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Numerous cell states are known to comprise the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). However, the developmental stemness and co-occurrence of these cell states remain poorly defined. Here, we performed single-cell RNA sequencing (scRNA-seq) on a cohort of treatment-naive PDAC time-of-diagnosis endoscopic ultrasound-guided fine needle biopsy (EUS-FNB) samples (n=25) and surgical resections (n=6), which were then combined with publicly available samples to increase statistical power (n=80). Following annotation into 25 distinct cell states, cells were scored for developmental stemness and a customized version of the Ecotyper tool was used to identify communities of co-occurring cell states in bulk RNA-seq samples (n=268). We discovered a tumor microenvironmental community comprised of aggressive basal-like malignant cells, tumor-promoting SPP1+ macrophages, and myofibroblastic cancer-associated fibroblasts associated with especially poor prognosis. We also found a developmental stemness continuum with implications for survival that is present in both malignant cells and cancer-associated fibroblasts (CAFs). We further demonstrated that high-dimensional analyses predictive of survival are feasible using standard-of-care, time-of-diagnosis EUS-FNB specimens. In summary, we identified novel tumor microenvironmental and developmental stemness characteristics from a high-dimensional gene expression analysis of PDAC using human tissue specimens including time-of-diagnosis EUS-FNB samples. These reveal new connections between tumor microenvironmental composition, CAF and malignant cell stemness, and patient survival that could lead to better upfront risk stratification and more personalized upfront clinical decision-making.

Bulk and single-cell analyses of the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) have revealed a largely immunosuppressive milieu. Thus far, efforts to utilize insights from TME features to facilitate more effective therapeutics have largely failed. Here, we performed single-cell RNA sequencing (scRNA-seq) on a cohort of treatment-naive PDAC time-of-diagnosis endoscopic ultrasound-guided fine needle biopsy samples (n=22) and surgical samples (n=6), integrated with 3 public datasets (n=49), resulting in ∼140,000 individual cells from 77 patients. Based on expression markers assessed by Seurat v3 and differentiation status assessed by CytoTRACE, we divided the resulting tumor cellular clusters into 5 molecular subtypes based on expression of previously reported marker genes: Basal, Mixed Basal/Classical, Classical Low, Classical High, and ADEX. We then queried these 5 tumor cell profiles, along with 15 scRNA-seq-derived tumor microenvironmental cellular profiles, in 391 bulk expression samples from 4 published datasets of localized PDAC with associated clinical metadata using CIBERSORTx. Through unsupervised clustering analysis of these 20 cell state fractions representing tumor, leukocyte and stromal cells, we identified 7 unique clustering patterns representing combinations of tumor cellular and microenvironmental cell states present in PDAC tumors. We termed these cell state patterns communities, and found them to correlate with overall survival, tumor ecotypes, and tumor cellular differentiation status. The community associated with worst overall survival contained basal tumor cells, exhausted CD4 and CD8 T cells, and was enriched for fibroblasts. In contrast, the highest overall survival was associated with a community high in immune cell enrichment. The differentiation state of tumor cells (assessed by CytoTRACE) was also correlated with survival in a dose-dependent fashion. Further, we identified a subset of PDAC samples that were significantly enriched for CD8 T and plasma cells that achieved a 2-year overall survival rate of 71%, suggesting we can identify PDAC patients with significantly improved prognoses and, potentially, higher sensitivity to immunotherapy.In summary, we identified novel tumor microenvironmental communities from high-dimensional analysis of PDAC RNA sequencing data that reveal new connections between tumor microenvironmental composition and patient survival that could lead to better upfront risk stratification and more personalized clinical decision-making.

Introduction: Severe immune-related adverse events (irAEs) occur in up to 60% of melanoma patients treated with immune checkpoint inhibitors (ICIs), causing substantial treatment-related morbidity and in the most severe cases, death. There is no clinical assay to predict who will develop severe ICI-induced irAEs and who will not. Using single-cell profiling assays applied to a retrospective melanoma cohort treated with ICIs, we recently showed that higher baseline levels of circulating CD4 effector memory T (TEM) cells were associated with severe irAE development, independent of the affected organ system (Lozano et al. Nature Medicine, 2022). As part of a prospective validation study of 100 melanoma patients, here we report our initial findings on the first 24 patients accrued to date. Methods: We prospectively collected pre-ICI blood from 24 metastatic melanoma patients from two academic medical centers from February 2021 onward. Peripheral blood was collected pre-treatment on the day of immunotherapy (cycle 1 day 1). We then isolated peripheral blood mononuclear cells (PBMCs) and applied mass cytometry by time of flight (CyTOF) to profile 38 leukocyte markers including markers specific to CD4 TEM cells. Cellular subpopulations were quantified using Cytobank v9. Patients underwent routine medical oncology follow-up during and after ICI treatment including grading of irAEs using the Common Terminology Criteria for Adverse Events v5. Results: Median follow-up time after pre-ICI blood collection was 9.1 months (range 2.6-18.5). Fifteen (63%) patients received combination (anti-PD1/anti-CTLA4) ICIs while the remainder received anti-PD1 monotherapy. Eight patients developed severe (grade 3+) irAEs at a median of 8.5 weeks (range 4-21) after treatment initiation, including 4 who developed life-threatening (grade 4) irAEs. Severe and life-threatening irAE development spanned 9 separate organ systems, most commonly gastrointestinal, dermatological, and hepatic. Using CyTOF, we found that circulating CD4 TEM cells were more abundant in patients who developed severe irAEs compared to those who did not (P = 0.01; AUC = 0.81), irrespective of the involved organ systems. Moreover, median-splitting the patient cohort into two groups based on pretreatment CD4 TEM levels revealed that patients with low CD4 TEMs had significantly longer freedom from severe irAE than those with high CD4 TEMs (not reached vs. 4.6 months; P = 0.013; HR = 6.7). There was no significant difference in pretreatment CD4 TEM levels between patients with and without durable clinical benefit to ICIs (P = 0.75). Conclusion: Circulating CD4 TEM levels measured by CyTOF were associated with severe irAE development, independent of response status, in patients with advanced melanoma. These findings could form the basis for pretreatment ICI risk stratification in the future. Citation Format: Abul Usmani, Noah Earland, Wubing Zhang, Peter K. Harris, Antonietta Bacchiocchi, Aishwarya Nene, David Y. Chen, Mario Sznol, Ruth Halaban, Aaron M. Newman, Aadel A. Chaudhuri. Association between circulating CD4 memory T cell levels and severe immune-related adverse events in melanoma patients treated with immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6670.

