PTCH1 Mutations Research Articles

Detection of SMO gene mutations in odontogenic keratocyst

Objective: To detect the SMO mutations in odontogenic keratocyst (OKC) and to explore the mechanism behind. Methods: Patients with OKC who received treatment in the Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology,Peking University, from September 2012 to June 2017 were enrolled. OKC samples from 10 patients diagnosed as naevoid basal cell carcinoma syndrome (NBCCS)-related OKC (4 females and 6 males) and 20 patients diagnosed as sporadic OKC (7 females and 13 males) were collected. Genomic DNAs were extracted from fibrous capsules and epithelial lining respectively. SMO mutations were detected and analyzed by Sanger sequencing. Results: Three SMO mutations were found in one NBCCS-associated OKC who carrying c.2081C>G (p.P694R) mutation) and two sporadic OKC who carrying c.907C>T (p.L303F) mutation and c.1247_1248delinsAA (p.G416E), respectively), among which the first two mutations were novel mutations that had not been reported before. Besides, two mutations in sporadic OKC were not paired with PTCH1 mutations. Conclusions: In addition to PTCH1 gene mutations, SMO gene mutations also exist in OKC which might be related to the development of OKC.

Novel PTCH1 and concurrent TP53 mutations in four patients with numerous non-syndromic basal cell carcinomas: The paradigm of oncogenic synergy.

There has been a significant increase in basal cell carcinoma (BCC) incidence, the most common cancer in humans and the age of presentation with the first diagnosis of BCC has decreased in past decades. In this study, we investigated the possibility of genetic markers that can lead to earlier and closer observation of patients at high risk for development of multiple BCCs. The overall goal is to decrease the morbidity and the economic burden of diagnosis and treatment of recurring and/or advanced BCCs. Four patients with numerous BCCs, some of them exceptionally large, were included in this study. A sample of representative BCCs, normal non-sun-exposed skin and blood samples were obtained from each patient. Whole-exome sequencing of DNA was conducted on all samples, and a series of bioinformatics filtering was performed to identify potentially pathogenic sequence variants. The analysis of the data resulted in detection of oncogenic mutations in PTCH1, two of which being novel, and concurrent mutations in TP53 in BCC tumours of all four patients. Such mutations may explain the numerous and postexcision recurring nature of the BCCs of exceptionally large size observed in all these patients, and they can be suggested to serve as a genetic marker for high-risk patients for early detection, prognostication and close follow-up.

Defining the Spectrum, Treatment and Outcome of Patients With Genetically Confirmed Gorlin Syndrome From the HIT-MED Cohort.

Gorlin syndrome is a genetic condition associated with the occurrence of SHH activated medulloblastoma, basal cell carcinoma, macrocephaly and other congenital anomalies. It is caused by heterozygous pathogenic variants in PTCH1 or SUFU. In this study we included 16 patients from the HIT2000, HIT2000interim, I-HIT-MED, observation registry and older registries such as HIT-SKK87, HIT-SKK92 (1987 – 2020) with genetically confirmed Gorlin syndrome, harboring 10 PTCH1 and 6 SUFU mutations. Nine patients presented with desmoplastic medulloblastomas (DMB), 6 with medulloblastomas with extensive nodularity (MBEN) and one patient with classic medulloblastoma (CMB); all tumors affected the cerebellum, vermis or the fourth ventricle. SHH activation was present in all investigated tumors (14/16); DNA methylation analysis (when available) classified 3 tumors as iSHH-I and 4 tumors as iSHH-II. Age at diagnosis ranged from 0.65 to 3.41 years. All but one patient received chemotherapy according to the HIT-SKK protocol. Ten patients were in complete remission after completion of primary therapy; four subsequently presented with PD. No patient received radiotherapy during initial treatment. Five patients acquired additional neoplasms, namely basal cell carcinomas, odontogenic tumors, ovarian fibromas and meningioma. Developmental delay was documented in 5/16 patients. Overall survival (OS) and progression-free survival (PFS) between patients with PTCH1 or SUFU mutations did not differ statistically (10y-OS 90% vs. 100%, p=0.414; 5y-PFS 88.9% ± 10.5% vs. 41.7% ± 22.2%, p=0.139). Comparing the Gorlin patients to all young, SHH activated MBs in the registries (10y-OS 93.3% ± 6.4% vs. 92.5% ± 3.3%, p=0.738; 10y-PFS 64.9%+-16.7% vs. 83.8%+-4.5%, p=0.228) as well as comparing Gorlin M0 SKK-treated patients to all young, SHH activated, M0, SKK-treated MBs in the HIT-MED database did not reveal significantly different clinical outcomes (10y-OS 88.9% ± 10.5% vs. 88% ± 4%, p=0.812; 5y-PFS 87.5% ± 11.7% vs. 77.7% ± 5.1%, p=0.746). Gorlin syndrome should be considered in young children with SHH activated medulloblastoma, especially DMB and MBEN but cannot be ruled out for CMB. Survival did not differ to patients with SHH-activated medulloblastoma with unknown germline status or between PTCH1 and SUFU mutated patients. Additional neoplasms, especially basal cell carcinomas, need to be expected and screened for. Genetic counselling should be provided for families with young medulloblastoma patients with SHH activation.

