Prevalence of aneuploidies in products of spontaneous abortion: interest of FISH and MLPA.

Spontaneous abortion (SA) is the loss of the conceptus before 22 weeks of gestation when fetal weight is less than 500 g. The genetic etiology accounts for more than two third of SA, and autosomal aneuploidies alone account for up to 70% fetal loss. The aim of this study was to highlight the most common chromosomal causes of fetal loss. In this study, 220 products of abortion and in utero fetal death were analyzed by using FISH (AneuVysion) on interphase nuclei from chorionic villus and by using MLPA (SALSA P036, P070 and P245 kits) on DNA extracted from fetal tissues. The gestational age ranged from the 7th to the 38th week of gestation. Of a total of 151 samples analyzed by using FISH, 10 chromosomal abnormalities were observed: four trisomies 21 (one of them was mosaic), a trisomy 18, a trisomy 13, three triploidies and one monosomy X (Turner). From the additional 69 samples analyzed by using MLPA, two anomalies were found: two monosomies X (Turner). FISH and MLPA are simple, rapid and sensitive tools for the detection of chromosomal aneuploidies. Avoiding the cell culture step necessary for karyotyping, they represent very interesting alternative methods to diagnose genomic disorders in products of abortion in which poor sample quality often leads to cell culture failure.

Molecular assessment of minimal residual disease in PBSC harvests provides prognostic information in neuroblastoma.

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.

Prenatal ultrasound diagnosis of a 48,XXYY syndrome.

The 48,XXYY syndrome is a rare uncommon gonosome aneuploidy and its incidence is estimated to be 1:18,000-1:40,000. The phenotype associated with this syndrome, classically described as Klinefelter variant, is extremely variable but developmental abnormalities are always present. Ultrasound signs during pregnancy are inconsistent, and only three prenatal cases have been described in the literature. Here, we report a case of 48,XXYY syndrome identified in prenatal period because of the presence of polyhydramnios and bilateral clubfeet on second trimester ultrasound. This observation shows the importance of chromosomal prenatal diagnosis in cases with bilateral clubfeet on morphologic ultrasound. This diagnosis is essential for further characterization of the prenatal phenotype and to improving genetic counselling.

[Trisomy 21 and cancers]. / Trisomie 21 et cancers.

Patients with trisomy 21, still called Down's syndrome (DS), present a particular tumoral profile compared to the general population with an increased incidence of leukaemia in the childhood and a low risk of solid cancer in the adulthood. DS children indeed present a 50-fold risk of developing a leukaemia compared to age-matched non-trisomic children and most of them develop a specific myelodysplasic disorder called transient myelodysplasic disorder. In spite of the low incidence of solid tumors, some are very rare as breast cancer, nephroblastoma, neuroblastoma and medulloblastoma, whereas the others remain more frequent as retinoblastoma, lymphoma and gonadal and extragonadal germ cell tumours. In this review, we present possible mechanisms which can favour, or on the contrary repress the formation and progression of tumours in DS patients, which are related to gene effect dosage of oncogenes or tumour repressors on chromosome 21, tumour angiogenesis, apoptosis and epithelial cell-stroma interactions.

NK cytotoxicity and alloreactivity against neuroblastoma cell lines in vitro: comparison of Europium fluorometry assay and quantification by RT-PCR.

UNLABELLED: 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.

Prenatal diagnosis of a rare de novo centromeric chromosome 6 variant.

Cytogenetic heteromorphisms are described as heritable variations at specific chromosomal regions without phenotypic effect. Polymorphisms of the size of heterochromatic centromeric regions of chromosomes 1, 9 and 16 have been well documented in humans but only four previous reports described centromeric polymorphism of chromosome 6. We present a prenatal diagnosis of a rare de novo centromeric chromosome 6 variant. Cytogenetic and molecular techniques were used to characterize this variant and confirm the de novo nature of this event. This case illustrates the importance of reporting unusual variant chromosomes for genetic counseling and for determination of the frequency of variant chromosomes in the general population.

De novo unbalanced translocation 2;4 characterized by metaphase CGH and array CGH in a child with mental retardation and dysmorphic features.

BACKGROUND: It is known from postnatal diagnosis that imbalances of the subtelomeric regions contribute significantly to idiopathic mental retardation. PATIENT AND METHODS: We report a case of a 4-year-old child with growth retardation, minor physical abnormalities, hypotonia and developmental delay associated with a derivative chromosome 4. Molecular cytogenetic investigations were performed to characterize the chromosomal rearrangement. RESULTS: Multi fluorescence in situ hybridization revealed the presence of chromosome 2 material on the derivative chromosome 4. Metaphase comparative genomic hybridization detected a terminal 4q34 deletion. Array CGH analysis could precise breakpoints with duplication 2q36 → qter. The clinical phenotype was similar to those described in cases with a trisomy 2qter. CONCLUSION: This study emphasizes the value of array CGH to detect or characterize chromosome rearrangements in mentally retarded patients. Unlike metaphase CGH, the high resolution of array CGH in subtelomeric regions allows an accurate description of chromosomal aberrations.

