Elena Sala

Biological heterogeneity of putative bladder cancer stem-like cell populations from human bladder transitional cell carcinoma samples

Transitional cell carcinoma (TCC) is the most common type of bladder cancer. Emerging evidence has suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells, the cancer stem‐like cells (CSCs) or tumor initiating cells. We report on the isolation and biological characterization of putative bladder CSC populations from primary TCCs. Isolated cells were induced to proliferate in stem cell culture conditions (serum‐free medium containing mitogenic growth factors). The proliferating cells formed spheroids (urospheres) and their abilities for extensive proliferation and self‐renewal were assayed. Their positivity for several stem cell markers (CD133, Oct‐3/4, nestin, and cytokeratins) was also assessed by immunofluorescence tests and they could have the potential to differentiate in the presence of serum. In stem cell culture conditions they gradually showed loss of proliferation, adherence to the substrate, and morphological changes, which might reflect their progressive acquisition of differentiative capacity and loss of self‐renewal ability. To evaluate if effective cell selection occurred after isolation, conventional cytogenetic studies on fresh chromosome spreads immediately after isolation and after culture were carried out. In addition, a molecular cytogenetic study by UroVysion assay was carried out on paraffin‐embedded tissue sections and on fresh and after culture nuclei preparations. The data collected indicated important karyotype changes and a positive selection for hypo‐ or near‐diploid cells, losing the complexity present in fresh tumors. (Cancer Sci 2009)

De novo balanced chromosome rearrangements in prenatal diagnosis

We surveyed the datasheets of 29 laboratories concerning prenatal diagnosis of de novo apparently balanced chromosome rearrangements to assess the involvement of specific chromosomes, the breakpoints distribution and the impact on the pregnancy outcome.

Cytogenetics of Premature Ovarian Failure: An Investigation on 269 Affected Women

The importance of X chromosome in the aetiology of premature ovarian failure (POF) is well-known but in many cases POF still remains idiopathic. Chromosome aneuploidy increase is a physiological phenomenon related to aging, but the role of low-level sex chromosome mosaicism in ovarian function is still undiscovered. Standard cytogenetic analysis was carried out in a total of 269 patients affected by POF: 27 chromosomal abnormalities were identified, including X chromosome and autosomal structural and numerical abnormalities. In 47 patients with 46,XX karyotype we performed interphase FISH using X alpha-satellite probe in order to identify X chromosome mosaicism rate. Aneuploidy rate in the patient group was significantly higher than the general population group. These findings underline the importance of X chromosome in the aetiology of POF and highlight the potential role of low-level sex chromosome mosaicism in ovarian aging that may lead to a premature onset of menopause.

Cytogenetic and molecular evaluation of 241 small supernumerary marker chromosomes: cooperative study of 19 Italian laboratories

Purpose: We evaluated the experiences of 19 Italian laboratories concerning 241 small supernumerary marker chromosomes (sSMCs) with the aim of answering questions arising from their origin from any chromosome, their variable size and genetic content, and their impact on the carriers phenotype.Methods: Conventional protocols were used to set up the cultures and chromosome preparations. Both commercial and homemade probes were used for the fluorescent in situ hybridization analyses.Results: A total of 113 of the 241 sSMCs were detected antenatally, and 128 were detected postnatally. There were 52 inherited and 172 de novo cases. Abnormal phenotype was present in 137 cases (57%), 38 of which were antenatally diagnosed. A mosaic condition was observed in 87 cases (36%). In terms of morphology, monocentric and dicentric bisatellited marker chromosomes were the most common, followed by monocentric rings and short-arm isochromosomes. The chromosomes generating the sSMCs were acrocentric in 132 cases (69%) and non-acrocentric chromosomes in 60 cases (31%); a neocentromere was hypothesized in three cases involving chromosomes 6, 8, and 15.Conclusion: The presented and published data still do not allow any definite conclusions to be drawn concerning karyotype–phenotype correlations. Only concerted efforts to characterize molecularly the sSMCs associated or not with a clinical phenotype can yield results suitable for addressing karyotype–phenotype correlations in support of genetic counseling.

