Diagnostic Utility of a Modified Reticulin Algorithm in Pediatric Adrenocortical Neoplasms.

Pediatric adrenocortical neoplasms (ACNs) are extremely rare tumors in contrast to their adult counterparts. Distinguishing benign from malignant is challenging based on pure morphologic grounds. Previously, 2 scoring systems were proposed in pediatric ACN, including the Wieneke criteria (WC) and its modified version (modified WC [mWC]). In adults, the reticulin algorithm (RA) has proven inexpensive, reliable, predictive, and reproducible; however, it has been validated only recently in children in a limited number of cases. This study aims to assess the RA utility compared with other scoring systems in a series of 92 pediatric ACNs. All cases were individually scored, and mitotic rate cutoffs were recorded. Reticulin alterations were classified as quantitative and qualitative. Outcome data were available in 59/92. The median age was 5 years (0.1 to 18 y) with an M:F of 0.6. Clinical presentation included virilization (39%), Cushing syndrome (21%), other symptoms (4%), and asymptomatic (36%). The reticulin framework was intact in 27% and altered in 73% of cases, showing qualitative (22%), quantitative (73%), and both (5%) alterations. In patients with favorable outcomes, 59% showed either intact reticulin or qualitative alteration compared with the unfavorable outcome group, where 90% showed quantitative alterations. All scoring systems WC ( P < 0.0001), mWC ( P = 0.0003), and the adult/pediatric RA ( P < 0.0001) had predictive value. The RA is comparable to WC and mWC, easier to apply, and is the most sensitive histopathological approach to identifying aggressive behavior in pediatric ACN. Its integration into the WC might be helpful in ACN of uncertain malignant potential and deserves further investigation.

Prolonged fasting times reap greater geroprotective effects when combined with caloric restriction in adult female mice.

Emerging new evidence highlights the importance of prolonged daily fasting periods for the health and survival benefits of calorie restriction (CR) and time-restricted feeding (TRF) in male mice; however, little is known about the impact of these feeding regimens in females. We placed 14-month-old female mice on five different dietary regimens, either CR or TRF with different feeding windows, and determined the effects of these regimens on physiological responses, progression of neoplasms and inflammatory diseases, serum metabolite levels, and lifespan. Compared with TRF feeding, CR elicited a robust systemic response, as it relates to energetics and healthspan metrics, a unique serum metabolomics signature in overnight fasted animals, and was associated with an increase in lifespan. These results indicate that daytime (rest-phase) feeding with prolonged fasting periods initiated late in life confer greater benefits when combined with imposed lower energy intake.

Mutant and curli-producing E. coli enhance the disease phenotype in a hSOD1-G93A mouse model of ALS.

The gut microbiome is a potential non-genetic contributing factor for Amyotrophic Lateral Sclerosis. Differences in gut microbial communities have been detected between ALS subjects and healthy controls, including an increase in Escherichia coli in ALS subjects. E. coli and other gram-negative bacteria produce curli proteins, which are functional bacterial amyloids. We examined whether long-term curli overexposure in the gut can exacerbate the development and progression of ALS. We utilized the slow-developing hSOD1-G93A mouse model of ALS with their C57BL/6J WT littermate controls, including males and females, with a total of 91 animals. These mice were on a normal chow diet and fed curli-producing or curli-nonproducing (mutant) E. coli in applesauce (vehicle) 3 times/week, from 1 through 7 months of age. Male hSOD1 mice demonstrated gradual slowing in running speed month 4 onwards, while females exhibited no signs of locomotive impairment even at 7 months of age. Around the same time, male hSOD1 mice showed a gradual increase in frequency of peripheral CD19+ B cells. Among the male hSOD1 group, chronic gut exposure to curli-producing E. coli led to significant shifts in α- and ß-diversities. Curli-exposed males showed suppression of immune responses in circulation, but an increase in markers of inflammation, autophagy and protein turnover in skeletal muscle. Some of these markers were also changed in mutant E. coli-exposed mice, including astrogliosis in the brainstem and demyelination in the lumbar spinal cord. Overall, chronic overexposure to a commensal bacteria like E. coli led to distant organ pathology in our model, without the presence of a leaky gut at 6 months. Mechanisms underlying gut-distant organ communication are of tremendous interest to all disciplines.

Daily caloric restriction limits tumor growth more effectively than caloric cycling regardless of dietary composition.

