Noninvasive Prenatal Diagnosis of a Paternally Inherited MEN1 Pathogenic Splicing Variant.

CONTEXT: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disease caused by mutations in the tumor suppressor gene MEN1. The uncertainty of pathogenicity of MEN1 variants complexifies the selection of the patients likely to benefit from specific care. OBJECTIVE: MEN1-mutated patients should be offered tailored tumor screening and genetic counseling. We present a patient with hyperparathyroidism for whom genetic analysis identified a variant of uncertain significance in the MEN1 gene (NM_130799.2): c.654G > T p.(Arg218=). Additional functional genetic tests were performed to classify the variant as pathogenic and allowed prenatal testing. DESIGN: Targeted next generation sequencing identified a synonymous variant in the MEN1 gene in a 26-year-old male with symptomatic primary hyperparathyroidism. In silico and in vitro genetic tests were performed to assess variant pathogenicity. RESULTS: Genetic testing of the proband's unaffected parents showed the variant occurred de novo. Transcript study showed a splicing defect leading to an in-frame deletion. The classification of the MEN1 variant as pathogenic confirmed the diagnosis of MEN1 and recommended an adapted medical care and follow-up. Pathogenic classification also allowed to propose a genetic counseling to the proband and his wife. Noninvasive prenatal diagnosis was performed with a personalized medicine-based protocol by detection of the paternally inherited variant in maternal plasmatic cell free DNA, using digital PCR. CONCLUSION: We showed that functional genetic analysis can help to assess the pathogenicity of a MEN1 variant with crucial consequences for medical care and genetic counseling decisions.

International external quality assurance of JAK2 V617F quantification.

External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

Impact of Graft-Versus-Graft Natural Killer Cell Alloreactivity on Single Unit Dominance After Double Umbilical Cord Blood Transplantation.

BACKGROUND: Natural killer (NK) cell alloreactivity is favored after double umbilical cord blood transplantation (dUCBT) in which cord blood (UCB) units and patients are often HLA class I mismatched. Generally, only 1 UCB unit persists after dUCBT. We hypothesize, that NK cell alloreactivity mediated by killer cell immunoglobulin-like receptor (KIR)-HLA interactions may explain the dominance of 1UCB unit over the other after dUCBT. METHODS: We investigated the impact of KIR NK cell alloreactivities on the dominance of 1 full UCB unit in 50 dUCBT. We analyzed the effects of the KIR/HLA genetic incompatibilities and studied cord blood cells at both the phenotypic and functional levels. RESULTS: The genetic combination of KIR3DL1 loser UCB unit/Bw4 winner UCB unit determined both the dominance of 1 UCB unit (hazards ratio, 2.88 [1.32-6.27], P = 0.0077) and correlated with an increased incidence of relapse (hazards ratio, 4.91 [1.39-17.3], P = 0.0134). It is interesting to note that cord blood cells exhibited extremely low HLA class I expression. Moreover, resting cord blood KIR3DL1 NK cells exhibited a basal alloreactivity against Bw4 target cells that increased upon activation, thus triggering death by apoptosis. CONCLUSIONS: Our unicentric study suggests, for the first time, the significant impact of KIR NK cell alloreactivity in the determination of which UCB unit will dominate in dUCBT.

Acute cytotoxicity of MIRA-1/NSC19630, a mutant p53-reactivating small molecule, against human normal and cancer cells via a caspase-9-dependent apoptosis.

