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Background: Many clinical trials have reported the use of mesenchymal stromal cells (MSCs) following the indication of severe SARS-CoV-2 infection. However, in the COVID19 pandemic context, academic laboratories had to adapt a production process to obtain MSCs in a very short time. Production processes, especially freezing/thawing cycles, or culture medium have impacts on MSC properties. We evaluated the impact of an intermediate cryopreservation state during MSC culture to increase production yields. Methods: Seven Wharton’s jelly (WJ)-MSC batches generated from seven different umbilical cords with only one cryopreservation step and 13 WJ-MSC batches produced with intermediate freezing were formed according to good manufacturing practices. The identity (phenotype and clonogenic capacities), safety (karyotype, telomerase activity, sterility, and donor qualification), and functionality (viability, mixed lymphocyte reaction) were analyzed. Results: No significant differences between MSC production processes were observed, except for the clonogenic capacity, which was decreased, although it always remained above our specifications. Conclusions: Intermediate cryopreservation allows an increase in the production yield and has little impact on the basic characteristics of MSCs.

Purpose: Genetic instability plays an important role in ovarian carcinogenesis. We investigated the level of telomere shortening and genomic instability in early and preinvasive stages of ovarian cancer, serous tubal intraepithelial carcinoma (STIC), and tubo-ovarian dysplasia (TOD).Experimental Design: Fifty-one TOD from prophylactic salpingo-oophorectomies with BRCA1 or 2 mutation, 12 STICs, 53 tubo-ovarian high-grade serous carcinoma, and 36 noncancerous controls were laser capture microdissected from formalin-fixed, paraffin-embedded sections, analyzed by comparative genomic hybridization (array CGH) and for telomere length (using quantitative real-time PCR based on the Cawthon's method). TOD and STICs were defined by morphologic scores and immunohistochemical expressions of p53, Ki67, and γH2AX.Results: TOD showed marked telomere shortening compared with noncancerous controls (P < 10−7). STICs had even shorter telomeres than TOD (P = 0.0008). Ovarian carcinoma had shorter telomeres than controls but longer than STICs and dysplasia. In TOD, telomeres were significantly shorter in those with BRCA1 mutation than in those with BRCA2 mutation (P = 0.005). In addition, γH2AX expression in TOD and STIC groups with short telomeres was significantly increased (P < 10−7). In dysplastic epithelium, we found subtle genomic alterations, in contrast to more important genomic imbalances in STICs. The total number of genetic alterations was the highest in ovarian cancers.Conclusions: These findings suggest that genetic instability occurs in early stages of ovarian tumorigenesis. STICs and noninvasive dysplasia are likely an important step in early serous ovarian neoplasia. Clin Cancer Res; 19(11); 2873–82. ©2013 AACR.

Supplementary Tables 1-3 - PDF file 74K, aCGH data; minimal regions of interest in tubo-ovarian dysplasia and in STIC; Table S1 : Minimal regions of interest in ovarian dyaplasia (n=5); Table S2 : Minimal regions of interest in the common losses and gains in tubal dysplasia (n=9) 2; Table S3 : Minimal regions of interest in the common losses and gains in STIC (n=12)

Telomere length appears to correlate with survival in early non-small-cell lung cancer (NSCLC), but the prognostic impact of telomere status in advanced NSCLC remains undetermined. Our purpose was to evaluate telomere parameters as prognostic and predictive biomarkers in advanced NSCLC. In 79 biopsies obtained before treatment, we analyzed the telomere length and expression of TERT and shelterin complex genes (TRF1, TRF2, POT1, TPP1, RAP1, and TIN2), using quantitative PCR. Non-responders to first-line chemotherapy were characterized by shorter telomeres and low RAP1 expression (p = 0.0035 and p = 0.0069), and tended to show higher TERT levels (p = 0.058). In multivariate analysis, short telomeres were associated with reduced event-free (EFS, p = 0.0023) and overall survival (OS, p = 0.00041). TERT and TRF2 overexpression correlated with poor EFS (p = 0.0069 and p = 0.00041) and OS (p = 0.0051 and p = 0.007). Low RAP1 and TIN2 expression-levels were linked to reduced EFS (p = 0.00032 and p = 0.0069) and OS (p = 0.000051 and p = 0.02). Short telomeres were also associated with decreased survival after nivolumab therapy (p = 0.097). Evaluation of telomere status in advanced NSCLC emerges as a useful biomarker that allows for the selection of patient groups with different clinical evolutions, to establish personalized treatment.

Correlations were reported between sperm telomere length (STL) and male fertility, sperm DNA fragmentation, and oxidation. Sperm freezing is widely used for assisted reproductive techniques, fertility preservation, and sperm donation. However, its impact on STL remains unknown. For this study, semen surplus from patients who underwent routine semen analysis were used. The impact of slow freezing on STL was analyzed by performing qPCR before and after freezing. Sperm populations with different STL were evaluated using Q-FISH. The relationship between sperm DNA oxidation, DNA fragmentation, and STL was assessed in fresh and frozen sperm samples. No significant impact of slow freezing on STL was observed, neither measured by qPCR nor Q-FISH. However, Q-FISH allowed for the distinguishing of sperm populations with different STLs within individual sperm samples. Slow freezing induced different STL distributions for some of the analyzed sperm samples, but no correlation was found between STL and sperm DNA fragmentation or oxidation. Slow freezing does not alter STL despite increasing sperm DNA oxidation and fragmentation. As STL alterations could be transmitted to offspring, the lack of impact of the slow freezing method on STL ensures the safety of this procedure.

