Histological Phenotype Research Articles

Multi-Constraint Latent Representation Learning for Prognosis Analysis Using Multi-Modal Data.

The Cox proportional hazard model has been widely applied to cancer prognosis prediction. Nowadays, multi-modal data, such as histopathological images and gene data, have advanced this field by providing histologic phenotype and genotype information. However, how to efficiently fuse and select the complementary information of high-dimensional multi-modal data remains challenging for Cox model, as it generally does not equip with feature fusion/selection mechanism. Many previous studies typically perform feature fusion/selection in the original feature space before Cox modeling. Alternatively, learning a latent shared feature space that is tailored for Cox model and simultaneously keeps sparsity is desirable. In addition, existing Cox-based models commonly pay little attention to the actual length of the observed time that may help to boost the model's performance. In this article, we propose a novel Cox-driven multi-constraint latent representation learning framework for prognosis analysis with multi-modal data. Specifically, for efficient feature fusion, a multi-modal latent space is learned via a bi-mapping approach under ranking and regression constraints. The ranking constraint utilizes the log-partial likelihood of Cox model to induce learning discriminative representations in a task-oriented manner. Meanwhile, the representations also benefit from regression constraint, which imposes the supervision of specific survival time on representation learning. To improve generalization and alleviate overfitting, we further introduce similarity and sparsity constraints to encourage extra consistency and sparseness. Extensive experiments on three datasets acquired from The Cancer Genome Atlas (TCGA) demonstrate that the proposed method is superior to state-of-the-art Cox-based models.

COMPARISON OF CLINICAL PARAMETERS AND PATHOLOGICAL FEATURES OF INTESTINAL AND PANCREATOBILIARY HISTOLOGIC PHENOTYPES OF PERIAMPULLARY CARCINOMA

Background: Identification of the exact origin in large sized tumors of periampullary region is very difficult grossly and microscopically. The purpose of the study was to compare the two histological phenotypes of periampullary carcinoma in terms of their preoperative clinical parameters and pathological features. Methods: This was a prospective observational study done in patients diagnosed as periampullary carcinoma in specimen after pancreatoduodenectomy, between May 2018 and July 2019 at a Tertiary Teaching Hospital of Nepal. Relevant patient clinical characteristics, biochemical parameters including CEA and CA 19.9, immuno-histochemical markers (CK-20 and MUC-1) expression and histopathological details were recorded. Student t-test, ANOVA, median test, Kruskal Wallis test, binary logistic regression tests were used for analysis in SPSS version 20. P-value < 0.05 was considered significant. Results: Thirty-nine patients with periampullary adenocarcinoma in Pancreatoduodenectomy specimen were included in the study with mean age of 58.6 years. Ampullary tumors (53.8%) were common in this study. Pancreatobiliary phenotype (61.5% versus 38.5%), was more common in head of pancreas and distal CBD tumors and was associated with significantly higher lymph node ratio (0.174 ± 0.173 versus 0.069±0.096), higher rates of lymphovascular invasion (66.7% versus 33.3%) and perineural invasion (75% versus 26.7%) on univariate analysis. On multivariate analysis, Perineural invasion was associated with Pancreatobiliary phenotype (Odds ratio of 10.46). Conclusions: Pancreatobiliary phenotype of periampullary tumors was associated with poor prognostic pathologic features like high lymph node ratio, lymphovascular invasion and perineural invasion. Perineural invasion had the strongest association of all.

Whole Slide Imaging-Based Prediction of TP53 Mutations Identifies an Aggressive Disease Phenotype in Prostate Cancer.

Deep learning models predicting TP53 mutations from whole slide images of prostate cancer capture histologic phenotypes associated with stromal composition, lymph node metastasis, and biochemical recurrence, indicating their potential as in silico prognostic biomarkers. See related commentary by Bordeleau, p. 2809.

