PDGFRA Research Articles

The Histopathological Types and Distribution Characteristics of Gastric Mixed Tumors

ObjectiveThe present study aimed to investigate the histopathological types and distribution characteristics of gastric mixed tumors.MethodsDetailed histological observations, together with related immunohistochemical and genetic tests, were analyzed on 960 surgically resected samples in 6 hospitals with gastric mixed tumors from May 2017 to May 2021 in this retrospective study.ResultsEpithelial-derived tumors accounted for 80.10% (769/960) of the gastric mixed tumor samples studied, and tumors of different tissue origins accounting for 10.83% (104/960), mesenchymal-derived tumors accounting for 6.25% (60/960), neuroendocrine tumors accounting for 2.40% (23/960), and lymphoma accounting for 0.42% (4/960). The histological types of gastric mixed tumors identified as most commonly were epithelial originated, followed by mixed tumors of different tissue originated, then mixed neuroendocrine, lymphoma, and mesenchymal originated in sequence. The histological number of gastric mixed tumors was ≤ 3 in 83.23% (799/960) of cases and > 4 in 16.77% (161/960) of cases. The mixed histological patterns of gastric mixed tumors were divided into three types: those with tumor cells interspersed with each other, those with incomplete fibrous tissue separation, and those without fibrous tissue separation. The gene target characteristics of gastric mixed tumors were the existence of multi-gene mutation, including human epidermalgrowth factor receptor-2 (HER2) gene amplification, key result areas (K-ras) and platelet-derived growth factor receptor alpha (PDGFRA).ConclusionGastric mixed tumors should be adequately sampled, each piece of tissue should be involved in the morphological proportional division of the tumor, and any independent histological component should be written into the pathological examination report.

Abstract 660: Predicting imatinib responses in exon 18 PDGFRA-mutant GIST

Abstract Gastrointestinal stromal tumor (GIST) is the most common GI sarcoma and frequently harbor oncogenic mutations in KIT or platelet derived growth factor receptor alpha (PDGFRA) receptor tyrosine kinases. While type II tyrosine kinase inhibitors (TKIs) revolutionized the treatment of KIT-mutant GIST, they are thought to be ineffective for many PDGFRA-mutant GIST cases. Over 80% of PDGFRA mutations in GIST occur in exon 18 and patients with the most common mutation, D842V, are resistant to imatinib and other type II TKIs, leading to poor outcomes. However, beyond D842V, numerous other PDGFRA exon 18 mutations have been observed. From curated data of a large cohort of PDGFRA-mutant GISTs, we have identified nearly 50 unique PDGFRA exon 18 mutations, including both point mutations and complex indels. Although it is largely unknown how these mutations respond to imatinib, currently most, if not all, PDGFRA exon 18 mutations are assumed to be imatinib resistant. Using cell models, we tested some of the most commonly observed mutations in this cohort. Contrary to dogma, we observed that some exon 18 mutations tested were imatinib sensitive. We also observed that imatinib sensitivity seemed to be dependent on the properties of the residue in the 842-codon position, which encodes a key auto-inhibitory residue. We then hypothesized that imatinib sensitivity of every known or future reported PDGFRA exon 18 mutation might be predicted based on the 842 residue. To test the predictive potential of the 842 residue, we created mutagenic libraries that included every amino acid substitution at the 842 position. We expressed each mutation in Ba/F3 cells and assessed imatinib sensitivity via immunoblotting for phosphorylated-PDGFRA. Overall, 40% of tested mutations were sensitive to imatinib, further debunking the belief that all PDGFRA exon 18 mutations are imatinib resistant. In agreement with our hypothesis, we found that any hydrophobic residue at the 842 position conferred imatinib resistance (IC50 > 100nM), and all other amino acids tested to date were imatinib sensitive (IC50 < 100nM). Based on these data alone, over 55% of the 50 unique mutations identified from our PDGFRA-mutant GIST patient cohort would be predicted to be imatinib sensitive, demonstrating the importance of understanding the biochemical properties of various exon 18 mutations to predict treatment outcomes. Our predictive model would help identify patients with PDGFRA exon 18 mutations that could benefit from using front-line imatinib as an alternative to avapritinib, which has been recently approved for first-line treatment for all PDGFRA exon 18 mutant GIST. Imatinib, which has been used clinically for over 20 years, has a superior safety and tolerability profile compared to avapritinib. This paradigm shifting approach has the potential to transform patient care and outcomes by optimizing the sequencing of TKIs for PDGFRA-mutant GIST patients. Citation Format: Homma M. Khosroyani, Lillian R. Klug, Johanna Falkenhorst, Sebastian Bauer, Michael C. Heinrich. Predicting imatinib responses in exon 18 PDGFRA-mutant GIST [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 660.

The Transfigured Ingot-Gastrointestinal Stromal Tumour

Gastrointestinal stromal tumour is a frequently discerned, mesenchymal neoplasm occurring within gastrointestinal tract. Predominantly incriminating gastric region, tumefaction arises from interstitial cells of Cajal which constitute myenteric plexus situated within muscularis propria. Previously designated as gastrointestinal smooth muscle tumour, gastrointestinal autonomic nerve tumour, leiomyoblastoma, smooth muscle tumour of uncertain malignant potential or gastrointestinal pacemaker cell tumour, gastrointestinal stromal tumour may exhibit malignant biological behaviour [1,2]. Of obscure aetiology, majority of neoplasms are sporadic. However, few familial neoplasms may occur. Chromosomal mutations of KIT and PDGFRA genes are singularly associated with the tumour. Neoplasm may be discovered incidentally during various imaging or endoscopic procedures of gastrointestinal tract [1,2].