Telomeres protect chromosome ends and control cell division and senescence. During organogenesis, telomeres need to be long enough to ensure the cell proliferation necessary at this stage of development. Previous studies have shown that telomere shortening is associated with growth retardation and congenital malformations. However, these studies were performed in newborns or postnatally, and data on telomere length (TL) during the prenatal period are still very limited. We measured TL using quantitative PCR in amniotic fluid (AF) and chorionic villi (CV) samples from 69 control fetuses with normal ultrasound (52 AF and 17 CV) and 213 fetuses (165 AF and 48 CV) with intrauterine growth retardation (IUGR) or congenital malformations diagnosed by ultrasound. The samples were collected by amniocentesis at the gestational age (GA) of 25.0 ± 5.4 weeks and by CV biopsy at 18.1 ± 6.3 weeks. In neither sample type was TL influenced by GA or fetal sex. In AF, a comparison of abnormal versus normal fetuses showed a significant telomere shortening in cases of IUGR (reduction of 34%, P

Cytogenetic aberrations are found in 65% of adults and 75% of children with acute leukemia. Specific aberrations are used as markers for the prognostic stratification of patients. The current standard cytogenetic procedure for acute leukemias is karyotyping in combination with FISH and RT-PCR. Optical genome mapping (OGM) is a new technology providing a precise identification of chromosomal abnormalities in a single approach. In our prospective study, the results obtained using OGM and standard techniques were compared in 29 cases of acute myeloid (AML) or lymphoblastic leukemia (ALL). OGM detected 73% (53/73) of abnormalities identified by standard methods. In AML cases, two single clones and three subclones were missed by OGM, but the assignment of patients to cytogenetic risk groups was concordant in all patients. OGM identified additional abnormalities in six cases, including one cryptic structural variant of clinical interest and two subclones. In B-ALL cases, OGM correctly detected all relevant aberrations and revealed additional potentially targetable alterations. In T-ALL cases, OGM characterized a complex karyotype in one case and identified additional abnormalities in two others. In conclusion, OGM is an attractive alternative to current multiple cytogenetic testing in acute leukemia that simplifies the procedure and reduces costs.

10039 Background: Children with relapsed and refractory neuroblastoma (RR-NBL) have poor outcomes. Early identification of children at greatest risk of relapse could mean timelier modifications of treatment to improve outcomes. High levels of adrenergic neuroblastoma mRNAs in blood of children with stage M neuroblastoma receiving frontline treatment predict poor outcome (Viprey PMID: 24590653). Since these markers have not been thoroughly studied in the RR-NBL population, we have prospectively evaluated the prognostic potential of the adrenergic neuroblastoma mRNAs paired-like homeobox 2B (PHOX2B ) and tyrosine hydroxylase (TH) in blood from children with RR-NBL treated in the BEACON-Neuroblastoma trial (NCT02308527). Methods: Blood samples collected at baseline from 88 children were analysed by reverse transcriptase polymerase chain reaction (RTqPCR) for PHOX2B and TH mRNAs. The prognostic power of these mRNAs was evaluated using Kaplan-Meier survival curves and Cox proportional hazards regression. Progression-free (PFS) and overall survival (OS) were calculated from the date that the blood sample was taken at screening to the date of an event; progression, disease recurrence, death or censored alive at the last clinical evaluation. Results: Of the children in this cohort, 58 (66%) had relapsed and 30 (34%) had refractory disease. Twenty-three (26%) had MYC-N amplified tumours. TH and PHOX2B mRNAs were detected in 55% and 60% of blood samples respectively; the correlation coefficient between TH and PHOX2B was 0.75. Higher levels of TH, PHOX2B mRNAs or both combined were associated with reduced PFS and OS (Table). For TH, median PFS for children with TH levels below the median was 12 months (95%CI, 4.6–13 months) versus 5.5 months (95%CI, 1.8–9.4 months) for those children with TH levels above the median. For PHOX2B, median PFS for children with PHOX2B levels below the median was 11.5 months (95%CI, 7.6–34 months), compared to 5.7 months (95%CI, 1.8–10.5 months) where levels were above the median. Conclusions: TH and PHOX2B mRNAs in blood collected at baseline identify children with refractory or relapsed neuroblastoma at greatest risk of progression or death. In the RR-NBL setting, this simple blood test could be used to stratify treatment strategies. Clinical trial information: NCT02308527. [Table: see text]

RNA splicing factors are frequently altered in cancer and can act as both oncoproteins and tumour suppressors. They have been found mutated or deregulated, justifying the growing interest in the targeting of splicing catalysis, splicing regulatory proteins, and/or specific, key altered splicing events. We recently showed that the DNA methylation alterations of CD34+CD15− chronic myeloid leukaemia (CML) cells affect, among others, alternative splicing genes, suggesting that spliceosome actors might be altered in chronic-phase (CP)-CML. We investigated the expression of 12 spliceosome genes known to be oncogenes or tumour suppressor genes in primary CP-CML CD34+ cells at diagnosis (n = 15). We found that CP-CML CD34+ cells had a distinct splicing signature profile as compared with healthy donor CD34+ cells or whole CP-CML cells, suggesting: (i) a spliceosome deregulation from the diagnosis time and (ii) an intraclonal heterogeneity. We could identify three profile types, but there was no relationship with a patient’s characteristics. By incubating cells with TKI and/or a spliceosome-targeted drug (TG003), we showed that CP-CML CD34+ cells are both BCR::ABL and spliceosome dependent, with the combination of the two drugs showing an additive effect while sparing healthy donors cells. Our results suggest that the spliceosome may be a new potential target for the treatment of CML.

