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BACKGROUND AND OBJECTIVES: The SLC35A2 gene, located at chromosome Xp11.23, encodes for a uridine diphosphate–galactose transporter. We describe clinical, genetic, neuroimaging, EEG, and histopathologic findings and assess possible predictors of postoperative seizure and cognitive outcome in 47 patients with refractory epilepsy and brain somatic SLC35A2 gene variants. METHODS: This is a retrospective multicenter study where we performed a descriptive analysis and classical hypothesis testing. We included the variables of interest significantly associated with the outcomes in the generalized linear models. RESULTS: Two main phenotypes were associated with brain somatic SLC35A2 variants: (1) early epileptic encephalopathy (EE, 39 patients) with epileptic spasms as the predominant seizure type and moderate to severe intellectual disability and (2) drug-resistant focal epilepsy (DR-FE, 8 patients) associated with normal/borderline cognitive function and specific neuropsychological deficits. Brain MRI was abnormal in all patients with EE and in 50% of those with DR-FE. Histopathology review identified mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy in 44/47 patients and was inconclusive in 3. The 47 patients harbored 42 distinct mosaic SLC35A2 variants, including 14 (33.3%) missense, 13 (30.9%) frameshift, 10 (23.8%) nonsense, 4 (9.5%) in‐frame deletions/duplications, and 1 (2.4%) splicing variant. Variant allele frequencies (VAFs) ranged from 1.4% to 52.6% (mean VAF: 17.3 ± 13.5). At last follow-up (35.5 ± 21.5 months), 30 patients (63.8%) were in Engel Class I, of which 26 (55.3%) were in Class IA. Cognitive performances remained unchanged in most patients after surgery. Regression analyses showed that the probability of achieving both Engel Class IA and Class I outcomes, adjusted by age at seizure onset, was lower when the duration of epilepsy increased and higher when postoperative EEG was normal or improved. Lower brain VAF was associated with improved postoperative cognitive outcome in the analysis of associations, but this finding was not confirmed in regression analyses. DISCUSSION: Brain somatic SLC35A2 gene variants are associated with 2 main clinical phenotypes, EE and DR-FE, and a histopathologic diagnosis of MOGHE. Additional studies will be needed to delineate any possible correlation between specific genetic variants, mutational load in the epileptogenic tissue, and surgical outcomes.

Single germline or somatic activating mutations of mammalian target of rapamycin (mTOR) pathway genes are emerging as a major cause of type II focal cortical dysplasia (FCD), hemimegalencephaly (HME) and tuberous sclerosis complex (TSC). A double-hit mechanism, based on a primary germline mutation in one allele and a secondary somatic hit affecting the other allele of the same gene in a small number of cells, has been documented in some patients with TSC or FCD. In a patient with HME, severe intellectual disability, intractable seizures and hypochromic skin patches, we identified the ribosomal protein S6 (RPS6) p.R232H variant, present as somatic mosaicism at ~15.1% in dysplastic brain tissue and ~11% in blood, and the MTOR p.S2215F variant, detected as ~8.8% mosaicism in brain tissue, but not in blood. Overexpressing the two variants independently in animal models, we demonstrated that MTOR p.S2215F caused neuronal migration delay and cytomegaly, while RPS6 p.R232H prompted increased cell proliferation. Double mutants exhibited a more severe phenotype, with increased proliferation and migration defects at embryonic stage and, at postnatal stage, cytomegalic cells exhibiting eccentric nuclei and binucleation, which are typical features of balloon cells. These findings suggest a synergistic effect of the two variants. This study indicates that, in addition to single activating mutations and double-hit inactivating mutations in mTOR pathway genes, severe forms of cortical dysplasia can also result from activating mutations affecting different genes in this pathway. RPS6 is a potential novel disease-related gene.