Myeloid/lymphoid neoplasm with eosinophilia and rearrangement of PDGFRA presenting as myeloid sarcoma.

A 32-year-old male patient with no significant past medical history presented with fatigue and left groin lymphadenopathy. The left groin lymph node was excised and measured 2.0 × 1.4 × 1.1 cm. On histologic examination, the lymph node architecture was effaced by atypical cells with intermediate to large nuclei, round nuclear contours, vesicular chromatin, prominent nucleoli, and moderate basophilic cytoplasm admixed with abundant eosinophils (Figure 1, top left, objective 40×). By immunohistochemistry, the neoplastic cells express CD13 (subset), CD33 (weak), CD34 (weak), CD45, CD68/PG-M1 (subset), CD71 (top right, objective 40×), CD117 (subset), CD123 (weak), and Hemoglobin A (subset). Given the extensive eosinophilic infiltration, FISH analysis for PDGFRA (4q12) and PDGFRB (5q33) was performed. A rearrangement of the PDGFRA (4q12) gene region with loss of 5′ PDGFRA and retention of 3′ oncogenic portion was identified in 63% of nuclei (Figure 1. bottom left, PDGFRA 5′(G) / 3′(R) [4q12], arrows indicate disruption of PDGFRA). No abnormality of PDGFRB was observed. FISH analyses for the ABL1, BCR, MYH11, and CBFB gene regions were within normal limits. Because of these findings, a peripheral blood smear, and bone marrow aspiration and biopsy were performed. The patient's blood smear demonstrated marked eosinophilia (59% of the white blood cell count differential) but no blasts. The bone marrow aspirate was hemodilute. The bone marrow core biopsy contained rare, atypical cells that were morphologically like those present in the lymph node, accompanied by numerous mature eosinophils (Figure 1, bottom right, objective 40×) and a small population of mast cells with spindled morphology. The atypical cells expressed the same antigens as their counterparts in the lymph node, and mast cells coexpressed CD25, CD117, tryptase but were negative for CD2. FISH performed on the peripheral blood showed a PDGFRA (4q12) gene rearrangement in 5% of nuclei. All other FISH studies were within normal limits (11q23 (MLL(KMT2A) sep), t(8;21) RUNX1T1::RUNX1 fusion, t(15;17) PML::RARA fusion, inv(16)/t(16;16) MYH11::CBFB fusion, -5q31(D5S630 × 2,EGR1 × 1), -5(D5S630,EGR1)x1,-7q31(D7Z1 × 2,D7S486 × 1), -7(D7Z1,D7S486)x1, inv(3) RPN1::MECOM fusion, t(6;9) DEK::NUP214 fusion, -17p13.1(TP53 × 1,D17Z1 × 2), -17(TP53,D17Z1)x1, and(9;22) ABL1::BCR fusion), and molecular analyses for KIT Asp816Val, JAK2 V617F, CALR exon 9, MPL exon 10 were negative for mutations. Next generation sequencing (NGS) for myeloid neoplasms revealed a variant of unknown significance (SH2B3: Chr12(GRCh37): g.111886029C > T; NM_005475.2(SH2B3): c.1651C > T; p.Arg551Trp (49%)). “Myeloid/lymphoid neoplasm with eosinophilia (M/LNs-Eo) and rearrangement of PDGFRA, PDGFRB or FGFR1, or with PCM1-JAK2” is recognized as a specific diagnostic category in the 2017 World Health Organization (WHO) classification. Recognition of these neoplasms is crucial given that genetic rearrangements of PDGFR1 and PDGFRB render them exquisitely sensitive to tyrosine kinase inhibitors. We present an unusual case of a PDGFRA-rearranged myeloid sarcoma with associated bone marrow involvement by a PDGFRA-rearranged chronic myeloid neoplasm. Spindle-shaped, CD25-positive mast cells are common in PDGFRA-rearranged chronic myeloid neoplasms of the bone marrow and are not considered to be indicative of systemic mastocytosis. The presence of eosinophils in both medullary and extramedullary sites should prompt testing for PDGFRA, PDGFRB, FGFR1, and PCM1- JAK2 rearrangements, in light of the potential therapeutic implications. The authors would like to thank Dr. Rhett P. Ketterling and Dr. Dong Chen at Mayo Clinic-Department of laboratory medicine and pathology for their continous help and support. The authors declare they have no conflicts of interest. At Mayo Clinic single participant case studies, or a case series with multiple participants that are prepared and disseminated for educational purposes, are not systematic investigations and, therefore, are not considered research. Mohammad Barouqa wrote the manuscript and took images. Dragan Jevremovic reviewed the lymph node and edited the manuscript. James Hoyer reviewed the bone marrow and took images. Patricia Theresa Greipp reviewed the cytogenetics and edited the manuscript. Kaaren Reichard reviewed the bone marrow and edited the NGS on the manuscript. Ellen McPhail supervised and edited the manuscript.

