CHRFAM7A diversifies human immune adaption through Ca2+ signalling and actin cytoskeleton reorganization.

BACKGROUND: Human restricted genes contribute to human specific traits in the immune system. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of the α7 nicotinic acetylcholine receptor (α7 nAChR), the highest Ca2+ conductor of the ACh receptors implicated in innate immunity. Understanding the mechanism of how CHRFAM7A affects the immune system remains unexplored. METHODS: Two model systems are used, human induced pluripotent stem cells (iPSC) and human primary monocytes, to characterize α7 nAChR function, Ca2+ dynamics and decoders to elucidate the pathway from receptor to phenotype. FINDINGS: CHRFAM7A/α7 nAChR is identified as a hypomorphic receptor with mitigated Ca2+ influx and prolonged channel closed state. This shifts the Ca2+ reservoir from the extracellular space to the endoplasmic reticulum (ER) leading to Ca2+ dynamic changes. Ca2+ decoder small GTPase Rac1 is then activated, reorganizing the actin cytoskeleton. Observed actin mediated phenotypes include cellular adhesion, motility, phagocytosis and tissue mechanosensation. INTERPRETATION: CHRFAM7A introduces an additional, human specific, layer to Ca2+ regulation leading to an innate immune gain of function. Through the actin cytoskeleton it drives adaptation to the mechanical properties of the tissue environment leading to an ability to invade previously immune restricted niches. Human genetic diversity predicts profound translational significance as its understanding builds the foundation for successful treatments for infectious diseases, sepsis, and cancer metastasis. FUNDING: This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti) and in part by NIH grant R01HL163168 (Yongho Bae).

Translational implications of CHRFAM7A, an elusive human-restricted fusion gene.

Genes restricted to humans may contribute to human-specific traits and provide a different context for diseases. CHRFAM7A is a uniquely human fusion gene and a negative regulator of the α7 nicotinic acetylcholine receptor (α7 nAChR). The α7 nAChR has been a promising target for diseases affecting cognition and higher cortical functions, however, the treatment effect observed in animal models failed to translate into human clinical trials. As CHRFAM7A was not accounted for in preclinical drug screens it may have contributed to the translational gap. Understanding the complex genetic architecture of the locus, deciphering the functional impact of CHRFAM7A on α7 nAChR neurobiology and utilizing human-relevant models may offer novel approaches to explore α7 nAChR as a drug target.

Impact of an NCCN-Compliant Multidisciplinary Conference on Treatment Decisions for Localized Prostate Cancer.

BACKGROUND: We sought to investigate the impact of an NCCN-compliant multidisciplinary conference on treatment decisions of patients with localized prostate cancer. METHODS: A retrospective review of our quality assurance localized prostate cancer database was performed. All patients with localized prostate cancer who sought a second opinion at Roswell Park Comprehensive Cancer Center between 2009 and 2019 were presented to the multidisciplinary Localized Prostate Cancer Conference (LPCC) that includes urologists, radiation oncologists, pathologists, and patient advocates. Multivariable regression models were fit to evaluate variables associated with concordance between community recommendations, LPCC recommendations, and treatment received by patients. RESULTS: A total of 1,164 patients were identified, of whom 26% had NCCN very low-/low-risk, 27% had favorable intermediate-risk, 25% had unfavorable intermediate-risk, and 22% had high-/very high-risk prostate cancer. Pathology changed in 11% of patients after genitourinary pathologist review, which caused disease reclassification in 9%. Concordance between community and LPCC recommendations occurred in 78%, with lowest concordance for androgen deprivation therapy (21%) and radiotherapy (53%). Concordance between community recommendations and treatment received occurred in 65%, with lowest concordance for androgen deprivation therapy and radiotherapy; among those who were recommended radiotherapy as the only option by their community urologist, only 26% received it. Concordance between LPCC recommendations and treatment received occurred in 92%. CONCLUSIONS: Community recommendations differed from the multidisciplinary NCCN-compliant recommendations in 22% of patients, primarily for radiotherapy. Multidisciplinary recommendations matched the treatment received in 92% of patients compared with 65% for community recommendations.