Clinicopathological factors and nomograms predicting nonsentinel lymph node metastases after neoadjuvant chemotherapy in breast cancer patients.

BACKGROUND: Studies have demonstrated the feasibility and accuracy of sentinel lymph node (SLN) biopsy after neoadjuvant chemotherapy (NAC) in breast cancer. Some SLN-positive patients have low risk of nonsentinel lymph node (non-SLN) involvement. Our goal was to determine clinicopathological factors correlating with the presence of non-SLN metastases in patients after NAC and to assess the validity of nomograms predicting additional axillary metastases. METHODS: Patients with infiltrating breast carcinoma (n = 132) were studied prospectively. All patients received NAC. At surgery, SLN biopsy followed by axillary lymph node dissection was performed. Lymphatic mapping was done using the isotope method. Fifty-one patients were SLN positive. RESULTS: In univariate analysis, tumor size (P = 0.016) and the size of SLN metastases (P = 0.0055) were significantly correlated with the presence of non-SLN metastases. In multivariate analysis, SLN macrometastases (P = 0.047) conferred significantly increased risk of non-SLN metastases. The Memorial Sloan-Kettering Cancer Center nomogram was not reliably predictive for non-SLN metastases (area under the receiver operating characteristic curve, AUC, of 0.542), whereas the MD Anderson (AUC 0.716) and Tenon scoring systems (AUC 0.778) were validated. CONCLUSION: Our results suggest that clinicopathological factors predicting non-SLN involvement in SLN-positive patients with and without NAC are essentially the same. The risk of involvement may be assessed using existing nomograms, but additional large prospective studies are needed to determine their accuracy in patients after NAC.

Allogeneic or haploidentical HSCT for refractory or relapsed solid tumors in children: toward a neuroblastoma model.

New concepts of allogeneic hematopoietic SCT (allo-HSCT) for neuroblastoma and other solid tumors do not rely on escalation of chemotherapy intensity and tumor load reduction but rather on a graft-vs-tumor effect. At this point, this is still an investigational and unusual application of allogeneic transplant, with 78 neuroblastoma patients reported to the European Group for Blood and Marrow Transplantation activity survey from 2002 to 2007 and less than 100 published cases. Two trends can be observed in the reviewed data: some teams have used allo-HSCT in children with refractory or progressive disease and significant tumor burden and other teams in children with CR, PR or minimal residual disease earlier in their disease process. Early studies of allo-HSCT in children with high-risk neuroblastoma suggest that this is a feasible approach that may improve outcome in this deadly disease. However, the proper timing for allo-HSCT during the disease course remains to be determined.

Familial deletion 11q14.3-q22.1 without apparent phenotypic consequences: a haplosufficient 8.5 Mb region.

We present the prenatal diagnosis of a chromosome 11q14.3-q22.1 deletion identified in three generations without apparent phenotypic consequences. A 25-year-old G2, P1 woman underwent amniocentesis at 15 weeks' gestation because of a positive result for Down syndrome maternal serum-screening test (1/70). The fetal karyotype revealed an interstitial deletion of the long arm of chromosome 11 confirmed by CGH and FISH: 46,XX,del(11)(q14.3q22.1). The mother and grandfather of the fetus presented the same interstitial deletion with a little if any phenotype effect. The pregnancy was carried to term and resulted in the birth of a normal girl. To our knowledge, only one case of a chromosome 11q14.3-q21 deletion without phenotypic anomalies has been reported. Our study allows the initially described haplosufficient region to be extended from 3.6 Mb to at least 8.5 Mb. This large deletion was compatible with fertility and apparently normal phenotype. Identification of such chromosomal regions is important for prenatal diagnosis and genetic counseling.

[Prognosis of chronic lymphocytic leukemia: focus on recent biomarkers]. / Pronostic de la leucémie lymphoïde chronique : mise au point sur les facteurs biologiques récents.

Early stages of chronic lymphocytic leukemia (CLL), which are the most frequent at diagnosis, have an extremely variable individual prognosis, as some patients remain stable for years whereas others develop aggressive forms of the disease less or more rapidly. Individual prognosis evaluation of early stages of CLL is then a challenge for physicians; also clinico-hematological stages are still the evaluation basis, numerous biological markers are helpful in providing independent information on patient prognosis. It is useful to distinguish the classical prognosis factors, described in the 1980s, and the recent markers described from the end of the 1990s, which are widely validated for certain, whereas for others further investigations are needed to confirm their prognostic impact. We propose to detail in this review these new prognostic factors of CLL, especially the different serum markers, cytogenetical abnormalities of pathologic lymphocytes, mutational status of the immunoglobulin genes (IgVH) and CD38 and ZAP-70 expression.

Gene dosage methods as diagnostic tools for the identification of chromosome abnormalities.

