Publications by authors named "Roberto Rosati"

24 Publications

  • Page 1 of 1

Non-syndromic Oculocutaneous Albinism: Novel Genetic Variants and Clinical Follow Up of a Brazilian Pediatric Cohort.

Front Genet 2020 28;11:397. Epub 2020 Apr 28.

Instituto de Pesquisa Pelé Pequeno Príncipe, Curitiba, Brazil.

Oculocutaneous albinism (OCA) is a genetic disorder characterized by skin, hair, and eye hypopigmentation due to a reduction or absence of melanin. Clinical manifestations include vision problems and a high susceptibility to skin cancer. In its non-syndromic form, OCA is associated with six genes and one chromosomal region. Because OCA subtypes are not always clinically distinguishable, molecular analysis has become an important tool for classifying types of OCA, which facilitates genetic counseling and can guide the development of new therapies. We studied eight Brazilian individuals aged 1.5-18 years old with clinical diagnosis of OCA. Assessment of ophthalmologic characteristics showed results consistent with albinism, including reduced visual acuity, nystagmus, and loss of stereoscopic vision. We also observed the appearance of the strabismus and changes in static refraction over a 2-year period. Dermatologic evaluation showed that no participants had preneoplastic skin lesions, despite half of the participants reporting insufficient knowledge about skin care in albinism. Whole-exome and Sanger sequencing revealed eight different mutations: six in the gene and two in the gene, of which one was novel and two were described in a population study but were not previously associated with the OCA phenotype. We performed two ophthalmological evaluations, 2 years apart; and one dermatological evaluation. To the best of our knowledge, this is the first study to perform clinical follow-up and genetic analysis of a Brazilian cohort with albinism. Here, we report three new OCA causing mutations.
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http://dx.doi.org/10.3389/fgene.2020.00397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198815PMC
April 2020

Negative effective excitonic diffusion in monolayer transition metal dichalcogenides.

Nanoscale 2020 Jan 11;12(1):356-363. Epub 2019 Dec 11.

Chalmers University of Technology, Department of Physics, 412 96 Gothenburg, Sweden.

While exciton relaxation in monolayers of transition metal dichalcogenides (TMDs) has been intensively studied, spatial exciton diffusion has received only a little attention - in spite of being a key process for optoelectronics and having already shown interesting unconventional behaviours (e.g. spatial halos). Here, we study the spatiotemporal dynamics in TMD monolayers and track optically excited excitons in time, momentum, and space. In particular, we investigate the temperature-dependent exciton diffusion including the remarkable exciton landscape constituted by bright and dark states. Based on a fully quantum mechanical approach, we show at low temperatures an unexpected negative effective diffusion characterized by a shrinking of the spatial exciton distributions. This phenomenon can be traced back to the existence of dark exciton states in TMD monolayers and is a result of an interplay between spatial exciton diffusion and intervalley exciton-phonon scattering.
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http://dx.doi.org/10.1039/c9nr07056gDOI Listing
January 2020

Penetrance of the R337H Mutation and Pediatric Adrenocortical Carcinoma Incidence Associated with Environmental Influences in a 12-Year Observational Cohort in Southern Brazil.

Cancers (Basel) 2019 Nov 16;11(11). Epub 2019 Nov 16.

Instituto de Pesquisa Pelé Pequeno Príncipe, Av. Silva Jardim, 1632, 80250-060 Curitiba PR, Brazil.

The R337H mutation is associated with increased incidence of pediatric adrenocortical tumor (ACT). The different environmental conditions where R337H carriers live have not been systematically analyzed. Here, the R337H frequencies, ACT incidences, and R337H penetrance for ACT were calculated using the 2006 cohort with 4165 R337H carriers living in Paraná state (PR) subregions. The effectiveness of a second surveillance for R337H probands selected from 42,438 tested newborns in PR (2016 cohort) was tested to detect early stage I tumor among educated families without periodical exams. Estimation of R337H frequencies and ACT incidence in Santa Catarina state (SC) used data from 50,115 tested newborns without surveillance, ACT cases from a SC hospital, and a public cancer registry. R337H carrier frequencies in the population were 0.245% (SC) and 0.306% (PR), and 87% and 95% in ACTs, respectively. The ACT incidence was calculated as ~6.4/million children younger than 10 years per year in PR (95% CI: 5.28; 7.65) and 4.15/million in SC (CI 95%: 2.95; 5.67). The ACT penetrance in PR for probands followed from birth to 12 years was 3.9%. R337H carriers living in an agricultural subregion (C1) had a lower risk of developing pediatric ACT than those living in industrial and large urban subregion (relative risk = 2.4). One small ACT (21g) without recurrence (1/112) was detected by the parents in the 2016 cohort. ACT incidence follows R337H frequency in each population, but remarkably environmental factors modify these rates.
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http://dx.doi.org/10.3390/cancers11111804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896071PMC
November 2019

The Prognostic Role of CD8 T Lymphocytes in Childhood Adrenocortical Carcinomas Compared to Ki-67, PD-1, PD-L1, and the Weiss Score.