Dravet syndrome is the most studied form of genetic epilepsy. It has now been clarified that the clinical spectrum of the syndrome does not have firmly established boundaries. The core phenotype is characterized by intractable, mainly clonic, seizures precipitated by increased body temperature with onset in the first year of life and subsequent appearance of multiple seizures types still precipitated by, but not confined to, hyperthermia. Cognitive impairment is invariably present when the full syndrome is manifested. This complex of symptoms is related to mutations in the SCN1A gene, which are often de novo and constitutional but can also be inherited from a parent with less severe clinical manifestations or be present as somatic mosaicism. Inheritance from less severely affected individuals, at times only having experienced a few febrile seizures, and differences in severity, even within the same family, with a subset of patients only showing fragments of the syndrome, testify to a remarkable phenotypic heterogeneity as far as severity, but less so clinical phenomenology, are concerned. This characteristic, together with underascertainment of SCN1A mutations due to human errors or technical limitations in uncovering alternative pathogenic molecular mechanisms, such as genomic rearrangements or poison exons, has contributed to making clinicians and geneticists suspicious that Dravet syndrome may be caused by more than one gene. This opinion has been further amplified by the description of other genetic disorders, such as PCDH19- or CHD2-related epilepsy, whose phenotypes have included fragments of the Dravet phenotypic spectrum, and by the suboptimal characterization of phenotypes associated with mutations in SCN1B, HCN1, KCN2A, GABRA1, GABRG2, and STXBP1. The SCN1A gene-Dravet syndrome association is in our opinion highly specific. However, because the syndrome spectrum is wide, fragments of it can at times also be manifested in other genetic epilepsy syndromes, thereby leading to overdiagnosis of Dravet syndrome beyond SCN1A. Dravet syndrome is in turn a severe SCN1A phenotype within a continuum of SCN1A-related clinical phenomenology.

Background Genetic polymorphisms in genes involved in pain modulation have been reported to be associated to opioid efficacy and safety in different clinical settings. Methods The association between COMT Val158Met polymorphism (rs4680) and the inter-individual differences in the response to opioid analgesic therapy was investigated in a cohort of 87 Italian paediatric patients receiving opioids for cancer pain (STOP Pain study). Furthermore, a systematic review of the association between opioid response in cancer patients and the COMT polymorphism was performed in accordance with the Cochrane Handbook and the Prisma Statement. Results In the 87 paediatric patients, pain intensity (total time needed to reach the lowest possible level) was significantly higher for G/G than A/G and A/A carriers (p-value = 0.042). In the 60 patients treated only with morphine, the mean of total dose to reach the same pain intensity was significantly higher for G/G than A/G and A/A carriers (p-value = 0.010). Systematic review identified five studies on adults, reporting that opioid dose (mg after 24 h of treatment from the first pain measurement) was higher for G/G compared to A/G and A/A carriers. Conclusions Present research suggests that the A allele in COMT polymorphism could be a marker of opioid sensitivity in paediatric cancer patients (STOP Pain), as well as in adults (Systematic Review), indicating that the polymorphism impact could be not age-dependent in the cancer pain context. Trial registration Registration number: CRD42017057831. Electronic supplementary material The online version of this article (10.1186/s12885-019-5310-4) contains supplementary material, which is available to authorized users.

Summary: Anaplastic large cell lymphoma (ALCL) accounts for approximately 15% of all pediatric non-Hodgkin lymphomas. It has distinct clinical features, including frequent involvement of extranodal sites and rare localization to the central nervous system. As some presenting features of ALCL are in common with the hemophagocytic syndrome, we previously analyzed a small series of patients with ALCL for PRF1 mutations and found that 27% of them carried mutations. We now expanded our preliminary study by increasing the cohort of ALCL patients to a total of 84 consecutive cases, in whom we extended mutation analysis to the genes SH2D1A, PRF1 e UNC13D, all related to familial HLH. Furthermore, perforin expression in tumor cells was investigated on paraffin-embedded tissues by immunohistochemical analysis. Mutations were observed in 23/84 patients (27.4%). Twenty-one patients (25%) carried a total of 10 different mutations of PRF1; they were monoallelic in 20 patients, biallelic in 1. No mutations were found in the gene SH2D1A. Two additional patients had missense mutations of the UNC13D gene. These data show that monoallelic germline mutations of PRF1 are frequent in patients with childhood ALCL, suggesting that partially impaired cytotoxic machinery may represent a predisposing factor for ALCL. Involvement is less frequent for UNC13D and absent for SH2D1A.