#5904 COMPARATIVE TRANSCRIPTOMICS OF THREE CANONICAL MOUSE MODELS OF CHRONIC KIDNEY DISEASE

Abstract Background and Aims A range of mouse models are available in preclinical chronic kidney disease (CKD) research but differ in their disease etiology and pathological hallmarks. To enable better selection of the mouse CKD model optimal for preclinical drug discovery studies, we characterized the kidney transcriptome signature of three well-established models of CKD induced by unilateral ureter obstruction (UUO), unilateral ischemic reperfusion injury (uIRI) and adenine-supplemented diet feeding (ADI). Method Male C57BL/6JRj mice were used in all studies. UUO or sham surgery was performed in 9 weeks old mice, which were terminated two weeks post-surgery. uIRI or sham surgery was performed in 10 weeks old mice terminated 6 weeks post-surgery. 12 weeks old mice received an adenine-supplemented diet or a control diet for 6 weeks. Endpoints included plasma biochemistry, kidney histology and RNA sequencing on kidney samples. Results Compared to corresponding controls, plasma urea was only increased in ADI mice. All three models presented with increased inflammation (F4/80, UUO>ADI>uIRI) and fibrosis (COl1A1, UUO = uIRI>ADI) in the histological analysis. Compared to corresponding controls, all three models demonstrated substantial increases in differential expressed genes (DEGs, UUO, n = 12,046; uIRI, n = 12,236; ADI, n = 11,468), with significant overlap in transcriptome signatures between the models. Gene expression markers of inflammation (e.g., Ccl2, Cd68 and Il1b), fibrogenesis (e.g., Tgfb1, Serpine1 and Col1a1) and kidney injury (e.g., Havcr1, Lcn2 and Spp1) supported histological findings in the three models. Interestingly, several molecular targets pursued in CKD drug discovery were significantly regulated, however with different directionality, in UUO (upregulated Atg7, Bax, Ednra/b, Gli2, Ifng, Shh; downregulated Glp1r, Nr3c2, Ptch1, Slc5a2), uIRI (upregulated Ednra, Gli2, Glp1r, Ifng, Nr3c2; downregulated Agt, Egfr, Slc5a2), and ADI mice (upregulated Agt, Atg7, Bax, Ednra; downregulated Ednrb, Egfr, Flt1, Nr3c2, Ptch1, Slc5a2). Conclusion UUO, uIRI and ADI mouse models demonstrate histopathological hallmarks of CKD, characterized by increased macrophage infiltration and fibrosis, which was corroborated by their individual transcriptome signatures. Despite similarities in histological phenotype, the models were distinguished based on kidney transcriptome changes, with several current drug targets regulated in a model-specific fashion. In conclusion, the current work will enable researchers to select the mouse model optimal for profiling individual preclinical test drugs with therapeutic potential in CKD. Also, our data may serve to enable further development of CKD mouse models with improved clinical translatability.

#6470 PATIENT SURVIVAL IN AN ASSOCIATED VASCULITIS COHORT – DATA FROM A REFFERAL CENTER

Abstract Background and Aims Patients with ANCA associated vasculitis (AAV) have variable survival rates depending on disease activity, complications and sometimes the choice of treatment. Still today there are a lot of differencies between various cohorts regardless of similarities in clinical presentation, serology or histology. Understanding risk factors for various outcomes in cohorts from different geographical areas might help build better and more uniform prediction models. We present data on patient survival from our refferal center. Method This study included 106 consecutive AAV patients with biopsy proven renal involvement in the period from 2007-2017. We analyzed clinical, laboratory and pathohistological data as predictors for death of the patients. Survival univariate analysis was performed using Kaplan-Meier method and log-rank (Mantel-Cox) test. Variables that had p<0,1 in univariate analysis were alongside age and gender included in multivariate Cox proportional hazard model. Results The study included 106 AAV patients with renal involvement: 66 (61,1%) MPA, 20 (18,5%) GPA, 20 (18,5%) RLV. There were 14 (13%) PR3-ANCA positive patients, 57 (52,8%) MPO ANCA positive, 5 (4,6%) PR3-ANCA+MPO-ANCA and 32 (29,6%) ANCA negative patients. Average SCr was 316,5 μmol/l (IQR 207,0-548,5), 24-hour proteinuria median was 1,7g/24h (IQR 0,8-2,8). Histologicaly (Berden classification) 43 (39,8%) patients had crescentic, 19 (17,6%) focal, 34 (31,5%) mixed and 12 (11,1%) sclerotic class. Follow up time was 1 to 127 months with median 21 months (IQR = 7 - 44) and medium follow up time of 28,6 months (SD = 26,6). All the patients recieved the same induction treatment (cyclophosphamide and glucocorticoids +/- acute haemodialysis and plasma exchange treatment) and remission maintenance treatment (azathioprine). During the follow up 21 patient (19,8%) died. Out of those 13 were females. Main causes of death were either infections or cardiovascular diseases. In follow up period patient survival was 83,9-81,2-79-74,7% at 12-24-36-60 months. There were no differences in survival rates between clinical, histological or serological phenotypes. In univariate analysis significant predictors for death of patients were: age (>68 years; p = 0,002), anaemia (Haemoglobin <96 g/l; p = 0,001), increased CRP levels (CRP > 63mg/l; p = 0,001), lower serum C3 levels (p = 0,001), BVAS > 18 (p = 0,003) and the need for dialysis (p = 0,06). In multivariate analysis however significant predictors for the death of patients were age (HR = 1,059, 95% CI = 1,001-1,120; p = 0,046), anaemia (HR = 0,952, 95% CI = 0,908-0,998; p = 0,040) and BVAS (HR = 1,093, 95% CI = 1,030-1,159; p = 0,003). Interestingly in additional analysis PLEX was, alongside acute haemodialysis, significant predictor in combined ESRD/death outcome (as well as for ESRD and relapse rate) but not for death alone. Conclusion In our data disease activity as defined by BVAS and anaemia alongside the patient age are predictors for the patient survival. Anaemia is not included either in BVAS or in other disease activity scores or prediction models. Considering its potential impact on multiple organ functions perhaps anaemia levels should be included either in BVAS or perhaps in VDI though anaemia itself is mostly not autoimmune phenomenon in AAV patients. Also in our cohort PLEX wasn't a significant predictor for patient survival.

