Comprehensive Analysis of Hypermutation in Human Cancer.

We present an extensive assessment of mutation burden through sequencing analysis of >81,000 tumors from pediatric and adult patients, including tumors with hypermutation caused by chemotherapy, carcinogens, or germline alterations. Hypermutation was detected in tumor types not previously associated with high mutation burden. Replication repair deficiency was a major contributing factor. We uncovered new driver mutations in the replication-repair-associated DNA polymerases and a distinct impact of microsatellite instability and replication repair deficiency on the scale of mutation load. Unbiased clustering, based on mutational context, revealed clinically relevant subgroups regardless of the tumors' tissue of origin, highlighting similarities in evolutionary dynamics leading to hypermutation. Mutagens, such as UV light, were implicated in unexpected cancers, including sarcomas and lung tumors. The order of mutational signatures identified previous treatment and germline replication repair deficiency, which improved management of patients and families. These data will inform tumor classification, genetic testing, and clinical trial design.

Clinical and biological landscape of constitutional mismatch-repair deficiency syndrome: an International Replication Repair Deficiency Consortium cohort study.

BACKGROUND: Constitutional mismatch repair deficiency (CMMRD) syndrome is a rare and aggressive cancer predisposition syndrome. Because a scarcity of data on this condition contributes to management challenges and poor outcomes, we aimed to describe the clinical spectrum, cancer biology, and impact of genetics on patient survival in CMMRD. METHODS: In this cohort study, we collected cross-sectional and longitudinal data on all patients with CMMRD, with no age limits, registered with the International Replication Repair Deficiency Consortium (IRRDC) across more than 50 countries. Clinical data were extracted from the IRRDC database, medical records, and physician-completed case record forms. The primary objective was to describe the clinical features, cancer spectrum, and biology of the condition. Secondary objectives included estimations of cancer incidence and of the impact of the specific mismatch-repair gene and genotype on cancer onset and survival, including after cancer surveillance and immunotherapy interventions. FINDINGS: We analysed data from 201 patients (103 males, 98 females) enrolled between June 5, 2007 and Sept 9, 2022. Median age at diagnosis of CMMRD or a related cancer was 8·9 years (IQR 5·9-12·6), and median follow-up from diagnosis was 7·2 years (3·6-14·8). Endogamy among minorities and closed communities contributed to high homozygosity within countries with low consanguinity. Frequent dermatological manifestations (117 [93%] of 126 patients with complete data) led to a clinical overlap with neurofibromatosis type 1 (35 [28%] of 126). 339 cancers were reported in 194 (97%) of 201 patients. The cumulative cancer incidence by age 18 years was 90% (95% CI 80-99). Median time between cancer diagnoses for patients with more than one cancer was 1·9 years (IQR 0·8-3·9). Neoplasms developed in 15 organs and included early-onset adult cancers. CNS tumours were the most frequent (173 [51%] cancers), followed by gastrointestinal (75 [22%]), haematological (61 [18%]), and other cancer types (30 [9%]). Patients with CNS tumours had the poorest overall survival rates (39% [95% CI 30-52] at 10 years from diagnosis; log-rank p<0·0001 across four cancer types), followed by those with haematological cancers (67% [55-82]), gastrointestinal cancers (89% [81-97]), and other solid tumours (96% [88-100]). All cancers showed high mutation and microsatellite indel burdens, and pathognomonic mutational signatures. MLH1 or MSH2 variants caused earlier cancer onset than PMS2 or MSH6 variants, and inferior survival (overall survival at age 15 years 63% [95% CI 55-73] for PMS2, 49% [35-68] for MSH6, 19% [6-66] for MLH1, and 0% for MSH2; p<0·0001). Frameshift or truncating variants within the same gene caused earlier cancers and inferior outcomes compared with missense variants (p<0·0001). The greater deleterious effects of MLH1 and MSH2 variants as compared with PMS2 and MSH6 variants persisted despite overall improvements in survival after surveillance or immune checkpoint inhibitor interventions. INTERPRETATION: The very high cancer burden and unique genomic landscape of CMMRD highlight the benefit of comprehensive assays in timely diagnosis and precision approaches toward surveillance and immunotherapy. These data will guide the clinical management of children and patients who survive into adulthood with CMMRD. FUNDING: The Canadian Institutes for Health Research, Stand Up to Cancer, Children's Oncology Group National Cancer Institute Community Oncology Research Program, Canadian Cancer Society, Brain Canada, The V Foundation for Cancer Research, BioCanRx, Harry and Agnieszka Hall, Meagan's Walk, BRAINchild Canada, The LivWise Foundation, St Baldrick Foundation, Hold'em for Life, and Garron Family Cancer Center.