Advances in single cell RNA sequencing (scRNA-seq) have provided the ability to describe individual cell profiles and query individual cell states, enabling a more in-depth analysis of the tumor microenvironment (TME) and tumor heterogeneity. We performed scRNA-seq of PDAC from standard-of-care endoscopic ultrasound guided fine needle biopsy (EUS-FNB) specimens at the time-of-diagnosis and from surgical samples obtained from tumor resections. In total, we acquired 32k cells across 25 independent PDAC samples for our in-house EUS-FNB cohort, and 12k cells from 6 independent PDAC samples for our in-house surgical cohort. To increase power, we then combined the in-house scRNA-seq data with three publicly available datasets, increasing our dataset to 80 samples. Cells were subsequently annotated based on a combination of known marker genes and gene set scores from the literature. Importantly, we partitioned malignant cells into Classical and Basal subtypes, CAFs into myofibroblast (myCAF) and inflammatory (iCAF) fibroblast, and TAMs into C1QC+, FCN1+, SPP1+, and proliferating TAMs based on similarity with previously published markers. We then determined the developmental status of the malignant and CAF subclusters with the CytoTRACE tool. We found that Basal malignant cells were less differentiated than their classical counterparts, agreeing with previous literature suggesting the aggressive basal subtype is more likely to undergo epithelial-mesenchymal transition (EMT). Interestingly, we also found that myCAFs are significantly less differentiated than iCAFs. We then extended our single cell expression profiles to 391 publicly available bulk expression samples with associated clinical metadata to find cell state patterns associated with patient survival. To this end, we modified the in-silico TME dissection tool EcoTyper and grouped significantly co-occurring cell states into ecotypes. Notably, one ecotype, termed PE3, showed consistently poor survival across all bulk RNA-seq datasets and harbored significant associations between Malignant - Basal, myCAF, and TAM - SPP1+ cell states, suggesting patients may benefit from treatment regimens that concurrently target TAM, CAF, and malignant cell dynamics in PDAC. In summary, we identified a novel TME community that revealed new connections between TME composition, developmental status, and patient survival that could lead to better upfront risk stratification and more personalized clinical decision-making. Citation Format: Erik Storrs, Prathamesh Chati, Abul Usmani, Li Ding, Ryan Fields, Koushik Das, Aadel Chaudhuri. High-dimensional deconstruction of pancreatic cancer identifies co-occurring TME features associated with survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1118.

Severe immune-related adverse events (irAEs) occur in up to 60% of patients with melanoma treated with immune checkpoint inhibitors (ICIs). However, it is unknown whether a common baseline immunological state precedes irAE development. Here we applied mass cytometry by time of flight, single-cell RNA sequencing, single-cell V(D)J sequencing, bulk RNA sequencing and bulk T cell receptor (TCR) sequencing to study peripheral blood samples from patients with melanoma treated with anti-PD-1 monotherapy or anti-PD-1 and anti-CTLA-4 combination ICIs. By analyzing 93 pre- and early on-ICI blood samples and 3 patient cohorts (n = 27, 26 and 18), we found that 2 pretreatment factors in circulation-activated CD4 memory T cell abundance and TCR diversity-are associated with severe irAE development regardless of organ system involvement. We also explored on-treatment changes in TCR clonality among patients receiving combination therapy and linked our findings to the severity and timing of irAE onset. These results demonstrate circulating T cell characteristics associated with ICI-induced toxicity, with implications for improved diagnostics and clinical management.

Infertility in men and women is a complex genetic trait with shared biological bases between the sexes. Here, we perform a series of rare variant analyses across 73,185 women and men to identify genes that contribute to primary gonadal dysfunction. We report CSMD1, a complement regulatory protein on chromosome 8p23, as a strong candidate locus in both sexes. We show that CSMD1 is enriched at the germ-cell/somatic-cell interface in both male and female gonads. Csmd1-knockout males show increased rates of infertility with significantly increased complement C3 protein deposition in the testes, accompanied by severe histological degeneration. Knockout females show significant reduction in ovarian quality and breeding success, as well as mammary branching impairment. Double knockout of Csmd1 and C3 causes non-additive reduction in breeding success, suggesting that CSMD1 and the complement pathway play an important role in the normal postnatal development of the gonads in both sexes., Many molecular and physiological mechanisms in the regulation of fertility are shared between female and male mammals. Here, Lee et al. report an association of CNVs in CSMD1 with early idiopathic menopause in women and show that loss of Csmd1 leads to gonadal dysfunction in both male and female mice.

