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BackgroundRhabdomyosarcoma (RMS) is rare cancer affecting children and adults. Pleomorphic RMS histology is almost exclusive to adult patients and often resistant to chemotherapy.ObjectiveWe report the case of a 19-year-old patient who presented with a metastatic chemoresistant pleomorphic RMS.MethodsConsidering the poor prognosis and the few systemic therapeutic options, we decided to carry out a whole exome sequencing (WES) of the tumour and germline DNA.ResultsWES identified a germline variation (c.1863_1864insT) in the MLH1gene corresponding to a pathogenic mutation: (p. Leu622Serfs*10), whereas the family history did not fit with classical criteria for Lynch syndrome. Loss-of-heterozygosity at MLH1locus was found in the tumour. Immunohistochemistry showed loss of MLH1 and PMS2 nuclear expression in the tumour cells. In view of the mismatch repair defects and a high programmed cell death ligand 1 (PD-L1) expression (60% of tumour cells expressed PD-L1), we administrated an anti-PD-1 antibody to the patient. He achieved a rapid complete response of the lung metastases, which appears sustained after a 1-year follow-up.ConclusionThis observation of an RMS revealing an unexpected Lynch syndrome underlines the overlap between tumorous and germline molecular genetics and emphasises the major impact of cancer genomic medicine in clinical practice for guiding treatment decision.

We report the case of a patient with a BRCA2germline mutation who developed a localized pleomorphic soft tissue sarcoma of the leg with poor prognostic features. BRCA2germline mutations were not previously reported to be associated with pleomorphic sarcoma. BRCA2loss-of-heterozygosity was found in the tumor, resulting in a complete BRCA2loss-of-function. BRCA2deficiency is associated with sensitivity to cisplatin-based chemotherapy in breast and ovarian cancer patients. We used a cisplatin-based chemotherapy. A rapid major partial response was obtained, which allowed a curative and conservative surgical resection of the sarcoma followed by adjuvant irradiation. This case illustrates that sarcoma patients may present unexpected but targetable genetic abnormalities and that BRCA2loss-of-function may be targetable in sarcoma as it is associated with enhanced sensitivity to cisplatin. Our observation emphasizes the input of genomic medicine in clinical practice, its importance for treatment decisions, and the overlap between constitutional and somatic genetics.

Pangenomic studies identified distinct molecular classes for many cancers, with major clinical applications. However, routine use requires cost-effective assays. We assessed whether targeted next-generation sequencing (NGS) could call chromosomal alterations and DNA methylation status. A training set of 77 tumors and a validation set of 449 (43 tumor types) were analyzed by targeted NGS and single-nucleotide polymorphism (SNP) arrays. Thirty-two tumors were analyzed by NGS after bisulfite conversion, and compared to methylation array or methylation-specific multiplex ligation-dependent probe amplification. Considering allelic ratios, correlation was strong between targeted NGS and SNP arrays (r = 0.88). In contrast, considering DNA copy number, for variations of one DNA copy, correlation was weaker between read counts and SNP array (r = 0.49). Thus, we generated TARGOMICs, optimized for detecting chromosome alterations by combining allelic ratios and read counts generated by targeted NGS. Sensitivity for calling normal, lost, and gained chromosomes was 89%, 72%, and 31%, respectively. Specificity was 81%, 93%, and 98%, respectively. These results were confirmed in the validation set. Finally, TARGOMICs could efficiently align and compute proportions of methylated cytosines from bisulfite-converted DNA from targeted NGS. In conclusion, beyond calling mutations, targeted NGS efficiently calls chromosome alterations and methylation status in tumors. A single run and minor design/protocol adaptations are sufficient. Optimizing targeted NGS should expand translation of genomics to clinical routine.

Neurofibromatosis type-1 is a genetic disorder caused by loss-of-function variants in the tumor-suppressor NF1. Approximately 4% to 11% of neurofibromatosis type-1 patients have a NF1locus complete deletion resulting from nonallelic homologous recombination between low copy repeats. Codeleted genes probably account for the more severe phenotype observed in NF1-deleted patients. This genotype–phenotype correlation highlights the need for a detailed molecular description. A droplet digital PCR (ddPCR) set along the NF1locus was designed to delimitate the three recurrent NF1deletion breakpoints. The ddPCR two mixes-set in 121 samples from nonrelated NF1-deleted patients was tested. Classification based on ddPCR versus multiplex ligation-dependent probe amplification (MLPA) was compared. In addition, microsatellites were analyzed to identify parental origin of deletions. ddPCR identified 77 type-1 (64%), 20 type-2 (16%), 7 type-3 (6%), and 17 atypical deletions (14%). The results were comparable with MLPA, except for three atypical deletions misclassified as type-2 using MLPA, for which the SUZ12gene was not deleted. A significant maternal bias (25 of 30) in the origin of deletions was identified. A fast and efficient ddPCR quantification to allow fine NF1deletion classification is proposed; ddPCR can be implemented easily into routine diagnosis to complement the techniques dedicated to NF1point variant identification. This new tool may help unravel the genetic basis conditioning phenotypic variability in NF1-deleted patients and offer tailored genetic counseling.

Churg-Strauss syndrome (CSS) is characterized by systemic vasculitis and blood and tissue eosinophilia. Blood eosinophilia correlates with disease activity, and activated T cells from CSS patients are predominantly T helper 2 (Th2). Interleukin (IL)-25 has been shown to link innate and adaptive immunity by enhancing Th2 cytokine production. We sought to determine the involvement of IL-25 and its receptor IL-17RB in the pathogenesis of CSS. We found increased levels of IL-25 in the serum of active CSS patients (952 ± 697 vs 75 ± 49 pg/mL in inactive patients and 47 ± 6 pg/mL in healthy donors). IL-25 was correlated with disease activity and eosinophil level. Eosinophils were the main source of IL-25, whereas activated CD4+ memory T cells were the IL-17RB–expressing cells in CSS. IL-25 enhanced the production of IL-4, IL-5, and IL-13 by activated peripheral blood mononuclear cells. IL-25 and IL-17RB were observed within the vasculitic lesions of patients with CSS, and IL-17RB colocalized with T cells. Increased expression of IL-17RB, tumor necrosis factor receptor–associated factor 6, and JunB in vasculitic lesions of CSS underscored the IL-25–mediated activation, whereas up-regulation of GATA3 and IL-10 supported Th2 differentiation. Our findings suggest that eosinophils, through the production of IL-25, exert a critical role in promoting Th2 responses in target tissues of CSS.

