MB Patients Research Articles

MEDB-09. Unraveling the role of unfolded protein response in medulloblastoma cancer stem cells

Medulloblastoma (MB) is the most common malignant childhood brain tumor. The current clinical approach consists of multimodal strategies with debilitating long-term effects and risk of tumor recurrence. Medulloblastoma stem cells (MBSCs) are a fraction of tumor cells with high proliferation potential and the capability to adapt to adverse/restrictive conditions in tumor milieu thus driving the refractoriness to conventional therapies. Recently, high basal levels of Unfolded Protein Response (UPR) molecules have been found in tumors of different tissue-origin and are correlated with poor prognosis and low patient survival. However, little is known about the role of UPR in MB. We investigated the expression and activation of UPR players in MBSCs. Human group 3 MB (G3MB) cell lines, specifically CHLA-01, D283- and D341-Med, were grown in Vitamin A and/or FBS or in stem selective medium (B27™) for 72 h before collection. Cells were fixed, stained with proper primary antibodies and images were acquired by confocal microscopy. The analysis of the transcription factors ATF-4 and CHOP revealed their elevated nuclear expression and co-localization, which resulted to be more marked in G3MB stem-like cells than in the differentiated ones. Also the ATF-6 branch was investigated, in differentiating conditions D283 and D341-Med showed a greater activation of ATF-6, represented by its nuclear localization, in respect to stem cells, while CHLA-01 did not show differences. Conversely XBP1, the transcription factor downstream IRE1 signaling, was not expressed in the three cell lines. Lastly, a Kaplan-Meier analysis on MB patients showed a worse prognosis with a shorter survival rate of patients expressing high ATF4 transcript levels. Our results reveal, even in resting conditions, preferential activation of the PERK branch in G3MB cells grown in stem-like condition suggesting that ATF-4 might be a promising therapeutic and prognostic factor to specifically target the stem compartment in aggressive MB.

MEDB-03. Medulloblastoma cerebrospinal fluid reveals hypoxic indicators (metabolites and lipids) and cancer-specific RNAs

Medulloblastoma (MB) is the most common malignant brain tumor in children. There remains an unmet need for diagnostics to sensitively detect the disease, particularly recurrences. Cerebrospinal fluid (CSF) provides a window into the central nervous system, and liquid biopsy of CSF could provide a relatively non-invasive means for disease diagnosis. There has yet to be an integrated analysis of the transcriptomic, metabolomic, and lipidomic changes occurring in the CSF of children with MB. CSF samples from patients with (n=40) or without (n=11; no cancer) MB were subjected to RNA-sequencing and high-resolution mass spectrometry to identify RNA, metabolite, and lipid profiles. Differentially expressed transcripts, metabolites, and lipids were identified and their biological significance assessed by pathway analysis. Multivariate analysis method DIABLO (R package mixOmics) was used to integrate the molecular changes characterizing the CSF of MB patients. Differentially expressed transcripts, metabolites, and lipids in CSF were discriminatory for the presence of MB but not the exact molecular subtype. One hundred ten genes and ten circular RNAs were differentially expressed in MB CSF compared to normal representing TGF-β signaling, TNF-a signaling via NF-kB, and adipogenesis pathways. Tricarboxylic acid cycle and other metabolites (malate, fumarate, succinate, α-ketoglutarate, hydroxypyruvate, N-acetyl-aspartate) and total triacylglycerols were significantly upregulated in MB CSF compared to normal CSF. Although the transcriptomic, metabolomic, and lipid signatures in CSF to differentiate MB subgroup separation was challenging, we were able to identify a group of omics signatures that could separate cancer from normal CSF. Metabolic and lipidomic profiles both contained indicators of tumor hypoxia. Our approach provides several candidate signatures that deserve further validation, including the novel circular RNA circ_463, and insights into the impact of MB on the CSF microenvironment.

EPID-07. Outcome disparities in children, adolescents and young adults with medulloblastoma: A population-based analysis