Purpose/objective(s) Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer type with a poor prognosis. There is a critical need for robust biomarkers to better risk-stratify patients and personalize treatment. Here, we utilize single-cell RNA sequencing (scRNA-seq) and CIBERSORTx deconvolution to genomically classify PDAC based on distinct tumor cell states in order to more accurately prognosticate patients. Materials/methods scRNA-seq was conducted on 24 PDAC patient samples prior to any systemic therapy at Washington University (19 core needle pancreatic biopsies from the time of diagnosis and 5 surgical PDAC resections) and from the public domain (n = 34). The resulting scRNA-seq data from 58 patients was integrated and clustered in order to identify distinct tumor cell states. Following gene expression profiling analysis to characterize each distinct tumor cell state, CIBERSORTx was used to deconvolve 125 bulk RNA-seq early-stage PDAC tumor samples from The Cancer Genome Atlas (TCGA), and each tumor was classified based on the dominant malignant cell state present. Following this, survival analysis was performed to compare between different groupings of TCGA patients based on their dominant malignant cell states. Results scRNA-seq data from 58 public and in-house samples were clustered and cell types were annotated based on known expression markers. We then clustered the malignant cell cluster (Epcam+) into 5 distinct tumor cell states. Fractions of these 5 malignant cell states were then quantified in TCGA via deconvolution using CIBERSORTx. TCGA samples were categorized into 5 groups based on the dominating malignant (mal) cell state fraction value - ranging from mal 0 to mal 4. In this fashion, we identified 39 patients as mal 0, 9 as mal 1, 6 as mal 2, 2 as mal 3, and 69 as mal 4. Gene expression profiling analysis revealed that the mal 0 cell state is representative of the "squamous-like" or "basal" subtype reported in prior literature, while mal 1-4 more likely represent the previously described "classical" subtype. We then aimed to perform survival analysis. To achieve meaningful statistical power, we grouped mal 1-3 into one category (called mixed) containing 17 patients. Kaplan-Meier analysis revealed the following median survival times (in months): mal 0 (9 months), mal 4 (16 months), and mixed (15 months). Log-rank statistical testing revealed that patients classified as mal 0 experienced significantly worse overall survival than those classified as other mal subtypes (P Conclusion We analyzed scRNA-seq data from 58 independent PDAC patients, which we used to identify 5 distinct tumor cell states. These tumor cell states appear to correspond with the previously reported "squamous-like" or "basal" vs. "classical" subtypes, correlate significantly with overall survival, and could potentially inform more personalized treatment strategies including radiotherapy in the future.

PURPOSE We hypothesized that circulating tumor DNA (ctDNA) molecular residual disease (MRD) analysis without prior mutational knowledge could be performed after neoadjuvant chemotherapy to assess oligometastatic colorectal cancer (CRC) treated surgically with curative intent. We also investigated urine as an alternative analyte for ctDNA MRD detection in this nongenitourinary setting. PATIENTS AND METHODS We applied AVENIO targeted next-generation sequencing to plasma, tumor, and urine samples acquired on the day of curative-intent surgery from 24 prospectively enrolled patients with oligometastatic CRC. Age-related clonal hematopoiesis was accounted for by removing variants also present in white blood cells. Plasma and urine ctDNA MRD were correlated with tumor cells detected in the surgical specimen, and adjuvant treatment strategies were proposed based on ctDNA-inferred tumor mutational burden (iTMB) and targetable alterations. RESULTS Seventy-one percent of patients were treated with neoadjuvant chemotherapy. Tumor-naive plasma ctDNA analysis detected MRD at a median level of 0.62% with 95% sensitivity and 100% specificity, and 94% and 77% sensitivity when only considering patients treated with neoadjuvant chemotherapy and putative driver mutations, respectively. In urine, ctDNA MRD detection specificity remained high at 100%, but sensitivity decreased to 64% with median levels being 11-fold lower than in plasma ( P < .0001). Personalized ctDNA MRD oncogenomic analysis revealed 81% of patients might have been candidates for adjuvant immunotherapy based on high iTMB or targeted therapy based on actionable PIK3CA mutations. CONCLUSION Tumor-naive plasma ctDNA analysis can sensitively and specifically detect MRD in patients with oligometastatic CRC after neoadjuvant chemotherapy. Urine-based ctDNA MRD detection is also feasible; however, it is less sensitive than plasma because of significantly lower levels. Oligometastatic patients with detectable MRD may benefit from additional personalized treatment based on ctDNA-derived oncogenomic profiling.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers worldwide. Bulk and single-cell technologies have recently been leveraged to better understand its genomic underpinnings. The PDAC tumor microenvironment (TME) has also been explored, revealing an immunosuppressive milieu. However, efforts to utilize TME features to facilitate more effective treatments have largely failed. Methods: Here, we performed single-cell RNA sequencing (scRNA-seq) on a cohort of treatment-naive PDAC biopsy samples (n=22) and surgical samples (n=6), integrated with 3 public datasets (n=49), resulting in ~150,000 individual cells from 77 patients. Based on expression markers assessed by Seurat v3 and differentiation status assessed by CytoTRACE, we divided the resulting tumor cellular clusters into 5 molecular subtypes: Basal, Mixed Basal/Classical, Less differentiated Classical, More differentiated Classical, and ADEX. We then queried these 5 tumor cell profiles along with 15 scRNA-seq-derived tumor microenvironmental cellular profiles in 391 bulk RNA-seq samples from 4 published datasets of localized PDAC with associated clinical metadata using CIBERSORTx. Through unsupervised clustering analysis of these 20 cell state fractions representing tumor, leukocyte and stromal cells, we identified 7 unique clustering patterns representing combinations of tumor cellular and microenvironmental cell states present in PDAC tumors, which we termed communities, and correlated these with survival, tumor ecotypes, and tumor cellular differentiation status. Results: We identified 7 distinct cellular communities in bulk RNA sequencing data after CIBERSORTx and unsupervised clustering. The community associated with worst overall survival contained basal tumor cells, exhausted CD4 and CD8 T cells, and was enriched for fibroblasts. In contrast, highest overall survival was associated with tumors enriched for differentiated classical tumor cells, NK cells and endothelial cells. The differentiation state of tumor cells (assessed by CytoTRACE) also correlated with survival in a dose-dependent fashion. We corroborated the community structures we identified with our unsupervised clustering approach with ecotypes obtained using Ecotyper and observed a significant correlation. We further identified a subset of PDAC samples that were significantly enriched for activated CD8 T cells that achieved a 3-year overall survival rate of 40%, suggesting we can identify PDAC patients with improved prognoses and with potentially higher sensitivity to immunotherapy. Conclusion: Discovered cellular communities from tumor bulk RNA-seq shed new insight into the composition of PDAC and could pave the way towards better upfront risk stratification and more personalized tumor biology-driven clinical decision-making. Citation Format: Erik P. Storrs, Abul Usmani, Prathamesh Chati, Bradley Krasnick, Li Ding, Ryan C. Fields, Koushik K. Das, Aadel A. Chaudhuri. High-dimensional analysis to deconstruct pancreatic ductal adenocarcinoma and identify tumor cellular communities with prognostic and potentially predictive value [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 477.