Churg-Strauss syndrome (CSS) is characterized by systemic vasculitis and blood and tissue eosinophilia. Blood eosinophilia correlates with disease activity, and activated T cells from CSS patients are predominantly T helper 2 (Th2). Interleukin (IL)-25 has been shown to link innate and adaptive immunity by enhancing Th2 cytokine production. We sought to determine the involvement of IL-25 and its receptor IL-17RB in the pathogenesis of CSS. We found increased levels of IL-25 in the serum of active CSS patients (952 ± 697 vs 75 ± 49 pg/mL in inactive patients and 47 ± 6 pg/mL in healthy donors). IL-25 was correlated with disease activity and eosinophil level. Eosinophils were the main source of IL-25, whereas activated CD4+memory T cells were the IL-17RB–expressing cells in CSS. IL-25 enhanced the production of IL-4, IL-5, and IL-13 by activated peripheral blood mononuclear cells. IL-25 and IL-17RB were observed within the vasculitic lesions of patients with CSS, and IL-17RB colocalized with T cells. Increased expression of IL-17RB, tumor necrosis factor receptor–associated factor 6, and JunB in vasculitic lesions of CSS underscored the IL-25–mediated activation, whereas up-regulation of GATA3 and IL-10 supported Th2 differentiation. Our findings suggest that eosinophils, through the production of IL-25, exert a critical role in promoting Th2 responses in target tissues of CSS.

During liver fibrogenesis, quiescent hepatic stellate cells switch their phenotype toward a myofibroblastic-like pattern with a gain in motility. Here, we show that SCG10 (superior cervical ganglia 10) mRNA expression, a microtubule-destabilizing protein that favors cell growth and motility in neurons, both increases and correlates with the stage of fibrosis in patients with chronic hepatitis C. We also show the de novoexpression of SCG10 mRNA in two rat models of liver fibrosis. We demonstrate that activated hepatic stellate cells appear to be the major cellular sources of SCG10 in the liver. Tracking of the SCG10 pathway in hepatic stellate cells shows that SCG10 initially accumulates in the perinuclear Golgi area then migrates in small vesicle-like structures along individual microtubules. Moreover, SCG10 vesicles cluster at the distal ends of microtubules in areas where tubules are spread and decompacted, suggesting their preferential association with destabilized and dynamic microtubules. Inhibition of SCG10 expression by gene-specific short interfering RNA in primary rat hepatic stellate cells is associated with a significant reduction in microtubule-dependent cellular functions, such as proliferation and migration. In conclusion, the de novoexpression of SCG10 by hepatic stellate cells may play a major role in cellular mechanisms associated with HSC activation, namely cell motility and division, through interference with microtubules. SCG10 may represent a potential molecular target for anti-fibrosis therapies.

Background The aim of this study was to assess the influence of hepatitis B virus (HBV) genotypes on serum hepatitis B surface antigen (HBsAg) kinetics in hepatitis B e antigen (HBeAg)-negative patients treated with pegylated interferon-a2a (PEG-IFN-a2a).Methods A total of 48 consecutive patients treated with PEG-IFN-a2a (180 µg/week) for 48 weeks were assessed. HBV genotype was determined. Serum HBV DNA and HBsAg were assessed at baseline, during treatment (weeks 12, 24 and 48) and during follow-up (weeks 72 and 96).Results The distribution of HBV genotype was A 27%, B 17%, C 12%, D 29% and E 14%. Mean ±sdpretreatment serum HBV DNA (6.9 ±1.5 log10copies/ml) was not different between genotypes and decreased under treatment in all genotypes without significant difference. Mean ±sdpretreatment serum HBsAg (3.6 ±0.6 log10 IU/ml) was significantly different between genotypes (P<0.001), with high levels in genotypes A and C, intermediate levels in genotypes D and E, and low levels in genotype B (4.0 ±0.3, 4.1 ±0.7, 3.6 ±0.5, 3.6 ±0.4 and 2.7 ±0.6 log10IU/ml, respectively). Serum HBsAg decreased under treatment in all genotypes with a significant difference. At the end of treatment, mean ±sddecrease was high in genotype A, intermediate in genotypes B and D, and low in genotypes C and E (1.3 ±1.6, 0.7 ±0.7, 0.6 ±0.9, 0.4 ±1.0 and 0.4 ±0.9 log10IU/ml, respectively; P<0.001). During follow-up, serum HBsAg continued to decrease in genotypes A and D, whereas rebound was observed in genotypes B, C and E.Conclusions HBV genotype has a strong influence on serum HBsAg kinetics during PEG-IFN-a2a therapy in HBeAg-negative patients.

In 5% to 10% of cases, neurofibromatosis type 1 is caused by microdeletions scattered across the entire NF1gene and various neighboring genes. The phenotype appears to be more severe in patients with NF1microdeletions than in patients with NF1single point mutations. We have developed a new method for detecting and characterizing NF1microdeletions based on a custom high-resolution oligonucleotide array comparative genomic hybridization by using the custom 8x15K Agilent array format. The array comprised a total of 14,207 oligonucleotide probes spanning the whole of chromosome 17, including 12,314 probes spanning an ∼8 Mb interval surrounding the NF1locus. We validated this approach by testing NF1microdeleted DNA samples previously characterized by means of microsatellites and real-time PCR methods. Our array comparative genomic hybridization provided enough information for subsequent long-range PCR and nucleotide sequencing of the microdeletion endpoints. Unlike previously described methods, our array comparative genomic hybridization was able to unambiguously differentiate between the three types of microdeletions (type I, type II, and atypical) and to characterize atypical microdeletions. Further comparative studies of patients with well-characterized genotypes and phenotypes and different microdeletions sizes and breakpoints will help determine whether haploinsufficiency of deleted genes and/or genes rearrangements influence clinical outcomes.

Retinal ischemia is a major cause of visual impairment and is associated with a high risk of subsequent ischemic stroke. The retina and its projections are easily accessible for experimental procedures and functional evaluation. We created and characterized a mouse model of global and transient retinal ischemia and provide a comprehensive chronologic profile of some genes that display altered expression during ischemia.

L’identification des mutations à l’origine de maladies génétiques chez l’homme a pris ces dernières années un essor considérable. Il est devenu possible d’établir le spectre des mutations délétères pour une maladie génétique donnée, et des bases de données internationales sont aujourd’hui accessibles viale réseau Internet. Le diagnostic génotypique des maladies héréditaires occupe actuellement une place prépondérante en matière de conseil génétique et de diagnostic prénatal. La connaissance du type de mutation délétère et des mécanismes en cause est essentielle pour déterminer la stratégie de diagnostic moléculaire adaptée à chaque situation. Cet article a pour objectif de présenter les différents types de mutations responsables de maladies génétiques (substitutions nucléotidiques, délétions ou insertions de petite taille, mutations dynamiques, grands remaniements…) et de récapituler les connaissances actuelles concernant les mécanismes moléculaires à l’origine de ces mutations. Leurs conséquences sur l’expression du gène (transcription et maturation du transcrit) et sur la fonction de la protéine sont également abordées dans cet article.