Pediatric Central Nervous System Cancers, Version 2.2023, NCCN Clinical Practice Guidelines in Oncology.

Central nervous system (CNS) cancers account for approximately one quarter of all pediatric tumors and are the leading cause of cancer-related death in children. More than 4,000 brain and CNS tumors are diagnosed each year in children and teens, and the incidence rate has remained stagnant in recent years. The most common malignant pediatric CNS tumors are gliomas, embryonal tumors consisting of predominately medulloblastomas, and germ cell tumors. The inaugural version of the NCCN Guidelines for Pediatric Central Nervous System Cancers focuses on the diagnosis and management of patients with pediatric diffuse high-grade gliomas. The information contained in the NCCN Guidelines is designed to help clinicians navigate the complex management of pediatric patients with diffuse high-grade gliomas. The prognosis for these highly aggressive tumors is generally poor, with 5-year survival rates of <20% despite the use of combined modality therapies of surgery, radiation therapy and systemic therapy. Recent advances in molecular profiling has expanded the use of targeted therapies in patients whose tumors harbor certain alterations. However, enrollment in a clinical trial is the preferred treatment for eligible patients.

Congenital Ewing Sarcoma Presenting as a Rapidly Growing Neck Mass in a Newborn.

BACKGROUND: Ewing sarcoma (EWS) is an aggressive soft-tissue and bone malignancy. Congenital EWS is extremely rare, and its presenting features can be unique from that of EWS occurring in older children. CLINICAL FINDINGS: A full-term female infant with a neck mass present at birth was admitted to a level I nursery with an otherwise well appearance and normal vital signs. After consultation with a neonatologist, she was transferred to a neonatal intensive care unit where she developed sudden respiratory collapse from rapid growth of the mass causing airway obstruction, leading to emergent intubation. Ultrasound and MRI scans of the neck mass demonstrated cystic and vascular components, and a timely biopsy revealed small round blue cells with diffuse CD99 expression and chromosomal translocation 11;22. PRIMARY DIAGNOSIS: Ewing sarcoma. INTERVENTIONS: An accelerated workup for EWS was done due to the patient's critical status. On day of life (DOL) 8, she was started on treatment of EWS as per the current standard-of-care AEWS0031. On DOL 24, she underwent tracheostomy placement. OUTCOMES: The patient completed 14 total cycles of chemotherapy and is more than 12 months old. Her tracheostomy was decannulated at 6 months of age. PRACTICE RECOMMENDATIONS: The rarity of EWS in neonates and its presentation as a neck mass make this disease difficult to recognize unless clinicians have a high index of suspicion. The aims of this case report are to increase awareness of malignancy as a potential cause of neck masses in neonates and to prompt nurses and physicians to prepare for airway stabilization at appropriate levels of care if a neck mass is present at birth.

Germline-driven replication repair-deficient high-grade gliomas exhibit unique hypomethylation patterns.