#6517 CLINICOHISTOLOGICAL CHARACTERISTICS, THERAPEUTIC APPROACH AND RENAL OUTCOME IN PATIENTS WITH C3 GLOMERULOPATHY: A SINGLE-CENTER EXPERIENCE

Abstract Background and Aims C3 glomerulopathy has been identified as a distinct disease in the last decade but it has already been associated with a high burden of end stage renal disease (ESRD). Due to the rarity and heterogeneity of the disease, data regarding the prognosis and optimal treatment are still lacking. We aimed to describe the clinical and histological phenotype of patients with C3 glomerulopathy followed at our center, the treatment regimens applied and their long-term renal outcome. Method We conducted a retrospective medical chart review of 19 patients with C3 glomerulopathy followed at our center and we reported clinical, histological and therapeutic parameters as well as their long-term renal outcome. Results Median age of patients at the time of diagnosis was 23 years (IQR 28), median proteinuria 2.6 g/d (IQR 2.8) and median eGFR 63ml/min (IQR 63). 26% of patients (5/19) presented with an eGFR <30ml/min. Low serum C3 levels were reported in 11/19 patients (58%). Α monoclonal paraprotein was detected in three patients and pathogenous mutations of CFH/CFI genes in 4 out of 10 patients who underwent genetic testing. 41% of patients (7/17) were treated with RAAS inhibitors only, while 59% (10/17) received immunosuppressive treatment. Immunosuppressive treatment consisted of different combinations of steroids with mycophenolic acid, cyclophosphamide, cyclosporine or rituximab and steroid monotherapy in one patient. Treatment data were not available for two patients. There was a significant difference in baseline proteinuria between patients treated with RAAS inhibitors only (median 1.5g/d, IQR 1.14) and those who were treated with immunosuppressants (median 3.95g/d, IQR 4.8) (p = 0.003). Remission (complete or partial) was achieved by 43% of patients at 6 months and by 50% at 12 months. Median follow up time was 45 months (IQR 66). Eight patients (42%) reached ESRD in a median time of 58.5 months (IQR 62.5) while other three patients (16%) ended up with a GFR decline >30%. Among patients with adverse renal outcome (ESRD, GFR decline >30%), 54.5% (6/11) had baseline proteinuria >3g/d, 73% (8/11) had low serum C3 and 54.5% (6/11) had interstitial fibrosis/tubular atrophy >25%, while the median percentage of glomerulosclerosis was 33% (IQR 58). On the contrary, in the group of patients with favorable renal outcome, baseline proteinuria >3g/d was reported in only 25% (2/8), low serum C3 in 37.5% (3/8), interstitial fibrosis/tubular atrophy >25% in 25% (2/8), while median glomerulosclerosis was only 8% (IQR 15). Attainment of complete remission was associated with favorable renal outcome (p = 0.001). Five of eight patients who reached ESRD underwent kidney transplantation from a living related donor. All of them received basiliximab, mycophenolic acid, tacrolimus and steroids. Recurrence of C3 glomerulopathy was reported in four of these patients and eculizumab was given in two of them. Graft loss due to disease recurrence was observed in one patient. Conclusion The present case series of patients with C3 glomerulopathy highlights the great heterogeneity in the clinical phenotype, therapeutic approach and renal prognosis of the disease. It also underscores the importance of attainment of complete remission regarding the long-term renal survival. Large studies are needed in order to better define clinical and histological predictors of adverse renal outcome as well as the optimal treatment regimens.

Predicting isocitrate dehydrogenase genotype, histological phenotype, and Ki-67 expression level in diffuse gliomas with an advanced contrast analysis of magnetic resonance imaging sequences.

The present study aimed to establish a robust predictive model based on a machine learning (ML) algorithm providing preoperative noninvasive diagnosis and to further explore the contribution of each magnetic resonance imaging (MRI) sequence to the classification to help select images for future model development. This was a retrospective cross-sectional study, and consecutive patients with histologically confirmed diffuse gliomas in our hospital from November 2015 to October 2019 were recruited. The participants were grouped into a training and testing set based on a ratio of 8:2. Five MRI sequences were employed to develop the support vector machine (SVM) classification model. An advanced contrast analysis of single-sequence-based classifiers was performed, according to which different sequence combinations were tested, and the best one was selected to form an ultimate classifier. Patients whose MRIs were acquired with other types of scanners formed an additional, independent validation set. A total of 150 patients with gliomas were used in the present study. Contrast analysis revealed that the contribution of the apparent diffusion coefficient (ADC) was the most significant [accuracies were as follows: histological phenotype, 0.640; isocitrate dehydrogenase (IDH) status, 0.656; and Ki-67 expression, 0.699] and that of T1 weighted imaging was limited (accuracies were as follows: histological phenotype, 0.521; IDH status, 0.492; and Ki-67 expression, 0.556). The ultimate classifiers for IDH status, histological phenotype, and Ki-67 expression achieved promising performances with area under the curve (AUC) values of 0.88, 0.93, and 0.93, respectively. The classifiers for the histological phenotype, IDH status, and Ki-67 expression correctly predicted 3 of 5 subjects, 6 of 7 subjects, and 9 of 13 subjects in the additional validation set, respectively. The present study showed satisfactory performance in predicting the IDH genotype, histological phenotype, and Ki-67 expression level. The contrast analysis revealed the contribution of different MRI sequences and suggested that the combination of all the acquired sequences was not the optimal strategy to build the radiogenomics-based classifier.