BackgroundHuman chorionic gonadotrophin beta (hCGβ) is expressed in several non-trophoblastic tumours, and this is usually associated with aggressive behaviour. Little is known about hCGβ expression in Barrett’s adenocarcinoma.Materials and methodsWe determined the hCGβ profile in a large series of surgically resected Barrett’s adenocarcinoma (a) at mRNA level using real-time quantitative reverse-transcription polymerase chain reaction analysis and (b) at protein level using immunohistochemistry with a polyclonal antibody and with a monoclonal antibody specific for free hCGβ. We then sought links between the hCGβ protein expression pattern and clinical and pathological parameters, including patient outcome as well as vascular endothelial growth factor (VEGF) expression.ResultshCGβ protein expression was observed in 43 of 76 (57%) Barrett’s adenocarcinomas. We showed a strong correlation between hCGβ protein abundance and CGB mRNA level. We observed a statistical link between hCGβ protein expression and infiltrative tumour type ( P=0.023), perineural neoplastic invasion ( P=0.007) and VEGF protein expression ( P=0.016). hCGβ expression tended to be associated with a poor outcome (16% versus 36% survival 8 years after resection).ConclusionExpression of hCGβ correlates with specific infiltrative characteristics and is associated with higher VEGF expression. Both molecules may play a co-ordinated role in the development of Barrett’s adenocarcinomas.

The PEA3/E1AF/ETV4 gene encodes an Ets-related transcription factor that is expressed in the epithelial cells of the mammary gland. Previous reports have shown that PEA3 can up-regulate promoter activities of many genes associated with tumorigenesis. A significant fraction of those encode matrix metalloproteinases (MMP genes) required for degradation of the extracellular matrix. To better obtain a molecular characterization of PEA3 expression in sporadic breast cancer, we quantified PEA3 mRNA by means of real-time reverse transcriptase–polymerase chain reaction assay in a large series of human primary breast tumors. PEA3 expression showed wide variations in tumor tissues, being under-expressed in 30 of 130 (23.1%) and over-expressed in 18 of 130 (13.8%) compared with normal breast tissues. High PEA3 mRNA levels correlated significantly with Scarff–Bloom–Richardson histopathological grade III (P = 0.018) but not with poor prognosis, suggesting that PEA3 is a marker of tumor aggressiveness rather than a prognostic factor in human breast cancer. We also observed positive links between the expression of PEA3 and those of MKI67 and ERBB2 (P = 0.034 and P = 0.045, respectively) and an inverse relationship with ERα (P = 0.0016). Our results do not support recent findings suggesting that PEA3 could be a tumor-suppressor gene that can act therapeutically in ERBB2 over-expressed tumors. Our results also suggest major roles of the MMP2, NRG1 and CGB genes (which encode type I gelatinase, heregulin and human chorionic gonadotropin β subunit, respectively) in the PEA3 pathway dysregulation observed in breast cancer. Taken together, the data confirm the role of the PEA3 gene in breast tumorigenesis, and suggest the existence of numerous other still unknown genes transactivated by the PEA3 transcription factor.

Benign prostatic hyperplasia (BPH) is characterized by a hyperplastic growth of epithelial and stromal cells in the prostate. Despite the high prevalence of the disease little is known regarding the molecular etiology of BPH. Therefore, a comparison of gene expression patterns between normal prostate, BPH and prostate cancer could provide insights into the pathogenic mechanisms of the disease and identify candidate genes that could be targeted for therapeutic use.

The aim of this study was to develop and validate a molecular index for the diagnosis of hepatocellular carcinoma (HCC) based on genes whose specificity and level of expression are the most discriminating for the diagnosis of HCC. The level of expression of 219 genes was assessed with a real-time reverse transcription-polymerase chain reaction approach in a training set of samples including normal livers (15), cirrhosis (12), and HCC (16). The most informative genes were selected for the molecular index. This index was prospectively validated in a new set of 40 samples (testing set) and in a set of 45 cirrhotic macronodules. 44 out of the 219 genes were differentially expressed in HCC. 13 out of these 44 genes were finally selected for the molecular index according to their diagnostic performance and after exclusion of most redundant genes. Using this index, 42 out of 43 samples of the training set and 39 out of the 40 samples of the testing set were correctly ranked as HCC or not HCC (normal liver or cirrhosis). The index also enabled correct ranking of 44 out of 45 cirrhotic macronodules into 2 groups: benign (including macroregenerative and dysplastic macronodules) and malignant macronodules. This molecular diagnostic index is an efficient tool both for identification of overt HCC as well as minute lesions (cirrhotic macronodules). It might be useful to correctly diagnose borderline lesion or small well-differentiated hepatocellular carcinomas whose diagnosis is often difficult on a histopathological basis.

Invasive cytotrophoblasts play a key role in the development of human placenta and is therefore essential for subsequent development of the embryo. Human implantation is characterized by a major trophoblastic invasion that offers a unique model of a controlled and oriented tumor-like process. The ligand-activated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) modulates cell growth and differentiation and might be therefore considered as a tumor suppressor. We have recently reported that PPARgamma, in synergy with its dimerization partner retinoid X receptor (RXR)alpha, controls the invasion of human primary cytotrophoblasts. Because these cells are unable to replicate in culture, we have, in the present study, transformed these primary cells with the simian virus 40 large T antigen for studying the role of PPARgamma in cell invasion process. Our results show that the cell line human invasive proliferative extravillous cytotrophoblast (HIPEC) 65 expressed markers of human invasive primary cytotrophoblast as determined by immunocytochemistry, immunobloting and real-time RT-PCR, and were highly invasive in vitro. We have next studied the role of PPARgamma/RXRalpha heterodimers in cell proliferation and invasion. Our results show that PPARgamma and RXRalpha are co-expressed by HIPEC 65 and that, as commonly observed, activation of PPARgamma/RXRalpha heterodimers with the specific PPARgamma agonist rosiglitazone induced lipid droplet accumulation as revealed by oil red O staining. Treatment with rosiglitazone or with the natural PPARgamma agonist 15-deoxy-delta-(12,14) PGJ2 did not modify cell growth, but interestingly, activation of PPARgamma by this synthetic (rosiglitazone) or natural (15d-PGJ2) ligand markedly inhibited cell invasion in a concentration-dependent manner. Finally, we showed that other potential natural PPARgamma ligand such as oxidized-but not native-low-density lipoprotein inhibited cell invasion. This proliferative and invasive human cytotrophoblast cell line from extravillous origin provides a new tool for studying specifically the role of PPARgamma in the control of cell invasion.

