A standardized treatment protocol for pilonidal disease can influence the health mindset of adolescents.

PURPOSE: Pilonidal disease (PD) significantly impacts patients' quality of life and requires regular maintenance behaviors to achieve cure. Health mindset is a psychological construct which can influence health behaviors and outcomes, with a growth mindset being associated with better outcomes than a fixed. We propose that participation in a standardized treatment protocol can affect the health mindset for adolescents with pilonidal disease. METHODS: PD patients' demographics, recurrence, and comorbidities were prospectively collected from 2019 to 2022. We assessed patients' mindset score at initial presentation using the validated Three-Item Mindset Scale (1-6) then reassessed during follow-up. t-test was used to compare baseline and follow-up mindset scores and stratified by recurrence or comorbidities. p ≤ 0.05 was considered significant. RESULTS: A total of 207 PD patients (108 males, 99 females) with mean age 18.2 ± 3.7 years were followed for 351 ± 327 days. Mean baseline mindset score (4.76 ± 1.27) was significantly lower than mean follow-up mindset score (5.03 ± 1.18, p = 0.049). Baseline mindset score was significantly lower among patients with PD recurrence (4.00 ± 0.66) compared to those without recurrence (4.8 ± 1.29, p = 0.05). Among patients with PD recurrence, mean baseline mindset score (4.00 ± 0.66) was significantly lower than mean follow-up mindset score (5.27 ± 0.93, p = 0.0038). Patient comorbidity did not affect the baseline or follow-up mindset score. CONCLUSIONS: Participation in a standardized treatment protocol is associated with the development of a stronger growth mindset over time for patients with PD. Furthermore, a growth mindset was linked to lower recurrence rate than a fixed mindset. Further investigations into how treatment approaches can work in concert with health mindset are proposed.

Standardized pilonidal protocol as rescue therapy for excision-refractory pilonidal disease.

PURPOSE: Severe pilonidal diseases have refractory symptoms despite multiple surgeries and optimal therapy remains unclear. We hypothesized that standardized minimally invasive protocol could be an effective rescue treatment. METHODS: We prospectively collected data from symptomatic patients who underwent ≥ 1 pilonidal excision prior to presentation at our clinic 2019-2023. We treated these patients with standardized protocol incorporating local wound care, regular manual/laser epilation, and selective debridement/pit trephination. RESULTS: We treated 34 refractory patients (23 males) with median follow-up 405 days. Median age of first symptoms was 17.1 years; presentation to our clinic 20.0 years. Prior to our clinic, 27 received one surgery (cleft lift-2, excision no closure-1, excision primary closure-18, wound vac after excision-3, excision flap closure-3); 7 had two surgeries (excision without closure + cleft lift-1, primary closure after excision twice-3, flap closure after excision twice-2, excision primary closure + excision without closure-1). We treated all patients with regular epilation ± local wound care. 14 (41%) underwent trephination ± debridement. All patients achieved complete resolution after median 52 days. Five (14.7%) recurred and were treated with trephination + debridement-2 or wound care alone-3. Symptom length had no correlation with resolution time, skin type, hair amount. CONCLUSIONS: Standardized minimally invasive protocol requiring only selective surgical intervention can treat refractory pilonidal disease with low recurrence rate.

Management and outcomes of pilonidal patients with secondary sinuses-a cohort study.