Replication repair deficiency (RRD) leading to hypermutation is an important driving mechanism of high-grade glioma (HGG) occurring predominantly in the context of germline mutations in RRD-associated genes. Although HGG presents specific patterns of DNA methylation corresponding to oncogenic mutations, this has not been well studied in replication repair-deficient tumors. We analyzed 51 HGG arising in the background of gene mutations in RRD utilizing either 450 k or 850 k methylation arrays. These were compared with HGG not known to be from patients with RRD. RRD HGG harboring secondary mutations in glioma genes such as IDH1 and H3F3A displayed a methylation pattern corresponding to these methylation subgroups. Strikingly, RRD HGG lacking these known secondary mutations clustered together with an incompletely described group of HGG previously labeled "Wild type-C" or "Paediatric RTK 1". Independent analysis of two comparator HGG cohorts showed that other RRD/hypermutant tumors clustered within these subgroups, suggesting that undiagnosed RRD may be driving some HGG clustering in this location. RRD HGG displayed a unique CpG Island Demethylator Phenotype in contrast to the CpG Island Methylator Phenotype described in other cancers. Hypomethylation was enriched at gene promoters with prominent demethylation in genes and pathways critical to cellular survival including cell cycle, gene expression, cellular metabolism, and organization. These data suggest that methylation arrays may provide diagnostic information for the detection of RRD HGG. Furthermore, our findings highlight the unique natural selection pressures in these highly dysregulated, hypermutant cancers and provide the novel impact of hypermutation and RRD on the cancer epigenome.

Inhibition of enhancer of zest homologue 2 is a potential therapeutic target for high-MYC medulloblastoma.

MYC amplification is common in Group 3 medulloblastoma and is associated with poor survival. Group 3 and Group 4 medulloblastomas are also known to have elevated levels of histone H3-lysine 27-tri-methylation (H3K27me3), at least in part due to high expression of the H3K27 methyltransferase enhancer of zest homologue 2 (EZH2), which can be regulated by MYC. We therefore examined whether MYC expression is associated with elevated EZH2 and H3K27me3 in medulloblastoma, and if high-MYC medulloblastomas are particularly sensitive to pharmacological EZH2 blockade. Western blot analysis of low (DAOY, UW228, CB SV40) and high (DAOY-MYC, UW228-MYC, CB-MYC, D425) MYC cell lines showed that higher levels of EZH2 and H3K27me3 were associated with elevated MYC. In fixed medulloblastoma samples examined using immunohistochemistry, most MYC positive tumors also had high H3K27me3, but many MYC negative ones did as well, and the correlation was not statistically significant. All high MYC lines tested were sensitive to the EZH2 inhibitor EPZ6438. Many low MYC lines also grew more slowly in the presence of EPZ6438, although DAOY-MYC cells responded more strongly than parent DAOY cultures with lower MYC levels. We find that higher MYC levels are associated with increased EZH2, and pharmacological blockade of EZH2 is a potential therapeutic strategy for aggressive medulloblastoma with elevated MYC.

Magnetic Resonance Fingerprinting to Characterize Childhood and Young Adult Brain Tumors.

OBJECT: Magnetic resonance fingerprinting (MRF) allows rapid, simultaneous mapping of T1 and T2 relaxation times and may be an important diagnostic tool to measure tissue characteristics in pediatric brain tumors. We examined children and young adults with primary brain tumors to determine whether MRF can discriminate tumor from normal-appearing white matter and distinguish tumor grade. METHODS: MRF was performed in 23 patients (14 children and 9 young adults) with brain tumors (19 low-grade glioma, 4 high-grade tumors). T1 and T2 values were recorded in regions of solid tumor (ST), peritumoral white matter (PWM), and contralateral white matter (CWM). Nonparametric tests were used for comparison between groups and regions. RESULTS: Median scan time for MRF and a sequence for tumor localization was 11 min. MRF-derived T1 and T2 values distinguished ST from CWM (T1: 1,444 ± 254 ms vs. 938 ± 96 ms, p = 0.0002; T2: 61 ± 22 ms vs. 38 ± 9 ms, p = 0.0003) and separated high-grade tumors from low-grade tumors (T1: 1,863 ± 70 ms vs. 1,355 ± 187 ms, p = 0.007; T2: 90 ± 13 ms vs. 56 ± 19 ms, p = 0.013). PWM was distinct from CWM (T1: 1,261 ± 359 ms vs. 933 ± 104 ms, p = 0.0008; T2: 65 ± 51 ms vs. 38 ± 8 ms, p = 0.008), as well as from tumor (T1: 1,261 ± 371 ms vs. 1,462 ± 248 ms, p = 0.047). CONCLUSIONS: MRF is a fast sequence that can rapidly distinguish important tissue components in pediatric brain tumor patients. MRF-derived T1 and T2 distinguished tumor from normal-appearing white matter, differentiated tumor grade, and found abnormalities in peritumoral regions. MRF may be useful for rapid quantitative measurement of tissue characteristics and distinguish tumor grade in children and young adults with brain tumors.