Abstract Medulloblastoma (MB) is the most common high-grade primary pediatric brain tumor. Recent registry-based studies in children with central nervous system (CNS) tumors have demonstrated that survival outcomes differ by race/ethnicity in multivariable analyses, with Hispanic patients having highest hazard of death overall. To investigate this finding in MB patients, we examined survival in children (0-14 years) and adolescent/young adults (15-39 years) with MB from 2007-2016 in the 2018 Surveillance Epidemiology and End Results Program database, using Kaplan Meier analysis, log-rank test and Cox proportional hazard ratios (HR) with 95% confidence intervals (CI). Race and ethnicity were categorized according to the U.S. Census, with Hispanic ethnicity (yes/no) analyzed separately from race (Black, White, Asian, Other). Among 1612 patients, 81% were White, 9% were Black, 8% were Asian or Pacific Islander, and 2% were from “other” or unknown racial groups. 28% of the cohort was of Hispanic ethnicity. Univariate analysis found that Black patients had a significantly higher hazard of death than White patients (HR=1.55, CI: 1.16 – 2.08, p=0.003). In contrast, Hispanic ethnicity was not significantly associated with outcome (HR=0.98, CI: 0.79-1.21, p=0.8). Medicaid or no insurance (vs. private) were each significantly associated with higher risk of death; Medicaid (HR =1.23, CI = 1.01 - 1.51, p=0.041); Uninsured (HR=2.07, CI=1.41-3.02, p=<0.001). Of the treatment modalities analyzed, patients who received neither chemotherapy nor radiation experienced higher hazard of death than patients who received both treatments (HR=3.63, CI 2.78-4.76, p=<0.001). Consistent with observations in other cancers, racial disparities are observed in patients with MB, with Black race conferring increased risk of death. Public insurance was also significantly associated with death, as was not receiving combined-modality therapy. Further work is needed to understand the multilevel factors impacting diagnosis, treatment and outcome among children and AYAs with MB and prospective studies are warranted.

MEDB-68. Analysis of telomeres length and Alternative Lengthening of Telomeres (ALT) in molecular subgroups of infant medulloblastoma

We investigated the association between the molecular profile and telomere length in a infant medulloblastoma (iMB) cohort, retrospectively studied. Activation of telomeres maintenance mechanisms was analyzed to determine whether the senescence escape triggered by telomere-elongation mechanisms could explain the aggressivity of some iMB belonging to the same molecular subgroup. Interestingly, several telomerase- and ALT-targeted therapies have recently been tested on pediatric cancers and might represent a promising strategy for the future treatment of aggressive telomerase- or ALT-positive iMB. We analyzed a cohort of 50 FFPE tissues from young MB patients (age ≤ 3); IHC, FISH, and an Illumina 850K methylation profile were used to identify molecular subgroups. Telomere length was measured using Telo-quantitative FISH, and image analysis was performed using TFL-Telo software. Three distinct telomere intensity categories (low (L), medium (M), and high (H)) were identified by comparing neoplastic- to endothelial-cell signals in each sample. ATRX loss and TERTp mutation/methylation were investigated using IHC and Sanger sequencing/methylation-specific PCR. SHH-MBs accounted for 59% of our cohort, while Group3/4-MBs accounted for 41%; no WNT-MBs were detected. ALT was found to be activated in 10% of iMBs and was not exclusive to any molecular subgroup, implying that it could be a potential mechanism associated with aggressive behaviour in a subset of iMBs. Promising results have been found in the telomere length distribution among the iMB molecular subgroups: SHH iMBs had a higher frequency of High (H) telomeres length (85%) than NON-SHH/NON-WNT iMBs (p=0.046), which were more frequently associated with Medium (M) telomeres length. CONCLUSIONS: ALT activation in infant MBs (10%) could be a novel target for risk-stratification and personalized therapy. It may be useful to examine ALT as a potential predictor of aggressive behaviour and as a promising novel therapeutic approach for a subset of these tumors in the diagnostic workup.

MEDB-31. THE CLINICAL SIGNIFICANCE OF EXTENT OF RESECTION IN MEDULLOBLASTOMA

Medulloblastoma (MB) patients determined to have a sub-total resection (STR), defined by >1.5cm2 post-surgical tumour residuum, receive intensified treatment regimes, but recently the designation of STR as a high risk feature is being questioned. We aimed to assess the clinical correlates of extent of resection (EOR) and its impact on survival, with particular consideration of EOR in relation to the four MB consensus molecular subgroups (WNT, SHH, Group 3, Group 4). We collected data from 1113 patients (n=419, UK CCLG institutions; n=694, published data) representing the largest ever combined cohort constructed to assess the impact of EOR in medulloblastoma. We performed association analyses and univariate/multivariate survival analysis using Kaplan-Meier, log-rank and Cox proportional hazard modelling, analysing overall survival (OS) cohort-wide and with reference to molecular subgroups and clinical features. Association analysis of the combined cohort evidenced that infant patients were more likely to have STR (p=0.02). In this whole-cohort analysis, EOR was significantly associated with survival in univariate analysis (HR 1.64, 95% CI 1.30-2.07, p=<0.001) but not in multivariate analysis. STR was variably prognostic in sub-cohort analyses of specific demographics and molecular subgroup; worse outcomes were observed in patients <5 years in SHH (p=0.044) and Group 4 (p=0.044). This was true for WNT patients >5 years old at diagnosis (p=0.034) although numbers were small and require validation in even larger cohorts. In this cohort of >1100 MBs, STR was significantly associated with a lower OS in univariate analysis, but this was driven by specific disease contexts (SHH and Group 4 patients <5 years old). STR was not independently prognostic overall or in any setting. We recommended that surgeons should continue to pursue maximal safe resection for all MB patients but suggest that consideration of STR as a high-risk feature should be disease context specific.