Introduction Our objective was to evaluate characteristics, treatment and outcome of vasculitis associated with myelodysplastic syndrome (MDS) and chronic myelomonicytic leukemia (CMML) Patients and Methods Retrospective descriptive analysis of MDS/CMML-related vasculitis and comparison with MDS/CMML patients without dysimmune features. Results Seventy patients with vasculitis and MDS/CMML were included, with median age of 71.5 [21–90] years and male/female ratio of 2.3. Vasculitis was diagnosed prior to MDS/CMML in 31 patients (44%), and after in 20 patients. In comparison with MDS/CMML without autoimmune/inflammatory features, vasculitis with MDS/MPN showed no difference in MDS/CMML subtypes distribution nor International Prognostic Scoring System and CMML-specific prognostic (IPSS/CPSS) scores. Vasculitis subtypes included Giant cell arteritis in 24 patients (34%), Behcet's-like syndrome in 11 patients (20%) and polyarteritis nodosa in 6 patients (9%). Glucocorticoids (GCs) were used as first-line therapy for MDS/CMML vasculitis in 64/70 patients (91%) and 41 (59%) received combined immunosuppressive therapies during the follow-up. After a median follow-up of 33.2 months [1–162], 31 patients (44%) achieved sustained remission. At least one relapse occurred in 43 patients (61%). Relapse rates were higher in patients treated with conventional Disease Modifying Anti-Rheumatic Drug (DMARDs) (odds ratio 4.86 [95% CI 1.38 - 17.10]), but did not differ for biologics (odds ratio 0.59 [95% CI 0.11–3.20]) and azacytidine (odds ratio 1.44 [95% CI 0.21–9.76]) than under glucocorticoids. Overall survival in MDS/CMML vasculitis was not significantly different from MDS/CMML patients without autoimmune/inflammatory features (p = 0.5), but acute leukemia progression rates were decreased (log rank Conclusion This study shows no correlation of vasculitis diagnoses with subtypes and severity of MDS/CMML, and no significant impact of vasculitis on overall survival. Whereas conventional DMARDs seem to be less effective, biologics or azacytidine therapy could be considered for even low-risk MDS/CMML vasculitis.

Background The 10q26 subtelomeric microdeletion syndrome is a rare and clinically heterogeneous disorder. The precise relationships between the causative genes and the phenotype are unclear. Case presentation We report two new cases of 860 kb deletion of 10q26.2 identified by array CGH in a fetus with intrauterine growth retardation and his mother. The deleted region encompassed only four coding genes, DOCK1, INSYN2, NPS and FOX12. The proband had dysmorphic facies characterized by a high forehead, malformed ears, a prominent nose, and retrognathia. He had bilateral club feet, clinodactily and mild psychomotor retardation. His mother had a short stature, microcephaly, a long face with a high forehead and bitemporal narrowing, arched and sparse eyebrows, strabismus, prominent nose and chin, a thin upper lip and large protruding ears, and mild intellectual disability. Conclusions This study presents the smallest 10q26.2 deletion so far identified, which further refines the minimal critical region associated with the 10q26 microdeletion syndrome. It focuses on three genes potentially responsible for the phenotype: DOCK1, which is the major candidate gene, and INSYN2 and NPS, which could be involved in cognitive functions.

Le syndrome microdeletionnel 10q26 correspond a un syndrome rare et cliniquement heterogene. La correlation genotype-phenotype reste aujourd’hui mal etablie. Nous rapportons deux nouvelles personnes (un garcon de 22 mois et sa mere) presentant une deletion 10q26.2 de 860 kb. L’ACPA avait ete realisee en prenatal en raison d’un retard de croissance intra-uterin. La region deletee contenait seulement 4 genes codants : DOCK1, INSYN2, NPS and FOX12. A la naissance, le proband presentait des particularites morphologiques faciales avec un front haut, des oreilles mal ourlees, un nez proeminent, et une retrognathie; ainsi que des pieds bots bilateraux et une clinodactylie. A l’âge de 22 mois, il presentait un retard psychomoteur global. Sa mere presentait une taille et un perimetre crânien a la limite inferieure de la normale, un visage allonge avec un front haut et une retraction bitemporale, des sourcils arques et clairsemes, un strabisme, un nez et un menton proeminents, une levre superieure fine et des oreilles larges et decollees. Elle a presente des difficultes d’apprentissage importantes. Cette deletion est la plus petite deletion decrite dans la litterature et permet d’affiner la region minimale critique du syndrome 10q26. Trois genes candidats pourraient participer au phenotype : le gene DOCK1, deja considere comme le gene candidat principal, et les genes INSYN2 and NPS qui pourraient etre impliques dans le phenotype cognitif.

International audience; Upregulation of the telomerase reverse transcriptase (TERT) gene in human cancers leads to telomerase activation, which contributes to the growth advantage and survival of tumor cells. Molecular mechanisms of TERT upregulation are complex, tumor-specific and can be clinically relevant. To investigate these mechanisms in breast cancer, we sequenced the TERT promoter, evaluated TERT copy number changes and assessed the expression of the MYC oncogene, a known transcriptional TERT regulator, in two breast cancer cohorts comprising a total of 122 patients. No activating TERT promoter mutations were found, suggesting that this mutational mechanism is not likely to be involved in TERT upregulation in breast cancer. The T349C promoter polymorphism found in up to 50% of cases was not correlated with TERT expression, but T349C carriers had significantly shorter disease-free survival. TERT gains (15-25% of cases) were strongly correlated with increased TERT mRNA expression and worse patient prognosis in terms of disease-free and overall survival. Particularly aggressive breast cancers were characterized by an association of TERT gains with MYC overexpression. These results evidence a significant effect of gene copy number gain on the level of TERT expression and provide a new insight into the clinical significance of TERT and MYC upregulation in breast cancer.

