Advantages of a next generation sequencing targeted approach for the molecular diagnosis of retinoblastoma.

BACKGROUND: Retinoblastoma (RB) is the most common malignant childhood tumor of the eye and results from inactivation of both alleles of the RB1 gene. Nowadays RB genetic diagnosis requires classical chromosome investigations, Multiplex Ligation-dependent Probe Amplification analysis (MLPA) and Sanger sequencing. Nevertheless, these techniques show some limitations. We report our experience on a cohort of RB patients using a combined approach of Next-Generation Sequencing (NGS) and RB1 custom array-Comparative Genomic Hybridization (aCGH). METHODS: A total of 65 patients with retinoblastoma were studied: 29 cases of bilateral RB and 36 cases of unilateral RB. All patients were previously tested with conventional cytogenetics and MLPA techniques. Fifty-three samples were then analysed using NGS. Eleven cases were analysed by RB1 custom aCGH. One last case was studied only by classic cytogenetics. Finally, it has been tested, in a lab sensitivity assay, the capability of NGS to detect artificial mosaicism series in previously recognized samples prepared at 3 different mosaicism frequencies: 10, 5, 1 %. RESULTS: Of the 29 cases of bilateral RB, 28 resulted positive (96.5 %) to the genetic investigation: 22 point mutations and 6 genomic rearrangements (four intragenic and two macrodeletion). A novel germline intragenic duplication, from exon18 to exon 23, was identified in a proband with bilateral RB. Of the 36 available cases of unilateral RB, 8 patients resulted positive (22 %) to the genetic investigation: 3 patients showed point mutations while 5 carried large deletion. Finally, we successfully validated, in a lab sensitivity assay, the capability of NGS to accurately measure level of artificial mosaicism down to 1 %. CONCLUSIONS: NGS and RB1-custom aCGH have demonstrated to be an effective combined approach in order to optimize the overall diagnostic procedures of RB. Custom aCGH is able to accurately detect genomic rearrangements allowing the characterization of their extension. NGS is extremely accurate in detecting single nucleotide variants, relatively simple to perform, cost savings and efficient and has confirmed a high sensitivity and accuracy in identifying low levels of artificial mosaicisms.

Hypoplastic left heart syndrome and 21q22.3 deletion.

Hypoplastic left heart syndrome (HLHS) is a rare congenital heart defect (CHD), associated with extracardiac anomalies in the 15-28% of cases, in the setting of chromosomal anomalies, mendelian disorders, and organ defects. We report on a syndromic female newborn with HLHS and terminal 21q22.3 deletion (del 21q22.3), investigated by Fluorescence In Situ Hybridization (FISH) using a panel of 26 contiguous BAC probes. Although rare, del 21q22.3 has been described in two additional patients with HLHS. In order to investigate the frequency and role of this chromosomal imbalance in the pathogenesis of left-sided obstructive heart defects, we screened for del 21q22.3 a series of syndromic and non-syndromic children with HLHS, aortic coarctation and valvular aortic stenosis, consecutively admitted to our hospital in a three-year period. Although none of the 56 analyzed patients were hemizygous for this region, the present case report and published patients argue that del 21q22 should be added to the list of chromosomal imbalances associated with HLHS. Accordingly, the presence of a cardiac locus mapping in the critical region cannot be excluded.

Diagnosis of Noonan syndrome and related disorders using target next generation sequencing.

BACKGROUND: Noonan syndrome is an autosomal dominant developmental disorder with a high phenotypic variability, which shares clinical features with other rare conditions, including LEOPARD syndrome, cardiofaciocutaneous syndrome, Noonan-like syndrome with loose anagen hair, and Costello syndrome. This group of related disorders, so-called RASopathies, is caused by germline mutations in distinct genes encoding for components of the RAS-MAPK signalling pathway. Due to high number of genes associated with these disorders, standard diagnostic testing requires expensive and time consuming approaches using Sanger sequencing. In this study we show how targeted Next Generation Sequencing (NGS) technique can enable accurate, faster and cost-effective diagnosis of RASopathies. METHODS: In this study we used a validation set of 10 patients (6 positive controls previously characterized by Sanger-sequencing and 4 negative controls) to assess the analytical sensitivity and specificity of the targeted NGS. As second step, a training set of 80 enrolled patients with a clinical suspect of RASopathies has been tested. Targeted NGS has been successfully applied over 92% of the regions of interest, including exons for the following genes: PTPN11, SOS1, RAF1, BRAF, HRAS, KRAS, NRAS, SHOC, MAP2K1, MAP2K2, CBL. RESULTS: All expected variants in patients belonging to the validation set have been identified by targeted NGS providing a detection rate of 100%. Furthermore, all the newly detected mutations in patients from the training set have been confirmed by Sanger sequencing. Absence of any false negative event has been excluded by testing some of the negative patients, randomly selected, with Sanger sequencing. CONCLUSION: Here we show how molecular testing of RASopathies by targeted NGS could allow an early and accurate diagnosis for all enrolled patients, enabling a prompt diagnosis especially for those patients with mild, non-specific or atypical features, in whom the detection of the causative mutation usually requires prolonged diagnostic timings when using standard routine. This approach strongly improved genetic counselling and clinical management.

