Targeting SAMHD1 with hydroxyurea in first-line cytarabine-based therapy of newly diagnosed acute myeloid leukaemia: Results from the HEAT-AML trial.

BACKGROUND: Treatment of newly diagnosed acute myeloid leukaemia (AML) is based on combination chemotherapy with cytarabine (ara-C) and anthracyclines. Five-year overall survival is below 30%, which has partly been attributed to cytarabine resistance. Preclinical data suggest that the addition of hydroxyurea potentiates cytarabine efficacy by increasing ara-C triphosphate (ara-CTP) levels through targeted inhibition of SAMHD1. OBJECTIVES: In this phase 1 trial, we evaluated the feasibility, safety and efficacy of the addition of hydroxyurea to standard chemotherapy with cytarabine/daunorubicin in newly diagnosed AML patients. METHODS: Nine patients were enrolled and received at least two courses of ara-C (1 g/m2 /2 h b.i.d. d1-5, i.e., a total of 10 g/m2 per course), hydroxyurea (1-2 g d1-5) and daunorubicin (60 mg/m2 d1-3). The primary endpoint was safety; secondary endpoints were complete remission rate and measurable residual disease (MRD). Additionally, pharmacokinetic studies of ara-CTP and ex vivo drug sensitivity assays were performed. RESULTS: The most common grade 3-4 toxicity was febrile neutropenia (100%). No unexpected toxicities were observed. Pharmacokinetic analyses showed a significant increase in median ara-CTP levels (1.5-fold; p = 0.04) in patients receiving doses of 1 g hydroxyurea. Ex vivo, diagnostic leukaemic bone marrow blasts from study patients were significantly sensitised to ara-C by a median factor of 2.1 (p = 0.0047). All nine patients (100%) achieved complete remission, and all eight (100%) with validated MRD measurements (flow cytometry or real-time quantitative polymerase chain reaction [RT-qPCR]) had an MRD level <0.1% after two cycles of chemotherapy. Treatment was well-tolerated, and median time to neutrophil recovery >1.0 × 109 /L and to platelet recovery >50 × 109 /L after the start of cycle 1 was 19 days and 22 days, respectively. Six of nine patients underwent allogeneic haematopoietic stem-cell transplantation (allo-HSCT). With a median follow-up of 18.0 (range 14.9-20.5) months, one patient with adverse risk not fit for HSCT experienced a relapse after 11.9 months but is now in second complete remission. CONCLUSION: Targeted inhibition of SAMHD1 by the addition of hydroxyurea to conventional AML therapy is safe and appears efficacious within the limitations of the small phase 1 patient cohort. These results need to be corroborated in a larger study.

Development of the SIOPE DIPG network, registry and imaging repository: a collaborative effort to optimize research into a rare and lethal disease.

Diffuse intrinsic pontine glioma (DIPG) is a rare and deadly childhood malignancy. After 40 years of mostly single-center, often non-randomized trials with variable patient inclusions, there has been no improvement in survival. It is therefore time for international collaboration in DIPG research, to provide new hope for children, parents and medical professionals fighting DIPG. In a first step towards collaboration, in 2011, a network of biologists and clinicians working in the field of DIPG was established within the European Society for Paediatric Oncology (SIOPE) Brain Tumour Group: the SIOPE DIPG Network. By bringing together biomedical professionals and parents as patient representatives, several collaborative DIPG-related projects have been realized. With help from experts in the fields of information technology, and legal advisors, an international, web-based comprehensive database was developed, The SIOPE DIPG Registry and Imaging Repository, to centrally collect data of DIPG patients. As for April 2016, clinical data as well as MR-scans of 694 patients have been entered into the SIOPE DIPG Registry/Imaging Repository. The median progression free survival is 6.0 months (95% Confidence Interval (CI) 5.6-6.4 months) and the median overall survival is 11.0 months (95% CI 10.5-11.5 months). At two and five years post-diagnosis, 10 and 2% of patients are alive, respectively. The establishment of the SIOPE DIPG Network and SIOPE DIPG Registry means a paradigm shift towards collaborative research into DIPG. This is seen as an essential first step towards understanding the disease, improving care and (ultimately) cure for children with DIPG.