BACKGROUND: Patients with pilonidal disease (PD) can present with concurrent draining secondary sinus at the superior gluteal cleft. The natural disease course in the setting of this severe phenotype is poorly characterized. We present the largest cohort of patients with PD and concurrent secondary sinus. METHODS: Patients with PD and concurrent secondary sinus who underwent Gips procedure with secondary sinus excision from 2019 to 2023 were prospectively followed. Patient demographics, drainage recurrence, symptom resolution, treatment, and follow-up period were recorded. Recurrent drainage from previous secondary sinus site was defined as isolated painless serous drainage after the wound had closed for > 3 weeks; recurrent PD was characterized as recurrent pain and bloody drainage after excision. RESULTS: One hundred and five patients (seventy-one males) with a median age of 17.2 years [interquartile range (IQR):15.4-19.0] underwent excision of their disease and were followed for a median of 367.0 days (IQR: 173.2-658.8). Without regular epilation, six patients (5.7%, five males, one female) had recurrent PD. With regular epilation, three patients (2.8%, three males) had recurrent PD. Eight patients (7.5%, six males, two females) had recurrent secondary sinus site drainage. Median time to recurrent drainage was 75.5 days (IQR: 65.2-216.2) after excision and for recurrent drainage to resolve was 72 days (IQR: 49-81). Recurrent secondary sinus site drainage was treated with antibiotics, silver nitrate, debridement, or no treatment. CONCLUSION: Patients who present with PD in the setting of concurrent secondary sinus have a unique, more severe disease phenotype. Excision can be complicated by recurrent drainage from the secondary sinus site that can resolve without repeat surgical excision.

Comorbidities are not associated with pain symptom or recurrence in patients with pilonidal disease.

BACKGROUND: Comorbidities can potentially impact the presentation or outcome of patients with pilonidal disease (PD) due to poor wound healing or increased inflammatory response. We hypothesized that certain comorbidities could lead to worse pain or higher recurrence rate. METHODS: A retrospective study was performed on all PD patients treated with standardized minimally invasive protocol at our clinic 2019-2022. Patients' demographics, comorbidities, initial/follow-up pain score, pain duration, and recurrence were recorded. Data were analyzed by t test and Chi-square test. RESULTS: Of 207 total PD patients (108 male, 99 female), 61 had comorbidities. Mean age was 18.2 years. The recurrence rate was 7%, and patients with recurrence were significantly younger. Associated comorbidities included mood/psychiatric disorders (31%), asthma/respiratory illness (30%), obesity-related illness (15%), gastrointestinal disorders (13%), diabetes (10%), thyroid disease (8%), cardiac disease (8%), musculoskeletal/connective tissue disorders (7%), immunologic disease (7%), inflammatory bowel disease (5%), and chest wall disorders (3%). The presence of comorbidities was not associated with PD recurrence. By dividing patients into adolescents (< 18 years) and adults (≥ 18 years), we found no association between comorbidity and recurrence in either group. 55% of patients had pain as an initial symptom. The initial pain score, pain duration, and pain score at follow-up were not associated with comorbidities. The comorbidities and recurrence were not associated with patient age or sex. CONCLUSIONS: Having comorbidities was not associated with pain symptoms or recurrence in PD patients. Even though patients with recurrence were younger, there was no association between comorbidity and recurrence in either adolescents or adults.

Concurrent application of interferon-gamma and vincristine inhibits tumor growth in an orthotopic neuroblastoma mouse model.

PURPOSE: Tumor-associated macrophages are present within neuroblastoma, and interferon-gamma (IFN-γ) can polarize macrophages into cancer-inhibiting M1 type. We hypothesize that treating neuroblastoma with interferon-gamma (IFN-γ) can suppress tumor growth, and the concurrent treatment with IFN-γ and vincristine can lead to enhanced tumor killing as compared to vincristine alone. METHODS: We loaded IFN-γ or vincristine into silk biomaterials and recorded the amount released over time. Orthotopic, syngeneic neuroblastoma xenografts were generated by injecting 9464D cells into adrenal gland of C57BL/6 mice, and IFN-γ-loaded and/or vincristine-loaded silk biomaterials were implanted into the tumor once the tumors reached 100 mm3. Drug release at different timepoints was measured and tumor growth after different treatments were compared. RESULTS: 1-2% of IFN-γ and 70% of vincristine were released from the biomaterials by the fifth day. Combining IFN-γ and vincristine significantly slowed tumor growth as compared to the controls (12.2 ± 2.7 days to reach 800 mm3 versus 5.7 ± 1.2 days, p = 0.01), and IFN-γ alone also delayed tumor growth as compared to the controls (10.9 ± 1.5 days versus 5.7 ± 1.2 days, p = 0.001). Hematoxylin and eosin staining demonstrated tumor necrosis adjacent to the drug-loaded silk biomaterials. CONCLUSION: Local delivery of sustained release IFN-γ can inhibit neuroblastoma tumor growth by itself and in combination with vincristine.