X-linked lymphoproliferative disease 1 (XLP1) is a rare congenital immunodeficiency caused by SH2D1A (Xq25) mutations resulting in lack or dysfunction of SLAM-associated protein adaptor molecule. In XLP1 patients, upon ligand (CD48) engagement, 2B4 delivers inhibitory signals that impair the cytolytic activity of NK (and T) cells. This causes the selective inability to control EBV infections and the occurrence of B-cell lymphomas. Here, we show that in the absence of SLAM-associated protein, co-engagement of 2B4 with different activating receptors, either by antibodies or specific ligands on target cells, inhibits different ITAM-dependent signaling pathways including activating killer Ig-like receptors. In XLP1 NK cells, 2B4 affected both the cytolytic and IFN-γ production capabilities, functions that were restored upon disruption of the 2B4/CD48 interactions. Notably, we provide evidence that 2B4 dysfunction does not affect the activity of DNAM-1 and NKG2D triggering receptors. Thus, while CD48+ B-EBV and lymphoma B cells devoid of NKG2D and DNAM-1 ligands were resistant to lysis, the preferential usage of these receptors allowed XLP1 NK cells to kill lymphomas that expressed sufficient amounts of the specific ligands. The study sheds new light on the XLP1 immunological defect and on the cross-talk of inhibitory 2B4 with triggering NK (and T) receptors.

Objective:To explore the prognostic value of initial clinical and mutational findings in infants with SCN1A mutations.Methods:Combining sex, age/fever at first seizure, family history of epilepsy, EEG, and mutation type, we analyzed the accuracy of significant associations in predicting Dravet syndrome vs milder outcomes in 182 mutation carriers ascertained after seizure onset. To assess the diagnostic accuracy of all parameters, we calculated sensitivity, specificity, receiver operating characteristic (ROC) curves, diagnostic odds ratios, and positive and negative predictive values and the accuracy of combined information. We also included in the study demographic and mutational data of the healthy relatives of mutation carrier patients.Results:Ninety-seven individuals (48.5%) had Dravet syndrome, 49 (23.8%) had generalized/genetic epilepsy with febrile seizures plus, 30 (14.8%) had febrile seizures, 6 (3.5%) had focal epilepsy, and 18 (8.9%) were healthy relatives. The association study indicated that age at first seizure and frameshift mutations were associated with Dravet syndrome. The risk of Dravet syndrome was 85% in the 0- to 6-month group, 51% in the 6- to 12-month range, and 0% after the 12th month. ROC analysis identified onset within the sixth month as the diagnostic cutoff for progression to Dravet syndrome (sensitivity = 83.3%, specificity = 76.6%).Conclusions:In individuals with SCN1A mutations, age at seizure onset appears to predict outcome better than mutation type. Because outcome is not predetermined by genetic factors only, early recognition and treatment that mitigates prolonged/repeated seizures in the first year of life might also limit the progression to epileptic encephalopathy.

To explore the prognostic value of initial clinical and mutational findings in infants withCombining sex, age/fever at first seizure, family history of epilepsy, EEG, and mutation type, we analyzed the accuracy of significant associations in predicting Dravet syndrome vs milder outcomes in 182 mutation carriers ascertained after seizure onset. To assess the diagnostic accuracy of all parameters, we calculated sensitivity, specificity, receiver operating characteristic (ROC) curves, diagnostic odds ratios, and positive and negative predictive values and the accuracy of combined information. We also included in the study demographic and mutational data of the healthy relatives of mutation carrier patients.Ninety-seven individuals (48.5%) had Dravet syndrome, 49 (23.8%) had generalized/genetic epilepsy with febrile seizures plus, 30 (14.8%) had febrile seizures, 6 (3.5%) had focal epilepsy, and 18 (8.9%) were healthy relatives. The association study indicated that age at first seizure and frameshift mutations were associated with Dravet syndrome. The risk of Dravet syndrome was 85% in the 0- to 6-month group, 51% in the 6- to 12-month range, and 0% after the 12th month. ROC analysis identified onset within the sixth month as the diagnostic cutoff for progression to Dravet syndrome (sensitivity = 83.3%, specificity = 76.6%).In individuals with

Santi R, Cetica V, Franchi A, Pepi M, Cesinaro A M, Miracco C, Paglierani M, De Giorgi V, Delfino C, Difonzo E M, Pimpinelli N, Bianchi S, Sardi I, Santucci M & Massi D (2011) Histopathology58, 455–466 Tumour suppressor gene TP53 mutations in atypical vascular lesions of breast skin following radiotherapy Aims: Atypical vascular lesions (AVL) occurring at the site of radiotherapy represent an uncommon but well-documented complication in the setting of breast-conserving therapy for breast carcinoma. Although the biological behaviour of AVL has been regarded as benign, it has been suggested that AVL may represent a precursor of angiosarcoma. A better understanding of the biology of AVL is essential in order to assess appropriate patient management. The aim of the present study was to investigate alterations of tumour suppressor gene TP53 in a series of radiation-induced AVL and angiosarcomas (AS). Methods and results: Direct sequencing analysis of the TP53 gene showed the presence of at least one variation in 10 of 12 (83.3%) AVL and in seven of eight (87.5%) AS. The most common alteration in both categories was the P72R polymorphism in exon 4. One angiosarcoma sample carried a pathogenetically relevant disruptive mutation c.592delG, a frameshift deletion in exon 6, causing a premature stop codon. Conclusions: The presence of TP53 alterations suggests that its mutational inactivation may be implicated in the pathogenesis of radiation-associated vascular proliferations. The common mutational pathway suggested by our data supports the hypothesis that AVL and AS are biologically related entities, most probably representing the extremes of a morphological continuum.