International audience; Sensitive and specific detection of Ewing sarcoma minimal residual disease in ovarian and testicular tissues Ewing sarcoma (EWS) is a common pediatric solid tumor with high metastatic potential. Due to toxic effects of treatments on reproductive functions, the cryopreservation of ovarian tissue (OT) or testicular tissue (TT) is recommended to preserve fertility. However, the risk to reintroduce residual metastatic tumor cells should be evaluated before fertility restoration. Our goal was to validate a sensitive and specific approach for EWS minimal residual disease (MRD) detection in frozen germinal tissues. Thawed OT (n=12) and TT (n=14) were contaminated with tumor RD-ES cells (10, 100 and 1000 cells) and EWS-FLI1 tumor-specific transcript was quantified with RT-qPCR. All contaminated samples were found to be positive, with the strong correlation between RD-ES cell numbers and EWS-FLI1 levels in OT (r=0.93) and TT (r=0.96) (P

Background Microdeletions encompassing chromosome bands 2q14.1q14.3 are rare. To date, eight reports of relatively large deletions of this region (∼20 Mb) but only two small deletions (

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International audience; In high-risk neuroblastoma (HR-NB), the clinical significance of long-term minimal residual disease (MRD) monitoring using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for neuroblastoma mRNAs has not been investigated. We report long-term MRD follow-ups of four patients with HR-NB throughout the disease (diagnosis, remission, and relapse) and treatment course (chemotherapy, autologous and allogeneic stem cell transplantation, and donor lymphocyte and natural killer cell infusions). The results showed the stability of mRNA marker expression after different treatments and demonstrated their validity to predict relapse and assess therapeutic response. This opens up the possibility of investigating the utility of long-term molecular monitoring of MRD in prospective multicenter studies.

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1 Background: The purpose of this study was to evaluate whether levels of neuroblastoma mRNAs in bone marrow and peripheral blood from stage M infants (≤12 months of age at diagnosis, MYCN amplified) and toddlers (between 12 and 18 months, any MYCN status) predict event‐free survival (EFS). 2 Methods: Bone marrow aspirates and peripheral blood samples from 97 infants/toddlers enrolled in the European High‐Risk Neuroblastoma trial were collected at diagnosis in PAXgene™ blood RNA tubes. Samples were analyzed by reverse transcription quantitative polymerase chain reaction according to standardized procedures. 3 Results: Bone marrow tyrosine hydroxylase (TH) or paired‐like homeobox 2b (PHOX2B) levels in the highest tertile were associated with worse EFS; hazard ratios, adjusted for age and MYCN status, were 1.5 and 1.8 respectively. Expression of both TH and PHOX2B in the highest tertile predicted worse outcome (p = 0.015), and identified 20 (23%) infants/toddlers with 5‐year EFS of 20% (95%CI: 4%–44%). Prognostic significance was maintained after adjusting for over‐fitting bias (p = 0.038), age and MYCN status. In peripheral blood, PHOX2B levels in the highest tertile predicted a two‐fold increased risk of an event (p = 0.032), and identified 23 (34%) infants/toddlers with 5‐year EFS of 29% (95%CI: 12%–48%). Time‐dependent receiver operating characteristic analysis confirmed the prognostic value of combined TH and PHOX2B in bone marrow and of PHOX2B in peripheral blood during the first year of follow‐up. 4 Conclusions: High levels of bone marrow TH and PHOX2B and of peripheral blood PHOX2B at diagnosis allow early identification of a group of high‐risk infant and toddlers with neuroblastoma who may be candidates for alternative treatments. Integration with additional biomarkers, as well as validation in additional international trials is warranted.

Nous presentons le cas de Camille, petit garcon, ne a terme a 41 + 6 SA par cesarienne pour stagnation du travail. Une hypotonie axiale et peripherique est apparue a la naissance dans un contexte d’anaoxo-ischemie neonatale (APGAR a 0-6-9). La grossesse s’est deroulee sans particularite. Le depistage combine au premier trimestre pour la trisomie 21 a ete calcule a 1/315 (clarte nucale a 0,7 mm). Le depistage sur ADN fœtal libre circulant etait negatif. Devant l’hypotonie majeure de Camille, sans point d’appel particulier, une Analyse sur Puce a ADN (ACPA) a ete prescrite. L’ACPA (Agilent®, puce Oligo 60 K, hg19) a montre des profils de fluorescence complexes au niveau des gonosomes. La realisation d’un caryotype et de FISH a permis de mettre en evidence dans toutes les cellules analysees 47 chromosomes avec presence de deux isodicentriques du bras court du chromosome Y (chromosome X a priori normal), a l’origine d’une tetrasomie de la region du chromosome Y depuis l’extremite du bras court jusqu’a la region Yq11.222 et d’une deletion terminale Yq11.222qter, de novo. Seul un petit nombre d’isodicentrique du bras court du chromosome Y a ete decrit dans la litterature. Les isodicentriques sont des remaniements de structure tres instable liee a la presence des deux centromeres. Ils sont frequemment observes en mosaique associes soit a un contingent 46,XY soit a un clone 45,X. Le tableau phenotypique associe a ces isodicentriques depend donc de la presence ou non d’une mosaique et de la proportion des differents clones mais egalement de la taille des segments concernes. Ici les deux isodicentriques semblent etre retrouves de maniere homogene suggerant une certaine stabilite et laisse envisager des retentissements phenotypiques pour cet enfant assez similaires a ceux dus a la presence d’une polysomie du chromosome Y (susceptibilite accrue au deficit intellectuel, retard des apprentissages, dysmorphie faciale, malformations squelettiques, perturbation de la spermatogenese) [1] , [2] .

