Charlie Teo Foundation Brain Tumour Bank

The role of the pediatrician is crucial in both the diagnosis and management of pediatric brain tumors, the most common solid tumor of childhood. Awareness of the presenting signs and symptoms of brain tumors can lead to timely diagnosis, and understanding the late effects of brain tumor treatment improves long-term management of childhood brain tumor survivors.After completing this article, readers should be able to: Recognize the presenting symptoms and physical examination findings suggestive of a childhood brain tumor and how these findings depend on tumor location.Review common brain tumor pathologies affecting children.Understand how molecular genetics plays a role in the diagnosis and treatment of childhood brain tumors.Recognize the late affects associated with the treatment of childhood brain tumors.Brain tumors are the most common solid malignancy in children and represent the leading cause of pediatric cancer-related deaths. Five thousand new brain tumors are diagnosed yearly in the United States in children ages 0 to 19 years, with an incidence of approximately 6 per 100,000 children. (1) Childhood brain tumors, more than half of which are malignant, vary in terms of biology, prognosis and treatment. Presenting signs and symptoms depend on tumor location, growth rate, and presence of obstructive hydrocephalus. Making the initial diagnosis of a brain tumor can be difficult because early symptoms, such as headaches or vomiting, are nonspecific to brain tumors and more frequently are associated with other etiologies, leading to delays in diagnosis. The pediatrician plays a crucial role in the timely diagnosis of patients with brain tumors as well as recognizing late effects resulting from tumor therapies. This review summarizes the presenting features on history and physical examination, tumor classification of common tumor types, genetic brain tumor predisposition syndromes, general management strategy, and late effects of therapy.Signs and symptoms of a pediatric brain tumor can be nonspecific, insidious, intermittent, and dependent on location within the central nervous system (CNS) and the anatomical pathways affected. Although headache is the most common presenting complaint overall, it is present in only approximately one-third of the children presenting with brain tumors, and, in the absence of other symptoms or physical examination findings, is not in itself predictive of a brain tumor. Elevated intracranial pressure (ICP) is present in approximately half of all children with brain tumors. In addition to headache, it can cause nausea/vomiting, abnormalities of gait and coordination, and papilledema. Vital sign abnormalities associated with increased ICP, known as the Cushing triad (bradycardia, hypertension, abnormal respirations), are late signs of acutely increased ICP but can be absent in those with chronically elevated ICP. In young children with an open fontanelle, macrocephaly, especially when progressive, can be suggestive of hydrocephalus and a potential mass-occupying lesion. (2)Presenting symptoms depend on tumor location (Fig 1), and certain constellations of symptoms can point to specific lesion locations. Table 1 lists commonly overlooked signs and symptoms that can lead to a delayed diagnosis. Wilne et al analyzed presenting features of more than 4,000 childhood brain tumors and found that for posterior fossa tumors, three-quarters presented with nausea and vomiting, two-thirds with headache, three-fifths with abnormal gait and coordination, and one-third with papilledema. (2) In contrast, headache, nausea, and vomiting were rare in patients presenting with supratentorial tumors. Instead, seizures were present in one-third of patients, along with focal neurologic deficits such as weakness or sensory deficits on the contralateral side if there is involvement of the cortical motor or sensory regions, respectively. (2) In cases of brainstem tumors, children can present with crossed findings of ipsilateral facial weakness and contralateral hemiparesis. More than 75% of patients with brainstem tumors present with abnormal gait and coordination, whereas cranial nerve (CN) palsies are present in more than half. Headache, however, is not common in patients with brainstem tumors and is present in less than one-quarter at the time of diagnosis. Thalamic tumors can cause coordination and motor difficulties or hemiplegia. (2)Patients with pituitary tumors or optic pathway tumors often present with visual deficits. It is not uncommon for even severe visual deficits in children to go unrecognized by the patient, parents, or pediatrician. (3) Because patients with neurofibromatosis (NF) type 1 are at increased risk for optic pathway glioma, they should have yearly ophthalmology evaluations. Children with pituitary or hypothalamic tumors often present with endocrine abnormalities, such as failure to thrive, excessive thirst, or central obesity.Children with spinal cord tumors most commonly present with back pain, present at diagnosis in approximately two-thirds of cases. Spinal cord tumors may occur in extradural, intramedullary, and extramedullary intradural locations. Although some children may present with scoliosis, most will not. Spinal cord compression causes signs such as gait and coordination abnormalities, focal weakness, or bowel and bladder dysfunction. (2)A comprehensive neurologic examination (summarized in Table 2) is crucial to identify abnormalities that might be suggestive of a CNS tumor. A normal neurologic examination does not exclude the diagnosis of a brain or spinal cord tumor and must be correlated with symptoms.Patients with acute hydrocephalus can display dramatic changes in their mental status, with increased sleepiness, decreased energy, and decreased responsiveness. However, those with chronic hydrocephalus might show only subtle signs, such as slowly declining school performance.A fundoscopic examination of the optic nerve, CN II, is crucial to assess for papilledema and optic nerve pallor, which can reveal information about hydrocephalus or tumors along the optic pathways. A fundoscopic examination can be difficult in young or uncooperative children, warranting referral to ophthalmology for a dilated examination. Vision should