Arginase 1 is a marker of protection against illness in contacts of leprosy patients

Leprosy household contacts are generally more prone to develop the disease compared to the general population. Previous studies have demonstrated that genes related to the alternative activation (M2) profile in macrophages are associated with the increased bacillary load in multibacillary leprosy patients (MB), and that contacts of MB patients have a higher risk of contracting the disease. In addition, positive serological responses to PGL-1 or LID-1 are associated with a higher risk of disease. We performed a 5-year follow-up of contacts of leprosy patients and evaluated the pattern of gene and protein expression in cells from contacts that developed leprosy during this period. Leprosy household contacts had decreased soluble CD163 and heme oxygenase 1 (HO-1) serum levels when compared with healthy donors and leprosy patients. In contrast, arginase 1 activities were higher in contacts when compared with both healthy donors and leprosy patients. Of the contacts, 33 developed leprosy during the follow-up. Gene expression analysis revealed reduced ARG1 expression in these contacts when compared with contacts that did not develop disease. Arginase activity was a good predictive marker of protection in contacts (sensitivity: 90.0%, specificity: 96.77%) and the association with serology for anti-PGL-1 and anti-LID-1 increased the sensitivity to 100%. Altogether, the data presented here demonstrate a positive role of arginase against leprosy and suggest that the evaluation of arginase activity should be incorporated into leprosy control programs in order to aid in the decision of which contacts should receive chemoprophylaxis.

Reduced vitamin D receptor (VDR) and cathelicidin antimicrobial peptide (CAMP) gene expression contribute to the maintenance of inflammatory immune response in leprosy patients

Leprosy is an infectious disease influenced by genetic, immunological, and environmental factors. Reduced gene expressions may be associated with the immunological response pattern and leprosy susceptibility. We investigated the direct and indirect effects of Vitamin D Receptor (VDR) and Cathelicidin Antimicrobial Peptide (CAMP) gene expressions on the serum levels of vitamin D, Cathelicidin, and cytokines in newly-diagnosed leprosy patients and post-six-months of multidrug therapy (MDT). Thirty-four leprosy patients were assessed, paucibacillary (PB; n = 14) and multibacillary (MB; n = 20) cases, untreated or having received six months of MDT, 18 healthy controls, and 25 household contacts. VDR and CAMP gene expression levels were strongly correlated to some important cytokines in both, untreated leprosy patients (PB, r = 0.9319; MB, r = 0.9569) and patients who had undergone MDT (PB, r = 0.9667; MB, r = 0.9569). We observed that both gene expressions directly influenced IL-2, IFN-γ, and IL-17F serum levels in leprosy patients compared to the household contacts and healthy individuals. VDR and CAMP gene expressions induced a persistent inflammatory response in PB and MB leprosy patients, even after six months of MDT, to fight the Mycobacterium leprae infection. Due to the persistent inflammatory profile, multidrug therapy is suggested to be maintained for more than six months, especially for MB patients. Vitamin D supplementation is recommended from the onset as a transcription factor to improve VDR and CAMP gene expression in leprosy patients.

Pattern of Leprosy in Post-Elimination Stage: A 20-Year Retrospective Study in The Largest Specialized Leprosy Hospital of Bangladesh

Background: Leprosy has affected humanity for over 4000 years. The global registered number of new leprosy cases in 2019 were 202256. Bangladesh has been achieved Elimination of leprosy in 1998, but a slow resurgence of the disease still continues in several parts of the country. Aims and objectives: Understanding the current magnitude of the disease is vital for the community, service providers and policy makers. The present study was undertaken to describe the pattern of the disease after leprosy has been declared eliminated. This will help for proper planning of patient- oriented leprosy services and judicial allocation of scarce resources. Materials and Methods: This descriptive study has been carried out using the registered record of patients attending the outpatient department (OPD) of leprosy hospital, Sylhet, Bangladesh during the period 2001- 2020. Results: The new case detection pattern was declining. Of the 1835 new cases, males (76.95%) outnumbered females (23.05%). The PB and MB cases were 27.74% and 72.26% respectively. Leprosy reactions developed in 31.39% cases and 18.09% cases presented with grade 2 disabilities. Smear positive cases were 19.89%. Mode of case detection was mainly passive (98.75%). The new case detection of childhood leprosy (≤14 years) was declining. Majority of patients (40.27%) were from &gt;40 age group. Borderline type (44.20%) was more common. Leprosy reactions and G2D were common in MB type of leprosy, 78.69% and 66.57% respectively. Conclusion: New case detection is declining but burden of leprosy in the community has not changed significantly. MB patients with grade 2 disabilities are still in upward trend. Although leprosy has been eliminated globally, the disease continues accompanying the significant cause of disabilities in Bangladesh. Community based surveillance could help to improve early detection, treatment, prevention of disabilities and stigma.