Research question Is sperm fluorescence in-situ hybridization (FISH) useful to evaluate the risk of chromosomally unbalanced gametes in interchromosomal reciprocal insertion (IRI) carriers? How do these imbalances lead to recurrent miscarriages? Design This study reports a clinical and molecular study of a rare familial balanced IRI resulting in recurrent spontaneous miscarriage. Sperm FISH was performed to estimate the number of unbalanced gametes. Results A 31-year-old healthy male (proband) and his 28-year-old female partner were referred to the Genetics Department for three spontaneous miscarriages occurring during the first trimester of pregnancy. FISH analysis of the proband with the LSI TRA/D (14q11.2) and DiGeorge N25 (22q11.2) break-apart probes showed the presence of a balanced IRI between 14q11.2 and 22q11.2 chromosomal regions. This IRI was also identified in the proband's father. Sperm FISH with the same probes showed that more than 40% of gametes of the proband were unbalanced for either 14q11.2 or 22q11.2, despite normal sperm parameters. FISH analysis of a product of conception indicated that unbalanced gametes result in a non-viable fetus. Conclusions This study shows the value of sperm FISH analysis in improving genetic reproductive advice for IRI carriers. Disruption of critical genes through this rearrangement and their consequent functional impairment could result in recurrent miscarriages. In this case, several genes located in the 14q11.2 region, particularly RNase 3, would be good candidates to explain the lethality of the imbalances.

International audience; Neuroblastoma (NB) is the most common type of extracranial solid tumor in children with a high prevalence in toddlers. For childhood cancer survivors, preservation of reproductive potential is an important factor for quality of life. The optimization of NB minimal residual disease (MRD) detection in testicular tissue is crucial to evaluate the risk of malignant cell reintroduction. The first step in the present study was to assess the accuracy of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect tyrosine hydroxylase (TH), paired-like homeobox 2b (PHOX2B) and doublecortin (DCX) mRNA expression in frozen/thawed testicular tissues of patients with non-obstructive azoospermia (NOA) contaminated (in vitro model) with an increasing number of IMR-32 and SK-N-SH NB cells. Testicular tissues were frozen by slow or snap freezing. The second step was to determine the expression levels of these markers in testicular samples from 4 pre-pubertal males (2 with stage IV NB and 2 with non-NB malignancy). The yield of extracted RNA was similar in testicular samples frozen by slow or snap freezing. In the in vitro model, TH and DCX transcripts were detected in uncontaminated testicular tissues, whereas PHOX2B mRNA was not detected. There was a strong positive association between the number of NB cells used for contamination and PHOX2B transcript levels. For IMR-32 and SK-N-SH NB cell lines, specificity and sensitivity rates of detection were 100% for PHOX2B following in vitro contamination with 10 tumor cells. In testicular samples from pre-pubertal males with and without NB, PHOX2B mRNA expression was not observed, but high expression levels of TH and DCX mRNA were detected, which were similar to expression detected in the in vitro model. Among the markers used in blood and bone marrow for NB MRD studies, the detection of PHOX2B transcripts by RT-qPCR may provide an accurate assessment of NB cells in testicular tissues from males who require fertility preservation.

Background Ovarian tissue cryopreservation (OTC) is the only option available to preserve fertility in prepubertal females with neuroblastoma (NB), a childhood solid tumor that can spread to the ovaries, with a risk of reintroducing malignant cells after an ovarian graft. Procedure We set out to determine whether the analysis of TH (tyrosine hydroxylase), PHOX2B (paired-like homeobox 2b), and DCX (doublecortin) transcripts using quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) could be used to detect NB contamination in ovarian tissue. Analyses were performed on benign ovarian tissue from 20 healthy women between November 2014 and September 2015 at the University Hospital of Clermont-Ferrand. Pericystic benign ovarian tissues were collected and contaminated with increasing numbers of human NB cells (cell lines IMR-32 and SK-N-SH) before detection using RT-qPCR. Results TH and DCX transcripts were detected in uncontaminated ovarian tissue from all the donors, hampering the detection of small numbers of tumor cells. By contrast, PHOX2B was not detected in any uncontaminated ovarian fragment. PHOX2B levels were significantly increased from 10 NB cells. Our study is the first to evaluate minimal residual disease detection using NB mRNAs in human ovarian tissue. Only PHOX2B was a reliable marker of NB cells contaminating ovarian tissue. Conclusions These results are encouraging and offer hope in the near future for grafting ovarian tissue in women who survive cancer, whose fertility has been jeopardized by treatment, and who could benefit from OTC without oncological risk.

Microdeletions of 17q12 encompassing TCF2 are associated with maturity-onset of diabetes of the young type 5, cystic renal disease, pancreatic atrophy, Mullerian aplasia in females and variable cognitive impairment. We report on a patient with a de novo 17q12 microdeletion, 1.8 Mb in size, associated with congenital diaphragmatic hernia (CDH). The 5-year-old male patient presented multicystic renal dysplasia kidneys, minor facial dysmorphic features and skeletal anomalies, but neither developmental delay nor behavioral abnormalities. CDH has been previously associated with the 17q12 microdeletion syndrome only in one prenatal case. The present study reinforces the hypothesis that CDH is part of the phenotype for 17q12 microdeletion and that 17q12 encompasses candidate(s) gene(s) involved in diaphragm development. We suggest that PIGW, a gene involved in an early step of GPI biosynthesis, could be a strong candidate gene for CDH. © 2014 Wiley Periodicals, Inc.

High proportion of disease-associated copy number variant maps to chromosome 17. Genomic studies have provided an insight into its complex genomic structure such as relative abundance of segmental duplication and intercepted repetitive elements. 17q21.31, 17q11.2 and 17q12 loci are well known on this chromosome and are associated with microdeletion and microduplication syndrome. No syndrome associated with 17q21.33 locus have been described. We report clinical, cytogenetic and molecular investigations of a 13 years-old girl admitted for evaluation of microcephaly, scoliosis, skeletal defects and learning difficulties. We carried out detailed analysis of the clinical phenotype of this patient and investigated the genetic basis using Agilent 180K Array Comparative Genomic Hybridization. We identified a ∼0.9 Mb de novo microduplication on chromosome 17q21.33. Four genes, COL1A1, SGCA, PPP1R9B and CHAD located within the duplicated region are possible candidates for clinical features present in our patients. Gene expression studies by real-time RT-PCR assay only showed an overexpression of SGCA (P < 0.01), a component of the dystrophin glycoprotein complex. Defect of SGCA was previously shown to lead to severe childhood autosomal recessive muscular dystrophy (LGMD2D) which result in progressive muscle weakness and can also be associated with hyperlordosis or scoliosis. Further cases with similar duplications are expected to be diagnosed. This will contribute to the delineation of this potential new microduplication syndrome and to improve genetic counseling.