Background Mutations of UNC13D are causative for familial haemophagocytic lymphohistiocytosis type 3 (FHL3; OMIM 608898). Objective To carry out a genotype–phenotype study of patients with FHL3. Methods A consortium of three countries pooled data on presenting features and mutations from individual patients with biallelic UNC13D mutations in a common database. Results 84 patients with FHL3 (median age 4.1 months) were reported from Florence, Italy (n=54), Hamburg, Germany (n=18), Stockholm, Sweden (n=12). Their ethnic origin was Caucasian (n=57), Turkish (n=10), Asian (n=7), Hispanic (n=4), African (n=3) (not reported (n=3)). Thrombocytopenia was present in 94%, splenomegaly in 96%, fever in 89%. The central nervous system (CNS) was involved in 49/81 (60%) patients versus 36% in patients with FHL2 (p=0.001). A combination of fever, splenomegaly, thrombocytopenia and hyperferritinaemia was present in 71%. CD107a expression, NK activity and Munc 13-4 protein expression were absent or reduced in all but one of the evaluated patients. 54 different mutations were observed, including 15 new ones: 19 missense, 14 deletions or insertions, 12 nonsense, nine splice errors. None was specific for ethnic groups. Patients with two disruptive mutations were younger than patients with two missense mutations (p

Rhabdoid meningioma is an uncommon meningioma variant categorized as WHO grade III. The majority of cases occur in adulthood. Herein, we describe a right fronto-temporal rhabdoid meningioma affecting a 3-year-old boy. The lesion measured approximately 4 cm in diameter and incorporated the ipsilateral middle cerebral artery. Sub-total surgical excision of the mass was performed. Histologically, the tumor was mainly composed of globoid plump cells with inclusion-like eosinophilic cytoplasm, peripheral nuclei, prominent nucleoli and occasional intra-nuclear cytoplasmic pseudo-inclusion. The cells appeared in many areas loosely arranged and focally disclosed a papillary architecture. At immunohistochemistry, the tumor cells were EMA, vimentin, HHF35, PgR, INI-1 and p53 positive. The proliferative index (Mib-1) was 15% in the most positive areas. Ultrastructurally, tumoral cells showed an abundant cytoplasm, which was filled with numerous intermediate filaments. Desmosomal junctions were seen. RT-PCR revealed the presence of NF2 gene expression. Molecular study did not indicate alterations of the INI-1 gene, whereas it showed the presence of Pro72Arg in exon 4 at heterozygous state in the TP53 gene. Morphologic features along with immunohistochemical, ultrastructural and molecular results were consistent with the diagnosis of rhabdoid meningioma. The patient was treated with chemotherapy. The lesion remained stable after 33 months of follow-up. Rhabdoid meningiomas rarely occur in children. Owing to its rarity, each new case should be recorded to produce a better clinical, pathological, molecular, prognostic and therapeutic characterization of this lesion.

Alkylating agents, commonly used for brain tumor therapy, induce DNA and RNA lesions that, if not repaired, drive cells to apoptosis. Thus, cellular mechanisms that are responsible for nucleic acid repair are possibly involved in drug resistance. This work analyzes hABH2 and hABH3, two human Fe(II)-dependent dioxygenases in pediatric brain tumors that are treated with alkylating agents. We analyzed 25 brain tumor samples for hABH2 and hABH3 mutations; a subset of samples was tested for quantitative expression with Real-Time PCR. Sequencing analysis showed two new mutations in two glioma patients, one of hABH2 coding sequence (I141 V) and the other of hABH3 (D189 N). The mutation at codon 189 falls in a crucial region of the protein. All subjects analyzed by Real-Time PCR showed an enhanced expression of the two genes, particularly of hABH2. This is the first study of hABH2 and hABH3 in pediatric brain tumors; further molecular investigations of their mutations and expression may help determine their role in response to chemotherapy.