Cancers (Basel) 2019 Nov 5;11(11). Epub 2019 Nov 5.

Pelé Pequeno Príncipe Research Institute, 1532 Silva Jardim, AV., Curitiba, PR 80250-200, Brazil.

Adrenocortical carcinoma (ACC) is a rare disease among children. Our goal was to identify prognostic biomarkers in 48 primary ACCs from children (2.83 ± 2.3 y; mean age ± SD) by evaluating the tumor stage and outcome for an age of diagnosis before or after 3 years, and association with ACC cluster of differentiation 8 positive (CD8) cytotoxic T lymphocytes (CD8-CTL) and Ki-67 immunohistochemical expression (IHC). Programmed death 1(PD-1)/Programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) in ACC was analyzed in a second, partially overlapping cohort ( = 19) with a similar mean age. All patients and control children were carriers of the germline R337H mutation. Survival without recurrence for less than 3 years and death unrelated to disease were excluded. Higher counts of CD8-CTL were associated with patients diagnosed with ACC at a younger age and stage I, whereas a higher percentage of the Ki-67 labeling index (LI) and Weiss scores did not differentiate disease free survival (DFS) in children younger than 3 years old. No PD-1 staining was observed, whereas weakly PD-L1-positive immune cells were found in 4/19 (21%) of the ACC samples studied. A high CD8-CTL count in ACC of surviving children is compelling evidence of an immune response against the disease. A better understanding of the options for enhancement of targets for CD8 T cell recognition may provide insights for future pre-clinical studies.
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http://dx.doi.org/10.3390/cancers11111730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896110PMC
November 2019

Exciton Propagation and Halo Formation in Two-Dimensional Materials.

Nano Lett 2019 10 23;19(10):7317-7323. Epub 2019 Sep 23.

Department of Physics , Chalmers University of Technology , 412 96 Gothenburg , Sweden.

The interplay of optics, dynamics, and transport is crucial for the design of novel optoelectronic devices, such as photodetectors and solar cells. In this context, transition-metal dichalcogenides (TMDs) have received much attention. Here, strongly bound excitons dominate optical excitation, carrier dynamics, and diffusion processes. While the first two have been intensively studied, there is a lack of fundamental understanding of nonequilibrium phenomena associated with exciton transport that is of central importance (e.g., for high-efficiency light harvesting). In this work, we provide microscopic insights into the interplay of exciton propagation and many-particle interactions in TMDs. On the basis of a fully quantum mechanical approach and in excellent agreement with photoluminescence measurements, we show that Auger recombination and emission of hot phonons act as a heating mechanism giving rise to strong spatial gradients in excitonic temperature. The resulting thermal drift leads to an unconventional exciton diffusion characterized by spatial exciton halos.
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http://dx.doi.org/10.1021/acs.nanolett.9b02948DOI Listing
October 2019

Spatial trends in congenital malformations and stream water chemistry in Southern Brazil.

Sci Total Environ 2019 Feb 5;650(Pt 1):1278-1291. Epub 2018 Sep 5.

Pelé Pequeno Príncipe Research Institute, Curitiba, PR, Brazil; Faculdades Pequeno Principe, Curitiba, PR, Brazil; Centro de Genética Molecular e Pesquisa do Câncer em Crianças (CEGEMPAC), Universidade Federal do Paraná, Curitiba, PR, Brazil; Departamento de Saúde Coletiva, Universidade Federal do Paraná, Curitiba, PR, Brazil. Electronic address:

The incidence of variable congenital malformation (CM) among 399 municipalities in the state of Paraná, southern Brazil, suggests the etiological role of environmental factors. This study examined a) environmental concentrations of chlorine anions (Cl) associated with organochlorines (OCs) and b) associations between these chemicals and agricultural output with CMs using a geographical information system. In one of the three years during the sampling period (2008, 2009 or 2010) Cl, dichlorodiphenyltrichloroethane (p,p'-DDT), dichlorodiphenyldichloroethylene (p,p'-DDE), dichlorodiphenyldichloroethane (p,p'-DDD), and endosulfan levels were measured in 465 (465/736, 63%) catchment basins. Agricultural outputs for crops during 2006-2010 were also evaluated (t/km). Further, CM kernel density for the 399 municipalities in Paraná during 2007-2014 was investigated. Cl levels increased significantly in one of the three years (2008, 2009 or 2010) in western catchment basins, compared to 1996 (p < 0.0001). The municipalities were divided according to the obtained Cl levels, where sub-region C2 (central-southern) < 1.8 mg/L ≤ sub-regions C1 (northern-western) and C3 (eastern-southern). We identified 8756 cases of CMs among 1,221,287 newborns (NB) in all sub-regions. C1 had higher DDT-DDE-DDD (p,p'-DDT + p,p'-DDE + p,p'-DDD) concentrations, agricultural output, and CM kernel density. C2 and C3 had minor agricultural outputs (per square kilometer) and CM densities. A 2.96 mg/L increase in Cl between sub-regions C1 and C2 was co-localized with a 45% increase in CM density (spatial relative risk = 1.45, CI 95%: 1.36-1.55). C1 had the highest log likelihood ratios (p = 0.001) identified via SaTScan clustering analyses. Organochlorines and other toxic chlorinated chemicals may contribute to CMs in humans, and these chemicals are ultimately transformed and release Cl in rivers. Higher Cl levels were correlated significantly with higher agricultural productivity, DDT-DDE-DDD levels, and CMs in some parts of the northern and western sub-regions (C1).
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http://dx.doi.org/10.1016/j.scitotenv.2018.09.061DOI Listing
February 2019

Impact of neonatal screening and surveillance for the TP53 R337H mutation on early detection of childhood adrenocortical tumors.

J Clin Oncol 2013 Jul 3;31(20):2619-26. Epub 2013 Jun 3.

Pelé Pequeno Príncipe Research Institute, Avenida Silva Jardim, 1632, Água Verde, Curitiba, PR 80.250-200, Brazil.

Purpose: The incidence of pediatric adrenocortical tumors (ACTs) is remarkably high in southern Brazil, where more than 90% of patients carry the germline TP53 mutation R337H. We assessed the impact of early detection of this mutation and of surveillance of carriers.

Patients And Methods: Free newborn screening was offered at all hospitals in the state of Paraná. Parents of positive newborns were tested, and relatives in the carrier line were offered screening. Positive newborns and their relatives age < 15 years were offered surveillance (periodic clinical, laboratory, and ultrasound evaluations). ACTs detected by imaging were surgically resected.

Results: Of 180,000 newborns offered screening, 171,649 were screened, and 461 (0.27%) were carriers. As of April 2012, ACTs had been diagnosed in 11 of these carriers but in only two neonatally screened noncarriers (P < .001); six patient cases were identified among 228 carrier relatives age < 15 years (total, 19 ACTs). Surveillance participants included 347 (49.6%) of 699 carriers. Tumors were smaller in surveillance participants (P < .001) and more advanced in nonparticipants (four with stage III disease; two deaths). Neonatally screened carriers also had neuroblastoma (n = 1), glioblastoma multiforme (n = 1), choroid plexus carcinoma (n = 2), and Burkitt lymphoma (n = 1). Cancer histories and pedigrees were obtained for 353 families that included 1,704 identified carriers. ACTs were the most frequent cancer among carrier children (n = 48).

Conclusion: These findings establish the prevalence of the TP53 R337H mutation in Paraná state and the penetrance of ACTs among carriers. Importantly, screening and surveillance of heterozygous carriers are effective in detecting ACTs when readily curable.
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http://dx.doi.org/10.1200/JCO.2012.46.3711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3808236PMC
July 2013

Controversies about the chromosomal stability of cultivated mesenchymal stem cells: their clinical use is it safe?

Curr Stem Cell Res Ther 2012 Sep;7(5):356-63

Bioprocess Engineering and Biotechnology Department, Federal University of Paraná, Curitiba, Paraná, Brazil.

The usefulness of adult stem cells in research and therapeutic applications highly relies on their genomic integrity and stability. Many laboratories including ours have addressed this concern using methods such as karyotyping, Qbanding, fluorescent in situ hybridization, array CGH, flow cytometry and Pap test to evaluate number and structure of chromosomes and cellular phenotype. This review attempts to summarize the findings reported so far for the studies on chromosomal aberrations in adult stem cells and warrant to perform certain basic tests before transplantation to avoid any adverse reactions, which will thus aid in better therapeutic output after cellular transplantation in the treatment of various diseases.
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http://dx.doi.org/10.2174/157488812802481472DOI Listing
September 2012

Identity by descent mapping of founder mutations in cancer using high-resolution tumor SNP data.

PLoS One 2012 2;7(5):e35897. Epub 2012 May 2.