Cancer incidence increases with age and is a leading cause of death. Caloric restriction (CR) confers benefits on health and survival and delays cancer. However, due to CR's stringency, dietary alternatives offering the same cancer protection have become increasingly attractive. Short cycles of a plant-based diet designed to mimic fasting (FMD) are protective against tumorigenesis without the chronic restriction of calories. Yet, it is unclear whether the fasting time, level of dietary restriction, or nutrient composition is the primary driver behind cancer protection. Using a breast cancer model in mice, we compare the potency of daily CR to that of periodic caloric cycling on FMD or an isocaloric standard laboratory chow against primary tumor growth and metastatic burden. Here, we report that daily CR provides greater protection against tumor growth and metastasis to the lung, which may be in part due to the unique immune signature observed with daily CR.

Fasting blood glucose as a predictor of mortality: Lost in translation.

Aging leads to profound changes in glucose homeostasis, weight, and adiposity, which are considered good predictors of health and survival in humans. Direct evidence that these age-associated metabolic alterations are recapitulated in animal models is lacking, impeding progress to develop and test interventions that delay the onset of metabolic dysfunction and promote healthy aging and longevity. We compared longitudinal trajectories, rates of change, and mortality risks of fasting blood glucose, body weight, and fat mass in mice, nonhuman primates, and humans throughout their lifespans and found similar trajectories of body weight and fat in the three species. In contrast, fasting blood glucose decreased late in life in mice but increased over the lifespan of nonhuman primates and humans. Higher glucose was associated with lower mortality in mice but higher mortality in nonhuman primates and humans, providing a cautionary tale for translating age-associated metabolic changes from mice to humans.

Pulmonary hypoplasia correlates with the length of anhydramnios in patients with early pregnancy renal anhydramnios (EPRA).

BACKGROUND: Early pregnancy renal anhydramanios (EPRA) occurs when the fetus is anuric before 22 weeks gestational age (GA) and is considered universally lethal. Serial amnioinfusions have successfully ameliorated the lethal pulmonary hypoplasia associated with EPRA and have resulted in cases of neonatal survival, peritoneal dialysis, and renal transplant. OBJECTIVE: We sought to evaluate the lung pathology of untreated fetuses and neonates that had EPRA. STUDY DESIGN: This is a retrospective case series of all fetuses and neonates diagnosed with isolated EPRA that underwent autopsy at a single tertiary care center between 1987 and 2018. Autopsy data were correlated with ultrasound findings and GA at delivery. Fetal weights, lung weights, and lung developmental stage were recorded. RESULTS: Nineteen cases met criteria for analysis and ranged from 16 to 38 weeks GA at termination or birth. The observed-to-expected (O/E) lung-to-body-weight ratio was significantly associated with GA (r = -0.51, p = 0.03), such that as GA increased the O/E ratio decreased. When limited to patients >22 weeks, this relationship strengthened (r = -0.75, p = 0.01). Importantly, overall O/E body weight had no relationship with GA. CONCLUSION: This study shows that the degree of pulmonary hypoplasia in EPRA increases with the length of anhydramnios. This suggests that amnioinfusions are likely to be of most benefit the soonest they can feasibly be initiated.

Differential Expression Profiles of Cell-to-Matrix-Related Molecules in Adrenal Cortical Tumors: Diagnostic and Prognostic Implications.

The molecular mechanisms of adrenocortical carcinoma development are incompletely defined. De-regulation of cellular-to-extracellular matrix interactions and angiogenesis appear among mechanisms associated to the malignant phenotype. Our aim was to investigate, employing PCR-based array profiling, 157 molecules involved in cell-to-matrix interactions and angiogenesis in a frozen series of 6 benign and 6 malignant adrenocortical neoplasms, to identify novel pathogenetic markers. In 14 genes, a significant dysregulation was detected in adrenocortical carcinomas as compared to adenomas, most of them being downregulated. Three exceptions-hyaluronan synthase 1 (HAS-1), laminin α3 and osteopontin genes-demonstrated an increased expression in adrenocortical carcinomas of 4.46, 4.23 and 20.32-fold, respectively, and were validated by immunohistochemistry on a series of paraffin-embedded tissues, including 20 adenomas and 73 carcinomas. Osteopontin protein, absent in all adenomas, was expressed in a carcinoma subset (25/73) (p = 0.0022). Laminin α3 and HAS-1 were mostly expressed in smooth muscle and endothelial cells of the vascular network of both benign and malignant adrenocortical tumors. HAS-1 was also detected in tumor cells, with a more intense pattern in carcinomas. In this group, strong expression was significantly associated with more favorable clinicopathological features. These data demonstrate that cell-to-matrix interactions are specifically altered in adrenocortical carcinoma and identify osteopontin and HAS-1 as novel potential diagnostic and prognostic biomarkers, respectively, in adrenal cortical tumors.