Cytophagic histiocytic panniculitis is a rare disease, associated with either nonmalignant conditions or subcutaneous panniculitis-like T-cell lymphoma, and often also associated with hemophagocytic lymphohistiocytosis (HLH). We report the case of a 11-year-old boy with a history of secondary HLH who, after a local trauma, developed a painful, indurated plaque over the right thigh associated with relapsing HLH. Histopathologic findings from skin biopsy specimens revealed significant lobular panniculitis with benign histiocytes showing hemophagocytosis. High-dose intravenous methylprednisolone and cyclosporine A treatment was highly effective. A genetic study after a new, relapsing episode of HLH revealed an heterozygous missense mutation on STX 11 gene inherited from the mother.

Background Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening, heterogeneous, hyperinflammmatory disorder. Prompt identification of inherited forms resulting from mutation in genes involved in cellular cytotoxicity can be crucial. X-linked lymphoproliferative disease 1 (XLP1), due to mutations in SH2D1A (Xq25) encoding signaling lymphocyte activation molecule–associated protein (SAP), may present with HLH. Defective SAP induces paradoxical inhibitory function of the 2B4 coreceptor and impaired natural killer (NK) (and T) cell response against EBV-infected cells. Objective To characterize a cohort of patients with HLH and XLP1 for SAP expression and 2B4 function in lymphocytes, proposing a rapid diagnostic screening to direct mutation analysis. Methods We set up rapid assays for 2B4 function (degranulation or 51 Cr-release) to be combined with intracellular SAP expression in peripheral blood NK cells. We studied 12 patients with confirmed mutation in SH2D1A and some family members. Results The combined phenotypic/functional assays allowed efficient and complete diagnostic evaluation of all patients with XLP1, thus directing mutation analysis and treatment. Nine cases were SAP − , 2 expressed SAP with mean relative fluorescence intensity values below the range of healthy controls (SAP dull ), and 1, carrying the R55L mutation, was SAP + . NK cells from all patients showed inhibitory 2B4 function and defective killing of B-EBV cells. Carriers with SH2D1A mutations abolishing SAP expression and low percentage of SAP + cells showed neutral 2B4 function at the polyclonal NK cell level. Three novel SH2D1A mutations have been identified. Conclusions Study of SAP expression is specific but may have insufficient sensitivity for screening XLP1 as a single tool. Combination with 2B4 functional assay allows identification of all cases.

Autoimmune lymphoproliferative syndrome (ALPS) is caused by genetic defects decreasing Fas function and is characterized by lymphadenopathy/splenomegaly and expansion of CD4/CD8 double-negative T cells. This latter expansion is absent in the ALPS variant named Dianzani Autoimmune/lymphoproliferative Disease (DALD). In addition to the causative mutations, the genetic background influences ALPS and DALD development. We previously suggested a disease-modifying role for the perforin gene involved in familial hemophagocytic lymphohistiocytosis (FHL). The UNC13D gene codes for Munc13-4, which is involved in perforin secretion and FHL development, and thus, another candidate for a disease-modifying role in ALPS and DALD. In this work, we sequenced UNC13D in 21 ALPS and 20 DALD patients and compared these results with sequences obtained from 61 healthy subjects and 38 multiple sclerosis (MS) patients. We detected four rare missense variations in three heterozygous ALPS patients carrying p.Cys112Ser, p.Val781Ile, and a haplotype comprising both p.Ile848Leu and p.Ala995Pro. Transfection of the mutant cDNAs into HMC-1 cells showed that they decreased granule exocytosis, compared to the wild-type construct. An additional rare missense variation, p.Pro271Ser, was detected in a healthy subject, but this variation did not decrease Munc13-4 function. These data suggest that rare loss-of-function variations of UND13D are risk factors for ALPS development.

Cytotoxic T lymphocytes, natural killer cells, and NKT cells are effector cells able to kill infected cells. In some inherited human disorders, a defect in selected proteins involved in the cellular cytotoxicity mechanism results in specific clinical syndromes, grouped under the name of familial hemophagocytic lymphohistiocytosis. Recent advances in genetic studies of these patients has allowed the identification of different genetic subsets. Additional genetic immune deficiencies may also induce a similar clinical picture. International cooperation and prospective trials resulted in refining the diagnostic and therapeutic approach to these rare diseases with improved outcome but also with improved knowledge of the mechanisms underlying granule-mediated cellular cytotoxicity in humans.