Syngeneic model of carcinogen-induced tumor mimics basal/squamous, stromal-rich, and neuroendocrine molecular and immunological features of muscle-invasive bladder cancer.

Introduction: Bladder cancer is a heterogenous disease and the emerging knowledge on molecular classification of bladder tumors may impact treatment decisions based on molecular subtype. Pre-clinical models representing each subtype are needed to test novel therapies. Carcinogen-induced bladder cancer models represent heterogeneous, immune-competent, pre-clinical testing options with many features found in the human disease. Methods: Invasive bladder tumors were induced in C57BL/6 mice when continuously exposed to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in the drinking water. Tumors were excised and serially passed by subcutaneous implantation into sex-matched syngeneic C57BL/6 hosts. Eight lines were named BBN-induced Urothelium Roswell Park (BURP) tumor lines. BURP lines were characterized by applying consensus molecular classification to RNA expression, histopathology, and immune profiles by CIBERSORT. Two lines were further characterized for cisplatin response. Results: Eight BURP tumor lines were established with 3 male and 3 female BURP tumor lines, having the basal/squamous (BaSq) molecular phenotype and morphology. BURP-16SR was established from a male mouse and has a stromal-rich (SR) molecular phenotype and a sarcomatoid carcinoma morphology. BURP-19NE was established from a male mouse and has a neuroendocrine (NE)-like molecular phenotype and poorly differentiated morphology. The established BURP tumor lines have unique immune profiles with fewer immune infiltrates compared to their originating BBN-induced tumors. The immune profiles of the BURP tumor lines capture some of the features observed in the molecular classifications of human bladder cancer. BURP-16SR growth was inhibited by cisplatin treatment, while BURP-24BaSq did not respond to cisplatin. Discussion: The BURP lines represent several molecular classifications, including basal/squamous, stroma-rich, and NE-like. The stroma-rich (BURP-16SR) and NE-like (BURP-19NE) represent unique immunocompetent models that can be used to test novel treatments in these less common bladder cancer subtypes. Six basal/squamous tumor lines were established from both male and female mice. Overall, the BURP tumor lines have less heterogeneity than the carcinogen-induced tumors and can be used to evaluate treatment response without the confounding mixed response often observed in heterogeneous tumors. Additionally, basal/squamous tumor lines were established and maintained in both male and female mice, thereby allowing these tumor lines to be used to compare differential treatment responses between sexes.

CHRFAM7A: A human specific fusion gene, accounts for the translational gap for cholinergic strategies in Alzheimer's disease.

BACKGROUND: Cholinergic neuronal loss is one of the hallmarks of AD related neurodegeneration; however, preclinical promise of α7 nAChR drugs failed to translate into humans. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of α7 nAChR and was unaccounted for in preclinical models. METHODS: Molecular methods: Function of CHRFAM7A alleles was studied in vitro in two disease relevant phenotypic readouts: electrophysiology and Aß uptake. Genome edited human induced pluripotent stem cells (iPSC) were used as a model system with the human context. Double blind pharmacogenetic study: We performed double-blind pharmacogenetic analysis on the effect of AChEI therapy based on CHRFAM7A carrier status in two paradigms: response to drug initiation and DMT effect. Mini Mental Status Examination (MMSE) was used as outcome measure. Change in MMSE score from baseline was compared by 2-tailed T-test. Longitudinal analysis of clinical outcome (MMSE) was performed using a fitted general linear model, based on an assumed autoregressive covariance structure. Model independent variables included age, sex, and medication regimen at the time of the first utilized outcome measure (AChEI alone or AChEI plus memantine), APOE4 carrier status (0, 1 or 2 alleles as categorical variables) and CHRFAM7A genotype. FINDINGS: The direct and inverted alleles have distinct phenotypes. Functional CHRFAM7A allele classifies the population as 25% non-carriers and 75% carriers. Induced pluripotent stem cell (iPSC) models α7 nAChR mediated Aß neurotoxicity. Pharmacological readout translates into both first exposure (p = 0.037) and disease modifying effect (p = 0.0048) in two double blind pharmacogenetic studies. INTERPRETATION: CHRFAM7A accounts for the translational gap in cholinergic strategies in AD. Clinical trials not accounting for this uniquely human genetic factor may have rejected drug candidates that would benefit 25% of AD. Reanalyses of the completed trials using this pharmacogenetic paradigm may identify effective therapy.