Risk of subsequent cancer following a primary CNS tumor.

Improvements in survival among central nervous system (CNS) tumor patients has made the risk of developing a subsequent cancer an important survivorship issue. Such a risk is likely influenced by histological and treatment differences between CNS tumors. De-identified data for 41,159 patients with a primary CNS tumor diagnosis from 9 Surveillance, Epidemiology and End Results (SEER) registries were used to calculate potential risk for subsequent cancer development. Relative risk (RR) and 95 % confidence interval (CI) of subsequent cancer was calculated using SEER*Stat 7.0.9, comparing observed number of subsequent cancers versus expected in the general United States population. For all CNS tumors studied, there were 830 subsequent cancers with a RR of 1.26 (95 % CI, 1.18-1.35). Subsequent cancers were observed in the CNS, digestive system, bones/joints, soft tissue, thyroid and leukemia. Radiotherapy was associated with an elevated risk, particularly in patients diagnosed with a medulloblastoma/primitive neuroectodermal tumor (MPNET). MPNET patients who received radiotherapy were at a significant risk for development of cancers of the digestive system, leukemia, bone/joint and cranial nerves. Glioblastoma multiforme patients who received radiotherapy were at lower risks for female breast and prostate cancers, though at an elevated risk for cancers of the thyroid and brain. Radiotherapy is associated with subsequent cancer development, particularly for sites within the field of radiation, though host susceptibility and post-treatment status underlie this risk. Variation in subsequent cancer risk among different CNS tumor histological subtypes indicate a complex interplay between risk factors in subsequent cancer development.

Exploring Barriers to Pediatric Cancer Clinical Trials: The Role of a Networked, Just-in-Time Study Program.

The Research to Accelerate Cures and Equity (RACE) for Children Act mandates that newly developed targeted oncology drugs be tested in children when molecular targets are relevant to pediatric cancers. In its first year, the RACE for Children Act was effective in creating novel drug development opportunities for children with cancer; however, significant barriers to clinical trial enrollment persist. Pediatric cancer clinical trials are impacted by challenges surrounding logistics, complexity, and access. As such, there is potential for a networked and centralized study approach to address these barriers. Here we discuss adapting a just-in-time clinical trial approach for adults to serve the pediatric oncology population. Through innovative patient matching solutions leveraging large, real-world datasets with high computational power, the Tempus Integrated Molecular Evaluation (TIME) for Kids Program aims to address barriers in the development of new therapies. This commentary explores the potential for reducing challenges in developing novel pediatric therapeutics, advancing equity in genomic biomarker testing for precision tailored treatment, and improving outcomes for pediatric oncology patients.

c-MYC expression sensitizes medulloblastoma cells to radio- and chemotherapy and has no impact on response in medulloblastoma patients.