Morphological and immunohistochemical diagnostic of extragastrointestinal stromal tumors - a 51 case series analysis.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the digestive tract, originating from structures differentiating towards Cajal cells. Due to their morphology and localization, the extragastrointestinal stromal tumors (EGISTs) can be a diagnostic challenge. We investigated a series of 51 EGISTs diagnosed in our institutions, aiming to explore the immunophenotypes and to analyze the process and the utility of the antibodies required for a positive diagnosis. Immunohistochemical examinations were done for pan-cytokeratin (pan-CK), Ki67, discovered on GIST1 (DOG1) protein and platelet-derived growth factor receptor alpha (PDGFRA), as necessary. The main tumor site was abdominal wall in 43 (84%) cases, most of the tumors showed spindle cell cellularity, followed by mixed and epithelioid type. Twenty-six cases revealed a full conventional immunohistochemical profile with DOG1 positivity. In 10 cases, c-KIT expression was absent but with the preservation of cluster of differentiation (CD)34 positivity, and eight cases were positive for PDGFRA. In our study, we found a subgroup of eight cases presenting in extra-abdominal settings (including one in lung and two in the head-and-neck area). We concluded EGISTs represent a histopathological and immunohistochemically challenging subgroup testing more often negative for c-KIT mutations and positive for PDGFRA compared to GIST. DOG1 remains the marker of choice regardless of tumor site, while CD34 and CD117 should be considered as adjuvants.

EPCT-09. ROVER: A phase 1/2 study of avapritinib in pediatric patients with solid tumors dependent on KIT or PDGFRA signaling

Abstract Prognosis for pediatric patients with advanced relapsed/refractory (R/R) solid (including central nervous system [CNS]) tumors is poor. KIT alterations are common in germ cell tumors and high-grade glioma (HGG); platelet-derived growth factor receptor alpha (PDGFRA) alterations are common in sarcoma and HGG. H3K27M-mutant gliomas are dependent on PDGFRA signaling. No KIT/PDGFRA targeted therapies are currently approved for pediatric patients with R/R solid or CNS tumors, or H3K27M-mutant gliomas. Selective KIT and PDGFRA inhibitor avapritinib demonstrated potent activity against KIT activation-loop (exon 17), juxtamembrane (exon 11), and extracellular-domain (exon 9) mutants (IC50<2 nM), and PDGFRA activation-loop (D842V) mutants (IC50=0.24 nM); cellular IC50 of PDGFRA wild-type was 95 nM. CNS penetration in preclinical models (steady-state brain-to-plasma ratios ranging from 0.74–1.00) has demonstrated potential for CNS antitumor activity. Avapritinib is approved in the USA to treat adults with unresectable/metastatic gastrointestinal stromal tumors (GIST) harboring PDGFRA exon 18 mutations (including D842V), and adults with advanced systemic mastocytosis. In the EU, avapritinib is approved for adults with unresectable/metastatic GIST harboring a PDGFRA D842V mutation. Objectives of ROVER 2-part phase 1/2, multicenter, open-label study (NCT04773782) are avapritinib safety, preliminary efficacy, and pharmacokinetics in pediatric patients aged 2 to <18 years with solid R/R tumors dependent on KIT or PDGFRA signaling, including H3K27M-mutant gliomas, and no alternative treatment options. Part 1 will enroll ≥12 patients; primary endpoint is confirmed age and body surface area physiologically based pharmacokinetic modeling predicted dose to provide equivalent exposure to the 300 mg adult avapritinib dose. Part 2 will enroll ≥25 patients at the recommended avapritinib dose from Part 1; the primary endpoint is objective response rate. Avapritinib once-daily will be administered in continuous 28-day cycles. Enrollment in this study is planned at 26 sites in 10 countries, including centers in North America, Europe, and Asia/Pacific.

The long-term analysis of prognostic role of mutational status in primary resectable gastrointestinal stromal tumors (GIST).

