Activation Is Necessary for the Sympathetic Response to Acute Intermittent Hypoxia.

Repetitive hypoxia is a key feature of obstructive sleep apnoea (OSA), a condition characterized by intermittent airways obstruction. Patients with OSA present with persistent increases in sympathetic activity and commonly develop hypertension. The objectives of this study were to determine if the persistent increases in sympathetic nerve activity, known to be induced by acute intermittent hypoxia (AIH), are mediated through activation of the pituitary adenylate cyclase activating polypeptide (PACAP) signaling system. Here, we show that the excitatory neuropeptide PACAP, acting in the spinal cord, is important for generating the sympathetic response seen following AIH. Using PACAP receptor knockout mice, and pharmacological agents in Sprague Dawley rats, we measured blood pressure, heart rate, pH, PaCO2, and splanchnic sympathetic nerve activity, under anaesthesia, to demonstrate that the sympathetic response to AIH is mediated via the PAC1 receptor, in a cAMP-dependent manner. We also report that both intermittent microinjection of glutamate into the rostroventrolateral medulla (RVLM) and intermittent infusion of a sub-threshold dose of PACAP into the subarachnoid space can mimic the sympathetic response to AIH. All the sympathetic responses are independent of blood pressure, pH or PaCO2 changes. Our results show that in AIH, PACAP signaling in the spinal cord helps drive persistent increases in sympathetic nerve activity. This mechanism may be a precursor to the development of hypertension in conditions of chronic intermittent hypoxia, such as OSA.