Development and Initial Testing of a Modified UroVysion-Based Fluorescence In Situ Hybridization Score for Prediction of Progression in Bladder Cancer.

OBJECTIVES: Our aim was to predict progression of non-muscle-invasive bladder urothelial carcinomas (NMIUCs) into muscle-invasive disease by assessing cytogenetic abnormality of tumors with a new UroVysion scoring system. METHODS: Seventy-five bladder cancer cases (including 57 NMIUCs) were classified according to the quantitatively assessed degree of UroVysion-detected chromosomal abnormalities into urine fluorescence in situ hybridization score (UFS) groups: UFS I, II, and III. Cox time-to-event, Kaplan-Meier, and C-statistics analyses were performed. RESULTS: UFS proved to be an independent prognostic factor of progression-free survival (PFS) and time to progression (TTP). NMIUCs with UFS III had a 34.05-fold increased hazard for progression to muscle-invasive cancer (TTP; 95% confidence interval, 5.841-198.5; P < .001) in comparison with UFS I to II cases. The addition of UFS to conventional risk scores increased the C-index for PFS and TTP. CONCLUSIONS: UFS can indicate an increased risk for progression into muscle-invasive disease in patients with NMIUC and improves prognostic accuracy of the current clinical risk assessment systems.

iPSC model of CHRFAM7A effect on α7 nicotinic acetylcholine receptor function in the human context.

The α7 nicotinic acetylcholine receptor (α7nAChR) has been a promising target for diseases affecting cognition and higher cortical functions; however, the effect observed in animal models failed to translate into human clinical trials identifying a translational gap. CHRFAM7A is a human-specific fusion gene with properties that enable incorporation into the α7nAChR and, being human specific, CHRFAM7A effect was not accounted for in preclinical studies. We hypothesized that CHRFAM7A may account for this translational gap and understanding its function may offer novel insights when exploring α7nAChR as a drug target. CHRFAM7A is present in different copy number variations (CNV) in the human genome with high frequency. To study the functional consequences of the presence of the CHRFAM7A, two induced pluripotent stem cell (iPSC) lines (0 copy and 1 copy direct) were developed. The 0 copy line was rescued with CHRFAM7A transfection to control for genetic heterogeneity. As readouts for genotype-phenotype correlation, α7nAChR synaptic transmission and amyloid beta 1-42 (Aß1-42) uptake were tested. Synaptic transmission in the presence of CHRFAM7A demonstrated that PNU-modulated desensitization of α7nAChR currents increased as a function of CHRFAM7A dosage. CHRFAM7A mitigated the dose response of Aß1-42 uptake suggesting a protective effect beyond physiological concentrations. Furthermore, in the presence of CHRFAM7A Aß1-42 uptake activated neuronal interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) without activating the canonical inflammasome pathway. Lead optimization may identify more potent molecules when the screen has a model harboring CHRFAM7A. Incorporating pharmacogenetics into clinical trials may enhance signals in efficacy measures.

Mucinous Cystadenoma of the Urachus and Review of Current Classification of Urachal Mucinous Cystic Neoplasms.

Urachal neoplasms are uncommon and represent a minor portion of bladder tumors. According to the recently updated World Health Organization classification (2016), these tumors are classified as adenomas, adenocarcinomas, nonglandular neoplasms, and mixed carcinomas. The mucinous cystic neoplasms represent a small percentage of urachal tumors with morphologic spectrum ranging from benign mucinous cystadenoma to borderline mucinous cystic tumor of low malignant potential and to malignant mucinous cystadenocarcinoma. Benign urachal mucinous cystic adenomas are exceedingly rare, and only a few cases have been reported in the literature to date. The goal of this review is to summarize the clinical features, histopathologic characteristics, treatment, and prognosis of urachal mucinous cystadenoma in light of differentiating them from mucinous cystic tumor of low malignant potential and mucinous cystadenocarcinoma.

A novel treatment approach prolonging survival in an uncommon metastatic primary bladder adenocarcinoma.