be assessed by confrontation in the 4 quadrants of each eye because different patterns of visual field deficits will suggest varying tumor locations. In younger children, assessment of visual fields can be performed using a colorful object for central fixation and introducing a second object in the periphery and watching for the eyes to track to that object.Eye movements are controlled by CNs III, IV, and VI. The nuclei of CNs III and IV are located in the midbrain, whereas the nucleus of CN VI is in the pons, and brainstem tumors can lead to abnormalities of extraocular movements. Large pineal tumors can cause Parinaud syndrome, characterized by upgaze palsy, convergence-retraction nystagmus, and poorly reactive pupils due to compression of the rostral midbrain. Nystagmus can also be seen in patients with cerebellar tumors or optic pathway tumors.CN V, the trigeminal nerve, has 3 divisions that give sensation to the face. The trigeminal nucleus is located in the pons, as is the nucleus of CN VII (the facial nerve), which controls facial movement. Facial asymmetry or decreased facial sensation should raise concern for a mass in this region. Hearing in each ear should be assessed to look for CN VIII dysfunction.The lower CNs (CNs IX, X, XII) exit from the medulla and are involved in phonation, swallowing, and tongue movement. Palatal asymmetry, change in voice quality, or unilateral glossal atrophy raises suspicion for a medullary lesion. CN XI, the accessory nerve, has the most distal nucleus, also in the medulla, and innervates the trapezius and sternocleidomastoid musculature.Motor function, sensation, and reflexes should be assessed with special attention to comparison with the contralateral findings. Asymmetry can indicate a lesion affecting corticospinal tracts (motor), spinothalamic tracts (temperature, pain, light touch), or dorsal columns (proprioception, vibratory sense). Asymmetrical hyporeflexia can indicate lower motor neuron injury, whereas hyperreflexia and the presence of a Babinski reflex are indicative of upper motor neuron dysfunction. In acute upper motor neuron injury, reflexes may be absent.Patients with cerebellar tumors can present with a wide-based ataxic gait and difficulty with tandem gait. A hemiparetic gait can suggest a tumor involving cortical motor areas, the thalamus, or the brain stem. Patients with cerebellar or brainstem tumors may exhibit abnormal coordination, elicited by testing rapid alternating movements, finger to nose testing, or finger (pointer to thumb) and toe tapping (on the floor) or asking a child to mirror the examiner's finger as the examiner moves the finger laterally and/or vertically.Although not technically part of the neurologic examination, a skin examination is important to assess for dermatologic manifestations of underlying tumor predispositions such as NF type 1 (predisposed to low-grade gliomas [LGGs], especially in optic pathways), NF type 2 (predisposed to acoustic schwannomas and meningiomas), tuberous sclerosis complex (predisposed to subependymal giant cell tumors), or, more rarely, constitutional mismatch repair deficiency syndrome. Patients with constitutional mismatch repair deficiency syndrome have a genetic defect in genes responsible for repairing a specific type of DNA damage known as mismatch repair. Abnormalities in these genes (MLH1, MSH2, MSH5, PMS2) make it more difficult for the body to repair normally occurring DNA damage, leading to mutations and predisposing these patients to many types of cancers at an early age, including brain tumors, most commonly high-grade gliomas (HGGs). (4)The child with a suspected brain tumor might require urgent interventions. Those with unstable vital signs, altered mental status, or concern for increased ICP warrant expedited evaluation, best managed initially in the emergency department. Although magnetic resonance imaging (MRI) with and without contrast is the gold standard imaging technique for optimal visualization for brain tumors and is often needed for neurosurgical planning, in the unstable child, a computed tomographic (CT) scan may be the best initial imaging choice. CT scans can provide information regarding acute hydrocephalus, impending herniation, or acute hemorrhage, all of which represent neurosurgical emergencies. They can also show the anatomical location of a mass, lesion size, presence of hydrocephalus, and whether the mass is compressing other brain structures, thereby helping to triage and plan a timeline for MRI, surgery, or other sedated procedures. When choosing the optimal initial imaging study for a young child who would require anesthesia to complete an MRI, the relative risks of anesthesia compared with the risk of exposure to ionizing radiation from a CT scan, which could be completed without sedation, must be weighed while taking into account the degree of suspicion for an abnormality and individual risk factors specific to that patient. (5)MRI with and without contrast is generally the preferred imaging modality for diagnosis and follow-up of brain tumors. MRI allows for more detailed characterization of the tumor itself and the surrounding anatomy, with more specialized sequences for visualization of edema, relationship to CNs, blood vessels, and perfusion. Furthermore, MRI does not expose children to ionizing radiation so is preferred over CT for repeated studies, as would be needed to follow a brain tumor. Most patients with a brain tumor require a spinal MRI to evaluate for evidence of leptomeningeal disease.When a diagnosis of a brain tumor is made based on imaging, in the absence of a neurosurgical emergency, patients should be managed in concert with neuro-oncology teams preoperatively. Early neuro-oncology consultation allows for additional baseline neurologic examination, can help inform surgical planning based on the working differential diagnosis and postoperative treatment options, and facilitates an opportunity for clinical trial enrollment where presurgical consent may be required.The care of the pediatric neuro-oncology patient requires a multidisciplinary team–based approach. In addition to an excellent primary care pediatrician, this team includes neuro-oncology, neuro-surgery, neurology, neuro-radiology, radiation