Although numerous studies have focused on the mechanisms of action of the candidate chemotherapeutic drug MIRA-1/NSC19630, initially described as a mutant p53-reactivating small molecule, the issue of its toxicological evaluation remains open. Here, we devised a strategy to examine the effects of MIRA-1 on a variety of human normal cells and cancer cell lines. First, we demonstrated a massive and rapid (within 2 hours) MIRA-1 apoptotic effect on human normal primary epithelial cells as shown using an intestinal mucosa explant assay. MIRA-1 was also cytotoxic to primary and subcultured human mesenchymal cells. Interestingly these effects were restricted to actively proliferating cells. Second, MIRA-1 acute toxicity was independent of p53, since it occurred in human normal cells with increased or silenced p53 expression level, in cancer cells derived from solid or liquid tumors, with either mutated or wt TP53, and in cancer cells devoid of p53. Third, combined pharmacological and genetic approaches showed that MIRA-1 acute cytotoxicity was mediated by a caspase-9-dependent apoptosis. In conclusion, our strategy unveils the limitations of the targeted action of a small molecule designed to reactivate mutant p53.

Complete donor T cell chimerism predicts lower relapse incidence after standard double umbilical cord blood reduced-intensity conditioning regimen allogeneic transplantation in adults.

Double umbilical cord blood (dUCB) allogeneic transplantation after a low-dose total body irradiation, cyclophosphamide, and fludarabine (TCF)-based reduced-intensity conditioning regimen (RIC) is increasingly used in adults lacking a suitable related or unrelated donor. Currently, there are little data regarding the long-term outcome of CD3(+) T cell chimerism (TCC) in this particular setting. Thirty-six adults with various hematological diseases who received dUCB allogeneic transplants conditioned with TCF were included in this retrospective study. Peripheral blood CD3(+) TCC was considered until day +100 after transplantation to determine the impact of full versus mixed chimerism on long-term outcomes. Twenty-nine and 7 patients were documented with full and mixed CD3(+) TCC, respectively, within the first 100 days after transplantation. With a median follow-up of 36 months, 3 year-overall survival (OS), disease-free survival (DFS), and cumulative incidence of relapse (CIR) were 61%, (95% confidence interval [CI], 43% to 75%); 50% (95% CI, 32.5% to 66%), and 28% (95% CI, 16% to 44%), respectively. In univariate analysis, a full CD3(+) TCC was associated with a better 3-year DFS: 59% (95% CI, 39% to 75.5%) versus 14% (95% CI, 7% to 46%); hazard ratio (HR), .24 (.09 to .65); P = .005 and a lower CIR: 24% (95% CI, 21.5% to 57%) versus 78% (95% CI, 52% to 99%); HR, .18 (.05 to .50); P = .004. In multivariate analysis, a full CD3(+) TCC remained associated with a lower CIR (HR, .17 [.028 to .99]; P = .049). CD3(+) TCC has no impact on graft-versus-host disease and nonrelapse mortality in this study. In conclusion, here, full CD3(+) TCC was independently associated with a lower risk of relapse in adults receiving a dUCB TCF RIC allogeneic transplantation. This highlights the need to develop immunotherapy approaches allowing for early conversion to full chimerism after this type of transplantation.

PRIMA-1Met induces myeloma cell death independent of p53 by impairing the GSH/ROS balance.

The aim of this study was to assess the efficiency of p53 reactivation and induction of massive apoptosis (PRIMA-1(Met)) in inducing myeloma cell death, using 27 human myeloma cell lines (HMCLs) and 23 primary samples. Measuring the lethal dose (LD50) of HMCLs revealed that HMCLs displayed heterogeneous sensitivity, with an LD50 ranging from 4 µM to more than 200 µM. The sensitivity of HMCLs did not correlate with myeloma genomic heterogeneity or TP53 status, and PRIMA-1(Met) did not induce or increase expression of the p53 target genes CDKN1A or TNFRSF10B/DR5. However, PRIMA-1(Met) increased expression of NOXA in a p53-independent manner, and NOXA silencing decreased PRIMA1(Met)-induced cell death. PRIMA-1(Met) depleted glutathione (GSH) content and induced reactive oxygen species production. The expression of GSH synthetase correlated with PRIMA-1(Met) LD50 values, and we showed that a GSH decrease mediated by GSH synthetase silencing or by and L-buthionine sulphoximine, an irreversible inhibitor of γ-glutamylcysteine synthetase, increased PRIMA-1(Met)-induced cell death and overcame PRIMA-1(Met) resistance. PRIMA-1(Met) (10 µM) induced cell death in 65% of primary cells independent of the presence of del17p; did not increase DR5 expression, arguing against an activation of p53 pathway; and synergized with L-buthionine sulphoximine in all samples. Finally, we showed in mouse TP53(neg) JJN3-xenograft model that PRIMA-1(Met) inhibited myeloma growth and synergized with L-buthionine sulphoximine in vivo.