Circulating tumor DNA (ctDNA) is a component of cell-free DNA that is shed by malignant tumors into the bloodstream and other bodily fluids. Levels of ctDNA are typically low, particularly in patients with localized disease, requiring highly sophisticated methods for detection and quantification. Multiple liquid biopsy methods have been developed for ctDNA analysis in solid tumor malignancies and are now enabling detection and assessment of earlier stages of disease, post-treatment molecular residual disease (MRD), resistance to targeted systemic therapy, and tumor mutational burden. Understanding ctDNA biology, mechanisms of release, and clearance and size characteristics, in conjunction with the application of molecular barcoding and targeted error correction, have increased the sensitivity and specificity of ctDNA detection techniques. Combinatorial approaches including integration of ctDNA data with circulating protein biomarkers may further improve assay sensitivity and broaden the scope of ctDNA applications. Circulating viral DNA may be utilized to monitor disease in some virally induced malignancies. In spite of increasingly accurate methods of ctDNA detection, results need to be interpreted with caution given that somatic mosaicisms such as clonal hematopoiesis of indeterminate potential (CHIP) may give rise to genetic variants in the bloodstream unrelated to solid tumors, and the limited concordance observed between different commercial platforms. Overall, highly precise ctDNA detection and quantification methods have the potential to transform clinical practice via non-invasive monitoring of solid tumor malignancies, residual disease detection at earlier timepoints than standard clinical and/or imaging surveillance, and treatment personalization based on real-time assessment of the tumor genomic landscape.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer type with a poor prognosis. Patients with the classical histologic subtype typically have a better prognosis than those with a squamous-like histology. Still, survival outcomes vary significantly, even in early-stage patients, making it challenging to personalize treatment via subtyping. Here, we utilize CytoTRACE to better classify PDAC based on tumor cell-intrinsic developmental states, to more accurately prognosticate patients from the time of initial surgical resection. METHODS: We performed core needle pancreatic biopsies in 13 patients and surgical PDAC resections in five patients, and analyzed the resulting single-cell RNA sequencing (scRNA-seq) data to identify tumor cell clusters. We then applied CytoTRACE for developmental state analysis. Following developmental state quantification, we classified PDAC tumor cells into 3 distinct subtypes: squamous-like, classical early developmental (ED), and classical late developmental (LD). We developed a gene signature for each subtype, which we then applied to two external bulk RNA-seq datasets - 1) The Cancer Genome Atlas (TCGA): 125 early-stage PDAC tumors, and 2) Bailey et al (Nature 2016): 86 predominantly early-stage PDAC tumors. RESULTS: scRNA-seq data was partitioned into two subtypes, classical and squamous-like, based on marker gene expression. The classical subtype was further partitioned into ED versus LD cell states using the developmental index from CytoTRACE. For the squamous-like group, we identified the top 20 differentially expressed genes (squamous-like gene signature). For the ED and LD subtypes, we identified the top 20 genes correlating with the CytoTRACE developmental index (ED gene signature). Using a multivariate cox proportional hazards regression, we showed that the squamous-like signature was associated with significantly worse overall survival in TCGA (HR = 6.8, P = .01). Strikingly, our newly derived ED cell state signature was also associated with inferior overall survival in TCGA (HR = 5.9, P = .02). Kaplan-Meier analysis using optimized cutpoints between squamous-like and classical subtype scores, and between ED and LD cell state scores, again showed that patients with predominantly squamous-like tumors had significantly worse survival (HR = 4.4, P = .04); and that predominantly classical tumors enriched for the ED cell state had significantly inferior overall survival compared to LD (median 15.0 vs. 22.0 months, HR = 4.6, P = .03). The same trends were observed in the less-powered Bailey et al cohort. CONCLUSION: We showed that three developmental cell states, learned through the analysis of PDAC scRNA-seq data, can prognosticate patients with bulk RNA-seq expression data. This could help facilitate more personalized risk-adapted approaches for PDAC in the future. Citation Format: Prathamesh Mandar Chati, Erik Storrs, Abul Usmani, Bradley Krasnick, Chris Wetzel, Thomas Hollander, Faridi Quium, Ian Sloan, Hephzibah Anthony, Badiyan Shahed, Gabriel D. Lang, Natalie D. Cosgrove, Vladimir M. Kushnir, Dayna S. Early, William G. Hawkins, Li Ding, Ryan C. Fields, Koushik K. Das, Aadel A. Chaudhuri. Pancreatic ductal adenocarcinoma developmental cell state signatures identified by single cell RNA sequencing are prognostic when applied to bulk RNA-seq data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 159.