Novel alterations in IFT172 and KIFAP3 may induce basal cell carcinoma

BackgroundBasal cell carcinoma (BCC) is the most commonly occurring neoplasm in patients with Gorlin syndrome. It is widely accepted that multiple basal cell carcinomas simultaneously develop in middle-aged patients with this syndrome. However, the presence of driver genes other than the PTCH1 in Gorlin syndrome has not been explored. This study aimed to identify common gene mutations other than PTCH1 in simultaneously occurring basal cell carcinomas in patients with Gorlin syndrome via exome sequencing analysis.MethodsNext-generation sequencing analysis was performed using four basal cell carcinoma samples, one dental keratinocyte sample, and two epidermoid cyst samples, which were surgically resected from one patient with Gorlin syndrome on the same day.ResultsOverall, 282 somatic mutations were identified in the neoplasms. No additional somatic mutations in PTCH1, PTCH2, TP53, and SMO were identified. However, enrichment analysis showed that multiple genes, such as IFT172 and KIFAP3, could regulate ciliary functions important for Hedgehog signaling.ConclusionThe development of BCCs in patients with Gorlin syndrome may be triggered by mutations that cause substantial dysfunction of cilia.

Subgroup and subtype-specific outcomes in adult medulloblastoma.

Medulloblastoma, a common pediatric malignant central nervous system tumour, represent a small proportion of brain tumours in adults. Previously it has been shown that in adults, Sonic Hedgehog (SHH)-activated tumours predominate, with Wingless-type (WNT) and Group 4 being less common, but molecular risk stratification remains a challenge. We performed an integrated analysis consisting of genome-wide methylation profiling, copy number profiling, somatic nucleotide variants and correlation of clinical variables across a cohort of 191 adult medulloblastoma cases identified through the Medulloblastoma Advanced Genomics International Consortium. We identified 30 WNT, 112 SHH, 6 Group 3, and 41 Group 4 tumours. Patients with SHH tumours were significantly older at diagnosis compared to other subgroups (p < 0.0001). Five-year progression-free survival (PFS) for WNT, SHH, Group 3, and Group 4 tumours was 64.4 (48.0-86.5), 61.9% (51.6-74.2), 80.0% (95% CI 51.6-100.0), and 44.9% (95% CI 28.6-70.7), respectively (p = 0.06). None of the clinical variables (age, sex, metastatic status, extent of resection, chemotherapy, radiotherapy) were associated with subgroup-specific PFS. Survival among patients with SHH tumours was significantly worse for cases with chromosome 3p loss (HR 2.9, 95% CI 1.1-7.6; p = 0.02), chromosome 10q loss (HR 4.6, 95% CI 2.3-9.4; p < 0.0001), chromosome 17p loss (HR 2.3, 95% CI 1.1-4.8; p = 0.02), and PTCH1 mutations (HR 2.6, 95% CI 1.1-6.2; p = 0.04). The prognostic significance of 3p loss and 10q loss persisted in multivariable regression models. For Group 4 tumours, chromosome 8 loss was strongly associated with improved survival, which was validated in a non-overlapping cohort (combined cohort HR 0.2, 95% CI 0.1-0.7; p = 0.007). Unlike in pediatric medulloblastoma, whole chromosome 11 loss in Group 4 and chromosome 14q loss in SHH was not associated with improved survival, where MYCN, GLI2 and MYC amplification were rare. In sum, we report unique subgroup-specific cytogenetic features of adult medulloblastoma, which are distinct from those in younger patients, and correlate with survival disparities. Our findings suggest that clinical trials that incorporate new strategies tailored to high-risk adult medulloblastoma patients are urgently needed.