Atypical vascular lesions (AVL) occurring at the site of radiotherapy represent an uncommon but well-documented complication in the setting of breast-conserving therapy for breast carcinoma. Although the biological behaviour of AVL has been regarded as benign, it has been suggested that AVL may represent a precursor of angiosarcoma. A better understanding of the biology of AVL is essential in order to assess appropriate patient management. The aim of the present study was to investigate alterations of tumour suppressor gene TP53 in a series of radiation-induced AVL and angiosarcomas (AS).Direct sequencing analysis of the TP53 gene showed the presence of at least one variation in 10 of 12 (83.3%) AVL and in seven of eight (87.5%) AS. The most common alteration in both categories was the P72R polymorphism in exon 4. One angiosarcoma sample carried a pathogenetically relevant disruptive mutation c.592delG, a frameshift deletion in exon 6, causing a premature stop codon.The presence of TP53 alterations suggests that its mutational inactivation may be implicated in the pathogenesis of radiation-associated vascular proliferations. The common mutational pathway suggested by our data supports the hypothesis that AVL and AS are biologically related entities, most probably representing the extremes of a morphological continuum.

Background Familial haemophagocytic lymphohistiocytosis (FHL) is a rare immune deficiency with uncontrolled inflammation; the clinical course usually starts within the first years of life, and is usually fatal unless promptly treated and then cured with haematopoietic stem cell transplant. FHL is caused by genetic mutations resulting in defective cell cytotoxicity; three disease related genes have been identified to date: perforin, Munc13-4 and syntaxin-11. A fourth gene, STXBP2 , has been identified very recently as responsible for a defect in Munc18-2 in FHL-5. Aims To describe the result of the screening of families with HLH and previously unassigned genetic defects. Methods Patients with HLH diagnosed according to current diagnostic criteria, and who lacked mutations in the PRF1, Munc13-4, and STX11 genes were sequenced for mutations in STXBP2. Functional study was performed when material was available. Results Among the 28 families investigated, 4 (14%) with biallelic STXBP2 mutations were identified. They originated from Italy, England, Kuwait and Pakistan. The p.Pro477Leu resulting from c.1430C>T, and p.Arg405Gln resulting from the single c.1214G>A nucleotide change are known, while we contribute two novel mutations: p.Glu132Ala resulting from c.395A>C, and p.Gly541Ser, resulting from c.1621G>A. The detrimental effect of the p.Gly541Ser mutation was documented biochemically and functionally in NK and CD8 cells. Additional polymorphisms are also described. Conclusion These data expand current knowledge on the genetic heterogeneity of FHL and suggest that patients with FHL5 may have different results in degranulation assays under different conditions.

Insufficient response to oral temozolomide (TMZ) in children with brain tumor may depend on the repair-action of inducible O6-methylguanine-DNA methyltransferase (MGMT). To investigate the clinical relevance of MGMT expression, we analyzed MGMT levels by qRT-PCR and immunohistochemistry, and the methylation of gene promoter in patients with relapsed or refractory brain tumor, enrolled in an off-label trial with oral temozolomide. The drug was administered at the dose of 200 mg/m(2)/day in patients with no prior cranio-spinal irradiation, and 180 mg/m(2)/day in those with previous radiotherapy and/or high-dose chemotherapy followed by autologous hematopoietic stem cell rescue. Nine patients with recurrent ependymoma (n=3), low grade glioma (n=3), glioblastoma (n=1), relapsed medulloblastoma (n=2) were enrolled in the study. Median absolute MGMT mRNA expression level standardized for GAPDH was 1.06 (range -0.453 to 3.932). The median relative expression level (RQ=2(-ddC)) was 4.29 (range 1.585 to 12.228). By immunohistochemistry, the score was 2+ in 6 of the 9 tumor samples, and 1+ in 3, while none was MGMT negative. Methylation of MGMT promoter was detected in only one ependymoma sample. The heterogeneous PFS in our patients treated with second line TMZ, indicates that MGMT expression alone is insufficient to predict the response to TMZ, presumably because of the DNA repair mechanisms involved.