[Prevalence of pain in the hospital: results from a survey among inpatients in a teaching hospital]. / Prevalenza del dolore in ospedale: risultati di una survey nella popolazione dei pazienti di un Policlinico Universitario.

INTRODUCTION: The aim of the study was to conduct a cross sectional study on pain among inpatients and outpatients in a large teaching hospital. METHODS: The study was carried out at the Policlinico "Umberto I", Rome, in October 2009, using a questionnaire developed by Gigi Ghirotti Foundation. The pain intensity was graded mild (VAS scale 1-3), moderate (VAS 4-6) and severe (7-10). RESULTS: The participants were 825 patients (response rate 75.8%), aged over or equal 18 years, 435 females (median age 54, range 18-95) and 390 males (median age 60, range 19-89). 420 patients (50.9%) declared to feel pain during the hospitalization, and among them 84.3% received adequate therapy. 23.3% of responders presented a pain score over 8, with patients admitted to internal medicine wards with highest score (p = 0.037). The pain control (score ³ 6) was obtained in 71.4% patients. Pain and severe pain perception was higher among females (55.6% and 36.1% vs 45.6% and 29.2%, respectively) (p = 0.004 and p = 0.036). The multivariate analysis showed that the following variables are positively associated to the dependent variable "how much pain do you feel in this moment?": female gender (p < 0.001), length of stay (p = 0.081), admission in medical wards (p = 0.028), being admitted in ordinary way (p = 0.004), while if the patients filled in the questionnaire was inversely associated (p < 0.001). CONCLUSIONS: The application of the guidelines on free pain hospital is increasing, however we must consider that almost one fifth of the patients who suffered pain within the hospital did not receive any treatment for that.

Acute colonic pseudo-obstruction in elderly Report of a case.

A case of acute colonic obstruction in an elderly patient is presented, with a brief discussion about peculiar aspects to pseudo-obstruction and particularly chronic idiopathic intestinal pseudo-obstruction (CIIP), in which it was classified by pathologists. Clinical and therapeutic implications of this classification are also discussed. In authors'opinion, interesting aspects of the reported case are represented by the acute presentation, without previous symptoms at medical history and above all by the evidence of a recto-sigmoid junction intraoperatively palpable mass, mimicking ring-like neoplastic disease. The preoperative and intraoperative features led surgeons to perform a total colectomy with ileo-rectal anastomosis and high ligation of mesenteric inferior artery, with complete regional lymphectomy according to oncologic standard, but at histological examination the mass revealed to be due to considerable muscular tissue thickening, therefore to a benign condition. These features probably suggest the need of a better clinical and pathological classification of this difficult and still controversial matter, in order to achieve better outcomes and to avoid misdiagnosis and overtreatment. KEY WORDS: Intestinal obstruction, Pseudo-obstruction, Total colectomy.

A familial chromosomal complex rearrangement confirms RUNX1T1 as a causative gene for intellectual disability and suggests that 1p22.1p21.3 duplication is likely benign.

BACKGROUND: Complex chromosomal rearrangements are constitutive structural aberrations involving three or more breaks. They can be balanced or unbalanced and result in different outcomes, depending on deletion/duplication of genomic material, gene disruption, or position effects. CASE PRESENTATION: We report on a patient presenting with severe anemia, splenomegaly, mild intellectual disability and facial dysmorphisms harboring a 4.3 Mb duplication at 1p22.1p21.3 and a 2.1 Mb deletion at 8q21.3q22.1, involving RUNX1T1 gene. The healthy brother presented the same duplication of chromosome 1p as at 1p22.1p21.3. CONCLUSIONS: The rearrangement found both these siblings resulted from malsegregation in the proband and recombination in her healthy brother of a balanced paternal complex chromosomal rearrangement. These results confirm RUNX1T1 as a causative gene for intellectual disability and suggest the 1p22.1p21.3 duplication is likely benign.