BACKGROUND: To study whether and how c-MYC expression determines response to radio- and chemotherapy in childhood medulloblastoma (MB). METHODS: We used DAOY and UW228 human MB cells engineered to stably express different levels of c-MYC, and tested whether c-MYC expression has an effect on radio- and chemosensitivity using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay, clonogenic survival, apoptosis assays, cell cycle analysis, and western blot assessment. In an effort to validate our results, we analyzed c-MYC mRNA expression in formalin-fixed paraffin-embedded tumor samples from well-documented patients with postoperative residual tumor and compared c-MYC mRNA expression with response to radio- and chemotherapy as examined by neuroradiological imaging. RESULTS: In DAOY - and to a lesser extent in UW228 - cells expressing high levels of c-MYC, the cytotoxicity of cisplatin, and etoposide was significantly higher when compared with DAOY/UW228 cells expressing low levels of c-MYC. Irradiation- and chemotherapy-induced apoptotic cell death was enhanced in DAOY cells expressing high levels of c-MYC. The response of 62 of 66 residual tumors was evaluable and response to postoperative radio- (14 responders (CR, PR) vs. 5 non-responders (SD, PD)) or chemotherapy (23 CR/PR vs. 20 SD/PD) was assessed. c-MYC mRNA expression was similar in primary MB samples of responders and non-responders (Mann-Whitney U test, p = 0.50, ratio 0.49, 95% CI 0.008-30.0 and p = 0.67, ratio 1.8, 95% CI 0.14-23.5, respectively). CONCLUSIONS: c-MYC sensitizes MB cells to some anti-cancer treatments in vitro. As we failed to show evidence for such an effect on postoperative residual tumors when analyzed by imaging, additional investigations in xenografts and larger MB cohorts may help to define the exact function of c-MYC in modulating response to treatment.

Molecular Profiling of Pediatric and Adult Glioblastoma.

OBJECTIVES: Although glioblastoma (GBM) is rare in the pediatric population, it is the most common cause of death among children with central nervous system neoplasms. Recent molecular profiling of these neoplasms has demonstrated distinct differences in comparison to their adult counterparts. Moreover, many pediatric GBMs occur within the context of cancer predisposition syndromes, such as constitutional mismatch repair deficiency syndrome (CMMRD). Children with CMMRD who develop GBM exhibit a high tumor mutational burden and may benefit from treatment with immune checkpoint inhibitors. METHODS: We performed next-generation sequencing and immunohistochemistry for mismatch repair proteins in our cohort of pediatric and adult GBMs to further characterize the molecular profiles of these groups. RESULTS: We examined a total of 11 pediatric and 11 adult GBMs. Pediatric patients had a higher number of alterations compared to their adult counterparts. They also had a higher frequency of alterations in the mismatch repair genes, which can be detected by immunohistochemistry (IHC). We also identified one pediatric patient with CMMRD syndrome. CONCLUSIONS: Our study highlighted the distinct molecular differences between pediatric and adult GBM. We also demonstrated that pediatric patients have a higher frequency of alterations in the mismatch repair genes, which may render them susceptible to treatment with immune checkpoint inhibitors. These alterations can be detected using routine IHC and should be performed on all pediatric GBM.

RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells.

BACKGROUND: With current treatment strategies, nearly half of all medulloblastoma (MB) patients die from progressive tumors. Accordingly, the identification of novel therapeutic strategies remains a major goal. Deregulation of c-MYC is evident in numerous human cancers. In MB, over-expression of c-MYC has been shown to cause anaplasia and correlate with unfavorable prognosis. METHODS: To study the role of c-MYC in MB biology, we down-regulated c-MYC expression by using small interfering RNA (siRNA) and investigated changes in cellular proliferation, cell cycle analysis, apoptosis, telomere maintenance, and response to ionizing radiation (IR) and chemotherapeutics in a representative panel of human MB cell lines expressing different levels of c-MYC (DAOY wild-type, DAOY transfected with the empty vector, DAOY transfected with c-MYC, D341, and D425). RESULTS: siRNA-mediated c-MYC down-regulation resulted in an inhibition of cellular proliferation and clonogenic growth, inhibition of G1-S phase cell cycle progression, and a decrease in human telomerase reverse transcriptase (hTERT) expression and telomerase activity. On the other hand, down-regulation of c-MYC reduced apoptosis and decreased the sensitivity of human MB cells to IR, cisplatin, and etoposide. This effect was more pronounced in DAOY cells expressing high levels of c-MYC when compared with DAOY wild-type or DAOY cells transfected with the empty vector. CONCLUSION: In human MB cells, in addition to its roles in growth and proliferation, c-MYC is also a potent inducer of apoptosis. Therefore, targeting c-MYC might be of therapeutic benefit when used sequentially with chemo- and radiotherapy rather than concomitantly.

Abnormal expression of REST/NRSF and Myc in neural stem/progenitor cells causes cerebellar tumors by blocking neuronal differentiation.