Regular epilation alone is an acceptable treatment for symptom-free pilonidal patients.

INTRODUCTION: Patients with mild pilonidal disease often experience symptom resolution without excision. We hypothesized that treating symptom-free/asymptomatic pilonidal patients with regular epilation alone had similar recurrence rate as patients who were also treated surgically. METHOD: Patient data were prospectively collected 2/2019-11/2022 at our Pilonidal Clinic. All patients received regular epilation; all patients presented before 12/2020 also underwent pit excision using trephines. Starting 1/2021, only symptomatic patients underwent pit excision; symptom-free patients at presentation received only regular epilation. Recurrence rates were statistically analyzed. RESULTS: 255 patients (male:54.4%, female:45.6%), median age 17.3years (IQR:15.8-19.1) were followed for median 612.5days (IQR:367.5-847). 44.1% identified as Hispanic, 36.5% Caucasian, 17.1% Asian, 2.4% Black. Median symptom duration at presentation was 180.5days (IQR:44.5-542.5). 160 patients were initially treated with surgical excision and regular epilation, while 95 patients with regular epilation only. The failure rate between patients who received surgical excision initially and recurred (9.4%) and patients who received epilation only and recurred (12.6%) was similar, after controlling for sex, race, age, comorbidities, skin type, hair color, hair thickness (p > 0.05). Patients who recurred after only undergoing regular epilation all underwent surgical excision, median 100days (IQR:59.5-123.5) after initial presentation. CONCLUSION: Regular epilation alone is an acceptable treatment for symptom-free pilonidal patients.

Burden of pilonidal disease and improvement in quality of life after treatment in adolescents.

PURPOSE: Pilonidal Disease (PD) affects adolescents in different aspects. We hypothesized that patients with different gender, ethnicity, and age have different quality of life (QOL) measurements which could improve with minimally invasive treatment (MIT). METHODS: 131 PD patients underwent MIT (laser epilation ± trephination) from 2019 to 2021. Patients' demographics were recorded. Before and after MIT, patients received QOL questionnaire consisting of four categories: daily activities, sports participation, school/work attendance, and socializing. Data were analyzed using Student and multivariate t test. P < 0.05 was considered statistically significant. RESULTS: 101 (51 male, 50 female) patients were included. 30 patients with incomplete data were excluded. 54% of patients were < 18 years old. 47.5% were Hispanic. Median symptom duration prior to presentation was 5.4 (1.3-15) months. Prior to MIT, patients' ability to perform daily activities, participate in sports, attend school/work, and socialize was moderately or severely impacted in 66%, 57%, 45%, and 23% of respondents, respectively; after MIT, only 7%, 8%, 2%, and 4% were affected (p < 0.01). Recurrence rate was 6%. Pre-MIT, older patients and non-Hispanics reported worse impact on their QOL. Symptom duration or PD recurrence did not correlate with patient's pre- or post-MIT QOL. CONCLUSION: Patients' ethnicity and age impacted QOL in PD. All patients' QOL significantly improved with MIT. Considering the importance of socializing, playing sports, and school/work attendance in adolescents, our study highlights importance of early treatment of PD.

Dexrazoxane Significantly Reduces Anthracycline-induced Cardiotoxicity in Pediatric Solid Tumor Patients: A Systematic Review.