Abstract 2421: PTCH1 C-terminal domain truncations in colorectal cancer increase mitogenic signalling and autophagy

Abstract The canonical Hedgehog (Hh) signaling pathway, driving activation of SMO and GLI1, has been implicated in colorectal cancer (CRC) stemness and resistance to chemotherapy. We recently reported that the C-terminal domain (CTD) of the Hh receptor PTCH1 limits autophagic flux and ERK signaling by non-canonical mechanisms (independently of SMO/GLI1). Given that those novel functions are implicated in tumorigenesis, we explored the existence of PTCH1 CTD mutations in cancer specimens. Analysis of the TCGA Cancer Atlas database and the Genomics England 100,000 Genomes Project revealed relatively frequent somatic mutations in the CTD of PTCH1 in CRC: S1203(fs) and R1308(fs), which result in premature truncations of the CTD in about 4% of cases. PTCH1 CTD mutations were strongly associated with BRAF mutations (61% samples) and with right-sided disease (10/10 of samples with sidedness information). Co-immunoprecipitation analysis of truncated PTCH1(S1203*) and PTCH1(R1308*) showed loss of interaction with ATG101 and GRB2, required for regulation of autophagic flux and ERK signaling, respectively. To determine the pathogenic role of those mutations, we engineered SW620 metastatic CRC cell lines using CRISPR/Cas9 to create two clones with indel mutations between S1203 and R1308. Truncation of the CTD increased basal ERK and AKT phosphorylation levels, proliferation, invasion and migration compared to isogenic cells expressing wild type PTCH1. The mutant clones also showed persistent proliferation in the presence of glycolysis inhibitors and of the autophagy inducer rapamycin. Moreover, both clones displayed an enhanced autophagic flux by analysis of the LC3BI/II and p62 markers in the presence and absence of Bafilomycin A1. RNA-seq analysis revealed that the CTD truncation resulted in increase in GO terms of metabolic pathways, pathways in cancer, mTOR signaling pathway, PI3K-AKT pathway, MAPK signaling pathway and regulation of actin cytoskeleton, among others. The clones morphology changed together with a remarkable cadherin switch and loss of vimentin at RNA and protein level, suggestive of mesenchymal-epithelial transition, a process necessary for establishment of metastasis following cancer cell spread. Interestingly, one clone showed enhanced apoptosis in response to FOLFOX (5-fluorouracil/lecovorin/oxaliplatin), the most effective chemotherapy for metastatic CRC, as determined by cleaved PARP and Caspase 7 activity, compared to the parental cells. These findings reveal important tumor suppressor functions of the CTD of PTCH1 acting to suppress autophagy and mitogenic signaling, which are dysregulated by CTD mutations in a subset of colon cancer patients. Our results provide mechanistic insights of the effect of PTCH1 mutations in CRC and identify potential therapeutic vulnerabilities. Citation Format: Begoña Caballero-Ruiz, Cintli C. Morales-Alcala, Henry M. Wood, Gianluca Canettieri, Natalia A. Riobo-Del Galdo. PTCH1 C-terminal domain truncations in colorectal cancer increase mitogenic signalling and autophagy [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 2421.

Hedgehog Pathway Mutations Drive Oncogenic Transformation in High-Risk T-Cell Acute Lymphoblastic Leukemia

Despite recent advances in improving outcome for patients with T-cell acute lymphoblastic leukemia (T-ALL), those who relapse or who have resistant disease are in urgent need of new therapeutic approaches. Recently, mutations in the Hedgehog pathway have been described in T-ALL, but the role of Hedgehog signaling in normal and malignant T-cell development is controversial, and whether these mutations are prognostic or drive oncogenic transformation is unknown, hindering the rationale for therapeutic intervention.Through targeted next generation sequencing on a cohort of 109 diagnostic lymphoblast specimens from children with newly diagnosed T-ALL enrolled on contemporary clinical trials (DFCI 05-001 and COG AALL0434), we identified mutations in the Hedgehog pathway in 16% of patients (n = 17/109); PTCH1, the major negative regulator of the pathway, was the most frequently mutated Hedgehog pathway gene with 8% of patients harboring heterozygous missense mutations (n = 9/109). Remission bone marrow samples were evaluable by Sanger sequencing for 5 of the 9 PTCH1 mutations, and revealed that 3 of these mutations were absent at remission, indicating a somatic origin, whereas, 2 of these mutations were detected at remission, suggestive of either an underlying cancer predisposition syndrome, or somatic mutations associated with clonal hematopoiesis. Hedgehog pathway mutations predicted resistance to induction chemotherapy, as defined by ≥ 5% residual lymphoblasts in the bone marrow by morphology or flow cytometry (P= 0.009).To investigate the role of Hedgehog signaling in T-ALL, we focused on PTCH1 because this was the most commonly mutated Hedgehog gene identified. Transduction of wild-type PTCH1 into a PTCH1 -mutant T-ALL cell line downregulated Hedgehog pathway activity, impaired viability (P < 0.001), and induced apoptosis (P= 0.005). The effect on both viability and apoptosis was reversed by downstream Hedgehog pathway activation using a small molecule Smoothened agonist (P < 0.001 and P= 0.007, respectively), indicating on-target toxicity. Most of the PTCH1 mutations identified in human T-ALL were significantly less toxic than wild-type PTCH1 when transduced into PTCH1 -mutant T-ALL cells, indicating these encode functionally defective alleles.To test whether PTCH1 mutations accelerate the onset of T-ALL in vivo, we turned to a CRISPR/Cas9 system for tissue-specific gene disruption in transgenic zebrafish. We first identified guide RNAs that effectively mutagenized zebrafish ptch1, or the locus syntenic to the human AAVS1 safe-harbor locus as a negative control. Guide RNAs were cloned into a vector that drives ubiquitous expression of the guide RNA, together with lineage-restricted expression of a Cas9-T2A-GFP self-cleaving protein under the control of the zebrafish rag2 promoter. Using this model, we found that inducing ptch1 mutations in thymocytesaccelerated the onset of notch1- induced T-ALL, with a median time to T-ALL onset of 12.3 weeks in the ptch1 -mutant group versus median not reached for the aavs1 -control group (P= 0.0004). These ptch1 -mutant leukemias were transplantable into irradiated wild-type recipients, and subsequent treatment of these animals with the Hedgehog pathway inhibitor cyclopamine had significant therapeutic activity (P= 0.002).In conclusion, our findings indicate that Hedgehog-activating mutations are associated with resistance to induction chemotherapy and drive oncogenic transformation in high-risk T-ALL. Together, these findings provide a molecular rationale for targeted therapy with Hedgehog pathway inhibitors for patients with PTCH1 -mutant high-risk T-ALL. DisclosuresNeuberg:Synta Pharmaceuticals: Other: Stock shares. Hunger:Jazz Pharmaceuticals: Honoraria; Novartis: Consultancy; Amgen: Consultancy, Equity Ownership; Erytech Pharmaceuticals: Consultancy.