Programme Cartes d'Identité des Tumeurs, Ligue Nationale Contre Le Cancer, Paris, France.

Dense genotype data can be used to detect chromosome fragments inherited from a common ancestor in apparently unrelated individuals. A disease-causing mutation inherited from a common founder may thus be detected by searching for a common haplotype signature in a sample population of patients. We present here FounderTracker, a computational method for the genome-wide detection of founder mutations in cancer using dense tumor SNP profiles. Our method is based on two assumptions. First, the wild-type allele frequently undergoes loss of heterozygosity (LOH) in the tumors of germline mutation carriers. Second, the overlap between the ancestral chromosome fragments inherited from a common founder will define a minimal haplotype conserved in each patient carrying the founder mutation. Our approach thus relies on the detection of haplotypes with significant identity by descent (IBD) sharing within recurrent regions of LOH to highlight genomic loci likely to harbor a founder mutation. We validated this approach by analyzing two real cancer data sets in which we successfully identified founder mutations of well-characterized tumor suppressor genes. We then used simulated data to evaluate the ability of our method to detect IBD tracts as a function of their size and frequency. We show that FounderTracker can detect haplotypes of low prevalence with high power and specificity, significantly outperforming existing methods. FounderTracker is thus a powerful tool for discovering unknown founder mutations that may explain part of the "missing" heritability in cancer. This method is freely available and can be used online at the FounderTracker website.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035897PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3342326PMC
September 2012

SNP array profiling of childhood adrenocortical tumors reveals distinct pathways of tumorigenesis and highlights candidate driver genes.

J Clin Endocrinol Metab 2012 Jul 26;97(7):E1284-93. Epub 2012 Apr 26.

Program Cartes d'Identité des Tumeurs, Ligue Nationale Contre Le Cancer, 14 rue Corvisart, 75013 Paris, France.

Context: Childhood adrenocortical tumors (ACT) are rare malignancies, except in southern Brazil, where a higher incidence rate is associated to a high frequency of the founder R337H TP53 mutation. To date, copy number alterations in these tumors have only been analyzed by low-resolution comparative genomic hybridization.

Objective: We analyzed an international series of 25 childhood ACT using high-resolution single nucleotide polymorphism arrays to: 1) detect focal copy number alterations highlighting candidate driver genes; and 2) compare genetic alterations between Brazilian patients carrying the R337H TP53 mutation and non-Brazilian patients.

Results: We identified 16 significantly recurrent chromosomal alterations (q-value < 0.05), the most frequent being -4q34, +9q33-q34, +19p, loss of heterozygosity (LOH) of chromosome 17 and 11p15. Focal amplifications and homozygous deletions comprising well-known oncogenes (MYC, MDM2, PDGFRA, KIT, MCL1, BCL2L1) and tumor suppressors (TP53, RB1, RPH3AL) were identified. In addition, eight focal deletions were detected at 4q34, defining a sharp peak region around the noncoding RNA LINC00290 gene. Although non-Brazilian tumors with a mutated TP53 were similar to Brazilian tumors, those with a wild-type TP53 displayed distinct genomic profiles, with significantly fewer rearrangements (P = 0.019). In particular, three alterations (LOH of chromosome 17, +9q33-q34, and -4q34) were significantly more frequent in TP53-mutated samples. Finally, two of four TP53 wild-type tumors displayed as sole rearrangement a copy-neutral LOH of the imprinted region at 11p15, supporting a major role for this region in ACT development.

Conclusions: Our findings highlight potential driver genes and cellular pathways implicated in childhood ACT and demonstrate the existence of different oncogenic routes in this pathology.
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http://dx.doi.org/10.1210/jc.2012-1184DOI Listing
July 2012

NPM1 deletion is associated with gross chromosomal rearrangements in leukemia.

PLoS One 2010 Sep 21;5(9):e12855. Epub 2010 Sep 21.

Hematology, University of Perugia, Perugia, Italy.

Background: NPM1 gene at chromosome 5q35 is involved in recurrent translocations in leukemia and lymphoma. It also undergoes mutations in 60% of adult acute myeloid leukemia (AML) cases with normal karyotype. The incidence and significance of NPM1 deletion in human leukemia have not been elucidated.