Cardiotoxicity is a dose-limiting and potentially lethal complication of anthracycline administration. Previous studies failed to determine definitive toxic doses or cardioprotective factors. Current dosing strategies may utilize unnecessarily high anthracycline doses, such that survival benefit may not outweigh increased toxicity rates. A systematic review of randomized controlled trials and prospective/retrospective studies investigating anthracycline treatment in pediatric solid tumors was performed from PubMed/MEDLINE and Cochrane databases. Generalized linear models mapping survival, cardiotoxicity, and cardiotoxicity-free survival adjusted for male-to-female ratio, follow-up time, and concomitant chemotherapeutic drugs or cardioprotective agents (dexrazoxane) were generated using R. Survival rose linearly with increasing cumulative anthracycline dose whereas cardiotoxicity demonstrated exponential increases both without (dose, >200 mg/m) and with (dose, >400 mg/m) dexrazoxane. Maximum cardiotoxicity-free survival was 268.2 mg/m without and 431.8 mg/m with dexrazoxane. Despite increasing cardiotoxicity-free dose by >150 mg/m, dexrazoxane minimally improved projected survival (71.9% vs. 75.4%). Cardiotoxicity increased linearly as a function of follow-up time with rates doubling from 5 to 20 years, without evidence of plateau. On the basis of our model, current dosing regimens-doxorubicin doses >375 mg/m without dexrazoxane-overvalue increased anthracycline administration and may contribute to devastating cardiotoxicity. The linear increase of cardiotoxicity without evidence of plateau confirms the necessity for lifelong cardiac monitoring.

Implantable chemotherapy-loaded silk protein materials for neuroblastoma treatment.

Neuroblastoma is the most common extracranial childhood solid tumor. Treatment of high risk tumors require intense multicycle chemotherapies, resulting in short- and long-term toxicities. Here, we present treatment of an orthotopic neuroblastoma mouse model, with silk fibroin materials loaded with vincristine, doxorubicin or the combination as a intratumoral, sustained release system. The materials, loaded with vincristine with or without doxorubicin, significantly decreased neuroblastoma tumor growth compared to materials loaded without drug or doxorubicin only as well as intravenous (IV) drug treatment. The intratumoral drug concentration was significantly higher with intratumoral delivery versus IV. Furthermore, intratumor delivery decreased the maximum plasma concentration compared to IV delivery, reducing systemic exposure and possibly reduing long-term side effects of chemotherapy exposure. Histopathologically, tumors with remission periods >25 days before recurrence transformed from a "small-round-blue cell" (SBRC) to predominantly "large cell" neuroblastoma (LCN) histopathology, a more aggressive tumor subtype with unfavorable clinical outcomes. These results show that intratumoral chemotherapy delivery may be a treatment strategy for pediatric neuroblastoma, potentially translatable to other focal tumors types. Furthermore, this treatment modality allows for a clinically relevant mouse model of tumor transformation that may be used for studying the phenotypical tumor recurrence and developing more effective treatment strategies for recurrent tumors.

Silencing Intersectin 1 Slows Orthotopic Neuroblastoma Growth in Mice.

Neuroblastoma accounts for 15% of all pediatric cancer deaths. Intersectin 1 (ITSN1), a scaffold protein involved in phosphoinositide 3-kinase (PI3K) signaling, regulates neuroblastoma cells independent of MYCN status. We hypothesize that by silencing ITSN1 in neuroblastoma cells, tumor growth will be decreased in an orthotopic mouse tumor model. SK-N-AS neuroblastoma cells transfected with empty vector (pSR), vectors expressing scrambled shRNA (pSCR), or shRNAs targeting ITSN1 (sh#1 and sh#2) were used to create orthotopic neuroblastoma tumors in mice. Volume was monitored weekly with ultrasound. End-point was tumor volume >1000 mm. Tumor cell lysates were analyzed with anti-ITSN1 antibody by Western blot. Orthotopic tumors were created in all cell lines. Twenty-five days post injection, pSR tumor size was 917.6±247.7 mm, pSCR was 1180±159.9 mm, sh#1 was 526.3±212.8 mm, and sh#2 was 589.2±74.91 mm. sh#1-tumors and sh#2-tumors were smaller than pSCR (P=0.02), no difference between sh#1 and sh#2. Survival was superior in sh#2-tumors (P=0.02), trended towards improved survival in sh#1-tumors (P=0.09), compared with pSCR-tumors, no difference in pSR tumors. Western blot showed decreased ITSN1 expression in sh#1 and sh#2 compared with pSR and pSCR. Silencing ITSN1 in neuroblastoma cells led to decreased tumor growth in an orthotopic mouse model. Orthotopic animal models can provide insight into the role of ITSN1 pathways in neuroblastoma tumorigenesis.