e23517 Background: Radical surgery is the primary treatment for GIST, but up to 50% of patients relapse, mainly in form of hepatic and peritoneal metastases. GISTs are a group of tumors with various pathological and molecular features as well as different clinical courses. The aim of the study was the long-term analysis of prognostic role of mutational status in primary GIST after radical resection. Methods: The material consisted of a group of 304 patients with primary GIST diagnosed till 04/2012, with known mutational status and treated surgically with curative intend without adjuvant imatinib. Data were collected prospectively as part of the GIST clinical register. Relapse-free survival (RFS) was calculated from the date of GIST resection to the date of local recurrence, distant metastatic disease or last follow-up. Overall survival (OS) was calculated from the date of resection to the date of last follow-up or death. Survival analyses were performed using the Kaplan-Meier method and log-rank test. Multivariate Cox regression was performed to asses impact of mutational status on RFS and OS. Results: The primary tumor (PT) locations were: gastric (57.6%) vs non-gastric (42.4%). The median of tumor size was 7cm (0.5-33 cm). Mitotic index was ≤5 in 54.3%, > 5 in 45.7%. Mutations in KIT gene exon 11 were as follow: del 557-558 in 22.7% or other (point mutations, other deletions or insertions) in 40.1% with similar rates in gastric and non-gastric GIST. KIT gene exon 9 dup 502-503 (7.2%) were more common in non-gastric vs gastric GIST (16.3% vs 0.6%). PDGFR D842V was observed in 26 patients (8.6%) while other mutations in PDGFRA gene in 17 (5.6%). The median follow-up was 25.1 months. Disease recurrence was observed in 124 cases (40.8%) resulting in median RFS of 84.4 (95%CI 48.3-120.5) months. The negative independent risk factors for RFS in univariate analysis were: primary tumor location, tumor size, mitotic index and KIT exon 9 dup 502-503 mutation (P < 0.05). In the multivariate analysis independent predictive factors for RFS were mitotic index > 5 (HR 4.38, 95%CI 2.82-6.78), PT location (HR 1.56, 95%CI 1.01-2.39). In multivariate analysis in patients with gastric GIST PDGFR D842V mutations were significantly correlated with better RFS (HR 0.16, 95%CI 0.04-0.61), what was not observed in non-gastric GIST. The median OS was 198.1 months. The negative independent risk factors for OS in univariate analysis were: primary tumor location, primary tumor size and KIT gene exon 9 dup 502-503 mutation. Mitotic index was also negative independent risk factors in a multivariate analysis (HR 1.70, 95%CI 1.15 -2.52). Conclusions: In the long-term analysis mutational status of primary resectable GIST is related to risk of relapse especially in gastric location, however it has no independent impact on overall survival. Genotype should be included in modern risk classification of GIST.

Platelet derived growth factor receptor alpha (PDGFRA) mutant gastrointestinal stromal tumours (GISTs): Clinicopathological characteristics and outcomes from a regional centre in the United Kingdom.

11534 Background: GISTs are the most common mesenchymal tumours of the gastrointestinal tract with an annual incidence of 10-15 per million. 10% of GISTS have activating mutations in the PDGFRA gene. We report our 13 year experience of all the PDGFRA-mutated GISTs in our regional centre in Cambridge. Methods: PDGFRA-mutant GISTs were identified from the Cambridge GIST database. Demographics, clinical and histopathological features, survival and response to tyrosine kinase inhibitors of all the patients from 2008-2021 were reviewed. Results: n = 50 (male:female 1.5:1) Median age 68 years (range 19-87). 8% of patients were under the age of 40 years. Tumour size ranged from 1-26 cm with a median of 5 cm. Mitotic index ranged from 1-51 with a median of 1 mitosis/5mm2. 52% of GISTs were located in the gastric body. Histological subtypes: 44% epithelioid, 36% mixed and 20% spindle cell. 38% of cases had high KIT expression (immunohistochemistry), whilst 48% had patchy expression and 14% were negative. Most were DOG1-positive (94%). 76% had radical surgery, 60% had laparoscopic resection. 24% were assessed as being high risk GISTs using the modified AFIP model contrasted to 13% being high risk with prognostic contour mapping. 13% developed metastatic disease with liver being the most common metastatic site. The table shows PDGFRA mutational analysis results: 58% had a D842V mutation. None had a KIT mutation. With a median follow up of 55.1 months, 82% were alive. 6 patients died from metastatic GIST and 3 from other causes. Median time to metastatic disease in resected GISTs was 30.1 months and median time from metastatic diagnosis to death was 18.5 months. Patients who presented with metastatic disease had poor survival compared with patients with localised disease ( p=0.001). 8 patients were treated with tyrosine kinase inhibitors including imatinib, sunitinib and regorafenib prior to 2020 with no objective responses. 3 patients were treated with Avapritinib since 2020 within the compassionate use programme. All 3 patients had partial responses and 2 patients are continuing Avapritinib with no grade 3 adverse events. Conclusions: We report the largest single centre PDGFRA-mutant GIST cohort from Europe. Male preponderance and exclusive gastric location were observed. KIT expression was patchy to negative in the majority of patients. 58% had PDGFRA D842V mutations for which Avapritinib has been recently approved. No objective responses were seen with imatinib, sunitinib or regorafenib. Our early experience with Avapritinib is promising.[Table: see text]

Tropomyosin-Related Kinase Fusions in Gastrointestinal Stromal Tumors.