Molecular alterations in basal cell carcinoma subtypes

A number of genes have been implicated in the pathogenesis of BCC in addition to the Hedgehog pathway, which is known to drive the initiation of this tumour. We performed in-depth analysis of 13 BCC-related genes (CSMD1, CSMD2, DPH3 promoter, PTCH1, SMO, GLI1, NOTCH1, NOTCH2, TP53, ITIH2, DPP10, STEAP4, TERT promoter) in 57 BCC lesions (26 superficial and 31 nodular) from 55 patients and their corresponding blood samples. PTCH1 and TP53 mutations were found in 71.9% and 45.6% of BCCs, respectively. A high mutation rate was also detected in CSMD1 (63.2%), NOTCH1 (43.8%) and DPP10 (35.1%), and frequent non-coding mutations were identified in TERT (57.9%) and DPH3 promoter (49.1%). CSMD1 mutations significantly co-occurred with TP53 changes (p = 0.002). A significant association was observed between the superficial type of BCC and PTCH1 (p = 0.018) and NOTCH1 (p = 0.020) mutations. In addition, PTCH1 mutations were significantly associated with intermittent sun exposure (p = 0.046) and the occurrence of single lesions (p = 0.021), while NOTCH1 mutations were more frequent in BCCs located on the trunk compared to the head/neck and extremities (p = 0.001). In conclusion, we provide further insights into the molecular alterations underlying the tumorigenic mechanism of superficial and nodular BCCs with a view towards novel rationale-based therapeutic strategies.

EMBR-19. SHH-DRIVEN MEDULLOBLASTOMA WITH CONCURRENT UNILATERAL RENAL AGENESIS

Case PresentationA 3-year-old female with insignificant past medical history presented with 6 weeks of headaches, emesis, and lethargy. MR imaging identified a heterogeneously enhancing right cerebellar hemispheric mass and obstructive hydrocephalus. Gross total resection was performed without complications; pathology revealed classical WHO grade 4 medulloblastoma (MB). MR imaging of the spine and cerebrospinal fluid testing were negative for disseminated disease. Treatment for standard risk medulloblastoma was initiated, comprising proton craniospinal irradiation with posterior fossa boost and concurrent vincristine, followed by adjuvant chemotherapy with vincristine, lomustine, cisplatin, and cyclophosphamide as standard of care. Next generation sequencing of the tumor tissue performed using a high-multiplex PCR-based NGS panel was negative for alteration of the SMO, PTCH1 and CTBNN1 genes. Further molecular characterization via methylation profiling demonstrated the sonic hedgehog (SHH) molecular subtype. Prior to initiation of chemotherapy, renal ultrasound was performed and identified congenitally absent right kidney; audiology evaluation was unremarkable. DiscussionIn patients with Gorlin syndrome, cases of unilateral renal agenesis in association with germline SHH-pathway mutations have been reported [1]. SHH signaling is implicated in multiple steps in the development of the urinary system [2]. Outside Gorlin syndrome, however, to the best of our knowledge unilateral renal agenesis coinciding with SHH-driven MB has not been reported. Our patient notably lacks any clinical stigmata of Gorlin syndrome (skeletal abnormalities, skin pits, macrocephaly) and does not exhibit the characteristic germline genetic abnormalities that define GS (PTCH1 mutation or 9q22.3 microdeletion). There are important treatment implications for patients with the constellation of abnormalities we describe here, particularly regarding the requisite frequent monitoring of renal function during multi-agent chemotherapy courses. Our patient tolerated chemoradiation well, and is currently on maintenance chemotherapy with favorable course to date.