Clinical Prognostic Implications of Wnt Hub Genes Expression in Medulloblastoma.

Medulloblastoma is the most common type of pediatric malignant primary brain tumor, and about one-third of patients die due to disease recurrence and most survivors suffer from long-term side effects. MB is clinically, genetically, and epigenetically heterogeneous and subdivided into at least four molecular subgroups: WNT, SHH, Group 3, and Group 4. We evaluated common differentially expressed genes between a Brazilian RNA-seq GSE181293 dataset and microarray GSE85217 dataset cohort of pediatric MB samples using bioinformatics methodology in order to identify hub genes of the molecular subgroups based on PPI network construction, survival and functional analysis. The main finding was the identification of five hub genes from the WNT subgroup that are tumor suppressors, and whose lower expression is related to a worse prognosis for MB patients. Furthermore, the common genes correlated with the five tumor suppressors participate in important pathways and processes for tumor initiation and progression, as well as development and differentiation, and some of them control cell stemness and pluripotency. These genes have not yet been studied within the context of MB, representing new important elements for investigation in the search for therapeutic targets, prognostic markers or for understanding of MB biology.

SMYD3 Promotes Cell Cycle Progression by Inducing Cyclin D3 Transcription and Stabilizing the Cyclin D1 Protein in Medulloblastoma.

Simple SummaryMedulloblastoma is the most common malignant pediatric brain tumor and is classified into four molecular subgroups: Wnt, Shh, Group 3, and Group 4. Of these subgroups, patients with Myc+ Group 3 MB have the worst prognosis. Using an RNAi functional genomic screen, we identified the lysine methyltransferase SMYD3 as a crucial epigenetic regulator responsible for promoting Group 3 MB cell growth. We demonstrated that SMYD3 drives MB cell cycle progression by inducing cyclin D3 transcription and preventing cyclin D1 ubiquitination. Using in vitro and ex vivo studies, we showed that SMYD3 suppression by shRNA and BCI-121 significantly impaired proliferation, resulting in the downregulation of cyclin D3, cyclin D1, and pRBSer795. Moreover, we are the first to show that SMYD3 methylates the cyclin D1 protein, indicating that the SMYD3 stabilizes cyclin D1 through post-translational modification. Collectively, our studies position SMYD3 as a promising treatment option for Group 3 Myc+ MB patients.Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Maximum safe resection, postoperative craniospinal irradiation, and chemotherapy are the standard of care for MB patients. MB is classified into four subgroups: Shh, Wnt, Group 3, and Group 4. Of these subgroups, patients with Myc+ Group 3 MB have the worst prognosis, necessitating alternative therapies. There is increasing interest in targeting epigenetic modifiers for treating pediatric cancers, including MB. Using an RNAi functional genomic screen, we identified the lysine methyltransferase SMYD3, as a crucial epigenetic regulator that drives the growth of Group 3 Myc+ MB cells. We demonstrated that SMYD3 directly binds to the cyclin D3 promoter to activate its transcription. Further, SMYD3 depletion significantly reduced MB cell proliferation and led to the downregulation of cyclin D3, cyclin D1, pRBSer795, with concomitant upregulations in RB in vitro. Similar results were obtained following pharmacological inhibition of SMYD3 using BCI-121 ex vivo. SMYD3 knockdown also promoted cyclin D1 ubiquitination, indicating that SMYD3 plays a vital role in stabilizing the cyclin D1 protein. Collectively, our studies demonstrate that SMYD3 drives cell cycle progression in Group 3 Myc+ MB cells and that targeting SMYD3 has the potential to improve clinical outcomes for high-risk patients.

GABAA receptor agonist suppresses pediatric medulloblastoma progression by inhibiting PKA-Gli1 signaling axis

The Sonic hedgehog-activated subgroup of medulloblastoma (SHH-MB) is one of the most common malignant pediatric brain tumors. Recent clinical studies and genomic databases indicate that GABAA receptor holds significant clinical relevance as a therapeutic target for pediatric MB. Herein, we report that "moxidectin," a GABAA receptor agonist, inhibits the proliferation of Daoy, UW426, UW228, ONS76, and PFSK1 SHH-MB cells by inducing apoptosis. Immunoblotting and immunofluorescence microscopy demonstrated that moxidectin significantly induced GABAA receptor expression and inhibited cyclic AMP (cAMP)-mediated protein kinase A (PKA)-cAMP response element-binding protein (CREB)-Gli1 signaling in SHH-MB. Gli1 and the downstream effector cancer stem cell (CSC) molecules such as Pax6, Oct4, Sox2, and Nanog were also inhibited by moxidectin treatment. Interestingly, moxidectin also inhibited the expression of MDR1. Mechanistic studies using pharmacological or genetic inhibitors/activators of PKA and Gli1 confirmed that the anti-proliferative and apoptotic effects of moxidectin were mediated through inhibition of PKA-Gli1 signaling. Oral administration of 2.5mg/kg moxidectin suppressed the growth of SHH-MB tumors by 55%-80% in subcutaneous and intracranial tumor models in mice. Exvivo analysis of excised tumors confirmed the observations made in the invitro studies. Moxidectin is an FDA-approved drug with an established safety record, therefore any positive findings from our studies will prompt its further clinical investigation for the treatment of MB patients.