Irreversible Electroporation as an Effective Technique for Ablating Human Metastatic Osteosarcoma.

Irreversible electroporation (IRE) induces apoptosis in tumor cells with electric energy, allowing treatment of unresectable tumors. One potential application is metastatic osteosarcoma (OS) in the pediatric population. A 12-year-old underwent thoracotomy with resection of metastatic OS. IRE was applied to 1 resected tumor section. Using 2 probes, 100 pulses with width of 90 ms were delivered. Efficacy was measured by increase in current draw during treatment. The treated sample was analyzed with hematoxylin and eosin and transmission electron microscopy. Default voltage of 1800 kV was ineffective. Voltage of 2700 kV caused excessive current draw and was aborted to prevent thermal injury. At 2200 kV, current draw rise was 9 amps, signifying successful treatment. Untreated specimen showed viable OS, normal surrounding lung tissue. Treated tumor had edema within the tumor and in surrounding lung tissue, with intra-alveolar hemorrhage and cellular architecture destruction. There was also evidence for cellular destruction such as disruption of lipid bilayer and release of intracellular fluid. Optimal voltage for treatment was 2200 kV, likely higher due to electrical conduction variation in the aerated lung. IRE may be an option for pediatric patients with unresectable metastatic OS.

Pilonidal disease in the pregnant patient: A case report.

INTRODUCTION: Pilonidal disease in the pregnant patient is underreported and may be instigated by significant shifts in hormone levels throughout pregnancy and the postpartum period. PRESENTATION OF CASE: An otherwise healthy primigravid 22-year-old woman developed pilonidal disease at the beginning of her pregnancy. While pregnant, her symptoms recurred once and resolved without treatment. She was managed non-operatively during pregnancy. Her symptoms recurred again in the postpartum period but they quickly resolved. Due to the recurrent symptoms, she underwent definitive management via minimally invasive Gips procedure in the postpartum period, without further recurrence. DISCUSSION: Pilonidal disease affects pregnant patients and can be managed non-operatively. The etiology of pilonidal disease in the setting of pregnancy may be related to hormonal shifts and warrants further investigation. CONCLUSION: We present the first report of pilonidal disease in a pregnant patient in the primary literature. The hormonal shifts associated with pregnancy may be associated with the development of pilonidal disease in the pregnant patient.

Using "Appointment Tickets" to Track Visit Duration as a Quality Improvement Initiative After Incorporating Laser Epilation in a Pilonidal Care Clinic.

BACKGROUND: Laser epilation (LE) is effective in decreasing pilonidal disease (PD) recurrence, but laser use has not been a standard practice in pediatric surgery clinic. We hypothesized that "appointment tickets" can 1) track utilization and clinic visit delays, 2) inform patients of their clinic progress in real time. METHODS: An observation study was performed on LE patients treated at our PD clinic 3/2021-7/2022. Two exam rooms were utilized for manual shaving and one for LE. After gluteal cleft hair shaving, various anesthetic (topical lidocaine, ice, or cryotherapy) was applied prior/during LE. At each visit, patient received an "appointment ticket" on which providers recorded the visit start/end time, manual shaving duration, local anesthetic application/wait time, LE duration. Visits were scheduled for 20 min-slots. Clinic staff recorded any delays (>20 min). RESULTS: 1317 visits were recorded. Mean number of visits per week was 18 ± 6. Mean total visit length was 60 ± 22 min, mean shaving time 15 ± 11 min, mean anesthetic application/wait time 16 ± 11 min, mean LE time 14 ± 9 min. Over the study period, average visit length has decreased, and average visits/clinic day has increased. Most delays occurred in months April, May, October-December. Delays due to patient late arrival occurred in May, July, and August of 2021, none in 2022. CONCLUSIONS: LE visits have multiple components that required close coordination to ensure no clinic delays. Clinic delays spiked prior to summer and winter holidays. "Appointment tickets" provided patients with real-time visit progress tracked clinic utilization to improve the quality of pilonidal care delivery. LEVEL OF EVIDENCE: Level IV.