Association of PIK3CA, PTCH1, FGF6, and NRAS mutations with venous thromboembolism in advanced lung and gastrointestinal cancer.

e15087 Background: Venous thromboembolism (VTE) in patients with gastrointestinal (GI) and non‐small cell lung cancer (NSCLC) is common and increases morbidity and mortality. In advanced GI and NSCLC, molecular subtyping has increased use of next-generation sequencing (NGS). The association between tumor mutation profile and VTE risk remains undetermined. Methods: We conducted a retrospective cohort study of consecutive GI/NSCLC patients from 2014-2019 with NGS and follow‐up at Brown Cancer Center. The NGS platform detected substitutions, indels, copy number alterations and select rearrangements in 324 genes. Patients with thrombophilia, anticoagulation use or &gt;1 malignancy were excluded. VTE was defined as deep vein thrombosis, pulmonary embolism or visceral vein thrombosis within 6 months prior to diagnosis or any time after. For statistical analysis SAS 9.5 was used with significance at alpha=0.05. Multinomial logistic regression was performed, in which the log odds of VTE was modeled as a linear combination of the genes. Odds ratios and 95% confidence intervals for VTE were generated. Results: A total of 364 patients were reviewed; after exclusions 326 patients were included comprising Stage III/IV NSCLC (58%), metastatic colorectal (33%) and other metastatic GI cancers - gastric, duodenal, esophageal, pancreatic and cholangiocarcinoma (9%). Approximately half (53%) were males with mean age of 59.1 yrs and 76.4% current/former smokers. There was a low level of microsatellite instability (0.9%). VTE occurred in 75 patients (23%) during a mean follow‐up of 24.5 months. Only 1 patient had surgery within 90 days prior to VTE. Of 248 genes mutated in the VTE group, 9 were more prevalent compared to those without VTE (Table). Statistical analysis showed PIK3CA, PTCH1, FGF6, and NRAS mutations significantly increased odds of VTE, with PIK3CA the most prevalent of these (Table). Conclusions: Patients with PIK3CA, PTCH1, FGF6 and NRAS mutations had significantly higher VTE risk. These tumor mutations and associated pathways may provide novel insight into risk stratification, prevention and targeted treatment of VTE in cancer. [Table: see text]

Radiation-Induced Lens Opacity and Cataractogenesis: A Lifetime Study Using Mice of Varying Genetic Backgrounds.

Recent epidemiological findings and reanalysis of historical data suggest lens opacities resulting from ionizing radiation exposures are likely induced at lower doses than previously thought. These observations have led to ICRP recommendations for a reduction in the occupational dose limits for the eye lens, as well as subsequent implementation in EU member states. The EU CONCERT LDLensRad project was initiated to further understand the effects of ionizing radiation on the lens and identify the mechanism(s) involved in radiation-induced cataract, as well as the impact of dose and dose-rate. Here, we present the results of a long-term study of changes to lens opacity in male and female adult mice from a variety of different genetic (radiosensitive or radioresistant) backgrounds, including mutant strains Ercc2 and Ptch1, which were assumed to be susceptible to radiation-induced lens opacities. Mice received 0.5, 1 and 2 Gy 60Co gamma-ray irradiation at dose rates of 0.063 and 0.3 Gy min-1. Scheimpflug imaging was used to quantify lens opacification as an early indicator of cataract, with monthly observations taken postirradiation for an 18-month period in all strains apart from 129S2, which were observed for 12 months. Opacification of the lens was found to increase with time postirradiation (with age) for most mouse models, with ionizing radiation exposure increasing opacities further. Sex, dose, dose rate and genetic background were all found to be significant contributors to opacification; however, significant interactions were identified, which meant that the impact of these factors was strain dependent. Mean lens density increased with higher dose and dose rate in the presence of Ercc2 and Ptch1 mutations. This project was the first to focus on low (<1 Gy) dose, multiple dose rate, sex and strain effects in lens opacification, and clearly demonstrates the importance of these experimental factors in radiobiological investigations on the lens. The results provide insight into the effects of ionizing radiation on the lens as well as the need for further work in this area to underpin appropriate radiation protection legislation and guidance.