Methodology And Principal Findings: Bone marrow samples from 145 patients with myelodysplastic syndromes (MDS) and AML were included in this study. Cytogenetically 43 cases had isolated 5q-, 84 cases had 5q- plus other changes and 18 cases had complex karyotype without 5q deletion. FISH and direct sequencing investigated the NPM1 gene. NPM1 deletion was an uncommon event in the "5q- syndrome" but occurred in over 40% of cases with high risk MDS/AML with complex karyotypes and 5q loss. It originated from large 5q chromosome deletions. Simultaneous exon 12 mutations were never found. NPM1 gene status was related to the pattern of complex cytogenetic aberrations. NPM1 haploinsufficiency was significantly associated with monosomies (p<0.001) and gross chromosomal rearrangements, i.e., markers, rings, and double minutes (p<0.001), while NPM1 disomy was associated with structural changes (p=0.013). Interestingly, in complex karyotypes with 5q- TP53 deletion and/or mutations are not specifically associated with NPM1 deletion.

Conclusions And Significance: NPM1/5q35 deletion is a consistent event in MDS/AML with a 5q-/-5 in complex karyotypes. NPM1 deletion and NPM1 exon 12 mutations appear to be mutually exclusive and are associated with two distinct cytogenetic subsets of MDS and AML.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0012855PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943467PMC
September 2010

Heterozygous TP53stop146/R72P fibroblasts from a Li-Fraumeni syndrome patient with impaired response to DNA damage.

Int J Oncol 2010 Apr;36(4):983-90

Departamento de Patologia Básica, UFPR, Brazil.

The Li-Fraumeni syndrome (LFS) is a rare autosomal dominant hereditary cancer syndrome, characterized by a wide spectrum of neoplasms, occurring in children and young adults. The identification of germline TP53 mutations in LFS has given rise to a number of in vitro studies using cultures of cancer cells and non-tumoral fibroblasts presenting germline TP53 mutations. In the present study, we performed a detailed documentation of the pedigree of an LFS family with a comprehensive analysis of genotype-phenotype correlations. We sequenced the TP53 gene and verified that the proband carries a germline nonsense mutation in codon 146 in one allele, the TP53Arg72Pro polymorphism in the second, and other intronic polymorphisms in the TP53 gene. In order to investigate the disruption of the p53 function in a patient presenting this mutation and the TP53Arg72Pro polymorphism who had so far suffered five malignant tumors and a benign meningioma, we tested her fibroblasts in response to DNA damage by evaluating the proliferation rate, apoptosis, and disruption of the TP53 pathway. The proband's heterozygous fibroblasts were not as efficient as control fibroblasts or those of her mother, who carried only the TP53Arg72Pro polymorphism, in causing cell arrest and cell death after DNA damage, which was correlated with diminished TP21 protein levels.
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http://dx.doi.org/10.3892/ijo_00000578DOI Listing
April 2010

A NUP98-positive acute myeloid leukemia with a t(11;12)(p15;q13) without HOXC cluster gene involvement.

Cancer Genet Cytogenet 2009 Sep;193(2):109-11

Hematology and Bone Marrow Transplantation Unit, University of Perugia, Ematologia e Trapianto di Midollo Osseo, Ospedale S.M. della Misericordia, (Padiglione B, piano -2), S. Andrea delle Fratte, 06156 Perugia, Italy.

We report a case of adult acute myeloid leukemia with a new t(11;12)(p15;q13) underlying a NUP98 rearrangement without HOXC cluster gene involvement. We designed a specific double-color double-fusion FISH assay to discriminate between this t(11;12)(p15;q13) and those producing NUP98-HOXC11 or NUP98-HOXC13. Our fluorescence in situ hybridization (FISH) showed that putative candidate partners mapping 600 kilobases centromeric to HOXC were RARG (retinoic acid receptor gamma), MFSD5 (major facilitator superfamily domain containing 5), and ESPL1 (extra spindle pole bodies homolog 1). It is noteworthy that so far only ESPL1 has been implicated in human cancers. This FISH assay is useful for diagnostic screening of NUP98-positive leukemias.
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http://dx.doi.org/10.1016/j.cancergencyto.2009.04.015DOI Listing
September 2009

Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(2;11)(p21;q23) translocation.

J Exp Med 2008 Oct 20;205(11):2499-506. Epub 2008 Oct 20.

Institut National de Santé et de Recherche Médicale, U563, Centre de Physiopathologie de Toulouse-Purpan, 31300 Toulouse, France.

Most chromosomal translocations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) involve oncogenes that are either up-regulated or form part of new chimeric genes. The t(2;11)(p21;q23) translocation has been cloned in 19 cases of MDS and AML. In addition to this, we have shown that this translocation is associated with a strong up-regulation of miR-125b (from 6- to 90-fold). In vitro experiments revealed that miR-125b was able to interfere with primary human CD34(+) cell differentiation, and also inhibited terminal (monocytic and granulocytic) differentiation in HL60 and NB4 leukemic cell lines. Therefore, miR-125b up-regulation may represent a new mechanism of myeloid cell transformation, and myeloid neoplasms carrying the t(2;11) translocation define a new clinicopathological entity.
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http://dx.doi.org/10.1084/jem.20080285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2571925PMC
October 2008

High frequency of loss of heterozygosity at 11p15 and IGF2 overexpression are not related to clinical outcome in childhood adrenocortical tumors positive for the R337H TP53 mutation.