Translation Animal Models of Diabetic Kidney Disease: Biochemical and Histological Phenotypes, Advantages and Limitations.

Animal models play a crucial role in studying the pathogenesis of diseases, developing new drugs, identifying disease risk markers, and improving means of prevention and treatment. However, modeling diabetic kidney disease (DKD) has posed a challenge for scientists. Although numerous models have been successfully developed, none of them can encompass all the key characteristics of human DKD. It is essential to choose the appropriate model according to the research needs, as different models develop different phenotypes and have their limitations. This paper provides a comprehensive overview of biochemical and histological phenotypes, modeling mechanisms, advantages and limitations of DKD animal models, in order to update relevant model information and provide insights and references for generating or selecting the appropriate animal models to fit different experimental needs.

AAV-mediated gene augmentation therapy of CRB1 patient-derived retinal organoids restores the histological and transcriptional retinal phenotype.

Retinitis pigmentosa and Leber congenital amaurosis are inherited retinal dystrophies that can be caused by mutations in the Crumbs homolog 1 (CRB1) gene. CRB1 is required for organizing apical-basal polarity and adhesion between photoreceptors and Müller glial cells. CRB1 patient-derived induced pluripotent stem cells were differentiated into CRB1 retinal organoids that showed diminished expression of variant CRB1 protein observed by immunohistochemical analysis. Single-cell RNA sequencing revealed impact on, among others, the endosomal pathway and cell adhesion and migration in CRB1 patient-derived retinal organoids compared with isogenic controls. Adeno-associated viral (AAV) vector-mediated hCRB2 or hCRB1 gene augmentation in Müller glial and photoreceptor cells partially restored the histological phenotype and transcriptomic profile of CRB1 patient-derived retinal organoids. Altogether, we show proof-of-concept that AAV.hCRB1 or AAV.hCRB2 treatment improved the phenotype of CRB1 patient-derived retinal organoids, providing essential information for future gene therapy approaches for patients with mutations in the CRB1 gene.

Prevalence and clinical significance of lymphadenopathy and its histological subtypes in patients with systemic lupus erythematosus: a retrospective cohort study

The objective of this study was toevaluate the prevalence and the clinical significance of lymphadenopathy and its histological subtypes in patients with systemic lupus erythematosus. We conducted a retrospective cohort study of patients with SLE diagnosed using the 1997 ACR criteria,who were followed at our institution between 2008 and 2022. Patients were grouped based on the presence of SLE-attributed LAD and its histological phenotype, then compared in terms of demographic, clinical and laboratory characteristics. Of the 255 patients, 33.7% had SLE-attributed, 0.8% lymphoma-related and 0.4% tuberculosis-related LAD. Univariate analysis identified significant associations between the presence of LAD and fever (p < 0.0001), weight loss (p = 0.009), pericarditis (p = 0.004), myocarditis (p = 0.003), myositis (p = 0.034), leukopenia (p = 0.004), lymphopenia (p = 0.003), membranous nephritis (p = 0.004), anti-RNP (p = 0.001), anti-Smith (p = < 0.0001), and SSB antibodies (p = 0.038), and hypocomplementemia (C3:p = 0.019; C4:p < 0.0001). Logistic regression confirmed the associations of LAD with fever (OR = 3.277, 95% C.I 1.657-6.481), pericarditis (OR = 4.146, 95% C.I:1.577-10.899), membranous nephritis (OR = 3.586, 95% C.I:1.305-9.854), and leukopenia (OR = 2.611, 95%C.I:1.319-5.166), but not withweight loss, myocarditis, or myositis. Biopsy ina subset of patients (33.7%of total) revealed reactive/proliferative (62.1%) or necrotizing (37.9%) histological patterns. When we compared the histologic patterns, necrotizing LAD was associated with fever (p = 0.052), sicca (p = 0.018), and malar rash (p = 0.005). Most patients received corticosteroids, hydroxychloroquine, and/or DMARDs with relatively quick clinical improvement. In conclusion,LAD is a common SLE manifestation, associated with constitutional symptoms, myo-/pericarditis, myositis, cytopenia, and membranous nephritis. Despite relatively high prevalence of LAD in SLE, a biopsy may still be needed to rule out lymphoma.