oncology, genetics, endocrinology, ophthalmology, audiology, neuropsychology, physical medicine and rehabilitation, palliative care, and social work.Upfront treatment of pediatric brain tumors generally includes surgery, radiotherapy, chemotherapy, or a combination of these modalities. For most tumor types, maximal safe surgical resection is pursued to obtain diagnosis and as the first step in definitive treatment. Some notable exceptions to this include tumors in eloquent locations where resection would result in significant morbidity or mortality. These locations include the brain stem, optic pathways, thalamus, internal capsule, sensory and motor cortices, visual cortex, or Broca and Wernicke areas, which are important for receptive and expressive language. In some cases, a small needle biopsy of these areas can be performed to obtain tissue for diagnostic purposes. For germ cell tumors, tumor markers can be diagnostic, obviating the need for upfront surgery. Some patients with low-grade–appearing lesions are followed with observation alone.Although some low-grade tumors can be treated with resection only, many low-grade and most high-grade tumors require additional postsurgical treatment. The standard of care for postsurgical management of pediatric brain tumors is constantly evolving based on emerging preclinical and clinical data. In many cases, enrollment in an open clinical trial is considered the standard of care. There are a variety of clinical trial consortia and cooperative groups with open protocols focused on pediatric brain tumors. A complete list of open clinical trials can be found on clinicaltrials.gov.There are more than 30 unique pathologies of CNS tumors in children. MRI characteristics of some common childhood brain tumors are shown in Fig 2. The advent of molecular genetics has enhanced our understanding of the biologic behavior of brain tumors, has changed tumor classification systems, and has had treatment implications.Medulloblastoma is the most common malignant brain tumor in children and is of embryonal origin. It generally presents as a posterior fossa mass and, due to its location, is often associated with obstructive hydrocephalus. Staging includes an MRI of the spine and a lumbar puncture looking for malignant cells in the cerebrospinal fluid (CSF). Histologically it is classified as classic, large cell anaplastic, or nodular desmoplastic. Overall, medulloblastoma has 5-year overall survival (OS) of approximately 70%. (6)Treatment depends on age at presentation, extent of resection, and presence of metastatic disease. Recent trials are accounting for molecular subtype in treatment decisions. Generally, treatment involves maximal tumor resection, craniospinal radiotherapy, and chemotherapy. Young patients undergo high-dose chemotherapy with autologous stem cell rescue to avoid or delay irradiation.Medulloblastoma has been classified into 4 principle molecular subgroups: WNT (wingless), SHH (sonic hedgehog), group 3, and group 4 (Table 3). (7) WNT-driven medulloblastomas are rarely metastatic and have the best overall prognosis, with greater than 90% OS. Current clinical trials are focused on reducing therapy in this subtype. SHH-driven tumors have a bimodal distribution presenting most commonly in infants or adolescents and young adults. They have an intermediate prognosis, although association with p53 mutations portends a poor prognosis. (9) Group 3 and group 4 tumors are known as non-WNT, non-SHH medulloblastoma subtypes. Although immunohistochemical studies can differentiate WNT and SHH medulloblastoma from the non-WNT and non-SHH medulloblastoma subtypes, other molecular methods, such as methylation studies, are needed to distinguish group 3 from group 4 tumors. Group 3 tumors can present in very young children, often have MYC amplification, are commonly metastatic at presentation, and have the poorest outcomes overall of any subgroup. Recent data suggest that group 3 tumors might benefit from intensified chemotherapy concurrent with radiotherapy. Group 4 tumors are the most common subgroup overall, presenting in children and adults and, similar to group 3 tumors, more commonly present in males than in females. (7) Group 4 tumors have an intermediate prognosis.Atypical teratoid rhabdoid tumors (ATRTs) are also embryonal tumors but can present in the posterior fossa or supratentorial region. These tumors have a very poor prognosis, with 3-year OS of approximately 25%. Survival trends improve with older age at diagnosis, with those older than 3 years faring better than younger patients. (10) Histologically, the loss of INI1, encoded by SMARCB1, is pathognomonic. Up to 35% of patients with ATRT have a germline mutation in SMARCB1 (or rarely SMARCA4), which predisposes them to the development of malignant rhabdoid tumors in other locations, most commonly the kidneys. Germline variants are more common in younger patients, and approximately two-thirds are sporadic. (11)Staging includes MRI of the brain and spine and lumbar puncture for CSF cytology. Treatment involves surgical resection, radiotherapy, and chemotherapy, with or without triple tandem autologous stem cell transplant. Recent clinical trial data showed improved survival outcomes compared with historical controls achieved with a regimen including radiotherapy for patients as young as 6 months and 3 cycles of high-dose chemotherapy with autologous stem cell rescue for all patients. (12) A meta-analysis including 130 patients with ATRT saw that survival correlated most strongly when patients were treated with regimens that included high-dose chemotherapy with autologous stem cell rescue. Treatment modalities of radiotherapy and intrathecal chemotherapy also lead to a statistically significant improvement in OS in this cohort. (10)ATRT tumors have also been classified based on molecular characteristics into 3 subgroups: ATRT–tyrosine (ATRT-TYR), ATRT–sonic hedgehog (ATRT-SHH), and ATRT–myelocytomatosis oncogene (ATRT-MYC), but further research is needed to delineate the prognostic and clinical implications of these subgroups. (13)Ependymoma represents the third most common brain tumor in children and arises from the ependymal cells lining the ventricles or the central canal of the spinal cord. Two-thirds of ependymomas present in the posterior fossa, with the