A novel anthelminthic drug suppresses the growth of medulloblastoma tumors by inhibiting PKA/Gli1 signaling axis

Pediatric brain tumor is one of the most malignant solid tumors in children and have profound impact on the morbidity and mortality in these patients. Statistically, brain tumors are one of the leading cause of cancer-related deaths in patients from 0-19 years of age. Medulloblastoma (MB) is one of the most common pediatric brain tumors occurring in children. Sonic hedgehog (Shh) activated subgroup of MB is considered to be highly aggressive and metastatic in nature. Shh-MB is characterized by mutations in PTCH1, SMO and SuFu along with amplified activation of Gli1, a major transcription factor of this signaling pathway. In the current study, we have evaluated the anti-cancer effects of an anthelminthic drug ‘moxidectin’. Several MB cell lines such as Daoy, UW426, UW228, ONS76, and PFSK1 were treated with moxidectin in a concentration and time dependent manner. Our results demonstrated that moxidectin treatment resulted in significantly reduced proliferation of MB cells. The IC50 of moxidectin in all the MB cell lines ranged 10-17μM after 24, 48 and 72 hours of treatment. Moreover, moxidectin was able to induce 3-4 fold increase of apoptosis in all the MB cell lines as evaluated by AnnexinV-FITC/PI assay, and increased cleavage of caspase 3 and PARP. Western blotting analysis demonstrated that moxidectin treatment significantly reduced non-canonical activation of Gli1 and its downstream effector molecules such as Pax-6, Oct-4, Sox-2 and Nanog. To our knowledge, this study for the first time focuses on GABAA receptor agonist mediated PKA inhibition and ultimately suppression of non-canonical Gli1 activation. Efficacy of moxidectin was evaluated in an in vivo tumor model by subcutaneously and intracranically implanting human Daoy MB cells. Our results demonstrated that 2.5 mg/kg moxidectin by oral administration everyday suppressed the growth of Daoy tumors by 50-55% in both subcutaneous and intracranial tumor models. Conclusively, our results indicate that moxidectin effectively reduces the growth of MB tumors by inhibiting PKA/Gli1 signaling. Most importantly, moxidectin is FDA approved drug and is already in clinical use for the treatment of river blindness in humans with an established safety record, therefore any positive findings from our studies will prompt further clinical investigation into repositioning moxidectin for the treatment of MB patients.

075 PTCH1 mutations in high-frequency basal cell carcinoma patients without Gorlin stigmata

075 PTCH1 mutations in high-frequency basal cell carcinoma patients without Gorlin stigmata

Medulloblastoma in Adults: Cytogenetic Phenotypes Identify Prognostic Subgroups.

Adult medulloblastomas (MB) are rare. We investigated the genetic landscape and prognostic impact of genetic aberrations in a cohort of 117 adult medulloblastomas. Histological features and pathway activation were evaluated at the protein level; 14.5% showed wingless-type activation, 63.3% SHH activation, and 22.2% were classified as non-WNT/non-SHH-MB. Genome-wide copy number analysis was performed by molecular inversion probe array technology. MB-related genes were sequenced in WNT- and SHH-activated MBs. 79.7% of SHH-MBs showed desmoplastic/nodular histology; all other MBs had classic histology. WNT-MBs carried oncogenic CTNNB1 mutations in 88.2% and had monosomy 6 in 52.9%. In SHH-MBs, TERT promoter mutations occurred in 97%, mutations in PTCH1 in 38.2%, SMO in 15.5%, SUFU in 7.4%, and TP53-mutations in 4.1%. In all, 84.6% of non-WNT/non-SHH-MBs had an isochromosome 17q. A whole chromosomal aberration (WCA) signature was present in 45.1% of SHH-TP53-wild type (wt)-MBs and 65.4% of non-WNT/non-SHH-MBs. In 98 cases with survival data, WNT-MBs had a 5-year overall survival (OS) of 68.6%. SHH-MBs TP53wt type and non-WNT/non-SHH-MBs showed 5-year OS of 80.4% and 70.8%, respectively. TP53-mutant SHH-MBs represented a prognostically unfavorable entity; all patients died within 5 years. Patients with a WCA signature showed significantly increased OS (p = 0.011 for SHH-TP53wt-MBs and p = 0.048 for non-WNT/non-SHH-MBs).

Basal Cell Nevus Syndrome caused by a new splice site mutation in PTCH1

Basal cell nevus syndrome is a rare inherited autosomal dominant syndrome characterized by developmental defects and tumor predisposition. There are more than 400 reported PTCH1 mutations, including frameshift, nonsense, missense, deletions, duplications, and splicing mutations. We report a 68-year-old Thai female presenting with multiple basal cell carcinoma scattered on the face and upper back and palmoplantar pits. Molecular diagnosis showed a novel heterozygous mutation in the splice site region c.746+1_746+4delGTAA, localized within the splice donor after exon 5 of PTCH1. Although the clinical manifestations are characteristic, this report adds another splice site mutation to the genotypic variation of BCNS patients and also highlights the importance of a multidisciplinary approach in the management of BCNS patients.