Simple SummaryThe canonical mutations in gastrointestinal stromal tumors (GISTs) are typically activating mutations in KIT and platelet-derived growth factor receptor alpha (PDGFRA). Imatinib, the treatment of choice for GISTs, shows a lower response in KIT/PDGFRA wild-type GISTs. Neurotrophic tyrosine receptor kinase (NTRK) fusion, which can be treated with an NTRK target agent, has been reported in KIT/PDGFRA wild-type GISTs, and, therefore, the Yonsei Cancer Center analyzed NTRK fusion incidence in KIT/PDGFRA wild-type GISTs. At the Yonsei Cancer Center, NTRK fusion was confirmed in 16% of cases. Confirmation of NTRK fusion in KIT/PDGFRA wild-type GISTs provides important information for improving therapeutic outcomes. NTRK fusion was confirmed in 16% of KIT/PDGFRA wild-type GIST cases at the Yonsei Cancer Center. Confirmation of NTRK fusion in KIT/PDGFRA wild-type GISTs will improve therapeutic outcomes.The canonical mutations in gastrointestinal stromal tumors (GISTs) are typically activating mutations in KIT and platelet-derived growth factor receptor alpha (PDGFRA). GISTs with non-canonical mutations are a heterogeneous group. Here, we examined tropomyosin-related kinase (TRK) fusion in GIST cases without KIT/PDGFRA mutations (KIT/PDGFRA wild-type (WT) GISTs). We retrospectively analyzed patients who were diagnosed with GISTs at the Yonsei Cancer Center, Severance Hospital, between January 1998 and December 2016. Thirty-one patients with KIT/PDGFRA WT GISTs were included in the analysis. TRK expression in tumor samples was assessed by pan-TRK immunohistochemistry (IHC), and the neurotrophic tyrosine receptor kinase (NTRK: the gene encoding TRK) rearrangement was analyzed by fluorescence in situ hybridization (FISH). IHC analyses revealed that five cases in this cohort exhibited a weak to moderate TRK expression. NTRK1 fusions were detected in three tumor samples, and two samples harbored NTRK3 fusions. The remaining 26 samples did not harbor NTRK fusions. Two types of NTRK fusions were detected, and the overall NTRK fusion frequency in KIT/PDGFRA WT GIST cases was 16% (5/31). Our data provide insights into the molecular alterations underpinning KIT/PDGFRA WT GISTs. More effort should be devoted to improve methods to identify this distinct disease subtype within the KIT/PDGFRA WT GIST group.

Prognostic Features of the Tumor Immune Microenvironment in Glioma and Their Clinical Applications: Analysis of Multiple Cohorts.

Glioma is the most common malignant tumor of the central nervous system. Tumor purity is a source of important prognostic factor for glioma patients, showing the key roles of the microenvironment in glioma prognosis. In this study, we systematically screened functional characterization related to the tumor immune microenvironment and constructed a risk model named Glioma MicroEnvironment Functional Signature (GMEFS) based on eight cohorts. The prognostic value of the GMEFS model was also verified in another two glioma cohorts, glioblastoma (GBM) and low-grade glioma (LGG) cohorts, from The Cancer Genome Atlas (TCGA). Nomograms were established in the training and testing cohorts to validate the clinical use of this model. Furthermore, the relationships between the risk score, intrinsic molecular subtypes, tumor purity, and tumor-infiltrating immune cell abundance were also evaluated. Meanwhile, the performance of the GMEFS model in glioma formation and glioma recurrence was systematically analyzed based on 16 glioma cohorts from the Gene Expression Omnibus (GEO) database. Based on multiple-cohort integrated analysis, risk subpathway signatures were identified, and a drug–subpathway association network was further constructed to explore candidate therapy target regions. Three subpathways derived from Focal adhesion (path: 04510) were identified and contained known targets including platelet derived growth factor receptor alpha (PDGFRA), epidermal growth factor receptor (EGFR), and erb-b2 receptor tyrosine kinase 2 (ERBB2). In conclusion, the novel functional signatures identified in this study could serve as a robust prognostic biomarker, and this study provided a framework to identify candidate therapeutic target regions, which further guide glioma patients’ clinical decision.

JUN and PDGFRA as Crucial Candidate Genes for Childhood Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder, characterized by marked genetic heterogeneity. In this study, two independent microarray datasets of cerebellum of ASD were integrative analyzed by NetworkAnalyst to screen candidate crucial genes. NetworkAnalyst identified two up-regulated genes, Jun proto-oncogene (JUN) and platelet derived growth factor receptor alpha (PDGFRA), as the most crucial genes in cerebellum of ASD patients. Based on KEGG pathway database, genes associated with JUN in the cerebellum highlight the pathways of Th17 cell differentiation and Th1 and Th2 cell differentiation. Genes associated with PDGFRA in the cerebellum were found enriched in pathways in EGFR tyrosine kinase inhibitor resistance and Rap1 signaling pathway. Analyzing all differentially expressed genes (DEGs) from the two datasets, Gene Set Enrichment Analysis (GSEA) brought out IL17 signaling pathway, which is related to the expression of JUN and PDGFRA. The ImmuCellAI found the elevated expression of JUN and PDGFRA correlating with increased Th17 and monocytes suggests JUN and PDGFRA may regulate Th17 cell activation and monocytes infiltrating. Mice model of maternal immune activation demonstrated that JUN and PDGFRA are up-regulated and related to the ASD-like behaviors that provide insights into the molecular mechanisms underlying the altered IL17 signaling pathway in ASD and may enable novel therapeutic strategies.

Imatinib Mesylate Reduces Voiding Frequency in Female Mice With Acute Cyclophosphamide-Induced Cystitis.