remainder in the supratentorial region or spinal cord. For pediatric ependymoma as a whole, OS at 10 years is approximately 64%, but cases achieving gross total resection followed by radiotherapy fare significantly better. Molecular subtype and gain of chromosome 1q has important prognostic implications as well. (14)Ependymoma is treated with maximal surgical resection followed by focal radiotherapy, except for spinal disease, in which gross total resection without adjuvant radiotherapy can be curative. The role of chemotherapy in ependymoma remains under clinical investigation. Studies have also explored the use of postoperative chemotherapy to delay or omit radiotherapy in patients younger than 3 years, but outcomes were inferior to regimens involving radiotherapy for children older than 12 months. (15)Ependymoma has been divided into 9 molecular subgroups, with 3 subgroups for each anatomical location: spinal, supratentorial, and posterior fossa. Only 6 of the molecular subtypes generally affect children. Pediatric ependymoma of the spine is divided into the subtype and the subtype spinal subtypes have a prognosis. In the posterior fossa, patients with have a prognosis than those with and in the supratentorial those with have OS than those with and are associated with OS less than and survival of approximately are a group of tumors that including tumors the most tumors as and tumors When represent the most common brain tumor in children and can present in many anatomical locations. are less to to other of the CNS than their malignant and in some cases gross total resection can be curative. However, resection is not in certain anatomical locations, such as in the brain stem or with optic pathway common in patients with NF type have a prognosis, with OS of and survival of in a study of with follow-up of when therapy is needed for the regimen of chemotherapy with or and although other chemotherapy regimens have as well. is not in the upfront management of due to for late of the molecular of has that most are by in the most commonly variants and NF type 1 have shown pediatric and have shown in tumors. contrast to pediatric have a prognosis. include high-grade tumors brainstem tumors and of pediatric is tumors may be to surgical is followed by radiotherapy and chemotherapy for these tumors, as a Group study showed improved survival when chemotherapy to radiotherapy compared with radiotherapy specific chemotherapy regimen has as a standard of care for upfront pediatric In contrast, for tumors such as glioma, chemotherapy to radiotherapy has not been shown to survival the to OS and the OS achieved with radiotherapy open based and clinical trials are patients, to improve outcomes for these patients. studies in pediatric that the of pediatric from that of mutations and in tumors, and in tumors, the of in pediatric and a poor prognosis. are from in older children, with significantly improved are more common in children younger than 1 and under are germ cell tumors represent approximately of pediatric brain tumors and are as and germ cell tumors They most commonly in the pineal region but can also present in the thalamus, or or or both or which can be in blood and/or can cause of in the CSF but not In some cases, diagnosis can be made based on CSF and tumor whereas biopsy is when tumor markers are have a better overall prognosis, with OS greater than 90% compared with to for are commonly treated with 4 cycles of chemotherapy followed by radiotherapy to the tumor and whereas are generally treated with 6 cycles of chemotherapy alternating with and craniospinal radiotherapy in many cases, although studies are whether radiotherapy can be in patients with to associated with craniospinal radiotherapy. is a tumor from the of the and solid Histologically, they are classified as tumors and are divided into and subtypes. to their location they can and treatment for is some a more neurosurgical in an to avoid radiotherapy, and an initial resection followed by upfront radiotherapy. are germline mutations that children to specific types of childhood brain tumors in the of tumor predisposition of these is important to the primary care who follow these patients In the child who presents with a brain especially in the of other history of tumors, history of tumors at a young age, or dermatologic findings, it is important to further for these predisposition Children with a known history of predisposition might require genetic for the presence of these syndromes, and specific tumor if found to of these Furthermore, the presence of certain underlying may the of therapy for the management of a brain tumor. Table 4 summarizes germline associated with specific brain tumor the treatment of different tumor types each of the commonly treatment modalities their risks and acute risks of include and damage to structures, as well as morbidity dependent on tumor For posterior fossa syndrome affects an to of patients who undergo resection of large posterior fossa tumors. fossa syndrome is characterized by a combination of or significant with and or motor occurring within 2 of cerebellar and symptoms can months to and many are with deficits. Patients with supratentorial tumors are at greater risk for postoperative seizures and are often on Children with tumors are at increased risk for postoperative visual deficits and tumors by or at a tumor to damage is over to a total to the are without mass, that when at a point to a certain they also radiation at a lower on both the and exit side of the have mass, so the radiation is to within the the of at that the that the effects of radiotherapy are due to the radiation in both and radiotherapy, patients can skin which generally over the treatment Patients intracranial radiotherapy often headache or radiotherapy can cause due to the by the body and can the growth of the resulting in loss of in younger radiotherapy is preferred over radiotherapy, for patients who require craniospinal radiotherapy, because it to important such as the and For patients focal radiotherapy, therapy may to important or result in a significantly overall radiation on tumor the radiation field and for a plan a plan can be to evaluate relative of over based on the brain that would a with each to the use of therapy are the of radiotherapy