Drivers Underlying Metastasis and Relapse in Medulloblastoma and Targeting Strategies.

Medulloblastomas comprise a molecularly diverse set of malignant pediatric brain tumors in which patients are stratified according to different prognostic risk groups that span from very good to very poor. Metastasis at diagnosis is most often a marker of poor prognosis and the relapse incidence is higher in these children. Medulloblastoma relapse is almost always fatal and recurring cells have, apart from resistance to standard of care, acquired genetic and epigenetic changes that correlate with an increased dormancy state, cell state reprogramming and immune escape. Here, we review means to carefully study metastasis and relapse in preclinical models, in light of recently described molecular subgroups. We will exemplify how therapy resistance develops at the cellular level, in a specific niche or from therapy-induced secondary mutations. We further describe underlying molecular mechanisms on how tumors acquire the ability to promote leptomeningeal dissemination and discuss how they can establish therapy-resistant cell clones. Finally, we describe some of the ongoing clinical trials of high-risk medulloblastoma and suggest or discuss more individualized treatments that could be of benefit to specific subgroups.

The potential of liquid biopsy for detection of the KIAA1549-BRAF fusion in circulating tumor DNA from children with pilocytic astrocytoma.

Background: Low-grade gliomas (LGGs) represent children's most prevalent central nervous system tumor, necessitating molecular profiling to diagnose and determine the most suitable treatment. Developing highly sensitive screening techniques for liquid biopsy samples is particularly beneficial, as it enables the early detection and molecular characterization of tumors with minimally invasive samples. Methods: We examined CSF and plasma samples from patients with pilocytic astrocytoma (PA) using custom multiplexed droplet digital polymerase chain reaction (ddPCR) assays based on whole genome sequencing data. These assays included a screening test to analyze BRAF duplication and a targeted assay for the detection of patient-specific KIAA1549::BRAF fusion junction sequences or single nucleotide variants. Results: Our findings revealed that 5 out of 13 individual cerebrospinal fluid (CSF) samples tested positive for circulating tumor DNA (ctDNA). Among these cases, 3 exhibited the KIAA1549::BRAF fusion, which was detected through copy number variation (CNV) analysis (n = 1) or a fusion-specific probe (n = 2), while 1 case each displayed the BRAF V600E mutation and the FGFR1 N577K mutation. Additionally, a quantitative analysis of cell-free DNA (cfDNA) concentrations in PA CSF samples showed that most cases had low cfDNA levels, below the limit of detection of our assay (<1.9 ng). Conclusions: While CNV analysis of CSF samples from LGGs still has some limitations, it has the potential to serve as a valuable complementary tool. Furthermore, it can also be multiplexed with other aberrations, for example, to the BRAF V600 test, to provide important insights into the molecular characteristics of LGGs.

Diagnostic yield and clinical impact of germline sequencing in children with CNS and extracranial solid tumors-a nationwide, prospective Swedish study.

Background: Childhood cancer predisposition (ChiCaP) syndromes are increasingly recognized as contributing factors to childhood cancer development. Yet, due to variable availability of germline testing, many children with ChiCaP might go undetected today. We report results from the nationwide and prospective ChiCaP study that investigated diagnostic yield and clinical impact of integrating germline whole-genome sequencing (gWGS) with tumor sequencing and systematic phenotyping in children with solid tumors. Methods: gWGS was performed in 309 children at diagnosis of CNS (n = 123, 40%) or extracranial (n = 186, 60%) solid tumors and analyzed for disease-causing variants in 189 known cancer predisposing genes. Tumor sequencing data were available for 74% (227/309) of patients. In addition, a standardized clinical assessment for underlying predisposition was performed in 95% (293/309) of patients. Findings: The prevalence of ChiCaP diagnoses was 11% (35/309), of which 69% (24/35) were unknown at inclusion (diagnostic yield 8%, 24/298). A second-hit and/or relevant mutational signature was observed in 19/21 (90%) tumors with informative data. ChiCaP diagnoses were more prevalent among patients with retinoblastomas (50%, 6/12) and high-grade astrocytomas (37%, 6/16), and in those with non-cancer related features (23%, 20/88), and ≥2 positive ChiCaP criteria (28%, 22/79). ChiCaP diagnoses were autosomal dominant in 80% (28/35) of patients, yet confirmed de novo in 64% (18/28). The 35 ChiCaP findings resulted in tailored surveillance (86%, 30/35) and treatment recommendations (31%, 11/35). Interpretation: Overall, our results demonstrate that systematic phenotyping, combined with genomics-based diagnostics of ChiCaP in children with solid tumors is feasible in large-scale clinical practice and critically guides personalized care in a sizable proportion of patients. Funding: The study was supported by the Swedish Childhood Cancer Fund and the Ministry of Health and Social Affairs.