BCL6B Contributes to Ocular Vascular Diseases via Notch Signal Silencing

Endothelial cell activation is tightly controlled by the balance between VEGF (vascular endothelial cell growth factor) and Notch signaling pathway. VEGF destabilizes blood vessels and promotes neovascularization, which are common features of sight-threatening ocular vascular disorders. Here, we show that BCL6B (B-cell CLL/lymphoma 6 member B protein), also known as BAZF, ZBTB28, and ZNF62, plays a pivotal role in the development of retinal edema and neovascularization. The pathophysiological physiological role of BCL6B was investigated in cellular and animal models mimicking 2 pathological conditions: retinal vein occlusion and choroidal neovascularization. An in vitro experimental system was used in which human retinal microvascular endothelial cells were supplemented with VEGF. Choroidal neovascularization cynomolgus monkey model was generated to investigate the involvement of BCL6B in the pathogenesis. Mice lacking BCL6B or treated with BCL6B-targeting small-interfering ribose nucleic acid were examined for histological and molecular phenotypes. In retinal endothelial cells, the BCL6B expression level was increased by VEGF. BCL6B-deficient endothelial cells showed Notch signal activation and attenuated cord formation via blockage of the VEGF-VEGFR2 signaling pathway. Optical coherence tomography images showed that choroidal neovascularization lesions were decreased by BCL6B-targeting small-interfering ribose nucleic acid. Although BCL6B mRNA expression was significantly increased in the retina, BCL6B-targeting small-interfering ribose nucleic acid suppressed ocular edema in the neuroretina. The increase in proangiogenic cytokines and breakdown of the inner blood-retinal barrier were abrogated in BCL6B knockout (KO) mice via Notch transcriptional activation by CBF1 (C promotor-binding factor 1) and its activator, the NICD (notch intracellular domain). Immunostaining showed that Müller cell activation, a source of VEGF, was diminished in BCL6B-KO retinas. These data indicate that BCL6B may be a novel therapeutic target for ocular vascular diseases characterized by ocular neovascularization and edema.

Presentation of orbital solitary fibrous tumours.

To evaluate presenting features of patients with orbital solitary fibrous tumours (SFTs), based on histological phenotype. A retrospective case-note review was performed for demographics and presenting features for patients with orbital SFTs. The tumours were classified as "Group IA" hypocellular SFT phenotype, "Group IB" haemangiopericytoma phenotype and low mitotic activity, and high-grade "Group II" haemangiopericytoma phenotype with high mitotic activity. Sixty-four patients (34 female; 53%) presented at a mean age of 42.2 years (median 38; range 19-82), with Group II patients presenting at an older age (mean 53 years). Median symptom duration was 12 months for Groups IA and IB, compared to 4 months for Group II, the commonest symptoms being proptosis (53%), diplopia (41%), periorbital swelling (31%), and altered vision (19%). Mean LogMAR was 0.17 (median 0.0; range -0.2-4), and 14% had ipsilateral optic neuropathy, with no significant difference between the three groups. Non-axial displacement was noted in 69%, a palpable mass in 45%, and reduced eye movements in 59%; choroidal folds and optic disc swelling were recorded in 12% and 9%. SFTs were mostly extraconal (59%), within the superior and superonasal quadrants (44%), with an average estimated tumour volume of 4.9 ml (median 3.6; range 0.31-14.5 ml). SFTs may present with impaired visual function (∼15%), fundal abnormalities (a fifth), globe displacement (two-thirds), and reduced ocular motility (over a half). High-grade tumours tend to present more than a decade later, with a shorter duration of symptoms.

Role of adjuvant chemotherapy on recurrence and survival in patients with resected ampulla of Vater carcinoma

Owing to rarity of disease and lack of prospective studies, data supporting the role of adjuvant chemotherapy in ampulla of Vater (AoV) carcinoma is limited. To evaluate whether adjuvant chemotherapy cases for AoV carcinoma had better disease-free survival (DFS) rates than cases of observation following curative surgery. We retrospectively analyzed the association between adjuvant chemotherapy and DFS and overall survival (OS) in patients with stage IB-III AoV carcinoma who underwent curative surgical resection. Fluorouracil-based adjuvant chemotherapy was administered after surgery at the discretion of the physician. Adjusted multivariate regression models were used to evaluate the association between adjuvant chemotherapy and survival outcomes. Of the total 104 patients who underwent curative surgery, 52 received adjuvant chemotherapy. Multivariate analysis revealed that higher histologic grade [hazard ratio (HR) = 2.24, P = 0.046], advanced tumor stage (HR = 1.85, P = 0.030), and vascular invasion (HR = 2.14, P = 0.010) were associated with shorter DFS. Adjuvant chemotherapy improved DFS compared to the observation group (HR = 0.50, P = 0.015) and tended to be associated with a longer OS, although the difference was not statistically significant (HR = 0.58, P = 0.098). Among patients with resected AoV carcinoma, the adjuvant chemotherapy group was not associated with a significant survival benefit compared to the observation group. However, on multivariate analysis adjusting for prognostic factors, adjuvant chemotherapy following surgery was an independent prognostic factor for DFS in patients with resected AoV carcinoma. Further studies are needed to investigate the effectiveness of adjuvant chemotherapy according to histologic phenotype.