BACKGROUND: Circulating tumor DNA (ctDNA) can detect molecular residual disease (MRD) in plasma after curative-intent treatment of non-metastatic solid tumor malignancy when prior mutational knowledge from either tumor or pre-treatment plasma is available. We hypothesized that ctDNA MRD analysis without prior mutational knowledge could be performed to assess oligometastatic colorectal cancer (CRC) treated with curative intent by applying an error-corrected ultra-deep targeted sequencing approach. We also investigated urine as an alternative analyte for ctDNA MRD detection in this non-genitourinary setting. METHODS: We applied AVENIO, a ctDNA technology derived from CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) with integrated digital error suppression (iDES), to plasma, tumor, and urine samples acquired on the day of surgery from 24 prospectively enrolled oligometastatic CRC patients. We sequenced patients' white blood cell DNA to remove age-related clonal hematopoiesis variants. Plasma and urine ctDNA MRD detection were then correlated with residual disease present in tumor tissue, and adjuvant treatment strategies were proposed based on plasma ctDNA-inferred tumor mutational burden and targetable genomic alterations. RESULTS: Tumor-naïve plasma ctDNA analysis detected MRD at a median level of 0.62% with 95% sensitivity and 100% specificity. Plasma ctDNA MRD levels correlated with tumor size at the time of surgery (ρ= 0.68, P = 0.0003) and were significantly lower in patients without evidence of disease in their surgical specimen compared to those with residual tumor cells (P = 0.02). In urine, ctDNA MRD detection specificity remained high at 100%, but sensitivity was lower at 64%, with median levels being 11-fold lower than in plasma (P < 0.0001). Cell-free DNA fragments containing mutations were shorter in urine with an average size of 150.1 bp, compared to 180.2 bp in plasma (P < 0.0001). Personalized ctDNA MRD oncogenomic analysis revealed that 81% of patients had a high inferred tumor mutational burden (>10 mutations/megabase), and 10% also had a targetable PIK3CA mutation. CONCLUSION: Tumor-naïve plasma ctDNA analysis can sensitively and specifically detect MRD in patients with oligometastatic CRC after neoadjuvant chemotherapy. Urine-based ctDNA MRD detection is also feasible in these patients; however, it is less sensitive than plasma. Analysis of oligometastatic CRC patients' ctDNA MRD also revealed potentially actionable results that might have implications for personalized adjuvant treatment regimens. Citation Format: Bruna Pellini, Nadja Pejovic, Wenjia Feng, Noah Earland, Peter K. Harris, Abul Usmani, Jeffrey J. Szymanski, Farid Qaium, Jacqueline Mudd, Hyun Kim, Benjamin Tan, Ryan C. Fields, Aadel A. Chaudhuri. ctDNA MRD detection from plasma and urine and personalized oncogenomic analysis in oligometastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 543.

Introduction: Recent work has described tumor heterogeneity in pancreatic ductal adenocarcinoma (PDAC). Here we use scRNA-seq followed by CIBERSORTx and CytoTRACE to profile specific cell states within PDAC tumors at time-of-diagnosis, and correlate these states with clinical outcomes. Methods: We performed diagnostic endoscopic ultrasound (EUS) guided core needle biopsies in ten patients with locally advanced/resectable PDAC, from which we generated scRNA-seq data to identify unique cell types and states. To validate the prognostic utility of these signatures, we utilized CIBERSORTx to deconvolve bulk RNA-seq data from two external datasets - 1) 170 cases from The Cancer Genome Atlas (TCGA) and 2) 51 cases from Kirby et al (Mol Oncol 2016). We also used CytoTRACE to predict tumor cell differentiation status from the scRNA-seq data. Results: From ~9,800 total cells in our single cell cohort, we identified 16 cell types and 25 cell states. Bulk RNA-seq deconvolution was performed using the expression profiles from these 25 cell states. Two groups of malignant ductal cells were found to negatively influence patient survival (p of .008 and .041), and a group CD14+ monocytes and CD4+ T cells were found to positively influence patient survival (p of .011 and .012). We performed further analysis on the two malignant ductal cell populations, which displayed gene expression profiles similar to PDAC subtypes (squamous-like and progenitor) previously described by RNA-seq gene set enrichment analysis (Bailey et al; Moffitt et al). On CytoTRACE analysis of our scRNA-seq data, the squamous-like tumor cell population remained distinct from the larger progenitor population, and was predicted to be significantly more differentiated. We stratified TCGA and Kirby samples based upon cell fraction of this squamous-like population. A Kaplan-Meier estimate was used to quantify survival over time of the two groups (>7% squamous-like vs. 7% group displayed a marked decrease in survival - log-rank p-values of .025 (TCGA) and .00002 (Kirby). When filtering for only TCGA samples that went on to receive chemotherapy this trend remained highly significant (p of .022), with 46% of the >7% group vs. 82% of the >7% group surviving beyond one year. Conclusion: scRNA-seq from EUS core biopsies identified distinct prognostic tumor cell state fractions that were corroborated by pre-treatment bulk RNA-seq through deconvolution. These novel investigational and digital cytometric approaches not only confirmed prior tumor subtypes and their prognoses, but also enabled the quantification of their fractions for additional prognostic value. Patients with certain cell state fraction profiles may benefit from combinatorial therapeutic interventions, especially in the context of clinical trials. Citation Format: Erik Storrs, Abul Usmani, Bradley Krasnick, Chris Wetzel, Thomas Hollander, Prathamesh Chati, Faridi Qaium, Hephzibah Anthony, Ian Sloan, Natalie Cosgrove, Gabriel Lang, Vladimir Kushnir, Daniel Mullady, Dayna Early, William Hawkins, Koushik Das, Ryan Fields, Aadel Chaudhuri. Time-of-diagnosis prognostication of pancreatic ductal adenocarcinoma based on single cell RNA-seq and digital cytometry [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4750.

e15545 Background: Cell-free DNA (cfDNA) analysis from blood is a useful liquid biopsy strategy. However, it requires phlebotomy and moderate volumes of plasma. Here, we explored the feasibility of urinary cfDNA (ucfDNA) analysis in patients with oligometastatic colorectal cancer (CRC). Methods: We applied error-corrected targeted next-generation sequencing (NGS) (AVENIO ctDNA surveillance assay) to 10 patients with oligometastatic CRC to the liver who underwent curative-intent surgery. 40 paired samples (tumor, plasma, urine supernatant, peripheral blood mononuclear cells (PBMCs)) were collected, from which DNA was extracted. Plasma and urine cfDNA samples (acquired preoperatively) were sequenced to a median de-duplicated depth of 8,065X with on-target rate of 71%. Similarly high depth targeted NGS was applied to tumor and PBMC samples. Variants were called from plasma cfDNA and tumoral DNA and queried in ucfDNA using methods derived from CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) with integrated digital error suppression (iDES). Clonal hematopoiesis was accounted for by removing mutations also present in PBMCs. Results: Patients had a median of 2 tumors resected (range 1-7) with average sum of longest diameters of 8.7 cm (range 0.6-11). We used an average of 10 ml of blood and 37 ml of urine for cfDNA extraction. Circulating tumor DNA (ctDNA) was detected in all preoperative plasma samples (n = 10) at a mean mutant allele fraction (MAF) of 2.35% (range 0.09-11.78). Plasma ctDNA MAFs correlated significantly with tumor SLD (Pearson r = 0.81, P = 0.03). The average number of single nucleotide variants detected in plasma cfDNA, tumor and ucfDNA was 8 (range 1-42), 4 (range 1-7) and 2 (range 0-11), respectively. Indicative of concordance, 79% of the mutations called in tumor were detected in plasma. Tumor-derived DNA was detected in the preoperative urine of 6 patients (60%) with a MAF of 0.05% (range 0.03-0.12), ~35-fold less than in plasma (MAF of 1.78%, range 0.09-7.94) (P = 0.004). 38% of mutations identified in tumor and 26% from plasma were detected in ucfDNA for these 6 patients. In two patients, targetable mutations (BRAF p.Asp594Gly and PIK3CA p.Glu545Lys) present in tumor were also detected in ucfDNA, with the latter not detected in plasma. Conclusions: Our results indicate that ucfDNA analysis in patients with oligometastatic CRC is feasible; however, the mutational levels and detection sensitivity were much lower than from plasma. Yet, there may be situations where actionable mutations are missed in plasma but detected in urine.