Primary bladder adenocarcinoma (PBA) is an epithelial malignancy with pure glandular differentiation, without evidence of typical urothelial (transitional cell) carcinoma. PBA is rare, accounting for 0.5%-2% of all malignant bladder neoplasms, and it is seen more frequently in men than in women and is commonly diagnosed in the sixth decade of life.¹â»³ Clinical presentation includes hematuria and symptoms of bladder irritation.² PBA is common in schistosomiasis-endemic regions and among patients with congenital bladder exstrophy (ectopia vesicae); it mostly arises in the trigone and posterior bladder wall.4 In contrast, urachal adenocarcinomas arise within urachal remnants (residual tissues from the embryonic allantoic stalk connecting the umbilicus and bladder), close to the dome and anterior wall of the bladder. Morphologically, PBA is classifed into enteric and nonenteric types, which includes mucinous, signetring cell variant, clear-cell type, hepatoid, and mixed forms.² Currently, there is no standard of care in the management of PBA. We present the case of a patient with metastatic PBA with intestinal differentiation and wild-type KRAS, who was treated with colorectal cancer regimens.

Clinical significance of prospectively assigned Gleason tertiary pattern 4 in contemporary Gleason score 3+3=6 prostate cancer.

OBJECTIVE: To determine the oncologic impact of prospectively assigned tertiary pattern 4 in contemporary Gleason score (GS) 3 + 3 = 6 radical prostatectomy (RP) specimens. PATIENTS AND METHODS: Oncologic outcomes were retrospectively reviewed for 720 consecutive patients from a single National Comprehensive Cancer Network (NCCN) center with at least 6 months follow-up after RP for GS3 + 3 = 6 (GS6, N = 222), GS6 with tertiary pattern 4 (GS6t4, N = 62), or GS3 + 4 = 7 (N = 436) prostate cancer, as prospectively graded since 2006 using the 2005 International Society of Urologic Pathologists criteria. Preoperative NCCN risk category, RP pathology, progression-free survival (PFS) and metastasis-free survival (MFS) were compared among the GS6, GS6t4, and GS3 + 4 = 7 groups using χ(2) , Kaplan-Meier, and log-rank analyses. RESULTS: The incidence of low NCCN preoperative risk classification for GS6t4 patients (63%) was less than that for GS6 patients (77%) while greater than that for GS3 + 4 = 7 patients (30%, P < 0.001). GS6t4 patients had RP pathologic features which were intermediate in risk between that of GS6 and GS3 + 4 = 7 based on extraprostatic extension (27% vs. 6% vs. 31%, respectively, P < 0.001) and mean percentage of prostate gland involvement (13% vs. 10% vs. 16%, respectively, P < 0.001). With a mean overall follow-up of 42 months, PFS for GS6t4 patients (5-year 85%) was intermediate between that of GS6 (5-year 93%) and GS3 + 4 = 7 (5-year 76%) patients (P < 0.001). The 5-year MFS rate was 100% for GS6 and GS6t4 patients compared to 97% for GS3 + 4 = 7 patients (P = 0.07). CONCLUSIONS: This study provides the longest follow-up to date for RP patients with prospectively assigned GS6t4 and supports a risk for adverse RP pathology and postoperative disease progression that is intermediate between GS6 and GS3 + 4 = 7. Whether a tertiary pattern 4 in GS6 disease increases the risk of metastasis is uncertain and requires longer term study. Given favorable oncologic outcomes, less stringent postoperative surveillance for both GS6 and GS6t4 patients may be warranted.

Radiation-induced Chondrosarcoma of the Bladder. Case Report and Review of Literature.

BACKGROUND: Chondrosarcoma of the bladder is an extremely rare disease. Only five previously described cases are known in the medical literature. PATIENTS AND METHODS: We present a chondrosarcoma developed 19 years after radiation treatment in a 73-year-old patient. A literature search of articles published from 1984 to 2014 was performed. RESULTS: This is the first reported case of post-radiation bladder chondrosarcoma. We compared the clinicopathological features of the previously reported cases and reviewed the medical literature of the bladder sarcomas and post-radiation sarcomas. CONCLUSION: The primary treatment for bladder mesenchymal neoplasms is surgical, preferably radical cystectomy with or without chemotherapy. Positive surgical margin is one of the most important factors negatively affecting disease-specific, recurrence-free and overall survival rates.