Integration of immunohistochemistry, RNA sequencing, and multiplex ligation-dependent probe amplification for molecular classification of pediatric medulloblastoma.

Medulloblastoma (MB) is commonly classified into four molecular groups, that is, WNT, SHH, group 3, and group 4, for prognostic and therapeutic purposes. Here we applied immunohistochemistry (IHC) and RNA sequencing (RNA-seq) for the molecular classification of MB, and utilized multiplex ligation-dependent probe amplification (MLPA) to determine chromosomal alterations and specific gene amplifications. We retrospectively enrolled 37 pediatric MB patients. Twenty-three had genomic material available for gene/RNA analysis. For IHC, β-catenin, GAB1, and YAP were the biomarkers to segregate MB into three subgroups, WNT (1/23), SHH (5/23), and non-WNT/non-SHH (17/23). However, four cases (17.3%) were found to be misclassified after analysis by RNA-seq. The result of MLPA revealed two group 3 tumors carrying MYC amplification, and three SHH tumors harboring MYCN amplification. While IHC provided rapid subgroup stratification, it might result in incorrect subgrouping. Thus, validation of the IHC result with genomic data analysis by RNA-seq or other tools would be preferred. In addition, MLPA can detect important genetic alterations and is helpful for the identifications of high-risk patients. Our study revealed that integration of these diagnostic tools can provide a precise and timely classification of MB, optimizing an individualized, risk-directed postoperative adjuvant therapy for these patients. This workflow can be applied in a countrywide fashion to guide future clinical trials for patients with MB.

Serum Interleukin 6 Level and Nutrition Status as Potential Predictors of Clinical Leprosy Development Among Household Contacts in Endemic Areas.

BackgroundLeprosy is a chronic infectious disease that can lead to severe lifelong disabilities. Close contacts of patients with leprosy have a higher risk of acquiring the disease. Nevertheless, there is a lack of reliable markers to predict Mycobacterium leprae infection. We aimed to identify new potential markers for developing clinical leprosy among contacts.MethodsSerum levels of interleukin (IL) 6, IL-8, IL-10, hemoglobin, ferritin, and transferrin saturation were measured in 67 patients with multibacillary leprosy (MB), 65 household contacts (HHCs) of MB patients, and 127 endemic controls (ECs). By means of multivariate logistic regression and receiver operating characteristic (ROC) analyses, we analyzed baseline variables and laboratory parameters that showed significant differences between MB in the HHC and EC groups and obtained the respective areas under the curve (AUC). Optimal cutoff values of the associated cytokines were also determined.ResultsElevated IL-6 level was observed in MB patients compared to HHCs and ECs (P = .022 and .0041, respectively). Anemia and iron deficiency were also higher in the MB group compared to HHCs or ECs (P < .001). Likewise, we observed an increased risk of having MB leprosy in underweight HHCs (odds ratio [OR], 2.599 [95% confidence interval {CI}, .991–6.820]) and underweight ECs (OR, 2.176 [95% CI, 1.010–4.692]). Further ROC analysis showed that high serum IL-6 level, underweight, anemia, and iron deficiency can discriminate leprosy from their HHCs (AUC, 0.843 [95% CI, .771–.914]; P = .000; optimal cutoff value of IL-6 = 9.14 pg/mL).ConclusionsOur results suggest that serum IL-6 and nutrition status could serve as potential prognostic markers for the development of clinical leprosy in infected individuals.

Dermlep Study Part 3: Post-RFT Events in Leprosy Patients Presenting to Dermatologists.