Therapeutic proteins are produced in microbes, mammalian cell lines, and body fluids by applying recombinant DNA technology. They are required for compensating the deficiency of essential proteins in patients. Animal bioreactors producing such valuable bio-pharmaceuticals in body fluids have lately emerged as efficient and cost-effective expression systems. Promoters, along with other regulatory elements of genes coding for milk proteins, have been cloned from few species for directing the expression of desired proteins in the milk of farm animals. However, buffaloes, which are the second largest source of milk production in the world, have remained unexplored for such use. Since mammary epithelial cell-specific β-casein is the most abundantly expressed protein found in buffalo milk, we have isolated the promoter region and the transcriptional regulatory element along with exon 1, Intron 1 and partial exon 2 of the β-casein gene from the genome of the Indian river buffalo (Bubalus bubalis) and have characterized the same (GenBank accession no. KF612339). Mammary epithelial cells of buffalo and human (MCF7) expressed Enhanced green fluorescent protein (EGFP) upon transfection with the construct where egfp was cloned under the β-casein promoter. Transfected HEK-293 cells failed to express EGFP. Transgenic female mice generated using this construct expressed EGFP in the milk gland during lactation, without leaky expression in any other organs. This promoter also drove expression of recombinant human Interferonγ suggesting its use for expressing recombinant bio-pharmaceuticals in the milk of buffalo or other farm animals. Additionally, this may also allow breast gland-specific gene expression for remediation of breast gland-associated diseases.

Male and female infertility are clinically managed and classified as distinct diseases, and relatively little is known about mechanisms of gonadal function common to both sexes. We used genome-wide genetic analysis on 74,896 women and men to find rare genetic variants that modulate gonadal function in both sexes. This uncovered an association with variants disrupting CSMD1, a complement regulatory protein located on 8p23, in a genomic region with an exceptional evolution. We found that Csmd1 knockout mice display a diverse array of gonadal defects in both sexes, and in females, impaired mammary gland development that leads to increased offspring mortality. The complement pathway is significantly disrupted in Csmd1 mice, and further disruption of the complement pathway from joint inactivation of C3 leads to more extreme reproductive defects. Our results can explain a novel human genetic association with infertility and implicate the complement system in the normal development of postnatal tissues.

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

Background Spermatogonial stem cell (SSC) transplantation (SSCT) has become important for conservation of endangered species, transgenesis and for rejuvenating testes which have lost germ cells (Gc) due to gonadotoxic chemotherapy or radiotherapy during the prepubertal phase of life. Creating a germ cell-depleted animal model for transplantation of normal or gene-transfected SSC is a prerequisite for such experimental studies. Traditionally used intraperitoneal injections of busulfan to achieve this are associated with painful hematopoietic toxicity and affects the wellbeing of the animals. Use of testicular busulfan has been reported recently to avoid this but with a very low success rate of SSCT. Therefore, it is necessary to establish a more efficient method to achieve higher SSCT without any suffering or mortality of the animals. Methods A solution of busulfan, ranging from 25 μg/20 μl to 100 μg/20 μl in 50 % DMSO was used for this study. Each testis received two diagonally opposite injections of 10 μl each. Only DMSO was used as control. Germ cell depletion was checked every 15 days. GFP-expressing SSC from transgenic donor mice C57BL/6-Tg (UBC-GFP) 30Scha/J were transplanted into busulfan-treated testis. Two months after SSCT, mice were analyzed for presence of colonies of donor-derived SSC and their ability to generate offspring. Results A dose of 75 μg of busulfan resulted in reduction of testis size and depletion of the majority of Gc of testis in all mice within 15 days post injection without causing mortality or a cytotoxic effect in other organs. Two months after SSCT, colonies of donor-derived Gc-expressing GFP were observed in recipient testes. When cohabitated with females, donor-derived offspring were obtained. By our method, 71 % of transplanted males sired transgenic progeny as opposed to 5.5 % by previously described procedures. About 56 % of progeny born were transgenic by our method as opposed to 1.2 % by the previously reported methods. Conclusions We have established an efficient method of generating a germ cell-depleted animal model by using a lower dose of busulfan, injected through two diagonally opposite sites in the testis, which allows efficient colonization of transplanted SSC resulting in a remarkably higher proportion of donor-derived offspring generation.