Human herpesvirus 6 reactivation before engraftment is strongly predictive of graft failure after double umbilical cord blood allogeneic stem cell transplantation in adults.

Our main objective was to determine new factors associated with engraftment and single-unit predominance after double umbilical cord blood (UCB) allogeneic stem-cell transplantation. Engraftment occurred in 78% of cases in this retrospective study including 77 adult patients. Three-year overall survival, disease-free survival, relapse incidence, and nonrelapse mortality were 55 ± 6%, 44 ± 6%, 33 ± 5%, and 23 ± 4%, respectively. In multivariate analysis, Human herpesvirus 6 reactivation during aplasia (hazard ratio [HR] = 2.63; 95% confidence interval [CI]: 1.64-4.17; p < 0.001), younger recipient age (<53 years) (HR = 1.97; 95% CI: 1.16-3.35; p = 0.012), and lower human leukocyte antigen matching between the two units (3 of 6 or 4 of 6) (HR = 2.09; 95% confidence interval: 1.22-3.59; p = 0.013) were the three factors independently associated with graft failure. Also, factors independently predicting the losing UCB unit were younger age of the UCB unit (odds ratio [OR] = 1.01; 95% CI: 1-1.02; p = 0.035), lower CD34(+) cell dose contained in the UCB unit (≤ 0.8 × 10(5)/kg) (OR = 2.55; 95% CI: 1.05-6.16; p = 0.04), and presence of an ABO incompatibility between the UCB unit and the recipient (OR = 2.53; 95% CI: 1.15-5.53; p = 0.02). Thus, Human herpesvirus 6 reactivation during aplasia, lower unit-unit human leukocyte antigen matching, and younger UCB unit age, as new unfavorable predictive factors, may represent new parameters to take into account after double UCB allogeneic stem-cell transplantation in adults. These results need to be confirmed prospectively, as they may influence unit selections and patient outcomes.

NOTCH2 and FLT3 gene mis-splicings are common events in patients with acute myeloid leukemia (AML): new potential targets in AML.

Our previous studies revealed an increase in alternative splicing of multiple RNAs in cells from patients with acute myeloid leukemia (AML) compared with CD34(+) bone marrow cells from normal donors. Aberrantly spliced genes included a number of oncogenes, tumor suppressor genes, and genes involved in regulation of apoptosis, cell cycle, and cell differentiation. Among the most commonly mis-spliced genes (>70% of AML patients) were 2, NOTCH2 and FLT3, that encode myeloid cell surface proteins. The splice variants of NOTCH2 and FLT3 resulted from complete or partial exon skipping and utilization of cryptic splice sites. Longitudinal analyses suggested that NOTCH2 and FLT3 aberrant splicing correlated with disease status. Correlation analyses between splice variants of these genes and clinical features of patients showed an association between NOTCH2-Va splice variant and overall survival of patients. Our results suggest that NOTCH2 and FLT3 mis-splicing is a common characteristic of AML and has the potential to generate transcripts encoding proteins with altered function. Thus, splice variants of these genes might provide disease markers and targets for novel therapeutics.

A genome-wide aberrant RNA splicing in patients with acute myeloid leukemia identifies novel potential disease markers and therapeutic targets.