Intermittent fasting: from calories to time restriction.

The global human population has recently experienced an increase in life expectancy with a mounting concern about the steady rise in the incidence of age-associated chronic diseases and socio-economic burden. Calorie restriction (CR), the reduction of energy intake without malnutrition, is a dietary manipulation that can increase health and longevity in most model organisms. However, the practice of CR in day-to-day life is a challenging long-term goal for human intervention. Recently, daily fasting length and periodicity have emerged as potential drivers behind CR's beneficial health effects. Numerous strategies and eating patterns have been successfully developed to recapitulate many of CR's benefits without its austerity. These novel feeding protocols range from shortened meal timing designed to interact with our circadian system (e.g., daily time-restricted feeding) to more extended fasting regimens known as intermittent fasting. Here, we provide a glimpse of the current status of knowledge on different strategies to reap the benefits of CR on metabolic health in murine models and in humans, without the rigor of continuous reduction in caloric intake as presented at the USU State of the Science Symposium.

Chronic Exposure to Cadmium Induces Differential Methylation in Mice Spermatozoa.

Cadmium exposure is ubiquitous and has been linked to diseases including cancers and reproductive defects. Since cadmium is nonmutagenic, it is thought to exert its gene dysregulatory effects through epigenetic reprogramming. Several studies have implicated germline exposure to cadmium in developmental reprogramming. However, most of these studies have focused on maternal exposure, while the impact on sperm fertility and disease susceptibility has received less attention. In this study, we used reduced representation bisulfite sequencing to comprehensively investigate the impact of chronic cadmium exposure on mouse spermatozoa DNA methylation. Adult male C57BL/J6 mice were provided water with or without cadmium chloride for 9 weeks. Sperm, testes, liver, and kidney tissues were collected at the end of the treatment period. Cadmium exposure was confirmed through gene expression analysis of metallothionein-1 and 2, 2 well-known cadmium-induced genes. Analysis of sperm DNA methylation changes revealed 1788 differentially methylated sites present at regulatory regions in sperm of mice exposed to cadmium compared with vehicle (control) mice. Furthermore, most of these differential methylation changes positively correlated with changes in gene expression at both the transcription initiation stage as well as the splicing levels. Interestingly, the genes targeted by cadmium exposure are involved in several critical developmental processes. Our results present a comprehensive analysis of the sperm methylome in response to chronic cadmium exposure. These data, therefore, highlight a foundational framework to study gene expression patterns that may affect fertility in the exposed individual as well as their offspring, through paternal inheritance.

Study of Longitudinal Aging in Mice: Presentation of Experimental Techniques.

Aging is associated with functional and metabolic decline and is a risk factor for all noncommunicable diseases. Even though mice are routinely used for modeling human aging and aging-related conditions, no comprehensive assessment to date has been conducted on normative mouse aging. To address this gap, the Study of Longitudinal Aging in Mice (SLAM) was designed and implemented by the National Institute on Aging (NIA/NIH) as the mouse counterpart to the Baltimore Longitudinal Study of Aging (BLSA). In this manuscript, we describe the premise, study design, methodologies, and technologies currently employed in SLAM. We also discuss current and future study directions. In this large population mouse study, inbred C57BL/6J and outbred UM-HET3 mice of both sexes are longitudinally evaluated for functional, phenotypic, and biological health, and collection of biospecimens is conducted throughout their life span. Within the longitudinal cohorts, a cross-sectional arm of the study has also been implemented for the well-controlled collection of tissues to generate a biorepository. SLAM and studies stemming from SLAM seek to identify and characterize phenotypic and biological predictors of mouse aging and age-associated conditions, examine the degrees of functional and biomolecular variability that occur within inbred and genetically heterogeneous mouse populations with age, and assess whether these changes are consistent with alterations observed in human aging in BLSA. The findings from these studies will be critical for evaluating the utility of mouse models for studying different aspects of aging, both in terms of interpreting prior findings and designing and implementing future studies.