Background Interstitial 2q36 deletion is a rare event. Only two previously published cases of 2q36 deletions were characterized using array-CGH. This is the first case diagnosed prenatally. Methods We report on the prenatal diagnosis of a 2q36.1q36.3 interstitial deletion in a fetus with facial dysmorphism, spina bifida, and cleft palate. Results Array-CGH analysis revealed a 5.6 Mb interstitial deletion of the long arm of chromosome 2q36.1q36.3, including the PAX3 and EPHA4 genes. Conclusion The present study reinforces the hypothesis that PAX3 haploinsufficiency may be associated with neural tube defects in humans and suggests that the EPHA4 gene might be implicated during palate development. This report also illustrates the added value of array-CGH to detect cryptic chromosomal imbalances in malformed fetuses and to improve genetic counseling prenatally. Birth Defects Research (Part A) 100:507–511, 2014. © 2014 Wiley Periodicals, Inc.

Introduction/Objectifs La dysfonction des telomeres, comme des mecanismes de reparation de l’ADN, joue un role majeur dans la progression tumorale. Nous avons etudie les parametres telomeriques et l’expression d’ERCC1, une proteine impliquee a la fois dans de nombreux mecanismes de reparation de l’ADN et dans la maintenance des telomeres, pour evaluer leur valeur pronostique dans 90 residus tumoraux de cancer du sein apres chimiotherapie neoadjuvante. Materiels/Patients et methodes Le statut d’ERCC1 a ete evalue a differents niveaux moleculaires (expression proteique, expression du gene et nombre de copie du gene) par immunohistochimie, qRT-PCR et QMF-PCR. Une analyse globale des parametres telomeriques a ete realisee par qPCR pour la longueur des telomeres et qRT-PCR pour l’expression de TERT et des genes codant la tankyrase (TNKS) et les proteines du complexe shelterin (TRF1, TRF2, POT1, TPP1, RAP1 and TIN2). Resultats Nous avons montre que des telomeres courts, une surexpression de TERT et de TNKS et une sous-expression d’ERCC1 sont des marqueurs de mauvais pronostic independants. Un clustering non supervise comprenant les parametres telomeriques et les differentes valeurs d’ERCC1 a permis d’identifier 2 sous-groupes associes, independamment du stade et du grade tumoral, a la survie sans maladie nettement diminuee (HR = 5,41, p = 0,004). Le 1er sous-groupe presente une sous-expression d’ERCC1, des alterations du nombre de copie d’ERCC1, des telomeres courts et une surexpression de TERT et le 2e une surexpression de TNKS et des genes codant les proteines du complexes shelterin. Conclusions Cette etude montre que la dysfonction simultanee des telomeres et des mecanismes de reparation de l’ADN peut contribuer de facon synergique a la resistance tumorale. Ces parametres ont une valeur pronostique dans le cancer du sein traite par chimiotherapie neoadjuvante et pourraient etre utilises cliniquement comme biomarqueurs utiles au choix de la chimiotherapie adjuvante.

Le role de la longueur des telomeres a ete largement etudie en cancerologie et dans plusieurs pathologies regroupees sous le terme de « telomeropathies » mais reste meconnu dans le developpement embryonnaire et la survenue de malformations congenitales. Plusieurs etudes ont montre un raccourcissement des telomeres dans les villosites choriales en cas de retard de croissance intra-uterin mais a ce jour, une seule etude a decrit l’association de telomeres courts avec des malformations congenitales [1] . Dans notre etude nous avons mesure la longueur des telomeres par PCR quantitative (Light cycler 480, technique SYBR Green I), a partir d’ADN extrait en direct sur des prelevements de liquide amniotique (n = 55) et de villosites choriales (n = 32) de fœtus « sains », hypotrophes ( [2] mais contrairement a ce qui avait ete decrit dans trois autres etudes [3] , [4] , [5] .

Purpose: Genetic instability plays an important role in ovarian carcinogenesis. We investigated the level of telomere shortening and genomic instability in early and preinvasive stages of ovarian cancer, serous tubal intraepithelial carcinoma (STIC), and tubo-ovarian dysplasia (TOD). Experimental Design: Fifty-one TOD from prophylactic salpingo-oophorectomies with BRCA1 or 2 mutation, 12 STICs, 53 tubo-ovarian high-grade serous carcinoma, and 36 noncancerous controls were laser capture microdissected from formalin-fixed, paraffin-embedded sections, analyzed by comparative genomic hybridization (array CGH) and for telomere length (using quantitative real-time PCR based on the Cawthon's method). TOD and STICs were defined by morphologic scores and immunohistochemical expressions of p53, Ki67, and γH2AX. Results: TOD showed marked telomere shortening compared with noncancerous controls (P < 10−7). STICs had even shorter telomeres than TOD (P = 0.0008). Ovarian carcinoma had shorter telomeres than controls but longer than STICs and dysplasia. In TOD, telomeres were significantly shorter in those with BRCA1 mutation than in those with BRCA2 mutation (P = 0.005). In addition, γH2AX expression in TOD and STIC groups with short telomeres was significantly increased (P < 10−7). In dysplastic epithelium, we found subtle genomic alterations, in contrast to more important genomic imbalances in STICs. The total number of genetic alterations was the highest in ovarian cancers. Conclusions: These findings suggest that genetic instability occurs in early stages of ovarian tumorigenesis. STICs and noninvasive dysplasia are likely an important step in early serous ovarian neoplasia. Clin Cancer Res; 19(11); 2873–82. ©2013 AACR.