Medulloblastoma, one of the most malignant brain tumors in children, is thought to arise from undifferentiated neural stem/progenitor cells (NSCs) present in the external granule layer of the cerebellum. However, the mechanism of tumorigenesis remains unknown for the majority of medulloblastomas. In this study, we found that many human medulloblastomas express significantly elevated levels of both myc oncogenes, regulators of neural progenitor proliferation, and REST/NRSF, a transcriptional repressor of neuronal differentiation genes. Previous studies have shown that neither c-Myc nor REST/NRSF alone could cause tumor formation. To determine whether c-Myc and REST/NRSF act together to cause medulloblastomas, we used a previously established cell line derived from external granule layer stem cells transduced with activated c-myc (NSC-M). These immortalized NSCs were able to differentiate into neurons in vitro. In contrast, when the cells were engineered to express a doxycycline-regulated REST/NRSF transgene (NSC-M-R), they no longer underwent terminal neuronal differentiation in vitro. When injected into intracranial locations in mice, the NSC-M cells did not form tumors either in the cerebellum or in the cerebral cortex. In contrast, the NSC-M-R cells did produce tumors in the cerebellum, the site of human medulloblastoma formation, but not when injected into the cerebral cortex. Furthermore, the NSC-M-R tumors were blocked from terminal neuronal differentiation. In addition, countering REST/NRSF function blocked the tumorigenic potential of NSC-M-R cells. To our knowledge, this is the first study in which abnormal expression of a sequence-specific DNA-binding transcriptional repressor has been shown to contribute directly to brain tumor formation. Our findings indicate that abnormal expression of REST/NRSF and Myc in NSCs causes cerebellum-specific tumors by blocking neuronal differentiation and thus maintaining the "stemness" of these cells. Furthermore, these results suggest that such a mechanism plays a role in the formation of human medulloblastoma.

Donor skin allograft survival after bone marrow transplantation: Case report and systematic review of the literature☆.

BACKGROUND: We present a case of skin allograft survival in a patient who previously received a bone marrow transplant from the same HLA-matched donor. DNA fingerprinting of skin biopsies showed mixed cellularity originating from the donor and recipient (68% and 32% donor DNA in the allograft skin and the native recipient's skin, respectively). Histologic sections demonstrated both grade 3/4 rejection and graft-versus-host-disease. We have conducted a systematic review in search for other cases of donor skin allograft survival after a bone marrow or hematopoietic stem cell transplantation. METHODS: All reported cases in English, Spanish, French, and German were captured using the electronic databases. Bibliographies of relevant articles were manually searched. RESULTS: Nineteen patients (12 females) who received skin allografts from their bone marrow or hematopoietic stem cell donors were identified. Average age was 27.2 years (range: 5 months to 64 years). Skin allografts were used to treat graft-versus-host-disease, Herlitz junctional epidermolysis bullosa, and to test tolerance before a kidney transplantation from the same donor. Eight cases were not receiving immunosuppressive therapy. Allografts survived in all patients. In three patients, skin punch biopsies were taken, and these biopsies demonstrated mixed donor and recipient cellularity. The pathology result is specified in two more cases, with no signs of rejection. CONCLUSIONS: The same donor skin allografts may be a safe option to treat severe cutaneous conditions in recipients of a bone marrow/hematopoietic stem cell transplantation. However, future studies are needed to confirm these results.

c-myc overexpression causes anaplasia in medulloblastoma.