Mitochondrial uncoupler and retinoic acid synergistically induce differentiation and inhibit proliferation in neuroblastoma.

Neuroblastoma is a leading cause of death in childhood cancer cases. Unlike adult malignancies, which typically develop from aged cells through accumulated damage and mutagenesis, neuroblastoma originates from neural crest cells with disrupted differentiation. This distinct feature provides novel therapeutic opportunities beyond conventional cytotoxic methods. Previously, we reported that the mitochondrial uncoupler NEN (niclosamide ethanolamine) activated mitochondria respiration to reprogram the epigenome, promoting neuronal differentiation. In the current study, we further combine NEN with retinoic acid (RA) to promote neural differentiation both in vitro and in vivo. The treatment increased the expression of RA signaling and neuron differentiation-related genes, resulting in a global shift in the transcriptome towards a more favorable prognosis. Overall, these results suggest that the combination of a mitochondrial uncoupler and the differentiation agent RA is a promising therapeutic strategy for neuroblastoma.

Increasing Amount of Hair Reduction using Laser Correlates with Lower Probability of Recurrence in Patients with Pilonidal Disease.

BACKGROUND: Hair at the gluteal cleft plays a key role in the development and recurrence of pilonidal disease (PD). We hypothesized that more hair reduction achieved using laser could correlate with lower chance of PD recurrence. METHODS: PD patients who underwent laser epilation (LE) were categorized by Fitzpatrick skin type, hair color, and hair thickness. Photos taken at LE sessions were compared to determine hair reduction amount. LE sessions completed prior to the recurrences were recorded. Groups were compared using multivariate T-test. RESULTS: 198 PD patients had mean age 18.1 ± 3.6 years. 21, 156, and 21 patients had skin types 1/2, 3/4, and 5/6, respectively. 47 patients had light- and 151 had dark-colored hair. 29 patients had fine hair, 129 medium, and 40 thick. Median follow-up was 217 days. 95%, 70%, 40%, and 19% of patients reached 20%, 50%, 75%, and 90% hair reduction after mean LE sessions of 2.6, 4.3, 6.6, 7.8 sessions, respectively. To reach 75% hair reduction, patients require a mean of 4.8-6.8 LE sessions, depending on different skin/hair characteristics. PD recurrence rate was 6%. Probability of recurrence after 20%, 50%, 75% hair reduction was decreased by 50%, 78%, 100%, respectively. Dark hair and skin type 5/6 were associated with higher recurrence rates. CONCLUSION: Patients with dark-color and thick hair require more LE sessions to achieve certain degree of hair reduction. Patients with dark hair and skin type 5/6 were more likely to recur; more hair reduction correlated with lower chance of recurrence. LEVEL OF EVIDENCE: Level IV.

Adoption of a standardized treatment protocol for pilonidal disease leads to low recurrence.