Introduction:Presently the leprosy program has no defined surveillance protocols for patients who complete the fixed duration multidrug therapy and are released from treatment (RFT). Hence, the information about the post-RFT events in these patients is sparse and qualitative and quantitative data regarding their health care requirements is missing. During the DermLep survey carried out by the Indian Association of Dermatologists,Venereologists and Leprologists (IADVL), a number of patients presented to dermatologists during the post RFT period for a variety of symptoms. This paper analyses the events in these patients during the post RFT period.Results:Out of a total of 3701 leprosy patients who presented to 201 dermatologists across India during the DermLep survey, 708 (26.2%) were in the post RFT period (488 males; 220 females). Of these, 21% were PB and 79% MB patients as per their treatment records. Majority were in the age group of 31-59 years (55.5%); however, a significant proportion of them (20.7%) were elderly (>60 years). Majority of the patients (45.5%) presented within the first year of RFT with variable symptoms; 28% were between 1-5 years, 5.5% between 5-10 years; and 11.0% presented more than 10 years after RFT. Most common presenting complaint being persistent skin lesions as perceived by patients in 21.2%, followed by neuritis in 14.5%; trophic ulcers in 13.8%; deformities in 67 (11.8%); lepra reactions in 66 (11.6%); and recurrence of original symptoms in 6.7%.Conclusion:The DermLep Survey highlights the importance of ‘post RFT’ patients as an important subset of leprosy patients who visit dermatologists for various health related issues. The most common complaints in this subset were active/persistent skin lesions, lepra reactions and neuritis. In these patients, who are a sub-group of ‘persons affected with leprosy’ the disease related issues can persist for many years post RFT. Hence, it is important to provide services in the programme to monitor and manage these complications for the prevention of impairments, disability and the related social issues.

Relationship between 25-Hydroxy Vitamin D Levels and Type of Morbus Hansen

Background: Morbus Hansen is a chronic granulomatous infection of Mycobacterium leprae (M. leprae) which is characterized by cardinal signs in the form of numbness of the skin, thickening of peripheral nerves and acid-fast bacteria (AFB) were found on bacterioscopic examination. WHO has classified MH into paucibasilar MH (MH-PB) and multibasilar MH (MH-MB) based on the number of skin lesions or bacterial index (IB). Vitamin D in MH is known to act as an immunomodulator. This study aims to determine the relationship between serum 25(OH)D levels with Morbus Hansen type at RSUP Dr. Mohammad Hoesin and Leprosy Hospital Dr. Rivai Abdullah Palembang.&#x0D; Methods: A laboratory observational study with a case series design was undertaken at the Dermatology and Venereology (DV) Polyclinic of Infectious Dermatology (DI) Mohammad Hoesin Hospital Palembang since December 2019 to January 2020. A sample of 33 patient MH met the inclusion criteria, consisting of 22 patients with Morbus Hansen (MH) type MB and 11 patients with Morbus Hansen (MH) type PB. Comparison of serum 25(OH)D levels between MB and PB type MH patients was analyzed using the Independent T Test, the relationship between serum 25(OH)D levels and MH type was analyzed using the Fisher Exact Test and the correlation between serum 25(OH)D levels with type of MH was analyzed using Spearman Rho's test. Data analysis using SPSS version 22.0.&#x0D; Results: In this study, there were no differences in demographic characteristics of gender, age, age category, body mass index, education and occupation between MB and PB type MH patients (p &gt; 0.05). There were no difference in duration of therapy (p = 0.155), ENL reaction (p = 0.276) and patient status (p = 0.304) between MB and PB type MH patients, but there were differences in bacterial index (p = 0.000) and clinical spectrum (p = 0.000) between MB and PB type MH patients. There is a difference in the mean level of 25(OH)D between MB and PB type MH patients (p = 0.006), there is a significant relationship between 25 (OH)D levels and MH type patients, (OR = 9.643 ; p = 0.010) and there is a significant moderate positive correlation between levels of 25 (OH)D and the type of MH (r = 0.467; p = 0.006).&#x0D; Conclusion: It can be concluded that there are differences in serum 25(OH)D levels among Morbus Hansen type. In addition, there is a significant relationship between serum 25(OH)D levels with Morbus Hansen type.

Relationship between 25-Hydroxy Vitamin D Levels and Type of Morbus Hansen

Background: Morbus Hansen is a chronic granulomatous infection of Mycobacterium leprae (M. leprae) which is characterized by cardinal signs in the form of numbness of the skin, thickening of peripheral nerves and acid-fast bacteria (AFB) were found on bacterioscopic examination. WHO has classified MH into paucibasilar MH (MH-PB) and multibasilar MH (MH-MB) based on the number of skin lesions or bacterial index (IB). Vitamin D in MH is known to act as an immunomodulator. This study aims to determine the relationship between serum 25(OH)D levels with Morbus Hansen type at RSUP Dr. Mohammad Hoesin and Leprosy Hospital Dr. Rivai Abdullah Palembang.&#x0D; Methods: A laboratory observational study with a case series design was undertaken at the Dermatology and Venereology (DV) Polyclinic of Infectious Dermatology (DI) Mohammad Hoesin Hospital Palembang since December 2019 to January 2020. A sample of 33 patient MH met the inclusion criteria, consisting of 22 patients with Morbus Hansen (MH) type MB and 11 patients with Morbus Hansen (MH) type PB. Comparison of serum 25(OH)D levels between MB and PB type MH patients was analyzed using the Independent T Test, the relationship between serum 25(OH)D levels and MH type was analyzed using the Fisher Exact Test and the correlation between serum 25(OH)D levels with type of MH was analyzed using Spearman Rho's test. Data analysis using SPSS version 22.0.&#x0D; Results: In this study, there were no differences in demographic characteristics of gender, age, age category, body mass index, education and occupation between MB and PB type MH patients (p &gt; 0.05). There were no difference in duration of therapy (p = 0.155), ENL reaction (p = 0.276) and patient status (p = 0.304) between MB and PB type MH patients, but there were differences in bacterial index (p = 0.000) and clinical spectrum (p = 0.000) between MB and PB type MH patients. There is a difference in the mean level of 25(OH)D between MB and PB type MH patients (p = 0.006), there is a significant relationship between 25 (OH)D levels and MH type patients, (OR = 9.643 ; p = 0.010) and there is a significant moderate positive correlation between levels of 25 (OH)D and the type of MH (r = 0.467; p = 0.006).&#x0D; Conclusion: It can be concluded that there are differences in serum 25(OH)D levels among Morbus Hansen type. In addition, there is a significant relationship between serum 25(OH)D levels with Morbus Hansen type.