Both anaplasia and increased c-myc gene expression have been shown to be negative prognostic indicators for survival in medulloblastoma patients. myc gene amplification has been identified in many large cell/anaplastic medulloblastoma, but no causative link between c-myc and anaplastic changes has been established. To address this, we stably overexpressed c-myc in two medulloblastoma cell lines, DAOY and UW228, and examined the changes in growth characteristics. When analyzed in vitro, cell lines with increased levels of c-myc had higher rates of growth and apoptosis as well as significantly improved ability to form colonies in soft agar compared with control. When injected s.c. into nu/nu mice, flank xenograft tumors with high levels of c-myc in DAOY cell line background were 75% larger than those derived from control. Overexpression of c-myc was required for tumor formation by UW228 cells. Most remarkably, the histopathology of the Myc tumors was severely anaplastic, with large areas of necrosis/apoptosis, increased nuclear size, and macronucleoli. Indices of proliferation and apoptosis were also significantly higher in Myc xenografts. Thus, c-myc seems to play a causal role in inducing anaplasia in medulloblastoma. Because anaplastic changes are often observed in recurrent medulloblastoma, we propose that c-myc dysregulation is involved in the progression of these malignant embryonal neoplasms.

Notch pathway inhibition depletes stem-like cells and blocks engraftment in embryonal brain tumors.

The Notch signaling pathway is required in both nonneoplastic neural stem cells and embryonal brain tumors, such as medulloblastoma, which are derived from such cells. We investigated the effects of Notch pathway inhibition on medulloblastoma growth using pharmacologic inhibitors of gamma-secretase. Notch blockade suppressed expression of the pathway target Hes1 and caused cell cycle exit, apoptosis, and differentiation in medulloblastoma cell lines. Interestingly, viable populations of better-differentiated cells continued to grow when Notch activation was inhibited but were unable to efficiently form soft-agar colonies or tumor xenografts, suggesting that a cell fraction required for tumor propagation had been depleted. It has recently been hypothesized that a small population of stem-like cells within brain tumors is required for the long-term propagation of neoplastic growth and that CD133 expression and Hoechst dye exclusion (side population) can be used to prospectively identify such tumor-forming cells. We found that Notch blockade reduced the CD133-positive cell fraction almost 5-fold and totally abolished the side population, suggesting that the loss of tumor-forming capacity could be due to the depletion of stem-like cells. Notch signaling levels were higher in the stem-like cell fraction, providing a potential mechanism for their increased sensitivity to inhibition of this pathway. We also observed that apoptotic rates following Notch blockade were almost 10-fold higher in primitive nestin-positive cells as compared with nestin-negative ones. Stem-like cells in brain tumors thus seem to be selectively vulnerable to agents inhibiting the Notch pathway.

Anti-proliferative activity of the quassinoid NBT-272 in childhood medulloblastoma cells.

BACKGROUND: With current treatment strategies, nearly half of all medulloblastoma (MB) patients die from progressive tumors. Accordingly, the identification of novel therapeutic strategies remains a major goal. Deregulation of c-MYC is evident in numerous human cancers. In MB, over-expression of c-MYC has been shown to correlate with anaplasia and unfavorable prognosis. In neuroblastoma--an embryonal tumor with biological similarities to MB--the quassinoid NBT-272 has been demonstrated to inhibit cellular proliferation and to down-regulate c-MYC protein expression. METHODS: To study MB cell responses to NBT-272 and their dependence on the level of c-MYC expression, DAOY (wild-type, empty vector transfected or c-MYC transfected), D341 (c-MYC amplification) and D425 (c-MYC amplification) human MB cells were used. The cells were treated with different concentrations of NBT-272 and the impact on cell proliferation, apoptosis and c-MYC expression was analyzed. RESULTS: NBT-272 treatment resulted in a dose-dependent inhibition of cellular proliferation (IC50 in the range of 1.7-9.6 ng/ml) and in a dose-dependent increase in apoptotic cell death in all human MB cell lines tested. Treatment with NBT-272 resulted in up to 90% down-regulation of c-MYC protein, as demonstrated by Western blot analysis, and in a significant inhibition of c-MYC binding activity. Anti-proliferative effects were slightly more prominent in D341 and D425 human MB cells with c-MYC amplification and slightly more pronounced in c-MYC over-expressing DAOY cells compared to DAOY wild-type cells. Moreover, treatment of synchronized cells by NBT-272 induced a marked cell arrest at the G1/S boundary. CONCLUSION: In human MB cells, NBT-272 treatment inhibits cellular proliferation at nanomolar concentrations, blocks cell cycle progression, induces apoptosis, and down-regulates the expression of the oncogene c-MYC. Thus, NBT-272 may represent a novel drug candidate to inhibit proliferation of human MB cells in vivo.