The human insulin-family genes regulate cell growth, metabolism, and tissue-specific functions. Among these different members, only INSL4gene shows a predominant placenta-specific expression. Here, we show that the human INSL4gene is tightly clustered with three other members of the human insulin superfamily (RLN1, RLN2, and INSL6) within a 176-kilobase genomic segment on chromosome region 9p23.3–p24.1. We also report evidence that INSL4is probably the only insulin-like growth factor gene to be primate-specific. We identified an unexpected human endogenous retrovirus (HERV) element inserted into the human INSL4promoter with a sequence similar to that of envgene, flanked by two long terminal repeats(LTRs). The emergence of INSL4gene and genomic insertion of HERV appear to have occurred after the divergence of New World and Old World monkeys (∼45 million years ago). Transient transfection experiments showed that the placenta-specific expression of INSL4is mediated by the 3′ LTR of the HERV element, and that the latter may have a major role in INSL4up-regulation during human cytotrophoblast differentiation into syncytiotrophoblast. Finally, we identified an INSL4alternatively spliced mRNA species that encodes putative novel INSL4-like peptides. These data support the view that ancient retroviral infection may have been a major event in primate evolution, especially in the functional evolution of the human placenta.

The human placenta is characterized by extensive trophoblast invasion of the uterus. Indeed, extravillous cytotrophoblast cells invade the decidua and the upper third of uterine spiral arteries in the myometrium. This invasion is reflected in situ by the expression of specific markers. In order to study this invasion process, we have established an in vitro culture model of human extravillous trophoblast isolated from first trimester chorionic villi. The aim of this study was to investigate the effect of a composite matrix, the Matrigel required for the culture of this homogenous population of extravillous trophoblasts (EVCT), on their in vitro differentiation. The effect of Matrigel was studied on different markers characterized by immunocytochemistry and by real-time polymerase chain reaction assay of transcripts. In addition, the expression of 12 different matrix metalloproteases and their inhibitors were investigated. We show that human extravillous cytotrophoblasts acquire an invasive phenotype on Matrigel associated with a specific pattern of protease gene expression. This in vitro model will be of interest to study the cellular mechanisms involved in abnormal trophoblast invasion observed in poor placentation and preeclampsia.

Epstein‐Barr virus (EBV) reactivation is more likely to occur in immunocompromised patients with subsequent higher susceptibility to EBV‐associated lymphoproliferations. In contrast to transplant recipients, limited data are available concerning the EBV load in HIV‐infected patients, with or without AIDS‐related non‐Hodgkin's lymphomas. We developed a TaqMan real‐time PCR assay, allowing both the EBV genome and a cellular gene to be quantified in order to obtain a reliable normalized measurement of the EBV load in peripheral blood mononuclear cells (PBMCs). With a wide 6‐log10quantification range and inter‐assay variations of less than 24%, this quantitative PCR was sufficiently accurate and reproducible for routine follow‐up. The EBV load was determined in PBMCs from 113 HIV‐infected patients, 11 patients with primary HIV infection and 24 HIV‐seronegative healthy controls. The rates of EBV detection were similar in the three groups. However, EBV loads were higher in the HIV‐infected group (P< 0.00001) except for the patients with primary HIV infection. Unexpectedly, EBV loads were not correlated with the clinical stages of HIV infection or HIV replication, and did not depend on the degree of immunodepression, as judged by CD4+counts. This study contributes towards the definition of the baseline EBV load during HIV infection and stresses the broad inter‐individual variability of the EBV load in HIV‐infected patients. Real‐time PCR provides a useful tool that can be used in further longitudinal studies to assess the relevance of the EBV load to identify HIV‐infected patients with a high risk of EBV‐associated lymphoproliferations. J. Med. Virol. 65:543–552, 2001. © 2001 Wiley‐Liss, Inc.

Human telomerase is a specialized reverse transcriptase that catalyses telomeric repeat addition at the ends of chromosomes. Activation of this enzyme is one of the key steps in cell immortalization and carcinogenesis, and one of its components, hTERT, is considered as the rate‐limiting factor. While telomerase activity was found to be prognostically relevant in various cancers, results obtained from renal cell carcinomas (RCC) failed to show any correlation with the usual prognostic factors. The aim of the study was to reassess the role of telomerase and its hTERT component in the biological behaviour of RCC using new quantitative techniques, such as the quantitative evaluation of hTERT mRNA level by a real‐time RT‐PCR procedure and the mesuring of telomerase activity by an ELISA TRAP assay. Since experimental evidence supports a relationship between cell proliferation or c‐myc expression and telomerase, the proliferation index and c‐myc mRNA levels were also studied. Forty‐one RCC (29 conventional renal cell carcinomas (CRCC), 10 papillary RCC and two urothelial carcinomas) were studied. In 73% of cases, normalized hTERT mRNA expression was significantly higher in the tumour sample than in the normal tissue. Telomerase activity was detected in 63% of RCC, while corresponding normal tissue was always negative. Analysis of correlations showed firstly that both telomerase activity and hTERT mRNA level were lower in the group of CRCC versus non‐CRCC (TRAP: 0.3±0.1 versus 0.6±0.2, p<0.05; hTERT/PO mRNA: 5±3 versus 37±8, p<0.001, respectively); secondly, that in the group of CRCC, hTERT mRNA expression level was correlated with the stage of the tumour (p=0.01); and thirdly, that no correlation was observed between c‐myc mRNA level and hTERT mRNA level. In conclusion, these results support the involvement of telomerase in RCC and the potential interest of hTERT mRNA quantification. Copyright © 2001 John Wiley & Sons, Ltd.