A case of unusual mast cell response with interstitial cystitis-like symptoms to neoadjuvant chemotherapy for muscle-invasive transitional cell carcinoma of the bladder.

Randomized trials support the use of neoadjuvant chemotherapy in muscle-invasive bladder cancer based on a noted survival advantage, which appeared to be strongly related to downstaging of the cancer to pT0 (complete pathologic response). This report presents a case of an unusual mast cell response along with bladder wall thickening after neoadjuvant chemotherapy. However, the final cystectomy specimen did not reveal any residual tumor (pT0). The authors hypothesize that neoadjuvant chemotherapy could have caused the diffuse mast cell response, and that this profound inflammatory response can be used as a biomarker of complete response to chemotherapy.

Gastrointestinal histoplasmosis in a hepatitis C-infected individual.

Gastrointestinal histoplasmosis is a rare manifestation of this fungal infection, typically identified in immunocompromised patients, such as those with HIV/AIDS. Here, we report a case of disseminated histoplasmosis with gastrointestinal involvement in a Hepatitis C-infected patient. The fungal agent was confirmed to be Histoplasma capsulatum by a DNA probe assay performed on a bone marrow sample. We propose that this fungal disease should be kept on the differential of patients infected with the Hepatitis C virus, as it has been reported to have numerous damaging effects on the adaptive immune system.

Automated signal pattern evaluation of a bladder cancer specific multiprobe-fish assay applying a user-trainable workstation.

OBJECTIVE: Signal pattern enumeration of Urovysion Fluorescence in Situ Hybridization test is tedious and requires great experience. Our aim was to eliminate human interaction by automating the process, using an adoptable, automated image acquisition, and analysis system. METHODS: For extensive analytical analysis control, cell populations were used, while preliminary clinical study was performed on 21 patients with clinical suspicion for bladder cancer. All investigations were carried out using an automated user-trainable workstation (Metafer4-Metacyte). RESULTS: The system identified nuclei with a specificity and sensitivity of 92.7 and 96.6%, respectively, while signal detection accuracy was 81.1% on average. Both analytical and diagnostic accuracy of automated analysis was comparable to manual approach (94.8 and 71% vs. 97.9 and 76%, respectively), but classification accuracy increased with degree of polysomy, thus diagnostic sensitivity in low grade, low stage cases was poor. CONCLUSION: It is possible to automate signal enumeration of Urovysion using a user-trainable system, and achieve efficiency comparable to manual analysis. Previously introduced automated immunophenotypic targeting should further increase diagnostic sensitivity, while resulting in a comprehensively automated method. However, the problem of reduced detection accuracy in cases featured with low polysomy is likely to remain a great challenge of automated signal enumeration.

Urovysion: Considerations on modifying current evaluation scheme, including immunophenotypic targeting and locally set, statistically derived diagnostic criteria.

Urovysion multitarget fluorescence in situ hybridization (FISH) assay is a promising tool for detection of bladder cancer, however, there is still no consensus regarding abnormal signal pattern and cut-off level, and the recommended targeting carries limitations similar to urine cytology. Aim of this study was to explore diagnostic benefits of a recently introduced method featuring target specific genotyping, as well as to investigate the feasibility of locally and statistically determined cut-off, compared with conventional evaluation scheme. Histology, cytology, and comparative FISH approaches were performed on 42 patients with high clinical suspicion for urothelial carcinoma (UC). FISH parallels were (1) Urovysion-alone (according to manufacturer's instruction); (2) Targeted-Urovysion (cytokeratin7 immunophenotyping followed by Urovysion), both of which evaluated by both conventional and statistical evaluation scheme. For statistical evaluation cut-offs and sufficient sample size were determined on controls and ratio of positive cells was recorded, whereas conventional evaluation relied on manufacturer's recommendations. The specificity of cytology, Urovysion-alone in general and targeted-Urovysion in general appeared 86%, 86%, and 100%, respectively. In the same comparison, overall sensitivity was 60%, 80%, and 93%, respectively. In superficial cases sensitivity was 48% for cytology, 72% for Urovysion-alone and 91% for targeted-Urovysion, while no prominent differences were seen in muscle invasive cases. The ratio of FISH positive cells was proportionate with both stage and grade, however, targeted genotyping could separate high grade/high stage cases more effectively. In conclusion, CK7 targeting raises diagnostic efficiency of Urovysion, and could be an ideal tool for identifying tumor cells in ambiguous cases or when other tumors are present. Statistical evaluation produces accuracy comparable with results of conventional evaluation, and with laboratories setting cut-offs individually but according harmonized protocol, it could aid method standardization. Furthermore, by providing additional quantitative information about tumor characteristics, is likely to have therapy relevant value in the future.