Investigating the role of X chromosome breakpoints in premature ovarian failure

The importance of the genetic factor in the aetiology of premature ovarian failure (POF) is emphasized by the high percentage of familial cases and X chromosome abnormalities account for 10% of chromosomal aberrations. In this study, we report the detailed analysis of 4 chromosomal abnormalities involving the X chromosome and associated with POF that were detected during a screening of 269 affected women. Conventional and molecular cytogenetics were valuable tools for locating the breakpoint regions and thus the following karyotypes were defined: 46,X,der(X)t(X;19)(p21.1;q13.42)mat, 46,X,t(X;2)(q21.33;q14.3)dn, 46,X,der(X)t(X;Y)(q26.2;q11.223)mat and 46,X,t(X;13)(q13.3;q31)dn. A bioinformatic analysis of the breakpoint regions identified putative candidate genes for ovarian failure near the breakpoint regions on the X chromosome or on autosomes that were involved in the translocation event. HS6ST1, HS6ST2 and MATER genes were identified and their functions and a literature review revealed an interesting connection to the POF phenotype. Moreover, the 19q13.32 locus is associated with the age of onset of the natural menopause. These results support the position effect of the breakpoint on flanking genes, and cytogenetic techniques, in combination with bioinformatic analysis, may help to improve what is known about this puzzling disorder and its diagnostic potential.

Role of FISH on Uncultured Amniocytes for the Diagnosis of Aneuploidies in the Presence of Fetal Anomalies

Objective: To assess the accuracy of fluorescent in situ hybridization (FISH) on amniocytes in fetuses affected by structural malformations suggestive of chromosomal anomalies. Methods: FISH of uncultured amniotic fluid cells and conventional cytogenetic analysis were performed on 48 pregnancies with ultrasonographic (US) evidence of fetal anomalies. The AneuVysion® assay (Vysis) with specific probes for chromosomes 13, 18, 21, X and Y, was used. Amniotic fluid samples were obtained between the 14th and 34th weeks of gestation. Results: In cases with a single abnormal US finding (n = 15), 5 aneuploidies were detected (1 case of trisomy 13 and 4 of trisomy 21). In the group with two or more malformations (n = 33) there were 15 aneuploidies (9 cases of trisomy 18, 2 of trisomy 21, 2 monosomy X, 1 trisomy 13, and 1 triploidy). In this group, conventional cytogenetic analysis revealed two additional chromosomal anomalies not detectable by FISH (1 trisomy 16 mosaic, and a terminal deletion 4p). No sex aneuploidies were observed. Conclusions: The lack of false-positive diagnosis in the FISH analysis in our sample prompts us to consider interphase FISH as a useful tool in pregnancies at high risk for chromosomal aneuploidies. When FISH analysis is normal, the overall risk of chromosomal abnormalities is significantly reduced. However, the finding of two chromosomal anomalies undetectable by AneuVysion® assay confirms the need for conventional chromosome analysis to complement FISH results. Moreover, the results collected here, in agreement with those already reported in the literature, indicate that FISH analysis on uncultured amniocytes can play an important role in counselling and decision-making, especially in cases at risk for aneuploidies, such as those with structural abnormalities at US.

Interstitial telomeres of an inv(9)(p11.2;q34) involved in a jumping translocation found in a woman through a stable unbalanced translocation in her malformed child

According to the original definition by Lejeune et al ,1 a constitutional jumping translocation (JT) is a non-reciprocal, unstable translocation of a specific chromosomal segment onto the ends of various chromosomes, which leads to a rearranged chromosome with interstitial telomeres. More than 20 cases of constitutional JTs have been reported so far,2–6 and JTs have also been detected in chromosomal instability syndromes7,8 and in about 20 tumours, mainly of the haematological type.9 Most cases of constitutional JTs involve acrocentric chromosomes as either donors or recipients, with a few balanced and unbalanced Robertsonian translocations. Whatever the partner chromosomes, the chromosomal regions around the majority of JT breakpoints are pericentromeric and subtelomeric and are built up of arrays of tandemly repeated sequences.4 In particular, the qh region of chromosome 1 seems be non-randomly involved in the JTs detected in most haematological neoplasms.9 We report here the case of a woman carrying JTs in which the donor segment originated from the short arm of chromosome 9. A particular aspect of this case is the presence of an inv(9)(p11.2;q34) chromosome in the constitutional karyotype, and the transmission of the most often represented JT (9;17)(p11.2;qter) to her malformed child. ### Case report The infant is the first and only child, spontaneously delivered at term after an uneventful pregnancy when the mother was 38 years old. His birth weight was 2720 g (<10th centile), length 49 cm (<25th centile), and head circumference 35 cm (50th centile). As a newborn, he showed facial dysmorphism including hypertelorism, a flat nasal bridge, and low set ears, bony abnormalities of both hands and feet (camptodactyly and talipes equinovarus with partial syndactyly of the second and third fingers), and unilateral cryptorchidism. During the first postnatal week, he experienced mild respiratory distress and oliguria. Echocardiography showed a …