Lamina propria interstitial cells that express the tyrosine kinase receptor, platelet-derived growth factor receptor alpha (PDGFRα) may play a role in urinary sensory signaling. Imatinib mesylate, also referred to as imatinib, is a tyrosine kinase inhibitor that can inhibit PDGFRα and has been widely used in urological research. We evaluated the functional effects of imatinib administration (via oral gavage or intravesical infusion) with two different experimental designs (prevention and treatment), in a cyclophosphamide (CYP)-induced cystitis (acute, intermediate, and chronic), male and female rodent model using conscious cystometry and somatic sensitivity testing. Imatinib significantly (0.0001 ≤ p ≤ 0.05) decreased voiding frequency and increased bladder capacity in acute CYP-induced cystitis, by the prevention (females) and treatment (females and males) designs. Imatinib was not effective in preventing or treating intermediate or chronic CYP-induced cystitis in either sex. Interestingly, in the prevention experiments, imatinib administration increased (0.0001 ≤ p ≤ 0.01) voiding frequency and decreased bladder capacity in control mice. However, in the treatment experiments, imatinib administration decreased (0.01 ≤ p ≤ 0.05) voiding frequency and increased bladder capacity in control mice. Bladder function improvements observed with imatinib treatment in acute CYP-induced cystitis mice remained and additionally improved with a second dose of imatinib 24 hours after CYP treatment. Imatinib administration did not affect pelvic somatic sensitivity in female mice with acute CYP-induced cystitis. Our studies suggest that (1) imatinib improves bladder function in mice with acute CYP-induced cystitis with a prevention and treatment design and (2) interstitial cells may be a useful target to improve bladder function in cystitis.

Transcriptome profiling of subepithelial PDGFRα cells in colonic mucosa reveals several cell-selective markers.

Subepithelial platelet-derived growth factor receptor alpha (PDGFRα)+ cells found in the colonic mucosal tissue come in close contact with epithelial cells, immune cells, neurons, capillaries, and lymphatic networks. Mucosal subepithelial PDGFRα+ cells (MuPαC) are important regulators in various intestinal diseases including fibrosis and inflammation. However, the transcriptome of MuPαC has not yet been elucidated. Using Pdgfra-eGFP mice and flow cytometry, we isolated colonic MuPαC and obtained their transcriptome data. In analyzing the transcriptome, we identified three novel, and selectively expressed, markers (Adamdec1, Fin1, and Col6a4) found in MuPαC. In addition, we identified a unique set of MuPαC-enriched genetic signatures including groups of growth factors, transcription factors, gap junction proteins, extracellular proteins, receptors, cytokines, protein kinases, phosphatases, and peptidases. These selective groups of genetic signatures are linked to the unique cellular identity and function of MuPαC. Furthermore, we have added this MuPαC transcriptome data to our Smooth Muscle Genome Browser that contains the transcriptome data of jejunal and colonic smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and smooth muscle resident PDGFRα+ cells: (https://med.unr.edu/physio/transcriptome). This online resource provides a comprehensive reference of all currently known genetic transcripts expressed in primary MuPαC in the colon along with smooth muscle resident PDGFRα cells, SMC, and ICC in the murine colon and jejunum.

FC041: Regeneration of Nephrons and Renal Stroma using the Renal Developmental Environment of Animal Fetuses

Abstract BACKGROUND AND AIMS The kidney is derived from three types of progenitor cells: nephron progenitor cells (NPCs), stromal progenitor cells (SPCs) and ureteric buds (UBs). Extensive research in regenerative medicine has made it possible to induce pluripotent stem cells (PSCs) to develop into NPCs and UBs in vitro and generate the organoids of nephrons and collecting ducts. Renal stroma, including mesangial cells and erythropoietin- and renin-producing cells, is essential for maintaining biological functions. However, to our knowledge, the induction of SPC differentiation from PSCs has not been established yet. Moreover, the renal stroma cannot mature in the in vitro environment, making it difficult to generate the whole kidney, including the renal stroma. In this study, we attempted to regenerate the renal stroma in the in vivo environment by transplanting rat SPCs into the nephrogenic zone of mouse fetuses to provide rat SPCs with the renal developmental mechanism of heterologous mouse fetuses. Furthermore, we developed an interspecies dual replacement of progenitor (i-DROP) system wherein two progenitor cell lines, NPCs and SPCs, were genetically modified and replaced with transplanted progenitor cells and examined the feasibility of the regeneration of kidneys, including nephrons and renal stroma. METHOD We crossed Foxd1GC/+ mice with R26RDTR/DTR mice to generate Foxd1GC/+; R26RDTR/+ mice (Foxd–DTR mice) can eliminate Foxd1-positive SPCs. A group of SPC-rich cells extracted from green fluorescent protein (GFP) rat metanephros targeting platelet-derived growth factor receptor alpha (PDGFRa) was transplanted into the kidneys of Foxd1–DTR mouse fetuses. Subsequently, the organ-cultured metanephros or the transplanted metanephros in immunodeficient NOD/Shi- scid IL2rγnull (NOG) mice or Sprague–Dawley (SD) rats were evaluated using immunostaining. Next, we assessed whether the i-DROP system could regenerate interspecies nephrons and stroma. We crossed Six2TGC/+; Foxd1GC/+ mice obtained from Six2TGC/+ mice and Foxd1GC/+ mice with R26RDTR/DTR mice to generate Six2TGC/+; Foxd1GC/+; and R26RDTR/+ mice (Six2/Foxd1–DTR mice) that can simultaneously eliminate Six2-positive NPCs and Foxd1-positive SPCs. The SPCs targeted to PDGFRa+ and NPCs targeted to PDGFRa−/integrin alpha 8 (Itga8)+ from GFP rat metanephros were simultaneously transferred to Six2/Foxd1–DTR mouse metanephros and evaluated similarly. RESULTS In the organ culture of Foxd1–DTR mouse metanephros, SPCs around the cap mesenchyme replaced from mouse to GFP rat. We achieved extensive rat stromal cell regeneration in mouse kidneys, including endocrine functions such as erythropoietin production in the rat in response to anemia in the host mouse. Next, in the organ culture of the hind kidney of Six2/Foxd1–DTR mice equipped with the i-DROP system, the NPCs and SPCs of GFP rats aggregated around the mouse UB and the cap mesenchyme reconstituted. In vivo, rat nephrons and renal stroma were regenerated simultaneously in the mouse kidneys. The newly generated glomeruli were composed of GFP-positive rat cells, both podocytes and mesangial cells of different lineages (Figure 1). Intravenous injection of fluorescently labeled dextran into host mice revealed that the regenerated nephrons had glomerular filtration and tubular reabsorption capabilities. Furthermore, taking the xenotransplantation of mouse metanephros into SD rats as a control, the transplantation of regenerated rat kidneys into SD rats using our method resulted in reduced xenogenic immune rejection. CONCLUSION The newly developed i-DROP system regenerated multifunctional kidneys, composed of nephrons and renal stroma originating from different species, with urine production and endocrine functions. Functional human kidneys could be regenerated in the future using the renal developmental mechanism of large heterologous animals, such as pigs.