Humans are widely exposed to a wide variety of DNA alkylating agents (AAs) either directly, for example via cigarette smoke or indirectly via their formation in the gastrointestinal tract. Certain AAs, such as the tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) are known human carcinogens but the role that other AAs may play in the aetiology of human cancers is still uncertain due to difficulties in assessing human exposure to these agents. The aim of this work was therefore to develop a screening method to assess exposure to AAs by measuring the formation of O6-alkylguanines (O6-AlkGs) in DNA and applying it to the analysis of human DNA samples. To do this we took advantage of the S. pombe alkyltransferase-like protein (Atl1), which binds to all O6-AlkGs so far studied, to develop a slot blot assay. For this, sonicated heat denatured DNA was bound to Hybond-N+ membranes and probed with horseradish peroxidase-conjugated maltose-binding protein-Atl1 fusion protein. The binding was quantitated via peroxidase-mediated enhanced chemiluminescence using TMZ-methylated calf thymus (CT) DNA standards previously calibrated using a tritium-based competition assay. Using 1 μg DNA, the limit of quantitation and detectability were approximately 1.0 and 0.5 fmoles O6-MeG/μg DNA, respectively. Prior treatment of the TMZ-CT DNA standards with the DNA repair protein, O6-methylguanine DNA methyltransferase, reduced the detectable signal to background levels, confirming that the assay detected O6-methylguanine (O6-MeG) in TMZ-CT DNA. Importantly, CT DNA reacted in vitro with the alkylating agents N-ethyl-N-nitrosourea, N-propyl-N-nitrosourea, N-butyl-N-nitrosourea, N-benzyl-N-nitrosourea, N-allyl-N-nitrosourea and potassium diazoacetate all produced strong signals in the assay demonstrating its ability to detect a range of different O6-AlkGs. In addition, a variety of human DNA samples have been analysed in pilot studies: of 14 human placental DNA samples, 8 had O6-AlkG levels in the range of 1.05-1.71 fmoles/μg DNA, 3 had trace levels and in 3 samples there was no detectable signal. Of 5 human breast tumour DNA samples, 4 had O6-AlkGs levels in the range of 1.07-1.30 fmoles/µg DNA and 1 had trace levels. Two DNA samples from normal human breast tissue had no detectable O6-AlkGs but DNA from the tumours of the same patients had levels >1 fmoles/μg DNA. Of 6 DNA samples from whole blood 2 had detectable levels of O6-AlkGs (1.23 & 1.56 fmoles/μg DNA), 3 had trace amounts and 1 had no detectable level. These data thus show the potential usefulness of this slot blot assay as a screening tool to detect and quantify total O6-alkGs adducts in human DNA samples. Citation Format: Hanum Yaakub, Anthony Howell, Geoffrey Margison, Andrew Povey. Developing a screen for O6-alkylguanines in human DNA using the damage-specific binding protein, Atl1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5780.