Little is known of the function and clinical significance of the androgen receptor (AR) in human breast cancer. Paradoxically, synthetic progestins, such as medroxyprogesterone acetate, are used for second line hormone therapy of breast cancer following tamoxifen failure. A sensitive and accurate assay for AR expression in breast tumors is thus required. Here we have developed and validated a real-time RT–PCR assay to quantify AR gene expression at the mRNA level in a series of 131 patients with unilateral invasive primary breast tumors. AR expression varied widely in tumor tissues (by at least 3 orders of magnitude), being underexpressed in 24/131 (18.3%) and overexpressed in 45/131 (34.4%) relative to normal breast tissues. We observed links (or trends) between AR status and age, menopausal status, Scarff–Bloom–Richardson histopathological grade, lymph node status and estrogen receptor α and progesterone receptor status. High AR mRNA levels were negatively linked to MYC gene overexpression (P = 8×10–6), confirming previous in vitro studies. Our results also suggest a role of the ARA70 gene (which encodes a major AR co-activator) in the AR pathway dysregulation observed in breast cancer. This simple, rapid and semi-automated method will be useful for screening cancer patients for altered AR expression and for predicting the response to androgen therapy in AR-related cancer patients.

Using a protein truncation assay, we have identified a new mutation in the neurofibromatosis type 1 (NF1) gene that causes a severe defect in NF1pre-mRNA splicing. The mutation, which consists of a G to A transition at position +1 of the 5′ splice site of exon 12a, is associated with the loss of both exons 11 and 12a in the NF1mRNA. Through the use of in vivoand in vitrosplicing assays, we show that the mutation inactivates the 5′ splice site of exon 12a, and prevents the definition of exon 12a, a process that is normally required to stimulate the weak 3′ splice site of exon 12a. Because the 5′ splice site mutation weakens the interaction of splicing factors with the 3′ splice site of exon 12a, we propose that exon 11/exon 12a splicing is also compromised, leading to the exclusion of both exons 11 and 12a. Our results provide in vivosupport for the importance of the exon definition model during NF1splicing, and suggest that the NF1region containing exons 11 and 12a plays an important role in the activity of neurofibromin.

The roles of the MYC, ERBB2, and CCND1genes in thyroid carcinogenesis are poorly known. We used realtime quantitative polymerase chain reaction (PCR) assays based on fluorescent TaqMan methodology to quantify MYC, ERBB2, and CCND1gene amplification and expression in 24 benign tumors (adenomas and goiter nodules) and 12 carcinomas (9 papillary, 2 follicular, and 1 anaplastic) of the thyroid. Real-time PCR is a recently developed method for nucleic acid quantification in homogeneous solutions, and has the potential to become a reference in terms of performance, accuracy, sensitivity, wide dynamic range, excellent interlaboratory agreement, and high throughput capacity, while avoiding the need for tedious post-PCR processing. Overexpression (>5 standard deviations above mean for normal thyroid tissues) of the ERBB2and CCND1genes was observed (3.2- to 5.2-fold and 3.8- to 8.4-fold, respectively) in 5 (14%) and 13 (36%) of 36 neoplastic thyroid RNA samples, respectively. Overexpression of the CCND1gene was observed in both the benign and malignant thyroid tumors, whereas the ERBB2gene was mainly overexpressed in malignant thyroid tumors. None of the neoplastic thyroid samples overexpressed MYC. No MYC, ERBB2, or CCND1gene amplification was identified. These results suggest that the CCND1gene plays an early role and the ERBB2gene a later role in thyroid tumorigenesis.

Expression of the telomerase catalytic sub-unit (htert) constitutes a key step in the development of human cancer. Although htert regulation is still unclear, several studies suggest that c-myc may activate its expression. Prostate cancer is one of the most common malignancies among men in Western countries. Since de-regulated expression of myc as well as telomerase activation may contribute to the pathogenicity of this cancer, we investigated this pathway in prostate tumorigenesis. For this purpose, myc- and htert-mRNA expression was quantified in 33 sporadic prostate tumors using a real-time quantitative PCR assay based on TaqMan methodology. myc over-expression was observed in 19 (58%) of 33 tumors, whereas telomerase status evaluated by htert expression was observed in 22 (67%). There was no correlation between myc over-expression or htert expression level and tumor stage or Gleason grade. A significant association (p = 0.0024) was found between myc over-expression and elevated htert expression, indicating that the up-regulation of telomerase activity often observed in prostate tumors might be conferred through transactivation of htert by myc. It is likely that the ability of c-myc protein to stimulate expression of htert and thereby enhance telomerase activity represents an important step in prostate tumorigenesis. Int. J. Cancer (Pred. Oncol.) 89:172–176, 2000. © 2000 Wiley-Liss, Inc.

La neurofibromatose de type 1 (NF1) est une maladie génétique caractérisée par le développement de tumeurs bénignes des gaines nerveuses, les neurofibromes (NF), qui peuvent se transformer en tumeurs malignes, les MPNST (malignant peripheral nerve sheath tumors). Transmise sur un mode autosomique dominant, la NF1 est causée par la perte de fonction du gène suppresseur de tumeur NF1. Malgré une pénétrance complète à l’âge de 8 ans, l’expressivité de la maladie est extrêmement variable, y compris au sein d’une même famille. De rares variants faux-sens du gène NF1ont pu être corrélés à des formes plus ou moins sévères de NF1, mais la plus grande part de la variabilité clinique de cette maladie reste inexpliquée. L’étude de modèles animaux et de corrélations phénotypiques intrafamiliales a suggéré l’existence de modificateurs génétiques. Nous présentons ici les résultats de la première étude d’association génome entier à la recherche de modificateurs génétiques des neurofibromes cutanés, sous-cutanés et plexiformes, réalisée en collaboration avec le réseau NF-France.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA on chromosomal ends. While telomerase is undetectable in most normal somatic tissues, telomerase activation has been detected by a polymerase chain reaction (PCR)-based assay (TRAP) in many immortal cell lines and various cancers, including prostate cancers. To investigate the role of telomerase in prostate cancer at the cellular level, the expression of one of the ribonucleoprotein complexes, the RNA component of human telomerase (hTR), was studied in normal, preneoplastic, and cancerous prostate tissues using a non-radioactive in situ hybridization procedure. Nine human prostates resected at the time of radical prostatectomy were studied. In each case, archival paraffin-embedded samples from normal tissue, prostatic intraepithelial neoplasia (PIN) lesions, the putative precancerous lesion, and prostate carcinomas were selected for in situ hybridization. hTR mRNA expression was detected in carcinomatous glands of seven out of the nine cancers (75 per cent). Furthermore, in seven out of the eight cases showing PIN lesions, the epithelial cells of PIN foci also expressed hTR mRNA. By contrast, in normal tissue, epithelial cells were negative, whereas hTR mRNA expression was detected in the basal cells. The detection of hTR mRNA in PIN lesions clearly strengthens the link between PIN and carcinomatous glands and suggests that telomerase expression occurs early in prostate carcinogenesis. Furthermore, this study confirms previous experimental data suggesting that the basal cell layer is the stem cell compartment in prostate. Copyright © 1999 John Wiley & Sons, Ltd.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA on chromosomal ends. While telomerase is undetectable in most normal somatic tissues, telomerase activation has been detected by a polymerase chain reaction (PCR)‐based assay (TRAP) in many immortal cell lines and various cancers, including prostate cancers. To investigate the role of telomerase in prostate cancer at the cellular level, the expression of one of the ribonucleoprotein complexes, the RNA component of human telomerase (hTR), was studied in normal, preneoplastic, and cancerous prostate tissues using a non‐radioactive in situhybridization procedure. Nine human prostates resected at the time of radical prostatectomy were studied. In each case, archival paraffin‐embedded samples from normal tissue, prostatic intraepithelial neoplasia (PIN) lesions, the putative precancerous lesion, and prostate carcinomas were selected for in situhybridization. hTR mRNA expression was detected in carcinomatous glands of seven out of the nine cancers (75 per cent). Furthermore, in seven out of the eight cases showing PIN lesions, the epithelial cells of PIN foci also expressed hTR mRNA. By contrast, in normal tissue, epithelial cells were negative, whereas hTR mRNA expression was detected in the basal cells. The detection of hTR mRNA in PIN lesions clearly strengthens the link between PIN and carcinomatous glands and suggests that telomerase expression occurs early in prostate carcinogenesis. Furthermore, this study confirms previous experimental data suggesting that the basal cell layer is the stem cell compartment in prostate. Copyright © 1999 John Wiley & Sons, Ltd.