Adult Medulloblastoma Demographic, Tumor and Treatment Impact since 2006: A Canadian University Experience.

Medulloblastoma is an aggressive primary brain tumor that is extremely rare in adults; therefore, prospective studies are limited. We reviewed the information of all MB patients treated at the CHUM between 2006 and 2017. We divided our cohort by age and further divided adult patients (53%) in two groups, those diagnosed between 2006–2012 and 2013–2017. In our adult population, median follow up was 26 months and SHH-activated MB comprised 39% of tumors. Adult 5yOS was 80% and first-line therapy led to a 5yPFS of 77%. The absence of radiosensitizing chemotherapy (100% vs. 50%; p = 0.033) negatively influenced 5yPFS. 96% of adult patients received radiotherapy and 48% of them received concomitant radiosensitizing chemotherapy. Complete surgical resection was performed on 85% of adults, but the extent of resection did not have a discernable impact on survival and did not change with time. Adjuvant chemotherapy did not clearly affect prognosis (5yOS 80% vs. 67%, p = 0.155; 5yPFS 78% vs. 67%, p = 0.114). From 2006–2012, the most common chemotherapy regimen (69%) was Cisplatinum, Lomustine and Vincristine, which was replaced in 2013 by Cisplatinum, Etoposide and Cyclophosphamide (77%) with a trend for worse survival. Nine patients recurred and seven of these (78%) were treated with palliative chemotherapy. In conclusion, we did not identify prognostic demographic or tumor factors in our adult MB population. The presence of radiosensitizing chemotherapy was associated with a more favorable PFS. Cisplatinum, Lomustine and Vincristine regimen might be a better adjuvant chemotherapy regimen.

Abstract 3049: Tumor treating fields induce DNA damage and apoptosis in medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant brain tumor in children and accounts for approximately 20% of all pediatric central nervous system tumors. Current multimodal treatment has led to a 70-90% five-year overall survival rate, however, the prognosis for patients with tumor dissemination and recurrent MB remains poor. Additionally, the majority of survivors exhibit long-term neurocognitive and neuroendocrine complications because of the cytotoxic drugs and high dose radiation. Consequently, more effective and less toxic treatments are necessary in order to raise the quality of life for these young patients. Tumor Treating Fields (TTFields) are an exciting new therapeutic opportunity for MB. TTFields are low intensity, alternating electric fields that disrupt cell division through physical interactions with key molecules during mitosis. The side effects from TTFields treatments are incredibly low, making it an ideal candidate for MB therapy. Currently TTFields were found to increase the survival of adult brain tumor patients by dysregulating the cytoskeletal network leading to mitotic arrest and as well as other varied multi-modal mechanisms of action. To determine if TTFields can be an effective treatment for MB, we conducted an in vitro study treating multiple MB cell lines with TTFields. We discovered that TTFields treatment reduced MB cell growth (Daoy, Med1-MB, D425, and D283) with an optimal frequency of 200-250 kHz. We also observed that TTFields treatment dysregulated actin polymerization that corresponded with a reduction in cell motility and invasion. TTFields treatment also caused DNA damage (γH2AX, 53BP1) that correlated with an increase in apoptotic cells. To expand on these initial findings, we conducted a combinatorial study with the small molecule inhibitor, CX-4945. CX-4945 is an inhibitor to casein kinase 2 and is currently being used in a phase I clinical trial to treat recurrent MB (NCT03904862). We discovered that CX-4945 enhanced the growth reduction of TTFields treatment. In addition, combining CX-4945 and TTFields increased the number of cells with dysregulated actin which correlated with a decrease in MB migration and invasion. Our findings demonstrate that TTFields may be a novel and less toxic method to treat MB patients. Citation Format: Ryan T. Nitta, Gordon Li. Tumor treating fields induce DNA damage and apoptosis in medulloblastoma [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 3049.