Should the solid variant of odontogenic keratocyst and keratoameloblastoma be classified as the same entity? A clinicopathological analysis of nine cases and a review of the literature

The solid variant of odontogenic keratocyst (SOKC) is an extremely rare odontogenic lesion, which remains poorly defined even in the 2017 World Health Organization odontogenic tumour classification. It is difficult to distinguish between SOKC and so called keratoameloblastoma (KAB), both rare lesions that have similarities in clinical, histological and biological characteristics. Here, we report clinicopathological data and results of molecular analysis of nine cases with a literature review. First, they were compared to previously reported cases of SOKC and/or KAB, and many overlaps were found in clinical and pathological characteristics. Second, we performed PCR analysis for BRAF V600E mutation. Although ameloblastoma-like epithelia were often encountered, none exhibited BRAF V600E mutation, which has been reported to occur frequently in ameloblastomas but not in odontogenic keratocysts (OKCs). One of two cases of SOKC in the present series from which fresh frozen tissue specimens were available was found to harbour PTCH1 mutations, indicating that these were more likely to be a subtype of OKC. Moreover, we also examined the differences between SOKC and primary intraosseous carcinoma (PIOC) with regard to the expression of cytokeratins (pan-CK, CK5/6, CK7, CK8/18, CK10, CK14 and CK19), p53 and Ki-67. The proportions of p53-and Ki-67-positive cells were significantly higher in PIOC than in SOKC. These findings suggest that immunostaining for p53 and Ki-67 would be useful to differentiate between SOKC and PIOC. We also conducted a review of SOKC and KAB cases reported in the English language literature.

Differences in RNA and microRNA Expression Between PTCH1- and SUFU-mutated Medulloblastoma.

Germline mutations in PTCH1 or SUFU in the sonic hedgehog (SHH) pathway cause Gorlin's syndrome with increased risk of developing SHH-subgroup medulloblastoma. Gorlin's syndrome precludes the use of radiotherapy (a standard component of treatment) due to the development of multiple basal cell carcinomas. Also, current SHH inhibitors are ineffective against SUFU-mutated medulloblastoma, as they inhibit upstream genes. In this study, we aimed to detect differences in the expression of genes and microRNAs between SUFU- and PTCH1-mutated SHH medulloblastomas which may hint at new treatment directions. We sequenced RNA and microRNA from tumors of two patients with germline Gorlin's syndrome - one having PTCH1 mutation and one with SUFU mutation - followed by bioinformatics analysis to detect changes in genes and miRNAs expression in these two tumors. Expression changes were validated using qRT-PCR. Ingenuity pathway analysis was performed in search for targetable pathways. Compared to the PTCH1 tumor, the SUFU tumor demonstrated lower expression of miR-301a-3p and miR-181c-5p, matrix metallopeptidase 11 (MMP11) and OTX2, higher expression of miR-7-5p and corresponding lower expression of its targeted gene, connexin 30 (GJB6). We propose mechanisms to explain the phenotypic differences between the two types of tumors, and understand why PTCH1 and SUFU tumors tend to relapse locally (rather than metastatically as in other medulloblastoma subgroups). Our results help towards finding new treatable molecular targets for these types of medulloblastomas.

Characterization of a new human astrocytoma cell line SHG140: cell proliferation, cell phenotype, karyotype, STR markers and tumorigenicity analysis.