Abstract LB171: WSI based prediction of TP53 mutations identifies aggressive disease phenotype in prostate cancer

Abstract In prostate cancer, there is an urgent need for objective prognostic biomarkers that identify a tumor’s metastatic potential at an early stage. While recent analyses indicated TP53 mutations as candidate biomarker, molecular profiling in a clinical setting is complicated by tumor heterogeneity. Deep learning models that predict the spatial presence of TP53 mutations in Whole Slide Images (WSIs) offer the potential to mitigate this issue. To assess the potential of WSIs as proxy for spatially resolved profiling or as biomarker for aggressive disease, we developed TiDo, a deep learning model that achieves state-of-the-art performance in predicting TP53 mutations from WSIs of primary prostate tumors. On an independent multi-focal cohort, we could show successful generalization of the model, both at patient and lesion level. Hence, the model offers insight into which lesions on a WSI most likely contain a TP53 mutation. Analysis of model predictions revealed that false positive (FP) predictions could at least partially be explained by TP53 deletions. This suggests that some FP carry another alteration of which the effect converges in the same histological phenotype. Comparative expression analysis and histological cell type analysis identified such common phenotype (related to stromal composition) in both TP and FP predictions. This indicates that WSI-based models might not be able to perfectly predict the spatial presence of individual TP53 mutations. However, we show they have the potential of capturing a tumor’s aggressive potential by observing a downstream phenotype of the tumor cells and TME associated with a biomarker of aggressive disease (TP53). Citation Format: Marija Pizurica, Maarten Larmuseau, Kim Van der Eecken, Louise de Schaetzen van Brienen, Francisco Carrillo-Perez, Simon Isphording, Nicolaas Lumen, Jo Van Dorpe, Piet Ost, Sofie Verbeke, Olivier Gevaert, Kathleen Marchal. WSI based prediction of TP53 mutations identifies aggressive disease phenotype in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB171.

Abstract 2048: Mutations in FOXA1 alter chromatin remodeling and cell fate in prostate organoids

Abstract Genomic analysis of targeted patient tumor sequencing identified frequent mutations, 41% in prostate cancer (Li, et al., 2020) in the gene FOXA1, a developmentally important pioneer transcription factor (TF) in mammary and prostate tissues. Previous work by our group and others has shown that these FOXA1 mutations alter global chromatin accessibility and promote growth in prostate cells (Adams, 2019), but the underlying molecular details, including the identity of partner TFs, remain unclear. To address this topic, we generated mouse prostate organoids expressing Foxa1 alleles harboring three distinct classes of mutations: (i) overexpression of WT Foxa1 (reflecting focal gene amplification seen in tumors), (ii) a series of mutants within the Wing2region of the forkhead binding domain (FHBD) and (iii) a mutant bearing a stop codon after the FHBD to represent a series of C-terminal truncation mutants. We performed single nucleus multiome sequencing to obtain gene expression (snRNA-seq) and chromatin accessibility (snATAC-seq) readouts from the same individual nuclei. Whereas each Foxa1 mutant has distinct, often mutant-specific features, several themes emerge. These include alterations in the relative proportion of stem-like (L2) luminal cells vs secretory (L1) luminal cells as well as changes in luminal or basal gene signatures, increased androgen receptor signaling output, and enrichment for motifs of distinct classes of partner TFs. For example, cells expressing the truncation mutant show gain in the accessibility of Gata3 and Pou2f1 TF binding motifs, as well as enhanced numbers of L1-like luminal cells. Functional studies demonstrate that Pou2f1 is specifically required for the pro-luminal phenotype in cells expressing the truncation mutant whereas Gata3 plays a more general pro-luminal role. Correlations in motif accessibility and transcription factor expression across single cells further revealed a composite androgen receptor (AR)-FOXA1 motif enriched in the pro-luminal truncation mutant, while the canonical AR motif was enriched in pro-basal cell mutants. Finally, Foxa1 mutants cooperative with Trp53 and Pten loss in orthotopic prostate tumorigenicity assays, most strikingly manifest by reversion of the basal-like features characteristic of Trp53/Pten loss tumors to Ck8+ luminal adenocarcinoma histology, mirroring that seen in Foxa1-mutant human prostate cancer tumors in mice. Thus, mutant Foxa1 alleles cooperate with canonical prostate cancer tumor suppressors and alter the histologic phenotype of prostate cancers in mice through the activation of basal or luminal lineage differentiation programs. Citation Format: Erik Ladewig, Abbas Nazir, Christina Leslie, Charles Sawyers. Mutations in FOXA1 alter chromatin remodeling and cell fate in prostate organoids [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 2048.