Hepatoblastoma in an 11-year-old: Case report and a review of the literature.

RATIONALE: Hepatoblastoma is a rare malignancy. Approximately 100 cases are diagnosed yearly in the United States. The highest incidence occurs in infants and in children younger than 5 years. Cases involving patients older than 5 years are very rare. We describe the case of a patient who was diagnosed with hepatoblastoma at an atypical age of presentation for this type of malignancy. We also performed Ovid MEDLINE search for hepatoblastoma and epidemiology reports occurring in children between the ages of 5 and 18 years. In this article we review the epidemiology and summarize case reports published between 1997 and 2012 of patients with hepatoblastoma, who were older than 5 years. PATIENT CONCERNS AND DIAGNOSIS: Our patient is an 11 year old boy with stage IV hepatoblastoma with lung and omental metastases at diagnosis. INTERVENTIONS: The patient received 7 cycles of chemotherapy following the treatment plan of COG protocol AHEP 0731, off study. He also had tumor resection and omentectomy and achieved complete remission. OUTCOMES: He later had disease recurrence and after undergoing treatment with different modalities, ultimately died of his disease. Review of SEER program data shows that the incidence of hepatoblastoma in children above the age of 5 years is too infrequent to be calculated. Literature review revealed 13 cases of patients diagnosed at age older than 5 years. Most cases were published due to unusual associations and/or complications. There are no obvious unifying characteristics for these cases, although there may be a slight male preponderance and many patients in this selected series presented with elevated Alpha-fetoprotein. LESSONS: The reported case is rare, given the very low incidence of hepatoblastoma outside of infancy. A systematic review of characteristics and outcomes for patients older than 5 years who are enrolled in cooperative group hepatoblastoma trials may reveal important information about the epidemiology and tumor biology in this rare patient population.

Mature salivary gland rests within sonic hedgehog-positive medulloblastoma: case report and insights into the molecular genetics and embryopathology of ectopic intracranial salivary gland analogs.

Intracranial ectopic salivary gland rests within dural-based lesions are reported very infrequently in the literature. The authors report the unique case of a 12-year-old boy with a cerebellar medulloblastoma positive for sonic hedgehog (Shh) that contained intraaxial mature ectopic salivary gland rests. The patient underwent clinical and radiological monitoring postoperatively, until he died of disseminated disease. An autopsy showed no evidence of salivary glands within disseminated lesions. The intraaxial presence of salivary gland rests and concomitant Shh positivity of the described tumor point to a disorder in differentiation as opposed to ectopic developmental foci, which are uniformly dural based in the described literature. The authors demonstrate the characteristic "papilionaceous" appearance of the salivary glands with mucicarmine stain and highlight the role of Shh signaling in explaining the intraaxial presence of seromucous gland analogs. This article reports the first intraaxial posterior fossa tumor with heterotopic salivary gland rests, and it provides molecular and embryopathological insights into the development of these lesions.

Cdk5 disruption attenuates tumor PD-L1 expression and promotes antitumor immunity.

Cancers often evade immune surveillance by adopting peripheral tissue- tolerance mechanisms, such as the expression of programmed cell death ligand 1 (PD-L1), the inhibition of which results in potent antitumor immunity. Here, we show that cyclin-dependent kinase 5 (Cdk5), a serine-threonine kinase that is highly active in postmitotic neurons and in many cancers, allows medulloblastoma (MB) to evade immune elimination. Interferon-γ (IFN-γ)-induced PD-L1 up-regulation on MB requires Cdk5, and disruption of Cdk5 expression in a mouse model of MB results in potent CD4(+) T cell-mediated tumor rejection. Loss of Cdk5 results in persistent expression of the PD-L1 transcriptional repressors, the interferon regulatory factors IRF2 and IRF2BP2, which likely leads to reduced PD-L1 expression on tumors. Our finding highlights a central role for Cdk5 in immune checkpoint regulation by tumor cells.