Cancer Genet Cytogenet 2008 Oct;186(1):19-24

Instituto de Pesquisa Pelé Pequeno Principe, Curitiba, Av. Silva Jardim, Paraná, Brazil.

A germline TP53 R337H mutation is present in childhood adrenocortical tumors (ACT) from southern Brazil. Other genetic alterations are also frequently found in these tumors. This study was designed to assess whether alterations of the 11p15 region exist in childhood ACT, accounting for IGF2 overexpression in these tumors, and how they are related to clinical outcome. Tumor DNA of 12 children with ACT (4 adenomas and 8 carcinomas) and from the blood of their parents was analyzed. All patients showed 11p15 loss of heterozygosity (LOH) in the tumor. In contrast to the single case of paternal LOH, IGF2 was overexpressed in tumors with maternal allele loss. Our data show that 11p15 LOH is a widespread finding in childhood ACT not related with malignancy, contrary to adult ACT. Alterations in the expression of other genes in the same region (e.g., CDKN1C) may contribute to ACT tumorigenesis.
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http://dx.doi.org/10.1016/j.cancergencyto.2008.05.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2603647PMC
October 2008

t(3;11)(q12;p15)/NUP98-LOC348801 fusion transcript in acute myeloid leukemia.

Haematologica 2008 Sep 4;93(9):1398-401. Epub 2008 Jul 4.

IbiT Foundation, Fondazione IRCCS Biotecnologie nel Trapianto, Hematology, University of Perugia, Perugia, Italy.

In a case of acute myeloid leukemia we report molecular cytogenetic findings of a t(3;11)(q12;p15), characterized as a new NUP98 translocation rearranging with LOC348801 at chromosome 3. NUP98 involvement was detected by fluorescence in situ hybridization. 3'-RACE-PCR showed nucleotide 1718 (exon 13) of NUP98 was fused in-frame with nucleotide 1248 (exon 2) of LOC348801. RT-PCR and cloning experiments detected two in-frame spliced NUP98-LOC348801 transcripts and the reciprocal LOC348801-NUP98. A highly specific double-color double-fusion FISH assay reliably detects NUP98-LOC348801.
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http://dx.doi.org/10.3324/haematol.12945DOI Listing
September 2008

Cryptic chromosome 9q34 deletion generates TAF-Ialpha/CAN and TAF-Ibeta/CAN fusion transcripts in acute myeloid leukemia.

Haematologica 2007 Feb;92(2):232-5

Hematology and Bone Marrow Transplantation Unit, University of Perugia, Policlinico Monteluce, via Brunamonti 51, 06122 Perugia, Italy.

In hematologic malignancies chromosome aberrations generating fusion genes include cryptic deletions. In a patient with acute myeloid leukemia and normal karyo-type we discovered a new cryptic 9q34 deletion and here report the cytogenetic and molecular findings. The 9q34 deletion extends 2.5 megabases and juxtaposes the 5' TAF-I to the 3' CAN producing a TAF-I/CAN fusion gene. TAF-I/CAN transcribes into two fusion proteins bearing either TAF-Ialpha or TAF-Ibeta moieties. We set up molecular assays to monitor the chimeric TAF-Ialpha/CAN and TAF-Ibeta/CAN transcripts which, after hematopoietic stem cell transplantation from an HLA-identical sibling, were no longer detected.
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http://dx.doi.org/10.3324/haematol.10538DOI Listing
February 2007

Immunohistochemistry predicts nucleophosmin (NPM) mutations in acute myeloid leukemia.

Blood 2006 Sep 23;108(6):1999-2005. Epub 2006 May 23.

Institute of Hematology, Policlinico Monteluce, 06122 Perugia, Italy.