Abstract 24: Oncogenic KrasG12D and Cdkn2a/p16 knockout in LGR5 expressing progenitor cells synergize to advance adenoma and adenocarcinoma phenotypes in murine small intestine

Abstract Background: KRAS activating mutations are reported in 42-54% and inactivation of CDKN2A/p16 in 14-30% of human small intestinal adenocarcinomas, suggesting a role in small intestine adenocarcinogenesis. The phenotypic effects of oncogenic KRAS and p16 loss have not been well characterized in small intestine (SI) adenocarcinogenesis. We targeted LGR5-expressing progenitor cells residing in intestinal crypts for conditional activation of Kras and/or Cdkn2a/p16 deletion to generate a murine model to study the single and combined effects of KRAS and p16 alterations driving gastrointestinal tumorigenesis. We hypothesized that oncogenic Kras and loss of Cdkn2a/p16 drives adenoma-adenocarcinoma sequence in murine SI. Methods: We generated mice with conditional knockout of Cdkn2a/p16 (p16del: IL1bwt/tg;Lgr5-Cre+/−;p16fl/fl;Kraswt/wt), conditional expression of oncogenic KrasG12D (IL1bwt/tg;Lgr5-Cre+/−;KrasG12D/wt), or both alterations (p16del+KrasG12D: IL1bwt/tg;Lgr5-Cre+/−;p16fl/fl;KrasG12D/wt), in mice carrying the human interleukin 1 beta (IL1B) and expressing modified Cre under the Lgr5 promoter. Our control murine model expresses wild type p16 and KRAS (p16wt+Kraswt: IL1bwt/tg;Lgr5-Cre+/−;p16wt/wt;Kraswt/wt). These mice were initially generated to study esophageal adenocarcinogenesis and express the IL1b transgene in esophagus but not in SI. All mice were given bile acid in drinking water daily and were treated with tamoxifen to activate Cre in Lgr5-positive gastrointestinal epithelial progenitor cells. Mice were euthanized and gross and histologic alterations in the SI were examined from 4 to 21 months of age. 3D organoids were isolated from the SI of each mouse model. Results: We examined 89 mice from 4 to 12 months of age. Mice with KRAS activation (KrasG12D) alone infrequently developed SI duodenal adenomas (6.25%: 1/16) compared to 50% (9/18) of mice carrying the combined P16del+KrasG12D genotypes (P=0.005). No adenomas were seen in 27 control and 28 p16del mice. Adenomas with high-grade dysplasia, intramucosal adenocarcinoma, and invasive adenocarcinoma were seen only in older p16del+KrasG12D mice (13-21 months). Single or multiple adenomas and adenocarcinomas were observed only in the duodenal segment of SI and not in the colon. 3D organoids isolated from each mouse model reproduce the intestinal histologic phenotype. Conclusions: Loss of Cdkn2a/p16 and KRASG12D expression synergistically advances adenoma-adenocarcinoma tumorigenesis in the SI duodenum, providing a murine model that recapitulates SI adenocarcinogenesis in vivo. 3D SI organoids provide useful in vitro models for mechanistic studies of SI adenocarcinogenesis. These in vivo and in vitro models will enable identification of key molecular pathways that may be targeted with novel therapies for this rare cancer. Citation Format: Tyler Seckar, Jing Sun, Elena V. Komissarova, Jorge L. Sepulveda, Timothy L. Wang, Antonia R. Sepulveda. Oncogenic KrasG12D and Cdkn2a/p16 knockout in LGR5 expressing progenitor cells synergize to advance adenoma and adenocarcinoma phenotypes in murine small intestine [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 24.

BlockmiR AONs as Site-Specific Therapeutic MBNL Modulation in Myotonic Dystrophy 2D and 3D Muscle Cells and HSALR Mice.

The symptoms of Myotonic Dystrophy Type 1 (DM1) are multi-systemic and life-threatening. The neuromuscular disorder is rooted in a non-coding CTG microsatellite expansion in the DM1 protein kinase (DMPK) gene that, upon transcription, physically sequesters the Muscleblind-like (MBNL) family of splicing regulator proteins. The high-affinity binding occurring between the proteins and the repetitions disallow MBNL proteins from performing their post-transcriptional splicing regulation leading to downstream molecular effects directly related to disease symptoms such as myotonia and muscle weakness. In this study, we build on previously demonstrated evidence showing that the silencing of miRNA-23b and miRNA-218 can increase MBNL1 protein in DM1 cells and mice. Here, we use blockmiR antisense technology in DM1 muscle cells, 3D mouse-derived muscle tissue, and in vivo mice to block the binding sites of these microRNAs in order to increase MBNL translation into protein without binding to microRNAs. The blockmiRs show therapeutic effects with the rescue of mis-splicing, MBNL subcellular localization, and highly specific transcriptomic expression. The blockmiRs are well tolerated in 3D mouse skeletal tissue inducing no immune response. In vivo, a candidate blockmiR also increases Mbnl1/2 protein and rescues grip strength, splicing, and histological phenotypes.

IL11 (Interleukin-11) Causes Emphysematous Lung Disease in a Mouse Model of Marfan Syndrome.