PURPOSE: Despite new treatments, acute myeloid leukemia (AML) remains an incurable disease. More effective drug design requires an expanded view of the molecular complexity that underlies AML. Alternative splicing of RNA is used by normal cells to generate protein diversity. Growing evidence indicates that aberrant splicing of genes plays a key role in cancer. We investigated genome-wide splicing abnormalities in AML and based on these abnormalities, we aimed to identify novel potential biomarkers and therapeutic targets. EXPERIMENTAL DESIGN: We used genome-wide alternative splicing screening to investigate alternative splicing abnormalities in two independent AML patient cohorts [Dana-Farber Cancer Institute (DFCI) (Boston, MA) and University Hospital de Nantes (UHN) (Nantes, France)] and normal donors. Selected splicing events were confirmed through cloning and sequencing analysis, and than validated in 193 patients with AML. RESULTS: Our results show that approximately 29% of expressed genes genome-wide were differentially and recurrently spliced in patients with AML compared with normal donors bone marrow CD34(+) cells. Results were reproducible in two independent AML cohorts. In both cohorts, annotation analyses indicated similar proportions of differentially spliced genes encoding several oncogenes, tumor suppressor proteins, splicing factors, and heterogeneous-nuclear-ribonucleoproteins, proteins involved in apoptosis, cell proliferation, and spliceosome assembly. Our findings are consistent with reports for other malignances and indicate that AML-specific aberrations in splicing mechanisms are a hallmark of AML pathogenesis. CONCLUSIONS: Overall, our results suggest that aberrant splicing is a common characteristic for AML. Our findings also suggest that splice variant transcripts that are the result of splicing aberrations create novel disease markers and provide potential targets for small molecules or antibody therapeutics for this disease.

New chondrosarcoma cell lines and mouse models to study the link between chondrogenesis and chemoresistance.

Chondrosarcomas are cartilage-forming, poorly vascularized tumors. They represent the second malignant primary bone tumor of adults after osteosarcoma, but in contrast to osteosarcoma they are resistant to chemotherapy and radiotherapy, surgical excision remaining the only therapeutic option. Few cell lines and animal models are available, and the mechanisms behind their chemoresistance remain largely unknown. Our goal was to establish new cell lines and animal cancer models from human chondrosarcoma biopsies to study their chemoresistance. Between 2007 and 2012, 10 chondrosarcoma biopsies were collected and used for cell culture and transplantation into nude mice. Only one transplanted biopsy and one injected cell line has engrafted successfully leading to conventional central high-grade chondrosarcoma similar to the original biopsies. In culture, two new stable cell lines were obtained, one from a dedifferentiated and one from a grade III conventional central chondrosarcoma biopsy. Their genetic characterization revealed triploid karyotypes, mutations in IDH1, IDH2, and TP53, deletion in CDKN2A and/or MDM2 amplification. These cell lines expressed mesenchymal membrane markers (CD44, 73, 90, 105) and were able to produce a hyaline cartilaginous matrix when cultured in chondrogenic three-dimensional (3D) pellets. Using a high-throughput quantitative RT-PCR approach, we observed that cell lines cultured in monolayer had lost expression of several genes implicated in cartilage development (COL2A1, COMP, ACAN) but restored their expression in 3D cultures. Chondrosarcoma cells in monolayer were sensitive to several conventional chemotherapeutic agents but became resistant to low doses of mafosfamide or doxorubicin when cultured in 3D pellets, in parallel with an altered nucleic accumulation of the drug. Our results indicate that the cartilaginous matrix produced by chondrosarcoma cells may impair diffusion of several drugs and thus contribute to chemoresistance. Therefore, 3D chondrogenic cell pellets constitute a more relevant model to study chondrosarcoma chemoresistance and may be a valuable alternative to animal experimentations.

The translocation t(4;14) can be present only in minor subclones in multiple myeloma.