Nucleophosmin (NPM) exon-12 mutations occur in 50% to 60% of adult acute myeloid leukemia (AML) with normal karyotype and are predictors of favorable prognosis. We evaluated bone marrow or peripheral blood samples from 450 adult patients with AML of the GIMEMA (Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto)/AML12 EORTC (European Organization for Research and Treatment of Cancer) trial to (1) search for new exon-12 NPM mutations; (2) determine whether NPM immunostaining on paraffin-embedded biopsies predicts NPM mutations; and (3) investigate altered nucleocytoplasmic NPM traffic in primary AML cells. Fourteen NPM mutations, including 8 new variants, were identified. All 200 AML cases expressing cytoplasmic NPM (NPMc(+) AML) carried NPM mutations. None of the 250 cases with nucleus-restricted NPM (NPMc(-) AML) was mutated. At the C-terminus, NPM leukemic mutants carried mutations of only tryptophan 290 or of both tryptophans 288 and 290 and a new nuclear export signal (NES) motif, which appear to underlie their nuclear export. The specific Crm1/exportin-1 inhibitor leptomycin-B relocated NPM mutants from cytoplasm to nucleus of primary NPMc(+) AML cells, demonstrating that nuclear export is NES dependent. NPM mutants bound and recruited wild-type NPM into leukemic cell cytoplasm. Because alterations at C-terminus of leukemic NPM mutants are similar, immunohistochemistry detects all exon-12 NPM mutations and is a valuable, inexpensive tool in the diagnostic-prognostic work-up of patients with AML with normal karyotype.
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http://dx.doi.org/10.1182/blood-2006-03-007013DOI Listing
September 2006

Denaturing high-performance liquid chromatography: a valid approach for identifying NPM1 mutations in acute myeloid leukemia.

J Mol Diagn 2006 May;8(2):254-9

Laboratory of Cytogenetics and Molecular Genetics, University of Perugia, Italy.

NPM1 gene mutations are the most frequent genetic lesion in the 60% of adult acute myeloid leukemias (AMLs) with normal karyotype and no evidence of typical fusion genes (BCR/ABL1, PML/RARA, AML1/ETO, CBFB/MYH11, DEK/CAN). Using direct sequencing we previously identified six different heterozygous mutants within exon 12 encoding the nucleophosmin C-terminus. Because of these mutations the shuttling protein nucleophosmin is aberrantly delocalized in the cytoplasm of leukemic cells (NPMc+). Here, we designed and tested a denaturing high-performance liquid chromatography (DHPLC) assay to detect NPM1 mutated variants. To assess specificity, sensitivity, reliability, and reproducibility, we analyzed DNA from 120 primary adult AMLs and compared DHPLC results with immunohistochemistry and sequencing. All electropherogram profiles in the 26 NPMc+ leukemias were different from the wild type, indicating 100% sensitivity. Sequencing categorized mutations A, B, and D, and all mutation A cases gave identical elution profiles. The other mutations showed typical chromatograms, with mutations B and D differing for one nucleotide. Elution profiles and sequencing also identified four new variants. Our results suggest that DHPLC detects NPM1mutations as well as direct sequencing and immunohistochemistry, providing a helpful approach in the diagnosis of NPMc+ AML.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1867590PMC
http://dx.doi.org/10.2353/jmoldx.2006.050098DOI Listing
May 2006

Both carboxy-terminus NES motif and mutated tryptophan(s) are crucial for aberrant nuclear export of nucleophosmin leukemic mutants in NPMc+ AML.

Blood 2006 Jun 2;107(11):4514-23. Epub 2006 Feb 2.

Institute of Hematology, University of Perugia, 06122 Perugia, Italy.

We recently identified aberrant cytoplasmic expression of nucleophosmin (NPM) as the immunohistochemical marker of a large subgroup of acute myeloid leukemia (AML) (about one-third of adult AML) that is characterized by normal karyotype and mutations occurring at the exon-12 of the NPM gene. In this paper, we have elucidated the molecular mechanism underlying the abnormal cytoplasmic localization of NPM. All 29 AML-associated mutated NPM alleles so far identified encode abnormal proteins which have acquired at the C-terminus a nuclear export signal (NES) motif and lost both tryptophan residues 288 and 290 (or only the residue 290) which determine nucleolar localization. We show for the first time that both alterations are crucial for NPM mutant export from nucleus to cytoplasm. In fact, the cytoplasmic accumulation of NPM is blocked by leptomycin-B and ratjadones, specific exportin-1/Crm1-inhibitors, and by reinsertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. NPM leukemic mutants in turn recruit the wild-type NPM from nucleoli to nucleoplasm and cytoplasm. These findings indicate that potential therapeutic strategies aimed to retarget NPM to its physiological sites will have to overcome 2 obstacles, the new NES motif and the mutated tryptophan(s) at the NPM mutant C-terminus.
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http://dx.doi.org/10.1182/blood-2005-11-4745DOI Listing
June 2006

Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.

N Engl J Med 2005 Jan;352(3):254-66

Institute of Hematology, University of Perugia, Perugia, Italy.

Background: Nucleophosmin (NPM), a nucleocytoplasmic shuttling protein with prominent nucleolar localization, regulates the ARF-p53 tumor-suppressor pathway. Translocations involving the NPM gene cause cytoplasmic dislocation of the NPM protein.

