ZSCAN1 Autoantibodies Are Associated with Pediatric Paraneoplastic ROHHAD.

OBJECTIVE: Rapid-onset Obesity with Hypothalamic Dysfunction, Hypoventilation and Autonomic Dysregulation (ROHHAD), is a severe pediatric disorder of uncertain etiology resulting in hypothalamic dysfunction and frequent sudden death. Frequent co-occurrence of neuroblastic tumors have fueled suspicion of an autoimmune paraneoplastic neurological syndrome (PNS); however, specific anti-neural autoantibodies, a hallmark of PNS, have not been identified. Our objective is to determine if an autoimmune paraneoplastic etiology underlies ROHHAD. METHODS: Immunoglobulin G (IgG) from pediatric ROHHAD patients (n = 9), non-inflammatory individuals (n = 100) and relevant pediatric controls (n = 25) was screened using a programmable phage display of the human peptidome (PhIP-Seq). Putative ROHHAD-specific autoantibodies were orthogonally validated using radioactive ligand binding and cell-based assays. Expression of autoantibody targets in ROHHAD tumor and healthy brain tissue was assessed with immunohistochemistry and mass spectrometry, respectively. RESULTS: Autoantibodies to ZSCAN1 were detected in ROHHAD patients by PhIP-Seq and orthogonally validated in 7/9 ROHHAD patients and 0/125 controls using radioactive ligand binding and cell-based assays. Expression of ZSCAN1 in ROHHAD tumor and healthy human brain tissue was confirmed. INTERPRETATION: Our results support the notion that tumor-associated ROHHAD syndrome is a pediatric PNS, potentially initiated by an immune response to peripheral neuroblastic tumor. ZSCAN1 autoantibodies may aid in earlier, accurate diagnosis of ROHHAD syndrome, thus providing a means toward early detection and treatment. This work warrants follow-up studies to test sensitivity and specificity of a novel diagnostic test. Last, given the absence of the ZSCAN1 gene in rodents, our study highlights the value of human-based approaches for detecting novel PNS subtypes. ANN NEUROL 2022;92:279-291.

Exploratory proteomic analysis implicates the alternative complement cascade in primary CNS vasculitis.

OBJECTIVE: To identify molecular correlates of primary angiitis of the CNS (PACNS) through proteomic analysis of CSF from a biopsy-proven patient cohort. METHODS: Using mass spectrometry, we quantitatively compared the CSF proteome of patients with biopsy-proven PACNS (n = 8) to CSF from individuals with noninflammatory conditions (n = 11). Significantly enriched molecular pathways were identified with a gene ontology workflow, and high confidence hits within enriched pathways (fold change >1.5 and concordant Benjamini-Hochberg-adjusted p < 0.05 on DeSeq and t test) were identified as differentially regulated proteins. RESULTS: Compared to noninflammatory controls, 283 proteins were differentially expressed in the CSF of patients with PACNS, with significant enrichment of the complement cascade pathway (C4-binding protein, CD55, CD59, properdin, complement C5, complement C8, and complement C9) and neural cell adhesion molecules. A subset of clinically relevant findings were validated by Western blot and commercial ELISA. CONCLUSIONS: In this exploratory study, we found evidence of deregulation of the alternative complement cascade in CSF from biopsy-proven PACNS compared to noninflammatory controls. More specifically, several regulators of the C3 and C5 convertases and components of the terminal cascade were significantly altered. These preliminary findings shed light on a previously unappreciated similarity between PACNS and systemic vasculitides, especially anti-neutrophil cytoplasmic antibody-associated vasculitis. The therapeutic implications of this common biology and the diagnostic and therapeutic utility of individual proteomic findings warrant validation in larger cohorts.

Functional Assessment of 2,177 U.S. and International Drugs Identifies the Quinoline Nitroxoline as a Potent Amoebicidal Agent against the Pathogen Balamuthia mandrillaris.