Background & Aims:Impact of hepatitis C treatment has never taken into account the dynamics of fibrosis progression. This study assessed the impact of interferon on liver fibrosis progression in patients with chronic hepatitis C according to 3-month aminotransferase activity response. Methods:We recruited 287 patients, 185 treated and 102 control, with paired biopsy specimens. Before follow-up, the fibrosis progression rate per year was estimated as the ratio between fibrosis stage in METAVIR units (1 U, 1 stage; 4 U, cirrhosis) and the duration of infection. During follow-up, fibrosis progression was assessed by the observed difference between stages divided by duration between biopsies. Results:The median fibrosis progression rate in treated patients decreased compared with the rate before treatment from 0.103 F METAVIR U/yr (95% confidence interval [CI], 0.087–0.120) to 0.000 (95% CI, 0.000–0.000; P≤ 0.0001). Among 91 treated responders, fibrosis stage worsened in 19 (22%), compared with 21 (22%) of 94 treated nonresponders and 57 of 102 controls (56%; P≤ 0.0001 compared with treated patients), and improved in 26 (29%), 17 (18%), and 8 (8%; P= 0.0002 compared with 29% and P= 0.03 compared with 18%), respectively. These observed differences persisted after genotype, viremia, sex, age at infection, duration of infection, and alcohol consumption were taken into account. Conclusions:Interferon treatment changes the natural fibrosis progression rate in patients with chronic hepatitis C independently of genotype and early response.

Renal angiomyolipomas are benign tumors composed of variable amounts of mature fat, smooth muscle, and thick-walled blood vessels. They occur either sporadically or in association with tuberous sclerosis. Such tumors are considered as hamartomas, but few data are available concerning their pathogenesis. Indeed, it is not known whether angiomyolipoma is a congenital malformation or a neoplastic process. To answer this question, we assessed the clonality of sporadic angiomyolipomas using molecular analysis. Seven women (mean age, 59 years) with renal angiomyolipomas were included. DNA of the tumor and the normal adjacent kidney was extracted from archival paraffin-embedded tissue. DNA methylation pattern at a polymorphic site on the HUMARA gene was studied by polymerase chain reaction (PCR) amplification after methylation-sensitive enzyme digestion. This procedure enables the differentiation between polyclonal and monoclonal lesions according to their X-chromosome inactivation pattern. Five of the seven women included were informative for the HUMARA gene. The mean size of the angiomyolipomas was 53 mm (range, 18 to 110). In one case, a tumor thrombus was observed in the inferior vena cava. Clonal analysis showed that all of the angiomyolipomas and the tumor thrombus studied were monoclonal. This study shows that sporadic angiomyolipomas are monoclonal lesions consistent with neoplastic disorders. This result strongly supports the hypothesis that angiomyolipomas arise from the donal proliferation of an uncommited cell, which will further evolve toward different cell types.

Summary: The cystic fibrosis (CF) gene was recently identified as a gene spanning 250 kilobases (kbp) and coding for a 1480 amino acid protein, cystic fibrosis transmembrane conductance regulator (CFTR). Approximately 70% of CF mutations involve a three-base-pair deletion in CFTR exon 10, resulting in the loss of a phenylalanine at position 508 in the gene product (ΔF508). In order to screen for other molecular defects, we have used a strategy based on denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified gene segments. This method, which permits rapid detection of any sequence change in a given DNA stretch, was used successfully to analyse 61 non-ΔF508 CF chromosomes from French CF patients. A study of CFTR exons 10, 11, 14a, 15 and 20 detected three mutations located in exons 14a, 15 and 20, along with several nucleotide sequence polymorphisms. These nucleotide changes were identified by direct sequencing of PCR fragments displaying altered electrophoretic behaviour, together with some of the polymorphisms and mutations previously characterized by others. The strategy presented here constitutes a valuable tool for the development of carrier testing for individuals or couples with a family history of cystic fibrosis, and will contribute to deciphering the functionally important regions of the CFTR gene.

Neurofibromatosis type 1 (NF1), a genetic disorder with neuroectodermal involvement, demonstrates phenotypic overlap in some patients with Noonan syndrome (NS), ultimately resulting in the so-called neurofibromatosis-Noonan syndrome (NF-NS). A strong association of the two phenotypic traits was recently illustrated by a four-generation family, although NF1 and NS were eventually demonstrated to segregate independently on the basis of polymorphic DNA markers [Bahuau et al., 1996: Am J Med Genet 66:347–355]. Identification of the causal NF1 mutation seemed a prerequisite to further dissecting this singular familial association. Using the protein truncation assay, a nonsense mutation (C2446T→R816X) of the neurofibromin gene was evidenced. This mutation occurred on a CpG dinucleotide within exon 16 and 5' to the GAP domain-specifying region of the gene. R816X creates a recognition site for endonuclease HphI, absent in 2 individuals with NS only. Screening 184 unrelated NF1 patients, three novel occurrences of the mutation were found in individuals diagnosed with classical NF1. Based on the assumption of genotype-phenotype correlation in these individuals, clinical and molecular analyses of this four-generation family demonstrated that the NF-NS phenotype was additive, being the result of both classical NF1 and NS. This particular observation also suggests the presence of an NS locus on 17q, which might be of interest for further linkage studies. Am. J. Med. Genet. 75:265–272, 1998. © 1998 Wiley-Liss, Inc.