A Unique Case of Eosinophilic Pancreatitis and Anencephaly in the Fetus of a Type I Diabetic Mother.

Pancreatic infiltration with eosinophils is an uncommon finding with numerous etiologies. While two rare cases of eosinophilic pancreatitis in infants born to Type I diabetic mothers have been reported once in the English literature and once in the French literature, we present the additional finding of anencephaly in a 34 week old fetus. Although the pancreas was grossly unremarkable, histological inspection demonstrated an eosinophilic infiltrate in the fibrous septae and islets of Langerhans along with hypertrophy and hyperplasia of the pancreatic islets.

Increased efficiency of detecting genetically aberrant cells by UroVysion test on voided urine specimens using automated immunophenotypical preselection of uroepithelial cells.

There is a steady search for procedure which could replace or at least reduce the frequency of the invasive cystoscopy in the surveillance of heterogeneous superficial transitional cell carcinoma (TCC) of the bladder. Recently, UroVysion FISH assay has been shown to provide with better sensitivity than the urine cytology except for the lowest stage pTa and grade I-II TCCs. Data indicate that this failure of the sensitive FISH might be due to mistargeting. Therefore, our aim was to elaborate a procedure enabling FISH analysis in phenotypically preselected urothelial cells, only. Cytokeratin 7 (CK-7) chromogenic immunolabeling was applied to various mixtures of negative and positive control cells as well as voided urine specimens. Cellular targets and CK-7 positive cells were identified by morphometric and pixel intensity indices using an automated microscope workstation. UroVysion FISH pattern was analyzed only in the subsequently relocalized CK-7 positive events. Automated phenotypical preselection of urothelial cells proved to have 97.3% sensitivity, 96.1% specificity, and 99.0% accuracy, whereas combined pheno- and genotyping revealed 93.3% sensitivity and 99.8% specificity, respectively. In clinical samples, the overall 20.4% FISH positivity gained by traditional target identification contrasted with the 55.6% positivity obtained by the combined method, by which the efficiency of identifying chromosomally aberrant cells proved to be two to threefold higher even in grade I lesions. FISH analysis of phenotypically preselected urothelial cells might represent a reliable asset in surveillance of low stage-low grade TCCs.

Functional, histopathologic and natural history study of neuropathy associated with EGR2 mutations.

Mutations in the EGR2 gene cause a spectrum of Charcot-Marie-Tooth disease and related inherited peripheral neuropathies. We ascertained ten consecutive patients with various EGR2 mutations, report a novel de novo mutation, and provide longitudinal clinical data to characterize the natural history of the peripheral neuropathy. We confirmed that respiratory compromise and cranial nerve dysfunction are commonly associated with EGR2 mutations and can be useful in guiding molecular diagnosis. We also contrast morphological studies in the context of the I268N homozygous recessive mutation affecting the NAB repressor binding site and the R359W dominant-negative mutation in the zinc-finger domain.

Histopathologic abnormalities of the lymphoreticular tissues in organic cation transporter 2 deficiency: evidence for impaired B cell maturation.

Immunohistology of lymphoreticular tissues of a fatal case of organic cation transporter 2 deficiency revealed inhibited proliferation with increased apoptosis in the germinal centers, resulting in "burned out" follicles. This is indicative of impaired antigen driven B cell affinity maturation. Defective humoral immune response might explain the recurrent infections in untreated organic cation transporter 2 deficiency.