As almost all patients with high-risk neuroblastomas have autograft, we aimed to determine if minimal residual disease (MRD) quantified by RT-PCR for tyrosine hydroxylase in PBSC is prognostic in neuroblastomas. PBSC harvests from 38 children were analyzed. Seven had harvests positive for TH-mRNA. Patients with a positive MRD had a lower 2-year-overall-survival compared to those with negative MRD (P = 0.04) regardless of whether or not PBSC were re-infused. Patients in CR/VGPR group with positive MRD have hazard ratio of death at 7.3 [1.3–40.5]. In conclusion, molecular MRD status in PBSC of good response group may be of interest as a survival prognostic factor in high-risk neuroblastomas. Pediatr Blood Cancer 2013;160:E109–E112. © 2013 Wiley Periodicals, Inc.

To better define the place of multiplex ligation-dependent probe amplification (MLPA) in routine cytogenetic diagnosis in chronic lymphocytic leukemia (CLL), we compared MLPA and fluorescence in situ hybridization (iFISH) data obtained in 77 CLL patients. Although MLPA detected most recurrent copy number genomic aberrations (90.9%), false-negative results were found in cases with small-size abnormal clones and false-positive MLPA findings resulting from point mutations (TP53) or an apparent lack of probe specificity (chromosome 19) were observed. Thus, MLPA may be a useful complementary but not alternative approach for iFISH testing of genomic aberration in CLL.

International audience; Dysfunctional telomeres and DNA damage repair (DDR) play important roles in cancer progression. Studies have reported correlations between these factors and tumour aggressiveness and clinical outcome in breast cancer. We studied the characteristics of telomeres and expression of ERCC1, a protein involved in a number of DNA repair pathways and in telomere homeostasis, to assess their prognostic value, alone or in combination, in 90 residual breast tumours after treatment with neoadjuvant chemotherapy (NCT). ERCC1 status was investigated at different molecular levels (protein and gene expression and gene copy-number variations) by immunohistochemistry, qRT-PCR and quantitative multiplex fluorescent-PCR (QMF-PCR). A comprehensive analysis of telomere characteristics was performed using qPCR for telomere length and qRT-PCR for telomerase (hTERT), tankyrase 1 (TNKS) and shelterin complex (TRF1, TRF2, POT1, TPP1, RAP1 and TIN2) gene expression. Short telomeres, high hTERT and TNKS expression and low ERCC1 protein expression were independently associated with worse survival outcome. Interestingly, ERCC1 gains and losses correlated with worse disease-free (p = 0.026) and overall (p = 0.043) survival as compared to survival of patients with normal gene copy-numbers. Unsupervised hierarchical clustering of all ERCC1 and telomere parameters identified four subgroups with distinct prognosis. In particular, a cluster combining low ERCC1, ERCC1 gene alterations, dysfunctional telomeres and high hTERT and a cluster with high TNKS and shelterin expression correlated with poor disease-free (HR= 5.41, p= 0.0044) and overall survival (HR= 6.01, p= 0.0023) irrespective of tumour stage and grade. This comprehensive study demonstrates that telomere dysfunction and DDR can contribute synergistically to tumour progression and chemoresistance. These parameters are predictors of clinical outcome in breast cancer patients treated with NCT and could be useful clinically as prognostic biomarkers to tailor adjuvant chemotherapy post-NCT.

International audience; Purpose: To analyze the dose rate influence in hyper-radiosensitivity (HRS) of human melanoma cells to very low doses of fast neutrons and to compare to the behaviour of normal human skin fibroblasts.Materials and methods: We explored different neutron dose rates as well as possible implication of DNA double-strand breaks (DSB), apoptosis, and energy-provider adenosine-triphosphate (ATP) levels during HRS.Results: HRS in melanoma cells appears only at a very low dose rate (VLDR), while a high dose rate (HDR) induces an initial cell-radioresistance (ICRR). HRS does not seem to be due either to DSB or to apoptosis. Both phenomena (HRS and ICRR) appear to be related to ATP availability for triggering cell repair. Fibroblast survival after neutron irradiation is also dose rate-dependent but without HRS.Conclusions: Melanoma cells or fibroblasts exert their own survival behaviour at very low doses of neutrons, suggesting that in some cases there is a differential between cancer and normal cells radiation responses. Only the survival of fibroblasts at HDR fits the linear no-threshold model. This new insight into human cell responses to very low doses of neutrons, concerns natural radiations, surroundings of accelerators, proton-therapy devices, flights at high altitude. Furthermore, ATP inhibitors could increase HRS during high-linear energy transfer (high-LET) irradiation.

Microdeletions of 8q21.3–8q22.1 have been identified in all patients with Nablus mask-like facial syndrome (NMLFS). A recent report of a patient without this specific phenotype presented a 1.6 Mb deletion in this region that partially overlapped with previously reported 8q21.3 microdeletions, thus restricting critical region for this syndrome. We report on another case of an 8q21.3 deletion revealed by array comparative genome hybridization (aCGH) in a 4-year-old child with global developmental delay, autism, microcephaly, but without Nablus phenotype. The size of the interstitial deletion was estimated to span 5.2 Mb. By combining the data from previous reports on 8q21.3–8q22.1 deletions and our case, we were able to narrow the critical region of Nablus syndrome to 0.5 Mb. The deleted region includes FAM92A1, which seems to be a potential candidate gene in NMLFS. © 2012 Wiley Periodicals, Inc.