KI, WU, and Merkel cell polyomavirus DNA was not detected in guthrie cards of children who later developed acute lymphoblastic leukemia.

BACKGROUND: Neonatal dried blood spots (Guthrie cards) have been used to demonstrate a prenatal origin of clonal leukemia-specific genetic aberrations in several subgroups of childhood acute lymphoblastic leukemia (ALL). One hypothesis suggests that an infectious agent could initiate genetic transformation already in utero. In search for a possible viral agent, Guthrie cards were analyzed for the presence of 3 newly discovered polyomavirus Karolinska Institutet polymavirus (KIPyV), Washington University polyomavirus (WUPyV), and Merkel cell polyomavirus (MCPyV). METHODS: Guthrie cards from 50 children who later developed ALL and 100 matched controls were collected and analyzed by standard or real-time polymerase chain reaction for the presence of the VP1 region of KIPyV, WUPyV, and MCPyV, and the LT region for MCPyV. RESULTS AND CONCLUSIONS: DNA from KIPyV, WUPyV, and MCPyV was not detected in neonatal blood samples from children with ALL or controls. Prenatal infections with these viruses are not likely to be etiological drivers for childhood leukemogenesis.

Dormant SOX9-Positive Cells Facilitate MYC-Driven Recurrence of Medulloblastoma.

Relapse is the leading cause of death in patients with medulloblastoma, the most common malignant pediatric brain tumor. A better understanding of the mechanisms underlying recurrence could lead to more effective therapies for targeting tumor relapses. Here, we observed that SOX9, a transcription factor and stem cell/glial fate marker, is limited to rare, quiescent cells in high-risk medulloblastoma with MYC amplification. In paired primary-recurrent patient samples, SOX9-positive cells accumulated in medulloblastoma relapses. SOX9 expression anti-correlated with MYC expression in murine and human medulloblastoma cells. However, SOX9-positive cells were plastic and could give rise to a MYC high state. To follow relapse at the single-cell level, an inducible dual Tet model of medulloblastoma was developed, in which MYC expression was redirected in vivo from treatment-sensitive bulk cells to dormant SOX9-positive cells using doxycycline treatment. SOX9 was essential for relapse initiation and depended on suppression of MYC activity to promote therapy resistance, epithelial-mesenchymal transition, and immune escape. p53 and DNA repair pathways were downregulated in recurrent tumors, whereas MGMT was upregulated. Recurrent tumor cells were found to be sensitive to treatment with an MGMT inhibitor and doxorubicin. These findings suggest that recurrence-specific targeting coupled with DNA repair inhibition comprises a potential therapeutic strategy in patients affected by medulloblastoma relapse. SIGNIFICANCE: SOX9 facilitates therapy escape and recurrence in medulloblastoma via temporal inhibition of MYC/MYCN genes, revealing a strategy to specifically target SOX9-positive cells to prevent tumor relapse.

Incidence of human papillomavirus (HPV) positive tonsillar carcinoma in Stockholm, Sweden: an epidemic of viral-induced carcinoma?

