Implementation of DNA Methylation Array Profiling in Pediatric Central Nervous System Tumors: The AIM BRAIN Project: An Australian and New Zealand Children's Haematology/Oncology Group Study.

DNA methylation array profiling for classifying pediatric central nervous system (CNS) tumors is a valuable adjunct to histopathology. However, unbiased prospective and interlaboratory validation studies have been lacking. The AIM BRAIN diagnostic trial involving 11 pediatric cancer centers in Australia and New Zealand was designed to test the feasibility of routine clinical testing and ran in parallel with the Molecular Neuropathology 2.0 (MNP2.0) study at Deutsches Krebsforschungszentrum (German Cancer Research Center). CNS tumors from 269 pediatric patients were prospectively tested on Illumina EPIC arrays, including 104 cases co-enrolled on MNP2.0. Using MNP classifier versions 11b4 and 12.5, we report classifications with a probability score ≥0.90 in 176 of 265 (66.4%) and 213 of 269 (79.2%) cases, respectively. Significant diagnostic information was obtained in 130 of 176 (74%) for 11b4, and 12 of 174 (7%) classifications were discordant with histopathology. Cases prospectively co-enrolled on MNP2.0 gave concordant classifications (99%) and score thresholds (93%), demonstrating excellent test reproducibility and sensitivity. Overall, DNA methylation profiling is a robust single workflow technique with an acceptable diagnostic yield that is considerably enhanced by the extensive subgroup and copy number profile information generated by the platform. The platform has excellent test reproducibility and sensitivity and contributes significantly to CNS tumor diagnosis.

Impact of supraphysiologic MDM2 expression on chromatin networks and therapeutic responses in sarcoma.

Amplification of MDM2 on supernumerary chromosomes is a common mechanism of P53 inactivation across tumors. Here, we investigated the impact of MDM2 overexpression on chromatin, gene expression, and cellular phenotypes in liposarcoma. Three independent regulatory circuits predominate in aggressive, dedifferentiated tumors. RUNX and AP-1 family transcription factors bind mesenchymal gene enhancers. P53 and MDM2 co-occupy enhancers and promoters associated with P53 signaling. When highly expressed, MDM2 also binds thousands of P53-independent growth and stress response genes, whose promoters engage in multi-way topological interactions. Overexpressed MDM2 concentrates within nuclear foci that co-localize with PML and YY1 and could also contribute to P53-independent phenotypes associated with supraphysiologic MDM2. Importantly, we observe striking cell-to-cell variability in MDM2 copy number and expression in tumors and models. Whereas liposarcoma cells are generally sensitive to MDM2 inhibitors and their combination with pro-apoptotic drugs, MDM2-high cells tolerate them and may underlie the poor clinical efficacy of these agents.

Chromatin complex dependencies reveal targeting opportunities in leukemia.

Chromatin regulators are frequently mutated in human cancer and are attractive drug targets. They include diverse proteins that share functional domains and assemble into related multi-subunit complexes. To investigate functional relationships among these regulators, here we apply combinatorial CRISPR knockouts (KOs) to test over 35,000 gene-gene pairings in leukemia cells, using a library of over 300,000 constructs. Top pairs that demonstrate either compensatory non-lethal interactions or synergistic lethality enrich for paralogs and targets that occupy the same protein complex. The screen highlights protein complex dependencies not apparent in single KO screens, for example MCM histone exchange, the nucleosome remodeling and deacetylase (NuRD) complex, and HBO1 (KAT7) complex. We explore two approaches to NuRD complex inactivation. Paralog and non-paralog combinations of the KAT7 complex emerge as synergistic lethal and specifically nominate the ING5 PHD domain as a potential therapeutic target when paired with other KAT7 complex member losses. These findings highlight the power of combinatorial screening to provide mechanistic insight and identify therapeutic targets within redundant networks.

Carnitine Palmitoyl Transferase Deficiency in a University Immunology Practice.

PURPOSE: This report describes the clinical manifestations of 35 patients sent to a University Immunology clinic with a diagnosis of fatigue and exercise intolerance who were identified to have low carnitine palmitoyl transferase activity on muscle biopsies. RECENT FINDINGS: All of the patients presented with fatigue and exercise intolerance and many had been diagnosed with fibromyalgia. Their symptoms responded to treatment of the metabolic disease. Associated symptoms included bloating, diarrhea, constipation, gastrointestinal reflux symptoms, recurrent infections, arthritis, dyspnea, dry eye, visual loss, and hearing loss. Associated medical conditions included Hashimoto thyroiditis, Sjogren's syndrome, seronegative arthritis, food hypersensitivities, asthma, sleep apnea, and vasculitis. This study identifies clinical features that should alert physicians to the possibility of an underlying metabolic disease. Treatment of the metabolic disease leads to symptomatic improvement.

Outcomes of Low-Intensity Treatment of Acute Lymphoblastic Leukemia at Butaro Cancer Center of Excellence in Rwanda.