Testicular Sertoli cells make a niche for the division and differentiation of germ cells. Sertoli cells respond to increased follicle-stimulating hormone (FSH) and testosterone (T) levels at the onset of puberty by producing paracrine factors which affect germ cells and trigger robust onset of spermatogenesis. Such paracrine support to germ cells is absent during infancy, despite Sertoli cells being exposed to high FSH and T within the infant testis. This situation is similar to certain cases of male idiopathic infertility where post-pubertal Sertoli cells fail to support germ cell division and differentiation in spite of endogenous or exogenous hormonal support. Defective Sertoli cells in such individuals may fail to express the full complement of their paracrine repertoire. Identification and supplementation with such factors may overcome Sertoli cells deficiencies and help trigger quantitatively and qualitatively normal differentiation of germ cells. To this end, we compared the transcriptome of FSH- and T-treated infant and pubertal monkey Sertoli cells by DNA microarray. Expression of Wnt3, a morphogen of the Wnt/β-catenin pathway, was higher in pubertal Sertoli cells relative to infant Sertoli cells. Transgenic mice were generated by us in which Wnt3 expression was curtailed specifically in post-pubertal Sertoli cells by shRNA. Subfertility and oligozoospermia were noticed in such animals with low Wnt3 expression in post-pubertal Sertoli cells along with diminished expression of Connexin43, a gap-junctional molecule essential for germ cell development. We report that the FSH- and T-targetedf Wnt3 governs Sertoli cell-mediated regulation of spermatogenesis and hence is crucial for fertility.

Our ability to decipher gene sequences has increased enormously with the advent of modern sequencing tools, but the ability to divulge functions of new genes have not increased correspondingly. This has caused a remarkable delay in functional interpretation of several newly found genes in tissue and age specific manner, limiting the pace of biological research. This is mainly due to lack of advancements in methodological tools for transgenesis. Predominantly practiced method of transgenesis by pronuclear DNA-microinjection is time consuming, tedious, and requires highly skilled persons for embryo-manipulation. Testicular electroporation mediated transgenesis requires use of electric current to testis. To this end, we have now developed an innovative technique for making transgenic mice by giving hypotonic shock to male germ cells for the gene delivery. Desired transgene was suspended in hypotonic Tris-HCl solution (pH 7.0) and simply injected in testis. This resulted in internalization of the transgene in dividing germ-cells residing at basal compartment of tubules leading to its integration in native genome of mice. Such males generated transgenic progeny by natural mating. Several transgenic animals can be generated with minimum skill within short span of time by this easily adaptable novel technique.

Our ability to decipher gene sequences has increased enormously with the advent of modern sequencing tools but the ability to divulge functions of new genes have not increased correspondingly. This has caused a remarkable delay in functional interpretation of several newly found genes in tissue and age specific manner, limiting the pace of biological research. This is mainly due to lack of advancements in methodological tools for transgenesis. Predominantly practiced method of transgenesis by pronuclear DNA-microinjection is time consuming, tedious and requires highly skilled persons for embryo-manipulation. Testicular electroporation mediated transgenesis requires use of electric current to testis. To this end, we have now developed an innovative technique for making transgenic mice by giving hypotonic shock to male germ cells for the gene delivery. Desired transgene was suspended in hypotonic Tris-HCl solution (pH 7.0) and simply injected in testis. This resulted in internalization of the transgene in dividing germ-cells residing at basal compartment of tubules leading to its integration in native genome of mice. Such males generated transgenic progeny by natural mating. Several transgenic animals can be generated with minimum skill within short span of time by this easily adaptable novel technique.

Background: Nonalcoholic fatty liver disease (NAFLD) is a well-recognized condition that includes a spectrum of clinicopathology conditions ranging from steatotosis to cirrhosis and liver failure. Available animal models are not ideal as they show only a partial resemblance to characteristic human NAFLD. Objective: This study was aimed at identifying a nonhuman primate model of NAFLD resembling features of human NAFLD, which will be useful in understanding the mechanism of the onset of this disease and for developing novel therapeutic modalities. Methods: The histological status of the liver and serum levels of triglycerides (TG), cholesterol, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of aged bonnet monkeys were compared with that of the aged rhesus and adult bonnet monkeys. Histopathology and immunostaining of liver sections and oil red ‘O’ confirmed NAFLD in aged bonnet monkeys. Results: Aged bonnet monkeys showed a significant (P

Spermatozoa are one of the few mammalian cell types that cannot be fully derived in vitro, severely limiting the application of modern genomic techniques to study germ cell biology. The current gold standard approach of characterizing single-gene knockout mice is slow as generation of each mutant line can take 6-9 months. Here, we describe an in vivo approach to rapid functional screening of germline genes based on a new nonsurgical, nonviral in vivo transfection method to deliver nucleic acids into testicular germ cells. By coupling multiplex transfection of short hairpin RNA (shRNA) constructs with pooled amplicon sequencing as a readout, we were able to screen many genes for spermatogenesis function in a quick and inexpensive experiment. We transfected nine mouse testes with a pilot pool of RNA interference (RNAi) against well-characterized genes to show that this system is highly reproducible and accurate. With a false negative rate of 18% and a false positive rate of 12%, this method has similar performance as other RNAi screens in the well-described Drosophila model system. In a separate experiment, we screened 26 uncharacterized genes computationally predicted to be essential for spermatogenesis and found numerous candidates for follow-up studies. Finally, as a control experiment, we performed a long-term selection screen in neuronal N2a cells, sampling shRNA frequencies at five sequential time points. By characterizing the effect of both libraries on N2a cells, we show that our screening results from testis are tissue-specific. Our calculations indicate that the current implementation of this approach could be used to screen thousands of protein-coding genes simultaneously in a single mouse testis. The experimental protocols and analysis scripts provided will enable other groups to use this procedure to study diverse aspects of germ cell biology ranging from epigenetics to cell physiology. This approach also has great promise as an applied tool for validating diagnoses made from medical genome sequencing, or designing synthetic biological sequences that can act as potent and highly specific male contraceptives.