In the county of Stockholm, between 1970 and 2002, we have previously reported a 3-fold parallel increase in the incidence of tonsillar squamous cell carcinoma (SCC) and the proportion of human papillomavirus (HPV) positive tonsillar SCC. Here, we have followed the above parameters in all patients (n = 120) diagnosed with tonsillar SCC during 2003-2007 in the same area, and also in correlation to our previous data. Ninety-eight pretreatment biopsies were available and presence of HPV DNA and HPV-16 E6 and E7 RNA were tested by polymerase chain reaction (PCR) and RT-PCR. Incidence data were obtained from the Swedish Cancer Registry. Data reported from 1970 to 2002 were also obtained for comparison. HPV DNA was present in 83 of 98 (85%) of the tonsillar SCC biopsies from 2003 to 2007 and 77 of these were HPV-16 positive. HPV-16 E6 and E7 RNA were found in 98% of 52 analyzed HPV-16 positive cases. The proportion of HPV-positive cancers had significantly increased both from 1970 to 2007 (p < 0.0001) as well from 2000 to 2007 (p < 0.01), with 68% (95% confidence interval (CI), 53-81) 2000-2002; 77% (95% CI, 63-87) 2003-2005; and 93% (95% CI, 82-99) 2006-2007. The incidence rate of HPV-positive tumors almost doubled each decade between 1970 and 2007, in parallel with a decline of HPV-negative tumors. In conclusion, the incidence of HPV-positive cancers is still increasing in the County of Stockholm, suggesting an epidemic of a virus-induced carcinoma, with soon practically all tonsillar SCC being HPV positive, as in cervical cancer.

DNA from BK virus and JC virus and from KI, WU, and MC polyomaviruses as well as from simian virus 40 is not detected in non-UV-light-associated primary malignant melanomas of mucous membranes.

The single most important causative factor for malignant melanomas of the skin is UV radiation. However, this is not true for melanomas on body surfaces sheltered from the sun; thus, it is important to seek new causative factors of melanoma genesis. Human papillomaviruses and gammaherpesviruses are associated with human skin cancer; for example, human papillomavirus types 5 and 8 are associated with epidermodysplasia verruciformis, and human herpesvirus 8 is associated with Kaposi's sarcoma. Recently, a newly described human polyomavirus, Merkel cell polyomavirus (MCPyV), has been associated with Merkel cell carcinoma, an unusual form of neurotropic skin cancer. Moreover, melanocytes are of neuroepithelial origin. This background impelled us to investigate if human polyomavirus DNA could play a role in the development of extracutaneous melanomas. Sixty-four extracutaneous melanomas were initially collected and dissected. Of these, 38 could be successfully used for further testing for the presence of the five human polyomaviruses known so far-BK virus (BKV), JC virus (JCV), KI polyomavirus (KIPyV), WU polyomavirus (WUPyV), and MCPyV-and of simian virus 40 (SV40). No polyomavirus DNA could be detected in any of the samples tested by use of a nested PCR detecting BKV, JCV, and SV40; a newly designed PCR detecting KIPyV and WUPyV; or a newly designed PCR for MCPyV. We conclude that since no human polyomavirus DNA was detected in primary malignant melanomas on non-sun-exposed body surfaces, these polyomaviruses presumably are not major factors for the development of extracutaneous melanomas.

Clinical, Radiologic, Pathologic, and Molecular Characteristics of Long-Term Survivors of Diffuse Intrinsic Pontine Glioma (DIPG): A Collaborative Report From the International and European Society for Pediatric Oncology DIPG Registries.

Purpose Diffuse intrinsic pontine glioma (DIPG) is a brainstem malignancy with a median survival of < 1 year. The International and European Society for Pediatric Oncology DIPG Registries collaborated to compare clinical, radiologic, and histomolecular characteristics between short-term survivors (STSs) and long-term survivors (LTSs). Materials and Methods Data abstracted from registry databases included patients from North America, Australia, Germany, Austria, Switzerland, the Netherlands, Italy, France, the United Kingdom, and Croatia. Results Among 1,130 pediatric and young adults with radiographically confirmed DIPG, 122 (11%) were excluded. Of the 1,008 remaining patients, 101 (10%) were LTSs (survival ≥ 2 years). Median survival time was 11 months (interquartile range, 7.5 to 16 months), and 1-, 2-, 3-, 4-, and 5-year survival rates were 42.3% (95% CI, 38.1% to 44.1%), 9.6% (95% CI, 7.8% to 11.3%), 4.3% (95% CI, 3.2% to 5.8%), 3.2% (95% CI, 2.4% to 4.6%), and 2.2% (95% CI, 1.4% to 3.4%), respectively. LTSs, compared with STSs, more commonly presented at age < 3 or > 10 years (11% v 3% and 33% v 23%, respectively; P < .001) and with longer symptom duration ( P < .001). STSs, compared with LTSs, more commonly presented with cranial nerve palsy (83% v 73%, respectively; P = .008), ring enhancement (38% v 23%, respectively; P = .007), necrosis (42% v 26%, respectively; P = .009), and extrapontine extension (92% v 86%, respectively; P = .04). LTSs more commonly received systemic therapy at diagnosis (88% v 75% for STSs; P = .005). Biopsies and autopsies were performed in 299 patients (30%) and 77 patients (10%), respectively; 181 tumors (48%) were molecularly characterized. LTSs were more likely to harbor a HIST1H3B mutation (odds ratio, 1.28; 95% CI, 1.1 to 1.5; P = .002). Conclusion We report clinical, radiologic, and molecular factors that correlate with survival in children and young adults with DIPG, which are important for risk stratification in future clinical trials.