PURPOSE: Although the translocation t(4;14) is supposed to be a primary event in multiple myeloma, we have been surprised to observe that in large relapse series of patients, the t(4;14) can be observed only in subpopulations of plasma cells, in contrast to what is seen at diagnosis. This observation raised the question of possible subclones harboring the translocation that would be observable only at the time of relapse. EXPERIMENTAL DESIGN: To address this issue, we analyzed by FISH a cohort of 306 patients for whom we had at least two samples obtained at different disease phases. RESULTS: We observed a "gain" of the t(4;14) in 14 patients, and conversely, a "loss" of the translocation in 11 patients. Two hypotheses were raised: either an acquisition of the translocation during evolution or the existence of small t(4;14)-positive subclones at the time of diagnosis. To address this question, we had the opportunity to analyze two patients at the time of diagnosis by RT-PCR (reverse transcription-polymerase chain reaction) to look for the chimeric Eµ-MMSET transcript, and one patient positive at diagnosis, but negative at relapse. The samples were positive, supporting the second hypothesis. Furthermore, the IGH sequences of two patients who "lose" the t(4;14) were identical at diagnosis and relapse, confirming the existence of a common ancestral clone. CONCLUSION: Thus, the conclusion of this study is that the t(4;14) is not a primary event in multiple myeloma and that it can be present in silent subclones at diagnosis, but also at relapse.

Comparison of outcomes after two standards-of-care reduced-intensity conditioning regimens and two different graft sources for allogeneic stem cell transplantation in adults with hematologic diseases: a single-center analysis.

Recent advances in allogeneic stem cell transplantation (allo-HSCT) have included the advent of reduced-intensity conditioning (RIC) regimens to decrease the toxicity of myeloablative allo-SCT and the use of double umbilical cord blood (dUCB) units as a graft source in adults lacking a suitable donor. The FB2A2 regimen (fludarabine 30 mg/kg/day for 5-6 days + i.v. busulfan 3.6 mg/kg/day for 2 days + rabbit antithymocyte globulin 2.5 mg/kg/day for 2 days) supported by peripheral blood stem cells (PBSCs) and the TCF regimen (fludarabine 200 mg/m² for 5 days + cyclophosphamide 50 mg/kg for 1 day + low-dose [2 Gy] total body irradiation) supported by dUCB units are currently the most widely used RIC regimens in many centers and could be considered standard of care in adults eligible for an RIC allo-SCT. Here we compared, retrospectively, the outcomes of adults patients who received the FB2A2-PBSC RIC regimen (n = 52; median age, 59 years; median follow-up, 19 months) and those who received the dUCB-TCF RIC regimen (n = 39; median age, 56 years; median follow-up, 20 months) for allo-SCT between January 2007 and November 2010. There were no significant between-group differences in patient and disease characteristics. Cumulative incidences of engraftment, acute grade II-IV and chronic graft-versus-host disease were similar in the 2 groups. The median time to platelet recovery, incidence of early death (before day +100), and 2-year nonrelapse mortality were significantly higher in the dUCB-TCF group (38 days versus 0 days [P <.0001]; 20.5% versus 4% [P = .05], and 26.5% versus 6% [P = .02], respectively). The groups did not differ in terms of 2-year overall survival (62% for FB2A2-PBSC versus 61% for dUCB-TCF), disease-free survival (59% versus 50.5%), or relapse incidence (35.5% versus 23%). In multivariate analysis, the presence of a lymphoid disorder was associated with a significantly higher 2-year overall survival (hazard ratio, 0.42; 95% confidence interval, 0.20-0.87; P = .02), whereas patients receiving a FB2A2-PBSC allo-SCT had a significantly lower 2-year nonrelapse mortality (hazard ratio, 0.24; 95% confidence interval, 0.1-0.7; P = .01). There were no factors associated with higher 2-year disease-free survival or lower relapse incidence. This study suggests that the dUCB-TCF regimen provides a valid alternative in adults lacking a suitable donor and eligible for RIC allo-SCT. Prospective and randomized studies are warranted to establish the definitive role of dUCB RIC allo-SCT in adults. In addition, strategies for decreasing nonrelapse mortality after dUCB RIC allo-SCT are urgently needed.