Mutations in the promoter region of the factor IX gene result in hemophilia B Leyden, which is characterized by considerable improvement in the disease after puberty. We have found that distinct nucleotide substitutions at the -6 position in the Leyden-specific (LS) region are associated with a different severity of hemophilia B. The proband (aged 2) from one family is a severe hemophiliac with factor IX activity (F.IXC) and antigen (F.IXAg) levels less than 1.0U/dl. F.IXC and F.IXAg levels in two affected uncles are approximately 30% of normal levels. The LS region was targeted for analysis because the phenotypes suggested the inheritance of a factor IX Leyden gene. An abnormal TaqI digestion pattern was found in amplified DNA from the proband, and sequencing showed a G (-6) to C transversion that was linked to the disease in the family. In another family, two brothers (aged 8 and 9) suffer from mild hemophilia with F.IXC ranging from 7 to 10 U/dl and F.IXAg from 3 to 4 U/dl. They are the only documented members of the family with a bleeding tendency. Denaturing gradient gel electrophoresis on amplified fragments from one of the patient's genomic DNA corresponding to the 8 exons and flanking sequences of the factor IX gene suggested a defect only in a segment from the 5' region. This segment showed an altered TaqI digestion pattern, and sequencing demonstrated a G(-6) to A transition that was traced to the patients's mother and a grandmother. The different phenotypes associated with the G (-6) to A purine nucleotide transition compared with a G(-6) to C transversion provide evidence that this area is directly involved in the regulation of the human factor IX gene expression in vivo by binding of regulatory factors. The ability to predict that the conditions of a hemophilia B patient will improve with age has important implications for genetic counseling of the family. Therefore, the LS region should always be included when scanning the factor IX gene for mutations.

Gene amplification is a common event in the progression of human cancers, and amplified oncogenes have been shown to have diagnostic, prognostic and therapeutic relevance. A kinetic quantitative polymerase-chain-reaction (PCR) method, based on fluorescent TaqMan methodology and a new instrument (ABI Prism 7700 Sequence Detection System) capable of measuring fluorescence in real-time, was used to quantify gene amplification in tumor DNA. Reactions are characterized by the point during cycling when PCR amplification is still in the exponential phase, rather than the amount of PCR product accumulated after a fixed number of cycles. None of the reaction components is limited during the exponential phase, meaning that values are highly reproducible in reactions starting with the same copy number. This greatly improves the precision of DNA quantification. Moreover, real-time PCR does not require post-PCR sample handling, thereby preventing potential PCR-product carry-over contamination; it possesses a wide dynamic range of quantification and results in much faster and higher sample throughput. The real-time PCR method, was used to develop and validate a simple and rapid assay for the detection and quantification of the 3 most frequently amplified genes (myc, ccnd1 and erbB2) in breast tumors. Extra copies of myc, ccnd1 and erbB2 were observed in 10, 23 and 15%, respectively, of 108 breast-tumor DNA; the largest observed numbers of gene copies were 4.6, 18.6 and 15.1, respectively. These results correlated well with those of Southern blotting. The use of this new semi-automated technique will make molecular analysis of human cancers simpler and more reliable, and should find broad applications in clinical and research settings. Int. J. Cancer 78:661–666, 1998. © 1998 Wiley-Liss, Inc.

Sequencing the complete factor IX gene of 2 sisters with hemophilia B with different phenotypes and no family history of hemorrhagic diathesis revealed a common 5' splice site mutation in intron 3 (T6704C) in both and an additional missense mutation (I344T) in one. The presence of dysfunctional antigen in the latter strongly suggested that these mutations are in trans. Neither mutation was found in leukocyte DNA from the asymptomatic parents, but the mother was in somatic mosaicism for the shared splice site mutation. This case illustrates the importance of defining the phenotype and considering somatic mosaicism in sporadic cases. It underlines the limitations of complete gene sequencing for the detection of mosaicism and has implication for genetic counseling.

Sequencing the complete factor IXgene of 2 sisters with hemophilia B with different phenotypes and no family history of hemorrhagic diathesis revealed a common 5′ splice site mutation in intron 3 (T6704C) in both and an additional missense mutation (I344T) in one. The presence of dysfunctional antigen in the latter strongly suggested that these mutations are in trans. Neither mutation was found in leukocyte DNA from the asymptomatic parents, but the mother was in somatic mosaicism for the shared splice site mutation. This case illustrates the importance of defining the phenotype and considering somatic mosaicism in sporadic cases. It underlines the limitations of complete gene sequencing for the detection of mosaicism and has implication for genetic counseling.

No relevant conflicts of interest to declare.

Abstract 163FN2This icon denotes a clinically relevant abstract

The hallmark of neurofibromatosis type 1 is the development of dermal and plexiform neurofibromas. Neurofibromatosis type 1 patients with plexiform neurofibromas are at risk of developing malignant peripheral nerve sheath tumors. We applied a 22,000-oligonucleotide microarray transcriptomic approach to a series of plexiform neurofibromas in comparison with dermal neurofibro-mas, and results were confirmed with real-time quantitative reverse transcription-polymerase chain reaction. Thirteen genes were up-regulated and 10 were downregulated in plexiform neurofibromas. The upregulated genes mainly encode molecules involved in cell adhesion, extracellular matrix, fibrogenesis, and angiogenesis. Several CCN gene family members were dysregulated in neuro-fibromatosis type 1 tumorigenesis; the angiogenic gene CCN1/CYR61 was specifically upregulated in the plexiform neurofibromas; CCN4/WISP1 was upregulated, and CCN3/NOV and CCN5/WISP2 were downregulated in paired comparisons of plexiform neuro-fibroma and malignant peripheral nerve sheath tumor from the same patients. CCN1 and CCN3 proteins were detected by immuno-histochemistry in neurofibromatosis type 1-associated tumors. Up-regulation of S100A8, S100A9, and CD36 was also observed and suggests a role of this pathway in inflammation-associated genesis of plexiform neurofibromas. In summary, a limited number of pathways are potentially involved in plexiform neurofibroma development. Some of the genes identified, particularly CCN1, might be useful diagnostic or prognostic markers or form the basis for novel therapeutic strategies.