Embryonal tumors are a group of malignant neoplasms that most commonly affect the pediatric population. Embryonal tumor with abundant neuropil and true rosettes is a recently recognized rare tumor. It is composed of neurocytes and undifferentiated neuroepithelial cells arranged in clusters, cords and several types of rosettes in a prominent neuropil-rich background. We describe a new case of this tumor. The patient, a 24-month-old female infant, was referred to the Meyer Children's Hospital with a history of right brachio-crural deficit associated with occasional episodes of headache and vomiting. Computed tomography scan and MRI revealed a large bihemispheric mass. The patient underwent two consecutive surgeries. The resultant surgical resection of the tumor was macroscopically complete. The postoperative period was uneventful. On light microscopy the tumor showed a composite morphology: embryonal tumor with abundant neuropil and true rosettes (specimen from the first surgery); medulloepithelioma with mesenchymal and epithelial areas (specimen from the second surgery). The immunohistochemistry evidenced the heterogeneous (neuronal, mesenchymal and epithelial) immunoprofile of tumoral cells. By real-time polymerase chain reaction (RT-PCR), the PTEN gene expression in the tumor was lower than in the five non-neoplastic brain tissues used as control. Mutation analysis did not show any variation in INI-1 and PTEN sequence while P53 analysis showed the presence of homozygote P72R variation. Fluorescent in situ hybridization analysis showed polysomy of chromosome 2 while amplification of N-MYC was not detected. Owing to the rarity of embryonal tumor with abundant neuropil and true rosettes, each new case should be recorded to produce a better clinical, pathological and molecular characterization of this lesion.

Microsatellite instability (MSI) is present in hereditary conditions due to mismatch repair (MMR) gene mutations. Following MSI analysis, tumor samples are classified into MSS (stable), MSI-L (low instability), and MSI-H (high instability) based on the fraction of unstable loci. Another MSI-based classification takes into account the size difference between mutant alleles in tumor DNA compared to wild-type alleles; two types of MSI, A and B, are recognized using this approach, type A being characterized by smaller, more subtle allelic shifts compared to type B. Biallelic mutations of MMR genes are associated with pediatric cancers, including glial tumors, in Turcot syndrome type 1 (TS1). However, most TS1-associated gliomas so far analyzed did not display MSI. We investigated the frequency of MSI in a series of 34 pediatric gliomas of different grade using a panel of five mononucleotide quasimonomorphic markers. Subtle qualitative changes were observed for the majority of markers in two glioblastomas (5.9% of the total series and 33.3% of glioblastomas). In both cases, family histories were compatible with TS1, and mutations of the PMS2 and MLH1 genes were identified. In one family, the MSI patterns were compared between the glioblastoma and a colon cancer from an affected relative, showing a clear qualitative difference, with the former displaying type A and the latter type B instability, respectively. These results were confirmed using additional microsatellite markers, indicating that knowledge of the association between TS1-related glial tumors and subtle type A MSI is important for full ascertainment of TS1 patients and appropriate counselling.

Patients who survive retinoblastoma are at risk for developing additional malignant neoplasms, including tumors of the sinonasal tract. We report 2 cases of secondary sinonasal malignancy arising in pediatric patients previously treated for retinoblastoma, with features of neuroendocrine carcinoma. Both lesions were characterized by a proliferation of round to oval cells arranged in solid sheets, trabeculae, and nests, diffusely infiltrating nasal mucosa and bone tissue. Immunohistochemically, they were diffusely positive for epithelial markers, as well as for neuroendocrine markers and for TP53 and retinoblastoma gene products. TP53 gene analysis showed the presence of a missense mutation P72R (CCC/CGC) and a single nucleotide polymorphism P36P (CCG/CCA) in exon 4 in 1 case. Literature review revealed 5 previously reported cases, all showing primitive undifferentiated morphology with variable expression of neural and epithelial markers. These tumors represent a peculiar subset of undifferentiated sinonasal neoplasms with extremely aggressive clinical behavior.

Background Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease affecting mostly children but also adults and characterized by hyperinflammatory features. A subset of patients, referred to as having familial hemophagocytic lymphohistiocytosis (FHL), have various underlying genetic abnormalities, the frequencies of which have not been systematically determined previously. Objective This work aims to further our understanding of the pathogenic bases of this rare condition based on an analysis of our 25 years of experience. Methods From our registry, we have analyzed a total of 500 unselected patients with HLH. Results Biallelic pathogenic mutations defining FHL were found in 171 (34%) patients; the proportion of FHL was much higher (64%) in patients given a diagnosis during the first year of life. Taken together, mutations of the genes PRF1 (FHL2) and UNC13D (FHL3) accounted for 70% of cases of FHL. Overall, a genetic diagnosis was possible in more than 90% of our patients with FHL. Perforin expression and the extent of degranulation have been more useful for diagnosing FHL than hemophagocytosis and the cytotoxicity assay. Of 281 (56%) patients classified as having "sporadic" HLH, 43 had monoallelic mutations in one of the FHL-defining genes. Given this gene dosage effect, FHL is not strictly recessive. Conclusion We suggest that the clinical syndrome HLH generally results from the combined effects of an exogenous trigger and genetic predisposition. Within this combination, different weights of exogenous and genetic factors account for the wide disease spectrum that ranges from HLH secondary to severe infection to FHL.