Establishment of a pure culture of immature Sertoli cells by PDGFRA staining and cell sorting.

Sertoli cells are key somatic cells in the testis that form seminiferous tubules and support spermatogenesis. The isolation of pure Sertoli cells is important for their study. However, it is a difficult effort because of the close association of Sertoli cells with peritubular myoid cells surrounding seminiferous tubules. Here, we propose a novel approach to the establishment of a pure Sertoli cell culture from immature mouse testes. It is based on the staining of testicular cells for platelet-derived growth factor receptor alpha (PDGFRA), followed by fluorescence-activated cell sorting and culturing of a PDGFRA-negative cell population. Cells positive for a Sertoli cell marker WT1 accounted for more than 96% of cells in cultures from 6 to 12 days postpartum (dpp) mice. The numbers of peritubular myoid cells identified by ACTA2 staining did not exceed 4%. Cells in the cultures were also positive for Sertoli cell proteins SOX9 and DMRT1. Amh and Hsd17b3 expression decreased and Ar and Gata1 expression increased in 12 dpp cultures compared to 6 dpp cultures, which suggests that cultured Sertoli cells at least partially retained their differentiation status. This method can be employed in various applications including the analysis of differential gene expression and functional studies.

Chronic Binge Alcohol Differentially Affects Extracellular Matrix Phenotype in Omental and Subcutaneous Adipose Tissue of SIV‐infected Macaques

The incidence of at‐risk alcohol use among people living with HIV (PLWH) is two times greater than in the general population. Though antiretroviral therapy (ART) has significantly reduced HIV‐associated mortality, increased survival of PLWH on ART is associated with increased incidence of metabolic comorbidities. Adipose tissue maladaptation is central to metabolic alterations in PLWH and at‐risk alcohol use. This involves adipokine disruption, adipocyte dysfunction, inflammation, and dysregulation of the extracellular milieu. Using a relevant preclinical model of HIV‐infection, the objective of our study was to investigate the effects of chronic binge alcohol (CBA) administration on omental adipose tissue (OmAT) and subcutaneous adipose tissue (SAT) extracellular matrix (ECM) phenotype. Rhesus macaques were administered CBA or isovolumetric water (VEH) through an intragastric catheter for 30 min, 5 days a week, with blood alcohol concentrations reaching 50‐60 mM. Three months after initiation of CBA or VEH administration, macaques were inoculated with SIVMac251, and 2.5 months later animals were initiated on ART. CBA or VEH administration was continued throughout the duration of the study (14.5 mo). Animals were euthanized at 11.5 mo of SIV infection, and adipose (OmAT and SAT) samples excised. Collagen content, adipocyte diameter and adipocyte numbers were measured by Picrosirius Red Staining (PSR). A custom PCR Array was used to assess differential expression of 41 ECM‐related genes. CBA administration significantly increased collagen expression and decreased adipocyte diameter in the OmAT without altering adipocyte numbers. There was a strong negative correlation of OmAT adipocyte diameter and collagen expression. CBA administration did not result in significant differences in collagen expression, adipocyte diameter or number in SAT. CBA administration significantly upregulated expression of transforming growth factor beta‐1 (TGFB1), platelet‐derived growth factor receptor alpha (PDGFRA) and secreted protein acidic and rich in cysteine (SPARC), mediators of a pro‐fibrotic milieu in OmAT. In contrast, CBA did not significantly alter expression of ECM‐related genes in the SAT. These data suggest that the maladaptive adipose changes associated with CBA in SIV infection are depot specific. We speculate that changes in the ECM contribute to altered gene expression and impaired adipocyte metabolic capacity.

Implantable Bioresponsive Hydrogel Prevents Local Recurrence of Breast Cancer by Enhancing Radiosensitivity.