Spontaneous chordoma: a case report on a female UM-HET3 mouse from the SLAM study.

A female UM-HET3 mouse from the Study of Longitudinal Aging in Mice (SLAM) was euthanized at 164 weeks of age due to hind limb weakness. Necropsy and histological analysis revealed that the most probable cause of the clinical finding was the compression of the thoracolumbar segment of the spinal cord by herniated intervertebral disks. In addition, a spontaneous chordoma was incidentally found in the coccygeal bones. Given the rarity of this type of tumor, bio-clinical annotations acquired throughout lifespan, detailed histopathological assessment, and comparative clinical-pathological correlations for this mouse are presented and discussed.

Detailed genomic characterization identifies high heterogeneity and histotype-specific genomic profiles in adrenocortical carcinomas.

Molecular characterization of adrenocortical carcinoma has been recently established, but the correlation between molecular profiles and clinical and pathological characteristics is still poorly defined with no data available about genetic heterogeneity along disease progression. In this scenario, a detailed molecular profile was correlated with clinical and pathological characteristics in adrenocortical carcinoma patients to identify potentially novel biomarkers. Targeted next-generation sequencing and copy number variation analyses for 18 most frequently altered genes in adrenocortical carcinoma were assessed on 62 adult cases (including 10 with matched primary and metastatic/recurrence samples) and results correlated with major clinical and pathological characteristics of tumors. A total of 433 somatic deleterious genetic alterations (328 gene mutations and 105 copy number variations) were identified in 57/62 cases, five resulted wild type for all genes tested. TERT, CDK4, ZNRF3,and RB1 were altered in more than 30% of cases. Among histological variants genotypes were significantly different. Lowest mutation burden was found in the oncocytic type (p = 0.006), whereas the highest with a prevalence of RB1 (p = 0.001) and CDK4 (p = 0.002) was found in the conventional and myxoid ones, respectively. None of the 10 cases with matched samples showed a stable genotype along tumor progression, although allelic frequencies or percentages of altered nuclei at fluorescence in situ hybridization were in most cases similar among different tumor samples for genes that were stable along tumor progression. Among individual genes, an altered p53/Rb1 pathway was the strongest adverse molecular signature, being associated with high Ki-67 index, high tumor stage, aggressive disease status, and shorter disease-free survival. The genomic signature in adrenocortical carcinoma is changing along tumor progression and is associated with specific clinical and pathological features, including histological variant and prognosis.

Analysis of histological and immunohistochemical patterns of benign and malignant adrenocortical tumors by computerized morphometry.

Diagnosis of benign and purely localized malignant adrenocortical lesions is still a complex issue. Moreover, histology-based diagnosis may suffer of a moment of subjectivity due to inter- and intra-individual variations. The aim of the present study was to assess, by computerized morphometry, the morphological features in benign and malignant adrenocortical neoplasms. Eleven adrenocortical adenomas (ACA) were compared with 18 adrenocortical cancers (ACC). All specimens were stained with H&E, cellular proliferation marker Ki-67 and reticulin. We generated a morphometric model based on the analysis of volume fractions occupied by Ki-67 positive and negative cells (nuclei and cytoplasm), vascular and inflammatory compartment; we also analyzed the surface fraction occupied by reticulin. We compared the quantitative data of Ki-67 obtained by morphometry with the quantification resulting from pathologist's visual reading. The volume fraction of Ki-67 positive cells in ACCs was higher than in ACAs. The volume fraction of nuclei in unit volume and the nuclear/cytoplasmic ratio in both Ki-67 negative cells and Ki-67 positive cells were prominent in ACCs. The surface fraction of reticulin was considerably lower in ACCs. Our computerized morphometric model is simple, reproducible and can be used by the pathologist in the histological workup of adrenocortical tumors to achieve precise and reader-independent quantification of several morphological characteristics of adrenocortical tumors.

Tissue Expression and Pharmacological In Vitro Analyses of mTOR and SSTR Pathways in Adrenocortical Carcinoma.