Leukocyte depletion and recovery with white blood cell sterile acrodisc® syringe filter

Bacterial contamination often occurs in human blood DNA samples, possibly due to bacteremia or an inappropriate procedure during sample preparation. This study aimed at analyzing the clinical significance of bacterial DNA contamination in human blood DNA samples and to assess its influence on experimental data. DNA samples (N = 1359) were randomly selected from population-based cohort samples to determine bacterial DNA contamination by polymerase chain reaction and direct DNA sequencing. Bacterial DNA contaminated samples (N = 150) were then assessed for experimental quality of single nucleotide polymorphism (SNP) chip data, compared with uncontaminated DNA samples (N = 1209). DNA sequencing data showed that a major source of bacterial contaminants was derived from Alcaligenes species. The occurrence of bacterial DNA contaminations was significantly associated with some clinical variables including a postprandial glucose level at 60 min, % body fat, and waist-to-hip ratio. It was also found that there was no difference of SNP call rates between bacterial DNA contaminated samples and uncontaminated DNA samples. This study showed that bacterial DNA contamination in human blood samples was related to donor's health condition, suggesting that the occurrence of bacterial DNA contamination may provide useful health information of blood donors and a potential tool for human disease genomics.

Previously, much of the perfluoroalkyl and polyfluoroalkyl substance (PFAS) research has focused on perfluoroalkyl carboxylates (PFCAs) or perfluoroalkane sulfonates (PFSAs). Recent studies indicate that known PFCAs and PFSAs accounted for 5-95% of the organofluorine (OF) in human and wild rat blood samples suggesting that a relatively large proportion of OF remained unknown. Until recently, some studies reported commercially available compounds such as polyfluoroalkyl phosphate diesters (diPAPs) and fluorotelomer sulfonates (FTSAs) in human blood and sludge samples. The present investigation is a pilot study aiming at surveying some newly identified PFASs such as diPAPs, FTSAs, and perfluorinated phosphinates (PFPiAs) in different environmental samples including surface water, sediment, sewage treatment plant influent and effluent, sludge, benthic worm, and human blood from Hong Kong. DiPAPs (6:2, 6:2/8:2, and 8:2) were detected in some of the samples at part-per-billion (ppb) levels in sludge, sub ppb levels in influent and effluent, sediment, worm, and human blood samples, and sub part-per-trillion (ppt) levels in surface waters. Sub ppt to ppb levels of 6:2 and 8:2 FTSAs were observed in worm, surface water, and human blood samples. PFPiAs were only observed in worm samples. The detected "new PFASs" accounted for a minor proportion (less than 5%) of the total PFASs in benthic worm and human blood, but up to 95% in sewage sludge samples from Hong Kong. This is the first report of commercial fluorosurfactants (PFPiAs, diPAPs, and FTSAs) in the samples from the environment and human blood in Hong Kong; further information on the distribution, fate, and transport of "new PFASs" in other Asian cities, as well as toxicity, is needed for further assessing the human exposure and risk.

Human StarD5 belongs to the StarD4 subfamily of START (for steroidogenic acute regulatory lipid transfer) domain proteins. We previously reported that StarD5 is located in the cytosolic fraction of human liver and binds cholesterol and 25-hydroxycholesterol. After overexpression of the gene encoding StarD5 in primary rat hepatocytes, free cholesterol accumulated in intracellular membranes. These findings suggested StarD5 to be a directional cytosolic sterol transporter. The objective of this study was to determine the localization of StarD5 in human liver. Western blot analysis confirmed StarD5's presence in the liver but not in human hepatocytes. Immunohistochemistry studies showed StarD5 localized within sinusoidal lining cells in the human liver and colocalized with CD68, a marker for Kupffer cells. Western blot analyses identified the presence of StarD5 in monocytes and macrophages as well as mast cells, basophils, and promyelocytic cells, but not in human hepatocytes, endothelial cells, fibroblasts, osteocytes, astrocytes, or brain tissue. Cell fractionation and immunocytochemistry studies on THP-1 macrophages localized StarD5 to the cytosol and supported an association with the Golgi. The presence of this cholesterol/25-hydroxycholesterol-binding protein in cells related to inflammatory processes provides new clues to the role of this protein in free sterol transport in the cells and in lipid-mediated atherogenesis.

Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR.