Marfan Syndrome (MFS) is an inherited connective tissue disorder caused by mutations in the FBN1 (fibrillin-1) gene. Lung abnormalities are common in MFS, but their pathogenesis is poorly understood. IL11 (interleukin-11) causes aortic disease in a mouse model of MFS and was studied here in the lung. We examined histological and molecular phenotypes in the lungs of Fbn1C1041G/+ mice (mouse model of Marfan Syndrome [mMFS]), an established mouse model of MFS. To identify IL11-expressing cells, we used immunohistochemistry on lungs of 4- and 16-week-old Fbn1C1041G/+:Il11EGFP/+ reporter mice. We studied the effects of IL11 inhibition by RT-qPCR, immunoblots and histopathology in lungs from genetic or pharmacologic models: (1) 16-week-old IL11 receptor (IL11RA) knockout mMFS mice (Fbn1C1041G/+:Il11ra1-/- mice) and (2) in mMFS mice administered IgG control or interleukin-11 receptor antibodies twice weekly from 4 to 24 weeks of age. mMFS lungs showed progressive loss and enlargement of distal airspaces associated with increased proinflammatory and profibrotic gene expression as well as matrix metalloproteinases 2, 9, and 12. IL11 was increased in mMFS lungs and localized to smooth muscle and endothelial cells in young mMFS mice in the Fbn1C1041G/+:Il11EGFP/+ reporter strain and in fibroblasts, in older mice. In mMFS mice, genetic (Fbn1C1041G/+:Il11ra1-/-) or pharmacologic (anti-interleukin-11 receptor) inhibition of IL11 signaling reduced lung emphysema, fibrosis, and inflammation. This protective effect was associated with reduced pathogenic ERK1/2 signaling and lower metalloproteinase 2, 9, and 12 expression. IL11 causes lung disease in mMFS. This reveals a shared IL11-driven disease mechanism in lung and aorta in MFS and suggests inhibition of IL11 signaling as a holistic approach for treating multiorgan morbidity in MFS.

Sertoli cell-only syndrome: advances, challenges, and perspectives in genetics and mechanisms.

Male infertility can be caused by quantitative and/or qualitative abnormalities in spermatogenesis, which affects men's physical and mental health. Sertoli cell-only syndrome (SCOS) is the most severe histological phenotype of male infertility characterized by the depletion of germ cells with only Sertoli cells remaining in the seminiferous tubules. Most SCOS cases cannot be explained by the already known genetic causes including karyotype abnormalities and microdeletions of the Y chromosome. With the development of sequencing technology, studies on screening new genetic causes for SCOS are growing in recent years. Directly sequencing of target genes in sporadic cases and whole-exome sequencing applied in familial cases have identified several genes associated with SCOS. Analyses of the testicular transcriptome, proteome, and epigenetics in SCOS patients provide explanations regarding the molecular mechanisms of SCOS. In this review, we discuss the possible relationship between defective germline development and SCOS based on mouse models with SCO phenotype. We also summarize the advances and challenges in the exploration of genetic causes and mechanisms of SCOS. Knowing the genetic factors of SCOS offers a better understanding of SCO and human spermatogenesis, and it also has practical significance for improving diagnosis, making appropriate medical decisions, and genetic counseling. For therapeutic implications, SCOS research, along with the achievements in stem cell technologies and gene therapy, build the foundation to develop novel therapies for SCOS patients to produce functional spermatozoa, giving them hope to father children.

Adult-Onset Genetic Focal Segmental Glomerulosclerosis: A Tale of Two Mutations

Focal segmental glomerulosclerosis (FSGS) is a kidney histologic lesion that may be caused by multiple aetiologies and pathophysiological mechanisms, with podocyte injury and depletion as the common denominator. FSGS may be subdivided into different subclasses: primary, secondary, genetic and unknown forms. Notwithstanding the overlapping clinical and histological characteristics across the different forms of FSGS, their management and response to treatment are strikingly different. Genetic FSGS may be suggested by the appearance of nephrotic syndrome during childhood, but it may also present in adulthood, where the diagnosis is rather challenging due to widely variable clinical and histological phenotypes. Herein we present the case of a 34-year-old female with a family history of chronic kidney disease of undetermined aetiology, referred for a Nephrology consultation due to haematoproteinuria and de novo arterial hypertension. Complementary evaluation revealed a urinary protein/creatinine ratio of 4.3 g/g and albumin/creatinine ratio of 3.9 g/g with hypoalbuminaemia. Kidney biopsy revealed lesions of FSGS, associated with extensive foot process effacement. The constellation of findings and family history of kidney disease raised the suspicion of a genetic cause, therefore genetic testing was performed. Two variants in the NPHS2 gene [c.686G&gt;A, p.(Arg229Gln) and c.855_856del, p(Arg286Thrfs*17)] were found in compound heterozygosity, compatible with the diagnosis of genetic FSGS. This case highlights the importance of a detailed evaluation of patients with FSGS lesions in order to identify the FSGS form, given its therapeutic and prognostic impact, including after kidney transplantation.