BACKGROUND: Pilonidal disease may present as acute abscesses or chronic draining sinuses. There is no standardized treatment and recurrence rates can be as high as 30%. Within our five-hospital network we have established a standardized treatment protocol including minimally invasive surgical trephination and aggressive epilation. We hypothesize that such a treatment protocol can be established across different hospital settings and lead to low overall recurrence. METHODS: Patients with pilonidal disease were enrolled in the study on presentation to our hospital network. Those that underwent initial surgery outside our hospital system or were noncompliant with our treatment protocol were excluded. Patients were grouped based on surgeon and treating facility. Frequency of recurrence per surgeon and per hospital was calculated and compared. RESULTS: Out of 132 patients, 80 patients were included (45 female, 35 male) while 52 were excluded because of initial surgery at a non-network hospital or for protocol noncompliance. Median age was 17 (16-19) years and median length of follow-up was 352 (261-496) days. There were 6 patients who experienced at least one recurrence. There was an overall 8% recurrence rate with no significant difference noted between surgeons or hospitals (p = 0.15, p = 0.64, respectively). CONCLUSIONS: We have successfully implemented a standardized treatment protocol for pilonidal disease across different hospital settings and by different surgeons, with an overall low recurrence rate. Our findings suggest that adoption of a standardized protocol for treatment of pilonidal disease can lead to low recurrence. LEVEL OF EVIDENCE: Level IV.

Mitochondrial Uncoupling Induces Epigenome Remodeling and Promotes Differentiation in Neuroblastoma.

The Warburg effect is the major metabolic hallmark of cancer. According to Warburg himself, the consequence of the Warburg effect is cell dedifferentiation. Therefore, reversing the Warburg effect might be an approach to restore cell differentiation in cancer. In this study, we used a mitochondrial uncoupler, niclosamide ethanolamine (NEN), to activate mitochondrial respiration, which induced neural differentiation in neuroblastoma cells. NEN treatment increased the NAD+/NADH and pyruvate/lactate ratios and also the α-ketoglutarate/2-hydroxyglutarate (2-HG) ratio. Consequently, NEN treatment induced promoter CpG island demethylation and epigenetic landscape remodeling, activating the neural differentiation program. In addition, NEN treatment upregulated p53 but downregulated N-Myc and ß-catenin signaling in neuroblastoma cells. Importantly, even under hypoxia, NEN treatment remained effective in inhibiting 2-HG generation, promoting DNA demethylation, and suppressing hypoxia-inducible factor signaling. Dietary NEN intervention reduced tumor growth rate, 2-HG levels, and expression of N-Myc and ß-catenin in tumors in an orthotopic neuroblastoma mouse model. Integrative analysis indicated that NEN treatment upregulated favorable prognosis genes and downregulated unfavorable prognosis genes, which were defined using multiple neuroblastoma patient datasets. Altogether, these results suggest that mitochondrial uncoupling is an effective metabolic and epigenetic therapy for reversing the Warburg effect and inducing differentiation in neuroblastoma. SIGNIFICANCE: Targeting cancer metabolism using the mitochondrial uncoupler niclosamide ethanolamine leads to methylome reprogramming and differentiation in neuroblastoma, providing a therapeutic opportunity to reverse the Warburg effect and suppress tumor growth. See related commentary by Byrne and Bell, p.167.

Telomerase-targeting compounds Imetelstat and 6-thio-dG act synergistically with chemotherapy in high-risk neuroblastoma models.