To the Editor: We read with great interest the article by Fouke et al.1 Malignant central nervous system tumors represent a public health issue because of their incurable nature and the morbidity they generate. The present project will focus on glioblastomas because they are the more common and most aggressive malignant primary brain tumors; they have a well-standardized treatment, allowing a large and relatively homogeneous cohort; and their poor prognosis allows complete survival data within a couple of years. Glioblastomas still create numerous difficulties because of the lack of reproducibility of the histopathological diagnosis, and they raise many questions on genetic/environmental risk factors and treatment. There is an urgent need for biomarkers of glioblastoma subtypes, prognosis, and treatment response, as well as genetic susceptibility to environmental risk factors. Only some large-scale translational programs based on a large clinic-biological database will enable the identification of these biomarkers. The Comprehensive Neuro-Oncology Data Repository (CONDR) project, which will collect an integrated data set that includes advanced imaging, pathology, and clinical data from up to 200 subjects, will answer this urgent need. With the same objective, the French glioblastoma biobank was funded in 2013 by the Institut National duCancer (INCa). It aims at establishing within 3 years a national, well-annotated, clinico-biological database containing at least 1200 patients, associated with a large collection of blood, tumor, and hair samples (http://www.french-glioblastoma-biobank.com). This database runs on Clinsight System of ENNOV, which is certified software for the management of clinical trials, meeting the recommendations of the US Food and Drug Administration (21CRF Part11) and the European Medicines Agency for IT security of clinical data. Basic epidemiological data, standardized histopathological analysis, clinical and imagery data, treatment, and follow-up are prospectively recorded via an electronic case report form via CSOnline. Biological samples (including at least a frozen specimen and formalin-fixed paraffin-embedded tissue of the tumor, blood sample, and hair) are conserved in the tumor banks and biological resource centers in each partner hospital center. With the help of the French pathological neurooncology network (RENOP), pathological specimens of each case included in the database will be reviewed by an expert panel of neuropathologists, and the immunohistochemical detection of IDH1R132H expression will be assessed in all cases. Unlike CONDR, data collected in French glioblastoma biobank will not be in open access, and use of clinical data/biological samples will be authorized by a steering committee after submission and evaluation of the scientific project. Currently, 23 centers are already engaged into the project, and 231 patients have been included. The second objective according to INCa directives will be the development of communication, interoperability, and harmonization with other brain tumor data banks comprising the 3 main ongoing rare brain tumor networks funded by INCa, POLA (Prise en charge des Oligodendrogiomes Anaplasique), and TUCERA (French acronym for Rare Cerebral Tumors). The POLA network, funded in 2010 (http://www.reseau-pola.org), is dedicated to high-grade oligodendroglial tumors. All information (clinical, radiological, histological, molecular markers) is collected in a dedicated database (https://www.reseaupola.com) hosted by GERCOR and using the LINCOLN management system. More than 800 patients are registered in this database. A correlation study between magnetic resonance imaging characteristics and molecular markers in anaplastic oligodendroglioma has been published by the POLA network, confirming the interest in clinico-biological databases.2,3 Underlining the importance of setting up perfect communication between brain tumors clinico-biological banks, interoperability between POLA and the French glioblastoma biobank is now operating for data exchange. The more recent TUCERA network, which covers primary central nervous system malignant tumors with specific histological subtypes, including medulloblastoma, grade 2 gliomas, atypical and anaplastic meningioma, and rare gliomas, is running on the same electronic case report form and informatics platform as the French glioblastoma biobank, thus facilitating data exchange. As in the French glioblastoma biobank, pathological specimens of each case included in the POLA and TUCERA databases will be reviewed by a RENOP expert panel. A unique informed consent is due to take place soon for all these clinico-biological data banks. Moreover, interoperability is in construction with 2 epidemiological registers (the French Brain Tumor database4 and the French Cancer Registers [FRANCIM network]). Communication and exchanges with international glioblastoma data banks are essential to progress in neuro-oncology. We would be delighted to discuss any kind of collaboration with the CONDR project, even if the choices of management are different (open access vs steering committee governance) and could generate governance issues. Disclosure The authors have no personal, financial, or institutional interest in any of the drugs, materials, or devices described in this article.

Enhanced Self-Renewal Capability in Hepatic Stem/Progenitor Cells Drives Cancer Initiation

Objective To establish a real-time quantitative polymerase chain reaction (RQ-PCR) assay for fast detection of Aspergillus fumigatus genome in human whole blood samples and explore its clinical application.Methods The primers and the TaqMan-probe were designed on the basis of the multi-copy ITS1-5. 8S region of the rDNA of Aspergillus fumigatus. The Aspergillus fumigatus genomic DNA were extracted with QIAamp(R) DNA Blood Mini Kit.A 20 μl RQ-PCR amplification system was established, and the simulated blood samples containing various given load of Aspergillus fumigatus genome and the 66 whole blood samples of the surgical febrile patients were examined. Results The detection limit of the RQ-PCR instrument is 10-1 genomes/μl DNA sample,namely 78 CFU/ml whole blood. The specificity and the sensitivity were 94. 25% and 99. 04% respectively; and the positive predictive value and negative predictive value were 97. 63% and 97. 62% respectively. The average relative error of the quantitative results was (3. 67 ±13. 19)%, and the intra- and the inter-assay average coefficients of variation were (12.38 ± 1. 53)% and (16. 27 ±2. 72)% , respectively. The average recovery rate of Aspergillus fumigatus genomic DNA in human whole blood samples was (107. 81 ±25. 92)% , and the average coefficient of variation of the average recovery rate was (26. 24 ± 5.62) % . No Aspergillus fumigatus genomic DNA was detected among the 66 blood samples of the surgical febrile patients. Conclusions The RQ-PCR assay for fast quantitative detection of Aspergillus fumigatus genome in human whole blood samples is of high sensitivity, specificity,accuracy and precision. The Aspergillus fumigatus genome was not detected in this group of surgical febrile patients. Key words: Real-time quantitative PCR; Aspergillus fumigalus; Human whole blood; Surgical infection

Recent advances in nanofluidic technologies have enabled the use of Integrated Fluidic Circuits (IFCs) for high-throughput Single Nucleotide Polymorphism (SNP) genotyping (GT). In this study, we implemented and validated a relatively low cost nanofluidic system for SNP-GT with and without Specific Target Amplification (STA). As proof of principle, we first validated the effect of input DNA copy number on genotype call rate using well characterised, digital PCR (dPCR) quantified human genomic DNA samples and then implemented the validated method to genotype 45 SNPs in the humpback whale, Megaptera novaeangliae, nuclear genome. When STA was not incorporated, for a homozygous human DNA sample, reaction chambers containing, on average 9 to 97 copies, showed 100% call rate and accuracy. Below 9 copies, the call rate decreased, and at one copy it was 40%. For a heterozygous human DNA sample, the call rate decreased from 100% to 21% when predicted copies per reaction chamber decreased from 38 copies to one copy. The tightness of genotype clusters on a scatter plot also decreased. In contrast, when the same samples were subjected to STA prior to genotyping a call rate and a call accuracy of 100% were achieved. Our results demonstrate that low input DNA copy number affects the quality of data generated, in particular for a heterozygous sample. Similar to human genomic DNA, a call rate and a call accuracy of 100% was achieved with whale genomic DNA samples following multiplex STA using either 15 or 45 SNP-GT assays. These calls were 100% concordant with their true genotypes determined by an independent method, suggesting that the nanofluidic system is a reliable platform for executing call rates with high accuracy and concordance in genomic sequences derived from biological tissue.