PURPOSE: Children with acute lymphoblastic leukemia (ALL) in low-income countries have disproportionately lower cure rates than those in high-income countries. At Butaro Cancer Center of Excellence (BCCOE), physicians treated patients with ALL with the first arm of the Hunger Protocol, a graduated-intensity method tailored for resource-limited settings. This article provides the first published outcomes, to our knowledge, of patients with ALL treated with this protocol. METHODS: This is a retrospective descriptive study of patients with ALL enrolled at BCCOE from July 1, 2012 to June 30, 2014; data were collected through December 31, 2015. Descriptive statistics were used to calculate patient demographics, disease characteristics, and outcomes; event-free survival was assessed at 2 years using the Kaplan-Meier method. RESULTS: Forty-two consecutive patients with ALL were included. At the end of the study period, 19% (eight) were alive without evidence of relapse: three completed treatment and five were continuing treatment. Among the remaining patients, 71% (30) had died and 10% (four) were lost to follow-up. A total of 83% (25) of the deaths were disease related, 3% (one) treatment-related, and 13% (four) unclear. Event-free survival was 22% (95% CI, 11% to 36%), considering lost to follow-up as an event, and 26% (95% CI, 13% to 41%) if lost to follow-up is censored. CONCLUSION: As expected, relapse was the major cause of failure with this low-intensity regimen. However, toxicity was acceptably low, and BCCOE has decided to advance to intensity level 2. These results reflect the necessity of a data-driven approach and a continual improvement process to care for complex patients in resource-constrained settings.

Pursuing equity in cancer care: implementation, challenges and preliminary findings of a public cancer referral center in rural Rwanda.

BACKGROUND: Cancer services are inaccessible in many low-income countries, and few published examples describe oncology programs within the public sector. In 2011, the Rwanda Ministry of Health (RMOH) established Butaro Cancer Center of Excellence (BCCOE) to expand cancer services nationally. In hopes of informing cancer care delivery in similar settings, we describe program-level experience implementing BCCOE, patient characteristics, and challenges encountered. METHODS: Butaro Cancer Center of Excellence was founded on diverse partnerships that emphasize capacity building. Services available include pathology-based diagnosis, basic imaging, chemotherapy, surgery, referral for radiotherapy, palliative care and socioeconomic access supports. Retrospective review of electronic medical records (EMR) of patients enrolled between July 1, 2012 and June 30, 2014 was conducted, supplemented by manual review of paper charts and programmatic records. RESULTS: In the program's first 2 years, 2326 patients presented for cancer-related care. Of these, 70.5% were female, 4.3% children, and 74.3% on public health insurance. In the first year, 66.3% (n = 1144) were diagnosed with cancer. Leading adult diagnoses were breast, cervical, and skin cancer. Among children, nephroblastoma, acute lymphoblastic leukemia, and Hodgkin lymphoma were predominant. As of June 30, 2013, 95 cancer patients had died. Challenges encountered include documentation gaps and staff shortages. CONCLUSION: Butaro Cancer Center of Excellence demonstrates that complex cancer care can be delivered in the most resource-constrained settings, accessible to vulnerable patients. Key attributes that have made BCCOE possible are: meaningful North-south partnerships, innovative task- and infrastructure-shifting, RMOH leadership, and an equity-driven agenda. Going forward, we will apply our experiences and lessons learned to further strengthen BCCOE, and employ the developed EMR system as a valuable platform to assess long-term clinical outcomes and improve care.

Diagnosis of cancer in rural Rwanda: early outcomes of a phased approach to implement anatomic pathology services in resource-limited settings.

OBJECTIVES: Adequate pathology services are a prerequisite to accurate cancer diagnoses and tailoring appropriate treatment. Limitations in skilled personnel and infrastructure are among the challenges faced by developing countries. We describe a stepwise implementation of anatomic pathology laboratory services at Butaro District Hospital, designated as a Cancer Center of Excellence in rural Rwanda. METHODS: The phased approach to developing pathology services up to December 2012 is described. A retrospective review of specimens submitted to Butaro District Hospital between July 1, 2012, and December 31, 2012, was conducted. Patient clinical characteristics and sociodemographics are also described. RESULTS: During the study period, a total of 437 tissue specimens were submitted. Among these, 143 (32.7%) were from male patients, 244 (55.8%) were confirmed as malignant, 163 (37.3%) were benign, 28 (6.4%) were inconclusive, and two (0.5%) results were not available at the time of analysis. The median time from specimen receipt at Butaro to final reporting was 32 days (range, 7-193 days; interquartile range, 23-44 days). CONCLUSIONS: Our experience demonstrates that anatomic pathology services can be established in resource-limited settings and local capacity can be built to support accurate diagnoses. Our approach included leveraging partnerships, volunteer experts, and task shifting and will be expanded to include telepathology.

Human glycinamide ribonucleotide transformylase: active site mutants as mechanistic probes.

Human glycinamide ribonucleotide transformylase (GART) (EC 2.1.2.2) is a validated target for cancer chemotherapy, but mechanistic studies of this therapeutically important enzyme are limited. Site-directed mutagenesis, initial velocity studies, pH-rate studies, and substrate binding studies have been employed to probe the role of the strictly conserved active site residues, N106, H108, and D144, and the semiconserved K170 in substrate binding and catalysis. Only two conservative substitutions, N106Q and K170R, resulted in catalytically active enzymes, and these active mutant enzymes gave pH-rate profiles and a steady-state kinetic mechanism essentially identical to those of the native enzyme. All inactive mutants were able to bind both substrates, ruling out disrupted formation of the ternary complex as the source of inactivity. Differences between human and Escherichia coli GART, previously used as a model for the human enzyme, were evident.