The incidence of hemorrhagic cystitis and BK-viruria in allogeneic hematopoietic stem cell recipients according to intensity of the conditioning regimen.

The influence of BK-viruria, donor background, and conditioning on the development of hemorrhagic cystitis was examined in 90 allogeneic hematopoetic stem cell transplant patients, of whom 15 developed hemorrhagic cystitis. Thirty-two patients had related and 58 had unrelated donors, while 44 received full, and 46 received reduced intensity conditioning (RIC). BK-viruria was more common in patients with hemorrhagic cystitis than in those without (p<0.01), and hemorrhagic cystitis was less common in patients with related donors than in those with unrelated donors (p=0.02). Finally, hemorrhagic cystitis and BK-viruria were less common in patients receiving RIC, rather than full conditioning (p<0.01 and p<0.01, respectively).

A related donor and reduced intensity conditioning reduces the risk of development of BK virus-positive haemorrhagic cystitis in allogeneic haematopoetic stem cell-transplanted patients.

The significance of the BK virus (BKV) and possible co-factors for the development of late onset haemorrhagic cystitis (HC) in allogeneic haematopoetic stem cell (HSCT)-transplanted patients is reviewed. BKV-associated HC causes significant morbidity and mortality in HSCT patients, however, BK-viruria cannot distinguish patients at risk of HC, since it is observed in patients with and without HC. Several studies have therefore attempted to identify co-factors for the development of HC. Acute graft versus host disease was in the past, though less so recently, reported to correlate to the incidence of HC. However, patients who had received grafts from unrelated donors (URD) and had had full conditioning prior to HSCT were shown to have an increased risk of HC, compared to patients who had received HSCT from a related donor (RD) or patients who had received reduced intensity conditioning. In conclusion, HSCT patients with BK-viruria, an URD and receiving full conditioning have an increased risk of developing HC.

DNA from KI, WU and Merkel cell polyomaviruses is not detected in childhood central nervous system tumours or neuroblastomas.

BACKGROUND: BK and JC polyomaviruses (BKV and JCV) are potentially oncogenic and have in the past inconclusively been associated with tumours of the central nervous system (CNS), while BKV has been hinted, but not confirmed to be associated with neuroblastomas. Recently three new polyomaviruses (KIPyV, WUPyV and MCPyV) were identified in humans. So far KIPyV and WUPyV have not been associated to human diseases, while MCPyV was discovered in Merkel Cell carcinomas and may have neuroepithelial cell tropism. However, all three viruses can be potentially oncogenic and this compelled us to investigate for their presence in childhood CNS and neuroblastomas. METHODOLOGY: The presence of KI, WU and MCPyV DNA was analysed, by a joint WU and KI specific PCR (covering part of VP1) and by a MCPyV specific regular and real time quantitative PCR (covering part of Large T) in 25 CNS tumour biopsies and 31 neuroblastoma biopsies from the Karolinska University Hospital, Sweden. None of the three new human polyomaviruses were found to be associated with any of the tumours, despite the presence of PCR amplifiable DNA assayed by a S14 housekeeping gene PCR. CONCLUSION: In this pilot study, the presence of MCPyV, KI and WU was not observed in childhood CNS tumours and neuroblastomas. Nonetheless, we suggest that additional data are warranted in tumours of the central and peripheral nervous systems and we do not exclude that other still not yet detected polyomaviruses could be present in these tumours.