Balamuthia mandrillaris is a pathogenic free-living amoeba that causes a rare but almost always fatal infection of the central nervous system called granulomatous amoebic encephalitis (GAE). Two distinct forms of B. mandrillaris-a proliferative trophozoite form and a nonproliferative cyst form, which is highly resistant to harsh physical and chemical conditions-have been isolated from environmental samples worldwide and are both observed in infected tissue. Patients suffering from GAE are typically treated with aggressive and prolonged multidrug regimens that often include the antimicrobial agents miltefosine and pentamidine isethionate. However, survival rates remain low, and studies evaluating the susceptibility of B. mandrillaris to these compounds and other potential therapeutics are limited. To address the need for more-effective treatments, we screened 2,177 clinically approved compounds for in vitro activity against B. mandrillaris The quinoline antibiotic nitroxoline (8-hydroxy-5-nitroquinoline), which has safely been used in humans to treat urinary tract infections, was identified as a lead compound. We show that nitroxoline inhibits both trophozoites and cysts at low micromolar concentrations, which are within a pharmacologically relevant range. We compared the in vitro efficacy of nitroxoline to that of drugs currently used in the standard of care for GAE and found that nitroxoline is the most potent and selective inhibitor of B. mandrillaris tested. Furthermore, we demonstrate that nitroxoline prevents B. mandrillaris-mediated destruction of host cells in cultured fibroblast and primary brain explant models also at pharmacologically relevant concentrations. Taken together, our findings indicate that nitroxoline is a promising candidate for repurposing as a novel treatment of B. mandrillaris infections.IMPORTANCEBalamuthia mandrillaris is responsible for hundreds of reported cases of amoebic encephalitis, the majority of which have been fatal. Despite being an exceptionally deadly pathogen, B. mandrillaris is understudied, leaving many open questions regarding epidemiology, diagnosis, and treatment. Due to the lack of effective drugs to fight B. mandrillaris infections, mortality rates remain high even for patients receiving intensive care. This report addresses the need for new treatment options through a drug repurposing screen to identify novel B. mandrillaris inhibitors. The most promising candidate identified was the quinoline antibiotic nitroxoline, which has a long history of safe use in humans. We show that nitroxoline kills B. mandrillaris at pharmacologically relevant concentrations and exhibits greater potency and selectivity than drugs commonly used in the current standard of care. The findings that we present demonstrate the potential of nitroxoline to be an important new tool in the treatment of life-threatening B. mandrillaris infections.

Can Radiologists Predict the Presence of Ductal Carcinoma In Situ and Invasive Breast Cancer?

OBJECTIVE: We hypothesize that radiologists' estimated percentage likelihood assessments for the presence of ductal carcinoma in situ (DCIS) and invasive cancer may predict histologic outcomes. MATERIALS AND METHODS: Two hundred fifty cases categorized as BI-RADS category 4 or 5 at four University of California Medical Centers were retrospectively reviewed by 10 academic radiologists with a range of 1-39 years in practice. Readers assigned BI-RADS category (1, 2, 3, 4a, 4b, 4c, or 5), estimated percentage likelihood of DCIS or invasive cancer (0-100%), and confidence rating (1 = low, 5 = high) after reviewing screening and diagnostic mammograms and ultrasound images. ROC curves were generated. RESULTS: Sixty-two percent (156/250) of lesions were benign and 38% (94/250) were malignant. There were 26 (10%) DCIS, 20 (8%) invasive cancers, and 48 (19%) cases of DCIS and invasive cancer. AUC values were 0.830-0.907 for invasive cancer and 0.731-0.837 for DCIS alone. Sensitivity of 82% (56/68), specificity of 84% (153/182), positive predictive value (PPV) of 66% (56/85), negative predictive value (NPV) of 93% (153/165), and accuracy of 84% ([56 + 153]/250) were calculated using an estimated percentage likelihood of 20% or higher as the prediction threshold for invasive cancer for the radiologist with the highest AUC (0.907; 95% CI, 0.864-0.951). Every 20% increase in the estimated percentage likelihood of invasive cancer increased the odds of invasive cancer by approximately two times (odds ratio, 2.4). For DCIS, using a threshold of 40% or higher, sensitivity of 81% (21/26), specificity of 79% (178/224), PPV of 31% (21/67), NPV of 97% (178/183), and accuracy of 80% ([21 + 178]/250) were calculated. Similarly, these values were calculated at thresholds of 2% or higher (BI-RADS category 4) and 95% or higher (BI-RADS category 5) to predict the presence of malignancy. CONCLUSION: Using likelihood estimates, radiologists may predict the presence of invasive cancer with fairly high accuracy. Radiologist-assigned estimated percentage likelihood can predict the presence of DCIS, albeit with lower accuracy than that for invasive cancer.

Inter-reader Variability in the Use of BI-RADS Descriptors for Suspicious Findings on Diagnostic Mammography: A Multi-institution Study of 10 Academic Radiologists.