Cytogenetics is the part of genetics that deals with chromosomes, particularly with numerical and structural chromosome abnormalities, and their implications in congenital or acquired genetic disorders. Standard karyotyping, successfully used for the last 50 years in investigating the chromosome etiology in patients with infertility, fetal abnormalities and congenital disorders, is constrained by the limits of microscopic resolution and is not suited for the detection of subtle chromosome abnormalities. The ability to detect submicroscopic chromosomal rearrangements that lead to copy-number changes has escalated progressively in recent years with the advent of molecular cytogenetic techniques. Here, we review various gene dosage methods such as FISH, PCR-based approaches (MLPA, QF-PCR, QMPSF and real time PCR), CGH and array-CGH, that can be used for the identification and delineation of copy-number changes for diagnostic purposes. Besides comparing their relative strength and weakness, we will discuss the impact that these detection methods have on our understanding of copy number variations in the human genome and their implications in genetic counseling.

Sentinel lymph node biopsy after neoadjuvant chemotherapy is accurate in breast cancer patients with a clinically negative axillary nodal status at presentation.

BACKGROUND: In breast cancer, neoadjuvant chemotherapy (NAC) is widely used in order to enable a conservative surgery. In patients treated with NAC, the use of sentinel lymph node (SLN) biopsy, which is a good predictor of the axillary nodal status in previously untreated patients, is still discussed. The aim of our study was to determine clinicopathological factors that may influence the accuracy of SLN biopsy after NAC. METHODS: Between March 2001 and December 2006, 129 patients with infiltrating breast carcinoma were studied prospectively. Preoperatively, all of them underwent NAC. At surgery, SLN biopsy followed by axillary lymph node (ALN) dissection was performed. Lymphatic mapping was done using the isotope method. RESULTS: The SLN identification rate was 93.8% (121/129). Fifty-six out of the 121 successfully mapped patients had positive ALN. Eight out of these 56 patients had tumor-free SLN (false-negative rate of 14.3%). The false-negative rate was correlated with larger tumor size (T1-T2 versus T3; P = 0.045) and positive clinical nodal status (N0 versus N1-N2; P = 0.003) before NAC. In particular, the false-negative rate was 0% (0/29) in N0 patients and 29.6% (8/27) in N1-N2 patients. Clinical and pathological responses to NAC did not influence the accuracy of SLN biopsy. CONCLUSION: Our results show that clinical nodal status is the main clinicopathological factor influencing the false-negative rate of SLN biopsy after NAC for breast cancer. SLN biopsy after NAC can predict the ALN status with a high accuracy in patients who are clinically lymph node negative at presentation.

Larsen-like phenotype associated with partial trisomy 3p and monosomy 5p.

BACKGROUND: We report on a fetus with radiographic features of Larsen Syndrome (LS) and unbalanced 3;5 translocation. Recently LS was shown to be caused by mutations in FLNB gene which maps on 3p14.3. METHODS: Comparative genomic hybridization (CGH) was performed to search for genomic imbalances. Fluorescence in situ analysis (FISH) was done with BAC clone RP11-754F19 probe from the FLNB gene region (3p14.3). RESULTS: CGH showed a large loss of the chromosome 5 short arm and a gain of half of the short arm of chromosome 3 resulting from a derivative chromosome 5. FISH analysis with FLNB probe demonstrated that it was not triplicated. Thus, we excluded the role of a gene dosage effect of FLNB in abnormal craniofacial development in this fetus. CONCLUSIONS: To our knowledge, this is the first report of Larsen-like phenotype associated with unbalanced translocation resulting in partial trisomy 3p and monosomy 5p.

Interleukin-6 gene amplification and shortened survival in glioblastoma patients.

Interleukin-6 (IL-6) is known to promote tumour growth and survival. We evaluated IL-6 gene amplification in tumours from 53 glioma patients using fluorescence in situ hybridisation. Amplification events were detected only in glioblastomas (15 out of 36 cases), the most malignant tumours, and were significantly associated with decreased patient survival.

Correlation between molecular metastases in sentinel lymph nodes of breast cancer patients and St Gallen risk category.

AIMS: To evaluate the clinical significance of tumour metastases detected using real-time reverse transcription-PCR (RT-PCR) in sentinel lymph nodes (SLN) of breast cancer patients. METHODS: Sixty-seven patients with T1-T2 primary breast cancer were included in a prospective study. SLN were analysed for the presence of metastatic tumour cells using standard histopathology staining, immunochemistry (IHC) and multimarker real-time RT-PCR assay for mammaglobin (MMG), carcinoembryonic antigen (CEA) and cytokeratin-19 (CK19) mRNA expression. Correlations between molecular metastases and traditional clinicopathological prognostic factors, including St Gallen risk categories were studied. RESULTS: Of the 67 patients, 15 (22.3%) had one or more pathology-positive SLN. Five (9.6%) pathology-negative SLN were positive by IHC and 19 (36.5%) by RT-PCR. Of note, RT-PCR analysis was also positive in all cases with pathology- or IHC-positive SLN. MMG was the most informative tumour marker in the panel. Molecularly detected metastases were significantly associated with intermediate St Gallen risk category (p=0.023). CONCLUSION: Molecular staging of SLN using real-time RT-PCR for early breast cancer could serve as a useful complement to standard clinicopathological risk factors. Studies with long-term follow-up are necessary to define the impact of molecular metastases on disease free survival and overall survival.