Microduplications 22q11.2 have been recently characterized as a new genomic duplication syndrome showing an extremely variable phenotype ranging from normal or mild learning disability to multiple congenital defects and sharing some overlapping features with DiGeorge/velocardiofacial syndrome (DGS/VCFS), including heart defects, urogenital abnormalities and velopharyngeal insufficiency. We present an atypical and inherited 0.8-Mb duplication at 22q11.2, in the distal segment of the DGS/VCFS syndrome typically deleted region (TDR), in a 3-year-old boy with motor delay, language disorders and mild facial phenotype. This 22q11.2 microduplication was identified by MLPA, designed to detect recurrent microdeletions and microduplications of chromosomal regions frequently involved in mental retardation syndromes and was further characterized by aCGH. The duplicated region encompasses 14 genes, excluding TBX1 but including CRKL, ZNF74, PIK4CA, SNAP29 and PCQAP known to contribute to several aspects of the DGS/VCFS phenotype. To the best of our knowledge, only one case of an isolated duplication in the distal segment of the TDR between chromosome 22-specific low-copy repeats B (LCR22-B) and D (LCR22-D) has been published, but the present report is the first one with a detailed description of physical and developmental features in a patient carrying this kind of atypical 22q11.2 duplication. This case illustrates the importance of reporting unusual 22q11.2 duplications to further evaluate the incidence of these rearrangements in the general population and to improve genotype-phenotype correlations and genetic counseling.

Telomeres are protective chromosomal end structures composed of G-rich nucleotide repeats and an associated protein complex termed shelterin. Because of incomplete replication, telomeric repeats are lost with every cell division, and this telomere attrition is involved in cell senescence and cancer (reviewed in Artandi and DePinho1). Short, unprotected telomeres are erroneously fused by the DNA-repair system, which gives rise to aneuploidies or structural chromosome rearrangements through chromosome fusion-bridge-breakage cycles. Thus, telomere dysfunction can drive genomic instability during tumorigenesis.1

New therapies for children with high risk neuroblastoma are needed, and haploidentical stem cell transplantation with NK post-graft injections is a potential option. To develop this strategy, we compared and correlated two methods of NK cytotoxicity assay. The aim of this work is to optimize in vitro NK cytotoxicity assays, investigate the effect of interleukin stimulation on NK cells and use of antiGD2 antibodies against tumor target cells and finally establish an in vitro model for haploidentical stem cell transplantation. Experimental design We evaluated NK cell cytotoxicity in vitro against NB cell lines (IMR-32 and SK-NSH) in different culture conditions using a Europium BATDA fluorescence test, and correlated the results with quantification of TH, Phox2B, and DCX transcripts evaluated by RT-PCR. Results Both IMR-32 and SK-N-SH neuroblastoma cell lines were sensitive to NK cells and particularly when NK cells were stimulated by interleukin IL-2 and IL-15 or when using anti-GD2 antibodies against tumor target cells. All these results were observed either with Europium fluorometry assay or with RT-PCR quantification. There is a clear correlation between the two methods, for the three transcripts at the ratio effector/target 50/1 (TH r = 0.75, Phox2B r = 0.79 and DCX r = 0.8), for all the values whatever the cell line. Besides for all three transcripts, the correlations were significantly independent of the cell line and the ratio E/T (all p values non-significant) even if the best correlation was observed for the ratio 50/1. After prolonged incubation times of effector and target cells (24 h), which could be evaluated only by RT-PCR, all the transcripts clearly decreased, confirming the haploidentical effect of NK against the two neuroblastoma cell lines in our two in vitro haploidentical models but no advantage of mismatch. Conclusions NK cytotoxicity against neuroblastoma cell lines can be evaluated by Europium assay and by RT-PCR with clear correlation for the three transcripts TH, Phox2B and DCX whatever the ratio E/T and cell line used. This new method of RT-PCR is simple and suitable for large-scale conditions like study of adherent tumor cells or prolonged incubations of target/effector cells which allowed us to observe haploidentical effect.

Little is known on strategies to prevent or to treat relapses occurring after haploidentical stem cell transplantation (haplo-HSCT) performed for the high-risk neuroblastoma (NB). We describe a 6-year-old male with refractory NB who relapsed 22 months after haplo-HSCT. A complete remission was obtained with a combination of immuno-chemotherapy based on donor NK cells transplants, IL2 infusions and temozolomide/topotecan. This case is an incentive to explore both the immediate therapeutic effect of haplo-graft provided via haplo-NK cells and the immunogenic platform that haplo-HSCT offers for future treatment. Our post-relapse strategy shows that chemo- and bio-treatment should be viewed as complementary therapeutic options. Pediatr Blood Cancer 2012;59:739–742. © 2011 Wiley Periodicals, Inc.

Background Graft contamination has been blamed for causing relapse in children with high-risk neuroblastoma (HRNB) after autologous hematopoietic stem cell transplantation (HSCT). Procedure We report the long-term results of hematopoietic reconstitution, post-transplant complications, and clinical outcome of 44 children with HRNB treated with busulfan/melphalan high-dose chemotherapy followed by transplantation of purged CD34+ immunoselected autologous peripheral HSCT. Minimal residual disease (MRD) of grafts was evaluated by anti-GD2 immunofluorescence or tyrosine hydroxylase reverse transcriptase-polymerase chain reaction (RT-PCR). Results Contaminating neuroblasts were found in 19/38 grafts (50%) before CD34+ positive selection, and none after (technique sensitivity of one cell in 105). A median of 6.5 × 106 CD34+ cells/kg (range 0.8–23.7) were transplanted with only 2% of TRM. Neutrophils and platelet recovery occurred within a median of 12 days (range 9–47) and 44 days (range 12–259), respectively, without any secondary graft failure. Twenty-three percents of patients experienced a sepsis (10/44) and 14% a pyelonephritis (6/44). Recurrence of varicella zoster virus occurred in 21% of patients (9/44). Negative RT-PCR MRD within the leukapheresis product and cis-retinoic acid therapy were significantly and independently associated to a better survival (P