Rare vascular lesions known as brain arteriovenous malformations (bAVMs) are caused by shunts between cerebral arteries and veins that do not involve the capillary bed. The majority of bAVMs do not cause any symptoms, however some can be controlled by seizures and result in potentially fatal brain hemorrhages. Over the past ten years, significant advancements in our understanding of bAVM pathogenesis have been accomplished, primarily through the use of genome sequencing and biomolecular analysis. An autosomal dominant vascular disorder called Hereditary Hemorrhagic Telangiectasia (HHT, also known as Rendu-Osler-Weber Syndrome) affects about 1 in 5000 people globally and is associated with about 5% of brain AVMs, while the remaining 95% are due to sporadic mutations. The major aim of the project is to investigate genetic causes of sporadic forms of AVMs in the brain by means of whole-exome sequencing of tissues with AVM lesions and blood samples. Analysis of somatic mutations will broaden the understanding of AVM pathogenesis. This study was carried out in accordance with the Declaration of Helsinki's tenets and approved by the National Center of Biotechnology 's ethics committee (№9, 7 November 2023, ) Astana). Based on clinical data, the age range of the patients at the onset of the disease also encompassed 25 to 40 years. To confirm the diagnosis, MRI and CAG data were used to validate the diagnosis. Each patient completed a specially designed questionnaire with medical information (such as age of debut and type of course) and demographic data (such as age, gender, and nationality). Three patients were classified as having a Spetzler-Martin grade II, while one patient was categorized as grade III. The sizes of arteriovenous malformations (AVMs) varied from 0 to 3 mm in one patient (grade II) to 3 to 6 mm in three patients (grades II-III). AVM tissue specimens were obtained during clinically necessary procedures. Brain AVM tissues were collected during surgery, which involved trepanation and excision of the AVM. Blood samples were also collected from these patients. Both DNA and RNA were isolated from these tissue and blood samples for subsequent exome sequencing an. Qubit 2.0 fluorometer was employed for more precise determination of DNA concentration, given that the nucleic acids will be utilized in future whole exome sequencing analysis. Whole exome sequencing was performed on six samples (blood and tissue) by Novaseq 6000 (Illumina). The annotated results were analyzed for the presence of single-nucleotide polymorphisms (SNP) and insertion-deletion mutations (Indels) between DNAs from the AVM tissue sample and blood. The GATK workflow was used for the analysis and annotation of the raw data, and vcf files were generated. Then, the variants were filtered for a quality ≥ 30 in Excel 1st blood 117554, 1st tissue 114715; 2nd blood 112972, 2nd tissue 116022; 3rd blood 112859, 3rd tissue 117168 and genotype differences between tissue and blood DNA samples 1st blood 114512, 1st tissue 111682; 2nd blood 110614, 2nd tissue 111982; 3rd blood 110564, 3rd tissue 112805. After selecting only non-synonymous, frameshift insertions and deletions variants with different genotypes in the blood and tissue samples, 1st blood 279, 1st tissue 277; 2nd blood 279, 2nd tissue 271; 3rd blood 275, 3rd tissue 268 variations were observed. Analysis of whole-exome data continues.

Identification of bacteria from clinical samples using Bartonella alpha-Proteobacteria growth medium

An improved extraction (ion pairing) and cleanup (ENVI-carb and solid phase extraction) method was developed for analysis of perfluorinated compounds (PFCs) in human whole blood samples from China. Ten PFCs including PFOS, PFHxS, PFOSA, PFDoDA, PFUnDA, PFDA, PFNA, PFOA, PFHpA, and PFHxA were detected in the blood samples (n=30) from five cities (Jintan, Nanjing, Guiyang, Beijing, and Shenyang). PFOS was found to be the dominant PFC ranging from 0.446-83.1 ng/mL. Total fluorine (TF) and extractable organic fluorine (EOF) also were measured in the blood samples using combustion ion chromatography for fluorine. Analysis of known PFCs and extractable organic fluorine showed that known PFCs could account for >70% of EOF in samples from Beijing, Shenyang, and Guiyang, whereas known PFCs could only account for approximately 30% of EOF in samples from Jintan. Results of the present study indicated the presence of substantial amounts of unidentified organic fluorine in human blood samples from Jintan. Characterization and identification of these unidentified fluorinated compounds will be instructive.

Analysis of omega-3 and omega-6 fatty acid-derived lipid metabolite formation in human and mouse blood samples