RATIONALE AND OBJECTIVES: The study aimed to determine the inter-observer agreement among academic breast radiologists when using the Breast Imaging Reporting and Data System (BI-RADS) lesion descriptors for suspicious findings on diagnostic mammography. MATERIALS AND METHODS: Ten experienced academic breast radiologists across five medical centers independently reviewed 250 de-identified diagnostic mammographic cases that were previously assessed as BI-RADS 4 or 5 with subsequent pathologic diagnosis by percutaneous or surgical biopsy. Each radiologist assessed the presence of the following suspicious mammographic findings: mass, asymmetry (one view), focal asymmetry (two views), architectural distortion, and calcifications. For any identified calcifications, the radiologist also described the morphology and distribution. Inter-observer agreement was determined with Fleiss kappa statistic. Agreement was also calculated by years of experience. RESULTS: Of the 250 lesions, 156 (62%) were benign and 94 (38%) were malignant. Agreement among the 10 readers was strongest for recognizing the presence of calcifications (k = 0.82). There was substantial agreement among the readers for the identification of a mass (k = 0.67), whereas agreement was fair for the presence of a focal asymmetry (k = 0.21) or architectural distortion (k = 0.28). Agreement for asymmetries (one view) was slight (k = 0.09). Among the categories of calcification morphology and distribution, reader agreement was moderate (k = 0.51 and k = 0.60, respectively). Readers with more experience (10 or more years in clinical practice) did not demonstrate higher levels of agreement compared to those with less experience. CONCLUSIONS: Strength of agreement varies widely for different types of mammographic findings, even among dedicated academic breast radiologists. More subtle findings such as asymmetries and architectural distortion demonstrated the weakest agreement. Studies that seek to evaluate the predictive value of certain mammographic features for malignancy should take into consideration the inherent interpretive variability for these findings.

Zika virus cell tropism in the developing human brain and inhibition by azithromycin.

The rapid spread of Zika virus (ZIKV) and its association with abnormal brain development constitute a global health emergency. Congenital ZIKV infection produces a range of mild to severe pathologies, including microcephaly. To understand the pathophysiology of ZIKV infection, we used models of the developing brain that faithfully recapitulate the tissue architecture in early to midgestation. We identify the brain cell populations that are most susceptible to ZIKV infection in primary human tissue, provide evidence for a mechanism of viral entry, and show that a commonly used antibiotic protects cultured brain cells by reducing viral proliferation. In the brain, ZIKV preferentially infected neural stem cells, astrocytes, oligodendrocyte precursor cells, and microglia, whereas neurons were less susceptible to infection. These findings suggest mechanisms for microcephaly and other pathologic features of infants with congenital ZIKV infection that are not explained by neural stem cell infection alone, such as calcifications in the cortical plate. Furthermore, we find that blocking the glia-enriched putative viral entry receptor AXL reduced ZIKV infection of astrocytes in vitro, and genetic knockdown of AXL in a glial cell line nearly abolished infection. Finally, we evaluate 2,177 compounds, focusing on drugs safe in pregnancy. We show that the macrolide antibiotic azithromycin reduced viral proliferation and virus-induced cytopathic effects in glial cell lines and human astrocytes. Our characterization of infection in the developing human brain clarifies the pathogenesis of congenital ZIKV infection and provides the basis for investigating possible therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic.

"Score the Core" Web-based pathologist training tool improves the accuracy of breast cancer IHC4 scoring.

Hormone receptor status is an integral component of decision-making in breast cancer management. IHC4 score is an algorithm that combines hormone receptor, HER2, and Ki-67 status to provide a semiquantitative prognostic score for breast cancer. High accuracy and low interobserver variance are important to ensure the score is accurately calculated; however, few previous efforts have been made to measure or decrease interobserver variance. We developed a Web-based training tool, called "Score the Core" (STC) using tissue microarrays to train pathologists to visually score estrogen receptor (using the 300-point H score), progesterone receptor (percent positive), and Ki-67 (percent positive). STC used a reference score calculated from a reproducible manual counting method. Pathologists in the Athena Breast Health Network and pathology residents at associated institutions completed the exercise. By using STC, pathologists improved their estrogen receptor H score and progesterone receptor and Ki-67 proportion assessment and demonstrated a good correlation between pathologist and reference scores. In addition, we collected information about pathologist performance that allowed us to compare individual pathologists and measures of agreement. Pathologists' assessment of the proportion of positive cells was closer to the reference than their assessment of the relative intensity of positive cells. Careful training and assessment should be used to ensure the accuracy of breast biomarkers. This is particularly important as breast cancer diagnostics become increasingly quantitative and reproducible. Our training tool is a novel approach for pathologist training that can serve as an important component of ongoing quality assessment and can improve the accuracy of breast cancer prognostic biomarkers.

Evolving paradigm for imaging, diagnosis, and management of DCIS.

Our understanding of the biology of breast cancer has dramatically expanded over the past decade, revealing that breast cancer is a heterogeneous group of diseases. This new knowledge can generate insights to improve screening performance and the management of ductal carcinoma in situ. In this article, the authors review the current state of the science of breast cancer and tools that can be used to improve screening and risk assessment. They describe several opportunities to improve clinical screening: (1) radiologists interpreting mammograms should aim to differentiate between the risk for invasive cancer and ductal carcinoma in situ to better assess the time frame for disease progression and the need for and optimal timing of biopsy; (2) imaging features associated with low risk, slow-growing cancer versus high risk, fast-growing cancer should be better defined and taught; and (3) as we learn more about assessing an individual's risk for developing breast cancer, we should incorporate these factors into a strategy for personalized screening to maximize benefit and minimize harm.