Advanced methods of cellular purification are required to apply genome technology to the study of spermatogenesis. One approach, based on flow cytometry of murine testicular cells stained with Hoechst-33342 (Ho-FACS), has been extensively optimized and currently allows the isolation of 9 germ cell types. This staining technique is straightforward to implement, highly effective at purifying specific germ cell types and yields sufficient cell numbers for high throughput studies. Ho-FACS is a technique that does not require species-specific markers, but whose applicability to other species is largely unexplored. We hypothesized that, due to the similar cell physiology of spermatogenesis across mammals, Ho-FACS could be used to produce highly purified subpopulations of germ cells in mammals other than mouse. To test this hypothesis, we applied Ho-FACS to 4 mammalian species that are widely used in testis research - Rattus norvegicus, Cavia porcellus, Canis familiaris and Sus scrofa domesticus We successfully isolated 4 germ cell populations from these species with average purity of 79% for spermatocytes, and 90% for spermatids and 66% for spermatogonia. Additionally, we compare the performance of mechanical and chemical dissociation for each species, and propose an optimized gating strategy to better discriminate round and elongating spermatids in the mouse, which can potentially be applied to other species. Our work indicates that spermatogenesis may be uniquely accessible among mammalian developmental systems, as a single set of reagents may be sufficient to isolate germ cell populations from many different mammalian species, opening new avenues in the fields of development and male reproductive biology. Funded by an FCT doctoral fellowship (SFRH/BD/51695/2011 to A.C.L.), grants from the United States National Institutes of Health (R01HD078641 and R01MH101810 to D.F.C.), and an FCT research contract (IF/01262/2014 to A.M.L.). The Siteman Cancer Center is supported in part by an NCI Cancer Center Support Grant #P30 CA91842.

Supplementary Information. (PDF 2480 kb)

The protocols in this chapter describe two techniques for the generation of transgenic mice by in vivo manipulation of spermatogonial stem cells (SSCs) with a high rate of success. SSCs in prepubescent animals can either be infected in vivo with recombinant lentiviruses expressing the transgene of interest or DNA can be injected into the testis followed by the application of an electric current resulting in integration of the linearized DNA containing a transgene downstream of the appropriate promoter into SSCs. All male pre-founder mice produced transgenic pups using both protocols with the transgene being heritable. Further, the pre-founder mice could be used in multiple mating experiments resulting in the generation of multiple progeny. These protocols could be extended to perform over-expression/knockdown screens in vivo using bar-coded lentiviruses/plasmid constructs, thus permitting the design of genetic screens in the mouse. Further, these protocols could be adapted to achieve transgenesis in other laboratory animals resulting in the generation of model systems that closely approximate human development and disease.

The protocols in this chapter describe two techniques for the generation of transgenic mice by in vivo manipulation of spermatogonial stem cells (SSCs) with a high rate of success. SSCs in prepubescent animals can either be infected in vivo with recombinant lentiviruses expressing the transgene of interest or DNA can be injected into the testis followed by the application of an electric current resulting in integration of the linearized DNA containing a transgene downstream of the appropriate promoter into SSCs. All male pre-founder mice produced transgenic pups using both protocols with the transgene being heritable. Further, the pre-founder mice could be used in multiple mating experiments resulting in the generation of multiple progeny. These protocols could be extended to perform over-expression/knockdown screens in vivo using bar-coded lentiviruses/plasmid constructs, thus permitting the design of genetic screens in the mouse. Further, these protocols could be adapted to achieve transgenesis in other laboratory animals resulting in the generation of model systems that closely approximate human development and disease.

Microinjection of foreign DNA in male pronucleus by in-vitro embryo manipulation is difficult but remains the method of choice for generating transgenic animals. Other procedures, including retroviral and embryonic stem cell mediated transgenesis are equally complicated and have limitations. Although our previously reported technique of testicular transgenesis circumvented several limitations, it involved many steps, including surgery and hemicastration, which carried risk of infection and impotency. We improved this technique further, into a two step non-surgical electroporation procedure, for making transgenic mice. In this approach, transgene was delivered inside both testes by injection and modified parameters of electroporation were used for in-vivo gene integration in germ cells. Using variety of constructs, germ cell integration of the gene and its transmission in progeny was confirmed by PCR, slot blot and immunohistochemical analysis. This improved technique is efficient, requires substantially less time and can be easily adopted by various biomedical researchers.

In certain forms of idiopathic infertility, there is failure of follicle stimulating hormone (FSH) and testosterone (T) to initiate spermatogenesis despite the presence of Sertoli cells and germ cells in the testis. In postnatal rats (up to 11 days of age) and infant monkeys (3–4 months old), robust division and differentiation of spermatogonial stem cells is not discerned, even though serum levels of FSH and T are similar to those found during adulthood. Lack of spermatogenesis together with normal hormone levels is a situation similar to that found in certain categories of male infertility. To investigate this intriguing situation, Sertoli cells were cultured from infant and pubertal rats and monkeys and differential gene expression by testicular Sertoli cells was evaluated by DNA microarray using the Agilent microarray system. To determine the role of candidate genes in regulation of spermatogenesis, transgenic animals over-expressing these genes must be generated. However, present techniques for generation of transgenic animals have limited utility for production of several transgenic animals within a short period of time. Therefore, we have developed a technique for making transgenic animals by the testicular route which is less labor intensive and less time consuming. This technique is also ethically superior since fewer mice are required than in existing alternative methods of transgenesis.

Nonalcoholic fatty liver disease (NAFLD) is a well-recognized condition that includes a spectrum of clinicopathology conditions ranging from steatotosis to cirrhosis and liver failure. Available animal models are not ideal as they show only a partial resemblance to characteristic human NAFLD.This study was aimed at identifying a nonhuman primate model of NAFLD resembling features of human NAFLD, which will be useful in understanding the mechanism of the onset of this disease and for developing novel therapeutic modalities.The histological status of the liver and serum levels of triglycerides (TG), cholesterol, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of aged bonnet monkeys were compared with that of the aged rhesus and adult bonnet monkeys. Histopathology and immunostaining of liver sections and oil red 'O' confirmed NAFLD in aged bonnet monkeys.Aged bonnet monkeys showed a significant (P0.01) increase in serum TG, AST and ALT compared with aged rhesus and adult bonnet monkeys. Histopathology of the liver of aged bonnet macaques showed diffused microvesicular and macrovesicular fatty changes, perivenular and portal and perisinusoidal fibrosis with fatty degeneration of hepatocytes, and immunostaining of liver sections was suggestive of NAFLD.The spontaneous occurrence of NAFLD in normal animals is rare, but aged bonnet monkeys may serve as a unique animal model for studies related to NAFLD because they mimic pathophysiological features of human NAFLD.