We report preliminary results in 3 children with cerebral X-linked adrenoleukodystrophy (ALD) who received in September 2006, January 2007 and June 2008 lentiviral vector transduced autologous hematopoietic stem cell (HSC). We have previously demonstrated that cerebral demyelination associated with cerebral ALD can be stopped or reversed within 12–18 months by allogeneic HSC transplantation. The long term beneficial effects of HCT transplantation in ALD are due to the progressive turn-over of brain macrophages (microglia) derived from bone-marrow cells. For the current HSC gene therapy procedure, we used mobilized peripheral blood CD34+ cells that were transduced ex vivo for 18 hours with a non-replicative HIV1-derived lentiviral vector (CG1711 hALD) at MOI25 and expressing the ALD cDNA under the control of the MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substituted) promoter, and in the presence of 4 human recombinant cytokines (Il- 3, Stem Cell Factor [SCF], Flt3-ligand and Megakaryocyte Growth and Differentiation Factor [MGDF]) and CH-296 retronectine. Transduced cells were frozen to perform the required (RCL) safety tests. After thawing and prior to reinjection, 50%, 30% and 40% of transduced CD34+ cells expressed the ALD protein with a mean of 0.7, 0.6 and 0.65 copies of integrated provirus per cell. Transduced CD34+ cells were infused to ALD patients after a conditioning regimen including full doses of cyclophosphamide and busulfan. Hematopoietic recovery occured at day 13–15 post-transplant and the procedure was uneventful. In patient P1 and P2, the percentage of lymphocytes and monocytes expressing the ALD protein declined from day 60 to 6 months after gene therapy (GT) and remained stable up to 16 months post-GT. In P1, 9 to 13% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein 16 months post-transplant. In P2 and at the same time-point after transplant, 10 to 18% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein. ALD protein was expressed in 18–20% of bone marrow CD34+ cells from patients P1 and P2, 12 months post-transplant. In patient P3, 20 to 23% of CD3+, CD14+ and CD15+ cells expressed ALD protein 2 months after transplant. Tests assessing vector-derived RCL and vector mobilization were negative up to the last followups in the 3 patients. Integration of the vector was polyclonal and studies of integration sites arein progress. At 16 months post-transplant, HSC gene therapy resulted in neurological effects comparable with allogeneic HSC transplantation in patient P1 and P2. These results support that: ex-vivo HSC gene therapy using HIV1-derived lentiviral vector is not associated with the emergence of RCL and vector mobilization; a high percentage of hematopoietic progenitors were transduced expressing ALD protein in long term; no early evidence of selective advantage of the transduced ALD cells nor clonal expansion were observed. (This clinical trial is sponsored by Institut National de la Santé et de la Recherche Médicale and was conducted in part under a R&D collaboration with Cell Genesys, Inc., South San Francisco, CA)

We report preliminary results in 3 children with cerebral X-linked adrenoleukodystrophy (ALD) who received in September 2006, January 2007 and June 2008 lentiviral vector transduced autologous hematopoietic stem cell (HSC). We have previously demonstrated that cerebral demyelination associated with cerebral ALD can be stopped or reversed within 12–18 months by allogeneic HSC transplantation. The long term beneficial effects of HCT transplantation in ALD are due to the progressive turn-over of brain macrophages (microglia) derived from bone-marrow cells. For the current HSC gene therapy procedure, we used mobilized peripheral blood CD34+ cells that were transduced ex vivofor 18 hours with a non-replicative HIV1-derived lentiviral vector (CG1711 hALD) at MOI25 and expressing the ALD cDNA under the control of the MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substituted) promoter, and in the presence of 4 human recombinant cytokines (Il- 3, Stem Cell Factor [SCF], Flt3-ligand and Megakaryocyte Growth and Differentiation Factor [MGDF]) and CH-296 retronectine. Transduced cells were frozen to perform the required (RCL) safety tests. After thawing and prior to reinjection, 50%, 30% and 40% of transduced CD34+ cells expressed the ALD protein with a mean of 0.7, 0.6 and 0.65 copies of integrated provirus per cell. Transduced CD34+ cells were infused to ALD patients after a conditioning regimen including full doses of cyclophosphamide and busulfan. Hematopoietic recovery occured at day 13–15 post-transplant and the procedure was uneventful. In patient P1 and P2, the percentage of lymphocytes and monocytes expressing the ALD protein declined from day 60 to 6 months after gene therapy (GT) and remained stable up to 16 months post-GT. In P1, 9 to 13% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein 16 months post-transplant. In P2 and at the same time-point after transplant, 10 to 18% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein. ALD protein was expressed in 18–20% of bone marrow CD34+ cells from patients P1 and P2, 12 months post-transplant. In patient P3, 20 to 23% of CD3+, CD14+ and CD15+ cells expressed ALD protein 2 months after transplant. Tests assessing vector-derived RCL and vector mobilization were negative up to the last followups in the 3 patients. Integration of the vector was polyclonal and studies of integration sites arein progress. At 16 months post-transplant, HSC gene therapy resulted in neurological effects comparable with allogeneic HSC transplantation in patient P1 and P2. These results support that:

Although World Health Organization (WHO) grade I pilocytic astrocytomas and grade II diffuse astrocytomas have been classified for decades as different clinicopathologic entities, few, if any, data are available on the biologic features explaining these differences. Although more than 50 microarray-related studies have been carried out to characterize the molecular profiles of astrocytic tumors, we have identified only 11 that provide sound data on low-grade astrocytomas. We have incorporated these data into a comparative analysis for the purpose of identifying the most relevant molecular markers characterizing grade I pilocytic and grade II diffuse astrocytomas. Our analysis has identified various interesting genes that are differentially expressed in either grade I or grade II astrocytomas when compared with normal tissue and/or high-grade (WHO grade III and IV) astrocytomas. A large majority of these genes encode adhesion, extracellular matrix, and invasion-related proteins. Interestingly, a group of 6 genes (TIMP4, C1NH, CHAD, THBS4, IGFBP2, and TLE2) constitute an expression profile characteristic of grade I astrocytomas as compared with all other categories of tissue (normal brain, grade II, and high-grade astrocytomas). The end products (proteins) of these genes act as antimigratory compounds, a fact that could explain why pilocytic astrocytomas behave as compact (well-circumscribed) tumors as opposed to all the other astrocytic tumor types that diffusely invade the brain parenchyma. Having validated these molecular markers by means of real-time reverse transcriptase-polymerase chain reaction, an integrated model was proposed illustrating how and why pilocytic astrocytomas constitute a distinct biologic and pathologic entity when compared with diffuse astrocytomas.