Methods: We used immunohistochemical methods to study the subcellular localization of NPM in bone marrow-biopsy specimens from 591 patients with primary acute myelogenous leukemia (AML). We then correlated the presence of cytoplasmic NPM with clinical and biologic features of the disease.

Results: Cytoplasmic NPM was detected in 208 (35.2 percent) of the 591 specimens from patients with primary AML but not in 135 secondary AML specimens or in 980 hematopoietic or extrahematopoietic neoplasms other than AML. It was associated with a wide spectrum of morphologic subtypes of the disease, a normal karyotype, and responsiveness to induction chemotherapy, but not with recurrent genetic abnormalities. There was a high frequency of FLT3 internal tandem duplications and absence of CD34 and CD133 in AML specimens with a normal karyotype and cytoplasmic dislocation of NPM, but not in those in which the protein was restricted to the nucleus. AML specimens with cytoplasmic NPM carried mutations of the NPM gene that were predicted to alter the protein at its C-terminal; this mutant gene caused cytoplasmic localization of NPM in transfected cells.

Conclusions: Cytoplasmic NPM is a characteristic feature of a large subgroup of patients with AML who have a normal karyotype, NPM gene mutations, and responsiveness to induction chemotherapy.
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http://dx.doi.org/10.1056/NEJMoa041974DOI Listing
January 2005

Cryptic insertion producing two NUP98/NSD1 chimeric transcripts in adult refractory anemia with an excess of blasts.

Genes Chromosomes Cancer 2004 Dec;41(4):395-9

Hematology and Bone Marrow Transplantation Unit, University of Perugia, 06123 Perugia, Italy.

We performed cytogenetic and molecular studies on an adult patient with refractory anemia with an excess of blasts with an add(11)(p15). Multicolor fluorescence in situ hybridization (FISH) identified the extra material on 11p as belonging to chromosome 15. Metaphase FISH with probes for chromosomes 5, 11, and 15 revealed a complex four-break rearrangement. Clone RP5-1173K1, containing exons 10-20 of the NUP98 gene, gave three fluorescence signals on the normal 11, the der(5), and the der(15). 3'-RACE-PCR identified an in-frame fusion between NUP98 and NSD1, which was confirmed by RT-PCR. Two different spliced forms, that is, NUP98 exon 11/NSD1 exon 6 and NUP98 exon 12/NSD1 exon 6, were detected. The reciprocal NSD1/NUP98 was not found. A dual-color experiment with RP5-1173K1 and CTC-549A4, spanning the entire NSD1 gene, indicated an insertion of NUP98 into the NSD1 locus. This is the first report of an adult with myelodysplastic syndrome (MDS) harboring an NUP98/NSD1 fusion resulting from insertion of 5'-NUP98 into the NSD1/5q35 locus.
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http://dx.doi.org/10.1002/gcc.20103DOI Listing
December 2004

Genetic profile of acute myeloid leukemia.

Rev Clin Exp Hematol 2002 Mar;6(1):3-25; discussion 86-7

Hematology and Bone Marrow Transplantation Unit, University of Perugia, Italy.

Understanding genomic events and the cascade of their effects in cell function is crucial for identifying distinct subsets of acute myeloid leukemia and developing new therapeutic strategies. Conventional cytogenetics, fluorescence in situ hybridization investigations and molecular studies have provided much information over the past few years. This review will focus on major genomic mechanisms in acute myeloid luekemia and on the genes implicated in the pathogenesis of specific subtypes.
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http://dx.doi.org/10.1046/j.1468-0734.2002.00060.xDOI Listing
March 2002

NUP98 is fused to the NSD3 gene in acute myeloid leukemia associated with t(8;11)(p11.2;p15).

Blood 2002 May;99(10):3857-60

Hematology and Bone Marrow Transplantation Unit, University of Perugia, Italy.

Fusion between the NUP98 and NSD3 genes in a patient with acute myeloid leukemia associated with t(8;11)(p11.2;p15), is reported for the first time. The t(8;11)(p11.2;p15) was identified by classical cytogenetics. Fluorescence in situ hybridization (FISH) analysis revealed a split signal with a mix of BAC 118H17 and 290A12, indicating the translocation disrupted NUP98. FISH restriction at 8p11-12 showed a split of BAC 350N15. Molecular investigations into candidate genes in this BAC showed the NUP98 fusion partner at 8p11.2 was the NSD3 gene. To date the NSD3 gene has never been implicated in hematologic malignancies.
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http://dx.doi.org/10.1182/blood.v99.10.3857DOI Listing
May 2002