Fetal trisomy 5 mosaicism: Case report and literature review

Fetal mosaicism can be suspected at prenatal diagnosis if a trisomy is detected in just a few cells in chorionic villus or colonies in amniocytes in at least two separate flasks and is confirmed if found in other analyzed fetal/neonatal tissues. Trisomic conditions for some chromosomes are clinically recognized whereas rare trisomy mosaicisms in amniocytes, such as trisomy 2, 3, 5, 9, 12, 14, 15, 16, 22, create some problems in interpretation. There are reports in the literature where only a few cells showing abnormal karyotype with a rare trisomy were seen later to be clinically significant in the newborn infant [Daniel et al., 2004]. The risk of abnormal pregnancy outcome varies depending on the chromosome involved in trisomic mosaic and on the presence of uniparental disomy (UPD). Phenotypic abnormalities in the offspring, IUGR and/or fetal demise or stillbirth, range frommoderate to severe [Hsu et al., 1997]. At the reference Regional Center for pregnancies complicated by IUGR (HSGerardo, Monza, Italy), we encountered a 28-year-old primiparous woman with insulin-dependent diabetes mellitus (IDDM type I) at the 26th week of gestation and showing ultrasound diagnosis of intrauterine growth retardation (IUGR) in a morphologically normal fetus. Amniotic fluid analysis showed 41 clones from 9 independent cultures with normal female karyotype (46,XX) and 5 clones from 3 independent cultures with trisomy 5 (47,XX,þ5). Fetal heart monitoring at 30 weeks showed severe variable decelerations. The patient delivered a female infant by Caesarean with Apgar scores of 5/1 and 9/5 and 7.33 cord blood pH. At birth trisomy 5 was also observed in metaphases from three placental biopsies whereas all analyzed metaphases from cord and peripheral blood (100 and 320, respectively) were normal. Dual color FISH analysis using D5S23 cosmidic probe and the a-satellite centromeric probe for chromosome 16 as control on interphasic placental nuclei confirmed the mosaicism: 64.2% (52/81) of nuclei showed three signals for chromosome 5 and two signals for chromosome 16 and 14.8% (12/81) of nuclei presented normal patterns. The peripheral blood showed disomy in all analyzed cells (174) (parents declined giving permission for biopsy of their baby’s skin). Because of the discrepancy observed between placental and fetal karyotypes, UPD5 was tested and excluded. Physical examination at birth showed: a weight of 740 g (3rd centile), a length of 34 cm (<10th centile), and a head circumference of 26 cm (10th centile). Clinodactyly of the fifth right toe and anterior anus were also noted in the baby. The histological examination of the complete placenta (150 g) showed a decidual vasculopathy. During admission to the neonatal intensive care unit the cerebral sonogram was normal, and a small interatrial shunt had completely disappeared at 11 months. The girl was discharged at 3 months of age, with an increase in length and weight but remaining within the 3rd centile with normal

Novel neurofibromatosis type 2 mutation presenting with status epilepticus

Neurofibromatosis type 2 (NF2) is a dominantly inherited syndrome caused by mutations of the tumour-suppressor NF2, which encodes the merlin protein. Mutations are associated with a predisposition to development of benign tumours in the central nervous system. Even though cerebral cortical lesions are frequently associated with seizures, epilepsy is rarely described in NF2. Here, we describe an adult case of NF2 in which the onset of symptoms was characterised by status epilepticus. In this patient, we identified the novel c.428_430delCTTdel mutation in NF2, involving the amino-terminal FERM domain, which is fundamental for the correct tumour suppressor function of the protein. Bioinformatic analyses revealed an important structural perturbation of the FERM domain, with a predicted impairment of the anti-tumour activity.

Using copy number alterations to identify new therapeutic targets for bladder carcinoma

Bladder cancer represents the ninth most widespread malignancy throughout the world. It is characterized by the presence of two different clinical and prognostic subtypes: non-muscle-invasive bladder cancers (NMIBCs) and muscle-invasive bladder cancers (MIBCs). MIBCs have a poor outcome with a common progression to metastasis. Despite improvements in knowledge, treatment has not advanced significantly in recent years, with the absence of new therapeutic targets. Because of the limitations of current therapeutic options, the greater challenge will be to identify biomarkers for clinical application. For this reason, we compared our array comparative genomic hybridization (array-CGH) results with those reported in literature for invasive bladder tumors and, in particular, we focused on the evaluation of copy number alterations (CNAs) present in biopsies and retained in the corresponding cancer stem cell (CSC) subpopulations that should be the main target of therapy. According to our data, CCNE1, MYC, MDM2 and PPARG genes could be interesting therapeutic targets for bladder CSC subpopulations. Surprisingly, HER2 copy number gains are not retained in bladder CSCs, making the gene-targeted therapy less interesting than the others. These results provide precious advice for further study on bladder therapy; however, the clinical importance of these results should be explored.