Abstract LB133: Targetting IL-6/gp130 signalling axis attenuates acquired drug resistance in medulloblastoma

Abstract Medulloblastoma (MB) is the most common pediatric brain tumour comprising of four distinct molecular subgroups exhibiting high level of intertumoural heterogeneity. The standard multimodal treatment yields a survival rate of 70% but the aggressive treatment affects the long-term sequelae of MB patients. The tumour microenvironment (TME) is a regulator of cancer progression and affects therapeutic efficacy in primary and metastatic brain malignancies. Mechanistic insights into the tumor-promoting role of the individual components of the brain TME will aid in identifying key survival pathways and design of potential therapeutics to combat drug resistance and pathogenesis of the disease. We hypothesize that tumor associated macrophages (TAMs) in the brain TME facilitate drug resistance via the IL-6/STAT3 signalling axis in MB. To understand the role of paracrine signaling resulting from TAMs, a co-culture system was used to evaluate the expression of pSTAT3 and correlated with drug sensitivity and/or resistance in Med8A cell line. When co-cultured with HMC3, a human microglia cell line, the chemosensitive Med8A-S cells exhibited enhanced pSTAT3 expression and acquired drug resistance. Interestingly, Med8A cells lacking expression of IL-6 receptor (IL6R-/-) also exhibited high pSTAT3 levels and drug resistance; suggesting that HMC3 may release soluble factors in addition to IL-6 sufficient to drive chemoresistance in Med8A variants. To assess this, we conditioned cells with stimulatory cytokines belonging to the IL-6 family. We found that oncostatin M (OSM), interleukin-11 (IL-11) and leukemia inhibitory factor (LIF) induced drug resistance and enhanced pSTAT3 levels in IL6R-/- cells. Receptors of the IL-6 family share a common signal transducing beta subunit, glycoprotein (gp130) that transduces the signal intracellularly and phosphorylates janus activated kinases (JAKs) and subsequently STAT3. Our results suggest that paracrine proinflammatory cytokines found in the TME can promote MB drug resistance by signalling through a common signal transducer gp130, hence targeting any one cytokine receptor of the IL-6 family of cytokines may not sufficiently abrogate acquired drug resistance. Currently, we are evaluating the efficacy of agents that target common downstream elements of the IL-6/STAT3 signalling axis, including gp130, JAKs and STAT3, as a potentially improved therapeutic strategy to circumvent acquired drug resistance in medulloblastoma. Citation Format: Lakshana Sreenivasan, Pascal Leclair, Chinten James Lim. Targetting IL-6/gp130 signalling axis attenuates acquired drug resistance in medulloblastoma [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 LB133.

Abstract LB254: PKA signaling: a friend or foe to pediatric medulloblastoma

Abstract Pediatric brain tumor is the leading cause of cancer-related deaths in pediatric patients ranging 0-19 years of age. Medulloblastoma is one of the most commonly occurring brain tumor in children. Sonic hedgehog (Shh) activated subgroup of medulloblastoma is considered to be highly aggressive and metastatic in nature. Recent studies and oncomine, a cancer microarray database has shown that GABAA receptor is down regulated in pediatric medulloblastoma. In this study, we have evaluated the anti-cancer effects of an anthelminthic drug ‘moxidectin' a GABAA receptor agonist. Treatment of medulloblastoma cells with moxidectin, resulted in reduced Protein kinase A (PKA) activity. Leading to the inhibition of Gli1, a major transcription factor of non-canonical Shh signaling. Moxidectin has inhibited the proliferation of Daoy, UW426, UW228, ONS76, and PFSK1 medulloblastoma cells and significantly induced apoptosis in a concentration and time dependent manner in all the cell lines. Western blotting and immunofluorescence microscopy demonstrated that moxidectin treatment significantly induced GABAA receptor expression and inhibited cAMP mediated PKA signaling. As a result, the non-canonical activation of Gli1 and its downstream effector molecules such as Pax-6, Oct-4, Sox-2 and Nanog were also inhibited by moxidectin treatment. Efficacy of moxidectin was also evaluated in subcutaneous and intracranial medulloblastoma tumor models. Our results demonstrated that daily oral administration of 2.5 mg/kg moxidectin suppressed the growth of medulloblastoma tumors by 55-70% in subcutaneous and intracranial models. Ex-vivo analysis of tumors by western blotting, IHC, TUNEL and H&amp;E staining revealed that moxidectin suppressed medulloblastoma tumor progression by inhibiting PKA/Gli1 signaling axis indicating the significance of this pathway in medulloblastoma progression. To our knowledge, this study for the first time focuses on GABAA receptor agonist mediated PKA/Gli1 signaling inhibition. Most importantly, moxidectin is an 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. Citation Format: Itishree Kaushik, Sanjay Srivastava. PKA signaling: a friend or foe to pediatric medulloblastoma [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 LB254.