Background: Primary tumor Cell was an important tool for tumor research. Here, a new astrocytoma cell line SHG-140 was established and its proliferation, phenotype, karyotype, STR authentication, pathological characteristics, and characteristics of the cells' intrancranial xenografts of nude mice were studied.Methods: Primary SHG-140 culture was performed in DMEM/F12 medium with 10% FBS. Cell proliferation, karyotype analysis, cell immunofluorescence and STR authentication of SHG140 cells were performed. HE staining and immunohistochemistry, Whole oncogene high flux sequencing of the patient sample were carried out. SHG140 cells were injected into the brain of nude mice, HE staining and immunohistochemistry of intracranial xenograft tumor were detected.Results: Cell immunofluorescence demonstrated that SHG140 cells were positive for A2B5 (Glial precursors ganglioside), GFAP (Glial fibrillary acidic protein), Nestin, S-100 (Acid calcium bingding protein), Olig2 (Oligodendrocyte transcription factor 2) and Ki67 (Nuclear-associated antigen), cells negatively stained for Vimentin. Cell proliferation curve revealed that SHG140 proliferated slightly within 48 h, which then significantly proliferated to the fourth day. Karyotype analysis demonstrated its total number of chromosomes was 55, having trisomy of chromosome 6, 7, 8, 9 and X, and tetrad of chromosome 1 and 21, chromosomal deletion and rearrangement were observed. STR markers analysis showed the cells were derived from human male. SHG140 cells had tumorigenic properties - the intracranial injection of these cells into nude mice gave rise to growing tumors. We found that the glioma tissue was diffusively positive for GFAP, Nestin, slightly positive for Olig2, S-100; the positive rate of Ki-67 was 65% and negative for Vimentin. SHG140 cells were tumorigenic, GFAP, Nestin, S-100 Olig-2, the proliferation marker Ki-67 were expressed in its intracranial xenograft, Vimentin was negative expressed. Whole oncogene high flux sequencing of the patient tissue showed TP53, PTEN, IDH1 and PTCH1 mutation were existed.Conclusions: Our study showed that SHG140 was an astrocytoma glioma continuous cell line derived from a human adult male, having a strong tumorigenicity in nude mice, which made it wound be a useful model for the study of human glioblastoma multiforme.

Clinical and molecular analysis of smoothened inhibitors in Sonic Hedgehog medulloblastoma.

BackgroundSmoothened inhibitors (SMOi) have shown activity in Sonic Hedgehog (SHH) medulloblastoma, however this therapeutic class was not developed in children due to severe effects reported on growth. We hereby report long-term follow-up of young patients treated with SMOi for recurrent medulloblastoma.MethodsClinical data on response and toxicity from patients treated with vismodegib or sonidegib from 2011 to 2019 for a SHH medulloblastoma were retrospectively reviewed. Methylation analysis and whole exome sequencing were performed whenever possible.ResultsAll patients with a somatic PTCH1 mutation responded to SMOi (6/8), including 2 prolonged complete responses. One patient was free of disease 8.2 years after treatment. SMOi was challenged again for 3 patients. Two of them had a response, one with SMOi alone, the other one in combination with temozolomide despite previous progression under monotherapy. SMO resistance mutations were found in patients from biopsy at relapse. Combination with temozolomide or surgery plus radiotherapy was associated with very long disease control in 2 patients. The most severe adverse events were myalgia and growth plate fusion with metaphyseal sclerosis. Normal growth velocity was recovered for 1 patient although her final height was below estimated target height.ConclusionsTargeting SMO in mutated PTCH1 is an interesting strategy for long-term responses. Combination of SMOi with chemotherapy or surgery and local radiotherapy is an appealing strategy to prevent early resistance and diminish SMOi exposure, especially in young patients. Inhibition of SHH pathway causes growth and development impairment but partial recovery of the growth velocity is possible.

PATH-24. MOLECULAR CLASSIFICATION OF HIGH RISK INFANT EMBRYONAL BRAIN TUMORS ENROLLED IN THE ACNS0334 TRIAL: A REPORT FROM THE CHILDREN’S ONCOLOGY GROUP

Young children with embryonal brain tumors including medulloblastoma (MB), supratentorial primitive neuro-ectodermal tumor, or pineoblastoma have historically been considered high-risk patients with poor outcomes despite the use of intensive radiation-sparing treatment. In the ACNS0334 phase III trial, 91 consented children <36 months old with the above diagnoses were randomized to intensive induction chemotherapy with or without methotrexate followed by consolidation with stem cell rescue. Here we present the results of a centralized integrated molecular analysis including global methylation profiling (65/91), and whole exome sequencing of tumor (46/91) and germline (35/91) DNA. Unsupervised clustering analyses of methylation profiles using multiple orthogonal methods against a reference dataset of 1200 pediatric brain tumors, revealed known and new molecular entities. For tumors diagnosed as MB on central pathology review, 7.3% (3/41) had a non-MB molecular diagnosis (2 embryonal tumor with multiple rosettes/ETMR, 1 group MYC pineoblastoma), with the remainder as MB Group SHH (11/41), Group3 (25/41), and Group4 (2/41). Among histologic non-MBs, 3/24 (12.5%) were molecular entities not intended for trial inclusion (1 each for ATRT, pleomorphic xanthoastrocytoma, and high-grade glioma). ETMR, historically considered a rare entity, was molecularly identified in a significant proportion (14/65; 21.5%) of samples. Among MB-SHH, we detected deleterious PTCH1 mutations in 6/9 tumors but none among 5 germline samples tested; a germline SUFU frameshift mutation with tumor LOH was also observed in MB-SHH. Correlation of these and other molecular features to the parallel clinical analysis will yield important markers of risk stratification and predictors of treatment response.