BACKGROUND: The majority of high-risk neuroblastomas harbor telomerase activity, and telomerase-interacting compounds, such as 6-thio-2'-deoxyguanosine (6-thio-dG), have been found to impair the growth of telomerase-positive neuroblastoma cell lines. It has remained unclear, however, how such drugs can be combined with other compounds used in current treatment concepts for neuroblastoma patients. METHODS: Growth-inhibitory effects of varying concentrations of 6-thio-dG in combination with etoposide, doxorubicin or ceritinib were determined in eight telomerase-positive neuroblastoma cell lines with distinct genetic backgrounds. Tumor growth inhibition of subcutaneous xenografts from three different cell lines was assessed upon treatment with 6-thio-dG, the competitive telomerase inhibitor imetelstat, etoposide, or combinations of these compounds. RESULTS: Robust synergistic anti-tumor effects were observed for combinations of 6-thio-dG and etoposide or doxorubicin, but not for 6-thio-dG and ceritinib, in telomerase-positive neuroblastoma cell lines in vitro. Treatment of mouse xenografts with combinations of 6-thio-dG and etoposide significantly attenuated tumor growth and improved mouse survival over etoposide alone in two of three cell line models. Treatment of xenograft tumors by imetelstat monotherapy decreased telomerase activity by roughly 50% and significantly improved survival over control in all three models, whereas treatment with imetelstat plus etoposide led to enhanced survival over etoposide monotherapy in one model. Mechanistically, the synergistic effect was found to be due to both increased apoptosis and cell cycle arrest. CONCLUSION: Our study indicates that telomerase is an actionable target in telomerase-positive neuroblastoma, and demonstrates that combination therapies including telomerase-interacting compounds may improve the efficacy of established cytotoxic drugs. Targeting telomerase may thus represent a therapeutic option in high-risk neuroblastoma patients.

Development of a dinutuximab delivery system using silk foams for GD2 targeted neuroblastoma cell death.

Neuroblastoma is the most common extracranial solid tumor of childhood and is associated with poor survival in high risk patients. Recently, dinutuximab (DNX) has emerged as an effective immunotherapy to treat patients with high risk neuroblastoma. DNX works through the induction of cell lysis via complement-dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC). However, one third of patients who undergo DNX treatment exhibit tumor relapse and the therapy is dose limited by side effects such as severe pain. To overcome delivery challenges of DNX, including large size and dose limiting side effects, we fabricated a delivery system capable of sustained local delivery of bioactive DNX utilizing silk fibroin. We evaluated the impact of silk properties (MW, crystallinity, and concentration) on release properties and confirmed the bioactivity of the release product. Additionally, we observed that the effectiveness of CDC induction by DNX could be correlated to the GD2 expression level of the target cells, with both the intravenous DNX formulation and the released DNX. Collectively, these data highlights a strategy to overcome delivery challenges and potentially improve therapeutic efficacy in cells expressing heterogenous levels of GD2.

Combining inhibitors of Brd4 and cyclin-dependent kinase can decrease tumor growth in neuroblastoma with MYCN amplification.

INTRODUCTION: High-risk neuroblastoma is a deadly disease; poor prognosticators are MYCN-amplification and TERT-overexpression. We hypothesized that Gene Set Enrichment Analysis (GSEA) could identify pathways associated with MYCN-amplification and that inhibition of these pathways could decrease tumor growth. METHODS: We analyzed the Neuroblastoma-Kocak dataset (GSE45547, n = 649) and identified pathways associated with MYCN-amplification. Inhibitors were selected from upregulated gene sets for in vitro cytotoxicity testing using ST16-patient-derived primary neuroblastoma cells and in vivo testing using orthotopic ST16-patient-derived xenografts (PDX) in mice. Tumor volume was measured with ultrasound and tumor sections examined after H&E staining. RESULTS: GSEA identified significantly overexpressed gene sets in MYCN-amplified tumors including MYC targets, cell cycle mitotic genes, TERT associated genes, loss of RB1 gene sets, and E2Fs targets. Several genes were potential Bromodomain-containing protein 4 (Brd4) targets, making Brd4 inhibitors - JQ1, AZD5153 - and cyclin-dependent kinase (Brd4's binding partner) inhibitors - dinaciclib - potential therapeutic agents. JQ1 and dinaciclib were synergistic in inducing cytotoxicity in vitro. Dinaciclib-AZD5153 in vivo decreased tumor size compared to control, and increased tumor lymphocyte infiltration and necrosis on histology. CONCLUSIONS: GSEA is a powerful approach to identify upregulated genes and potential therapeutic targets. Dinaciclib-AZD5153 combination therapy can be effective against MYCN-amplified and TERT-overexpressing neuroblastoma tumors.