New therapies for advanced adrenocortical carcinoma (ACC) are urgently needed, as the majority of the patients experience a rapid and inexorable progression despite surgery and adjuvant mitotane. In vitro data suggest that somatostatin receptors (SSTRs) and mTOR pathway might represent reasonable targets for novel therapies, being involved in functionality and growth of ACC cells. However, in vitro analysis of combination treatments targeting both mTOR and SSTR as compared to mitotane are poorly explored in ACC. This study aimed to investigate in vitro the effects on cell growth of pasireotide, everolimus, and mitotane, alone or combined, in the two ACC cell lines H295R and SW13 (mitotane sensitive and resistant, respectively). Moreover, the tissue expression of mTOR pathway molecules and SSTR (types 1-5) was assessed in 58 ACCs. In both cell lines, only everolimus induced a significant inhibition of cell growth. Conversely, the combinations among mitotane, pasireotide, and everolimus produced antagonistic effects on mitotane-induced growth inhibition on H295R cell line. A heterogeneous profile of mTOR-related molecules and SSTR expression was observed in ACC samples, being the mTOR pathway found activated in approximately 30% of cases. In conclusion, our data suggest caution in designing combinations of mitotane with other drugs potentially active in ACC, such as mTOR inhibitors or somatostatin analogs.

Caveolin 1 expression favors tumor growth and is associated with poor survival in primary lung adenocarcinomas.

Despite the consolidated clinico-pathological correlates of Caveolin 1 expression in non-small cell lung cancer, the available data on the role of Caveolin 1 in relation to proliferation, migration, and metastasis in lung adenocarcinoma cells is still scant. Here, we aimed to confirm whether Caveolin 1 may act as a promoter of cell growth in human lung adenocarcinoma using in vitro and in vivo models, supported by a survival analysis of Caveolin 1 expression in a series of 116 primary lung adenocarcinomas. The silencing of endogenous Caveolin 1 expression in H522 lung adenocarcinoma cells through stable shRNA transfection significantly inhibited cellular proliferation in vitro and in vivo, in a lung adenocarcinoma xenograft mouse model. The bioluminescence imaging analysis revealed that tumors derived from Caveolin 1 shRNA-transfected cells grew slower than control xenografts. However, this difference progressively diminished over time and was definitively lost after 21 days. This was consistent with a progressive Caveolin 1 re-expression, which started at day 7. The association between the restored expression of Caveolin 1 and the restart of tumor growth in vivo supports the booster role of Caveolin 1 in lung adenocarcinoma progression. To further confirm this role, Caveolin 1 expression was assessed by immunohistochemistry in a series of 116 human lung adenocarcinomas. Positive Caveolin 1 tumors accounted for 20% of cases and were associated with a significantly worse overall survival compared to Caveolin 1-negative cancers. Taken together, these data highlight that Caveolin 1 expression confers a proliferative advantage in lung adenocarcinoma cells, thus fostering increased tumor aggressiveness.

Validation of the prognostic role of the "Helsinki Score" in 225 cases of adrenocortical carcinoma.

Adrenocortical carcinoma patient prognosis is extremely variable and poorly predictable. The newly introduced Helsinki Score is the first so far proposed diagnostic and prognostic system based on the combined evaluation of morphological (mitoses and necrosis) and immunohistochemical (Ki-67) parameters. The aim of the study was to validate the prognostic role of the Helsinki Score for adrenocortical carcinoma characterization. Thus, 225 adrenocortical carcinomas were reclassified using the Weiss Score and the Helsinki Score (3× mitotic count + 5 × necrosis + Ki-67 index). At univariate analysis, statistically significant prognostic values were observed at the log-rank test for mitotic count (cutoff values: <6 and ≥55; P<.0001), Ki-67 (cutoff values: <20 and ≥50; P<.0001), Weiss Score (cutoff values: <5 and ≥8; P<.0001), Helsinki Score (cutoff values: <13 and ≥19; P<.0001), histological variant (conventional versus oncocytic; P=.009), necrosis (P=.001), and stage (P=.005). Cox multivariate analysis using a backward stepwise selection method retained only Helsinki Score and Weiss Score as predictors of poor prognosis (P<.0001 and P=.0005, respectively). Helsinki Score (with a threshold of 28.5 points; area under the curve [AUC]=0.729, 95% confidence interval=0.66-0.79) and Ki-67 (with a threshold of 20.5%; AUC=0.727, 95% confidence interval=0.66-0.79) showed the best and equivalent AUCs predicting disease-related deaths determined using receiver operating characteristic statistics. In conclusion, the Helsinki Score is a valuable system to predict prognosis in adrenocortical carcinoma, outperforming the currently established prognostic parameters.