Breast cancer is one of the most common types of cancer. Patients are often concerned about regional recurrence after breast cancer surgery. Radiotherapy plays a vital role in reducing recurrence and prolonging the survival of patients undergoing breast-conserving surgery and high-risk mastectomy. However, 8–15% of patients still have disease progression due to radiation resistance. Therefore, new strategies for combination radiotherapy sensitization must be investigated. In this study, an implantable drug loading system, sunitinib nanoparticles @ matrix metalloproteinases -response hydrogel (NSMRH), uses enzyme-sensitive hydrogel as a carrier to load sunitinib nanoparticles, was identified. The releasing profile demonstrated that sunitinib nanoparticles may be continuously released from the hydrogels. Functional experiments revealed that, when paired with NSMRH, radiation may significantly inhibit tumor cell proliferation, migration, and invasion in vitro. Further animal experiments showed that NSMRH combined with radiotherapy could more effectively control the recurrence of subcutaneous xenograft tumors, prolong the survival time, and have no obvious toxicity in nude mice. Finally, by studying the molecular mechanism of NSMRH, it was hypothesized that in breast cancer cells, NSMRH cooperated with sensitized radiotherapy, mainly due to significantly blocking the G2/M phase, reducing the DNA repair efficiency, inhibiting tumor angiogenesis, promoting apoptosis, and reversing the abnormal expression of platelet-derived growth factor receptor alpha (PDGFRA) after radiotherapy. These findings suggest that NSMRH’s radiation sensitization and anti-tumor activity may aid in the development of a novel method in future clinical applications.

Evaluation of the therapeutic potential of masitinib and expression of its specific targets c-Kit, PDGFR-α, PDGFR-β, and Lyn in canine prostate cancer cell lines.

Canine prostate cancer is classified into adenocarcinoma, transitional cell carcinoma with prostatic involvement, and mixed forms. Early metastatic spread leads to poor prognosis and limited treatment options. Masitinib is approved for the treatment of canine mast cell tumours and inhibits tyrosine kinase c-Kit, tyrosine-protein kinase Lyn (Lyn), and platelet-derived growth factor receptors alpha and beta (PDGFR-α, PDGFR-β), which are known to be expressed in canine prostate cancer. The aim of this study was to evaluate masitinib in an in vitro model consisting of cell lines from primary prostate adenocarcinoma, the associated lymph node metastasis of the same patient, and transitional cell carcinoma. To assess the suitability of the model system, the targets of masitinib were investigated by immunocytochemistry in the cell lines and by immunohistochemistry in the respective formalin-fixed, paraffin-embedded (FFPE) original neoplastic tissue. After exposure to masitinib, cell viability, cell count, apoptosis induction, and protein expression of c-Kit, Lyn, PDGFR-α, and PDGFR-β were assessed. To hedge the efficacy, two application protocols of masitinib (single application or 12-h double-dose regimen) were compared. Immunocytochemical and immunohistochemical analysis revealed increased Lyn, PDGFR-α, and PDGFR-β expression in cell lines and FFPE original neoplastic tissue compared to healthy prostate tissue. Masitinib exposure increased apoptosis, while the cell counts and cell viability decreased in a dose- and application interval-dependent manner, with increased impact in the 12-h double-dose regimen. These in vitro effects of masitinib in canine prostate cancer and associated metastasis support further in vivo research and modifications of the clinical treatment protocol in future studies.

Rare case of giant extra gastrointestinal stromal tumor of wild variant

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms of gastrointestinal tract. The interstitial cells of Cajal (pacemaker cells) act as normal counterpart of tumor. They are rare cancers with incidence of 1.5 in 100000. KIT and PDGFRA gene mutations are the most common pathogenetic causes, whereas a minority of GISTs do not harbour either of the mutations, referred to as wild type GISTs. A minority of cases are extra gastrointestinal (EGIST), arising from mesentry, omentum and retroperitoneum. We present a wild type EGIST measuring 32×24×15 cm and weighing approximately 6 kg.

Role of detrusor PDGFR\u03b1+ cells in mouse model of cyclophosphamide-induced detrusor overactivity

Cyclophosphamide (CYP)-induced cystitis is a rodent model that shares many features common to the cystitis occurring in patients, including detrusor overactivity (DO). Platelet-derived growth factor receptor alpha positive (PDGFRα+) cells have been proposed to regulate muscle excitability in murine bladders during filling. PDGFRα+ cells express small conductance Ca2+-activated K+ channels (predominantly SK3) that provide stabilization of membrane potential during filling. We hypothesized that down-regulation of the regulatory functions of PDGFRα+ cells and/or loss of PDGFRα+ cells generates the DO in CYP-treated mice. After CYP treatment, transcripts of Pdgfrα and Kcnn3 and PDGFRα and SK3 protein were reduced in detrusor muscle extracts. The distribution of PDGFRα+ cells was also reduced. Inflammatory markers were increased in CYP-treated detrusor muscles. An SK channel agonist, CyPPA, increased outward current and hyperpolarization in PDGFRα+ cells. This response was significantly depressed in PDGFRα+ cells from CYP-treated bladders. Contractile experiments and ex vivo cystometry showed increased spontaneous contractions and transient contractions, respectively in CYP-treated bladders with a reduction of apamin sensitivity, that could be attributable to the reduction in the SK conductance expressed by PDGFRα+ cells. In summary, PDGFRα+ cells were reduced and the SK3 conductance was downregulated in CYP-treated bladders. These changes are consistent with the development of DO after CYP treatment.