MAS is a severe complication of rheumatic diseases, mostly sJIA. Clinical and laboratory features are similar to those of FHL resulting from mutations in selected genes involved in the cytotoxicity pathway.

Macrophage Activation Syndrome (MAS) is a severe complication of rheumatic diseases, frequently associated with systemic juvenile idiopathic arthritis (sJIA), but also described in others pediatric inflammatory disorders including Juvenile Systemic Lupus Erythematosus (SLE) and Kawasaki disease. Due to the close resemblance to a group of histiocytic disorders collectively known as hemophagocytic lymphohistiocytosis (HLH) it is currently classified among the secondary or acquired forms of HLH, and the term rheu-HLH has been proposed.

Introduction. Pax-5 gene codifies for a transcription factor central to B-cell differentiation and function, expressed during the early stage of differentiation and alternatively spliced during B-cell development. In addition to the full-length isoform (Pax-5a), isoforms arising from the inclusion or exclusion of exons 2, 7, 8, and/or 9, and lacking the DNA-binding and the transactivating domains, have been described. These isoforms and their levels of expression increase during B-cell maturation. In particular, Pax-5b isoform (deleted of exon 2), resulting in protein with partial DNA-binding domain, is related with the differentiation stage. Recently, mutations of Pax-5 gene were reported in 31.7% of 242 children (Mullighan, Nature 2007) and 30% of 119 adults (Familiades, ASH 2007 abs. 2806) with B-cell precursor acute lymphoblastic leukemia (ALL). We investigated Pax-5 gene mutations and isoforms expression in patients with ALL at the diagnosis and in selected cell populations from control subjects. Methods. Pax-5 mutations and mRNA isoforms were investigated by sequence analysis. Pax-5a and Pax-5b expression levels were evaluated by quantitative PCR in leukemic cells. Total RNA was obtained from leukemic blasts of 95 patients with B-cell precursor ALL at diagnosis, 45 adults and 50 children. These last had been selected according to the presence of adverse translocations: BCR/ABL p190 (n=10), E2A-PBX1 (n=10), TEL/AML (n=10), MLL/AF4 (n=10), or none (n=10). mRNA was obtained from purified peripheral blood CD19+ lymphocytes (6 samples), from bone marrow CD34+ hematopoietic progenitor cells (6 samples), and pro-B CD34+CD19+ cells (3 samples) from a total of 8 healthy bone marrow donors. Results. Pax-5 gene point mutations were found in 8/45 adults (18%) and 14/50 children (28%) (p=n.s.), resulting in a total of 20 different mutations. Mutations were located as follows: exon 2 (53G>C, 76delG, 101C>G, 113G>A, 197G>A), exon 3 (223A>G, 247delA, 296T>C), exon 4 (446A>T, 526G>A), exon 5 (580A>C, 601G>A), exon 6 (625C>T, 716G>A), exon 7 (836C>A), exon 8 (955G>A, 964insC, 971A>G), exon 9 (1058C>T), exon 10 (1133G>A). There was no correlation between the presence of mutations and the listed genetic subtypes. Pax-5 alternative splicing was observed in 47/95 cases (49%): 29/45 adults (64%) and 18/50 children (36%) (p=n.s.). Alternative splicing resulted in increased frequencies of the Pax-5b isoform and the deletion of exons 8 and/or 9; furthermore, a novel isoform resulting from the skipping of exon 5 was documented. On the contrary, only the fulllenght isoform was present in CD34+ progenitors and CD34+CD19+ cells from healthy donors, in which deleted isoforms were detected only at the stage of mature B-lymphocytes. Conclusion: Pax-5 mutations are frequent in childhood and adult ALL and are scattered all over the gene. We extend the current knowledge on their pathogenic role by demonstrating that alternative splicing is a common event in these patients. Imbalance between the fulllength and the Pax-5b isoforms is expected to affect the maturation of the B-cell and its propensity to apoptosis (Robichaud Nucleic Acids Res 2008), thus contributing to the process of leukemogenesis. Based on our findings, this mechanism acts broadly and is independent of the most common ALL genetic translocations.