Array-CGH Analysis in a Cohort of Phenotypically Well-Characterized Individuals with "Essential" Autism Spectrum Disorders.

Copy-number variants (CNVs) are associated with susceptibility to autism spectrum disorder (ASD). To detect the presence of CNVs, we conducted an array-comparative genomic hybridization (array-CGH) analysis in 133 children with "essential" ASD phenotype. Genetic analyses documented that 12 children had causative CNVs (C-CNVs), 29 children had non-causative CNVs (NC-CNVs) and 92 children without CNVs (W-CNVs). Results on clinical evaluation showed no differences in cognitive abilities among the three groups, and a higher number of ASD symptoms and of non-verbal children in the C-CNVs group compared to the W-CNVs and NC-CNVs groups. Our results highlighted the importance of the array-CGH analyses and showed that the presence of specific CNVs may differentiate clinical outputs in children with ASD.

Nevoid melanoma of the vagina: report of one case diagnosed on thin layer cytological preparations.

BACKGROUND: Primary melanoma of the vagina is an extremely rare neoplasm with approximately 250 reported cases in the world literature 1234. In its amelanotic variant this lesion may raise several differential diagnostic problems in cytological specimens 5. In this setting, the usage of thin layer cytopathological techniques (Liquid Based Preparations = LBP) may enhance the diagnostic sensitivity by permitting immunocytochemical study without having to repeat the sampling procedure. The aim of this paper is to describe the cytomorphological presentation of primary vaginal melanoma on LBP since it has not previously been reported up to now, to our knowledge. CASE PRESENTATION: a 79-y-o female complaining of vulvar itching and yellowish vaginal discharge underwent a complete gynaecological evaluation during which a LBP cytological sample was taken from a suspicious whitish mass protruding into the vaginal lumen. A cytopathological diagnosis of amelanotic melanoma was rendered. The mass was radically excised and the patient was treated with alpha-Interferon. CONCLUSION: amelanotic melanoma may be successfully diagnosed on LBP cytological preparations. Thin layer preparations may enhance the diagnostic cytomorphological clues to its diagnosis and may permit an adequate immunocytochemical characterization of the neoplasm.

Fine needle aspiration cytolog of perineal alveolar rhabdomyosarcoma: cytohistopathologic and immunocytochemical correlations.

OBJECTIVE: To describe the cytopathologic findings in a case of alveolar rhabdomyosarcoma (ARMS) of the perineum in a 13-year-old girl and to compare the cytopathologic findings with the histopathologic and immunohistochemical features observed on the corresponding fragments obtained from core biopsy of the mass. STUDY DESIGN: The cytopathologic findings observed in fine needle aspiration biopsy of a vulvar mass were analyzed with reference to their predictive diagnostic value in pathologic evaluation of the lesion. RESULTS: Following a prospective cytopathologic diagnosis of ARMS, a cutting needle core biopsy was performed. Histopathologic and immunohistochemical study of the tissue fragments confirmed the cytopathologic diagnosis. CONCLUSION: Careful cytopathologic evaluation of optimal cell samples from ARMS may elicit a correct diagnosis provided that immunocytochemical staining for markers of myogenic differentiation is performed in a pretherapeutic phase.

Sometimes it is better to wait: First Italian case of a newborn with transient abnormal myelopoiesis and a favorable prognosis.

Congenital leukemia is rare disease with an incidence of one to five cases per million births. Transient abnormal myelopoiesis (TAM), also called transient myeloproliferative disorder, is a pre-leukemia disorder that may occur in Down syndrome (DS) or non-DS infants. TAM may enter spontaneous remission; however, continual monitoring is required, as this disorder has been observed to develop into acute megakaryoblastic leukemia in 16-30% of cases. In the literature, 16 cases of TAM in non-DS infants have been reported. The case presented in the current study is, to the best of our knowledge, the first case of an Italian non-DS newborn presenting with clinical manifestations of acute leukemia at five days after birth, exhibiting a normal karyotype, trisomy 21 only in blast cells, and spontaneous remission. Chromosomal analyses on peripheral blood cells, bone marrow cells and dermal fibroblasts were conducted using a G-banding technique, and fluorescence in situ hybridization (FISH) was used to identify the critical regions of DS. Amplification of GATA binding protein 1 (GATA1) exon 2 genomic DNA was performed using polymerase chain reaction. Cytogenetic analysis of 50 peripheral blood cells and dermal fibroblasts from the patient revealed a normal karyotype: 46, XX. Conversely, cytogenetic analysis of the patient's bone marrow revealed an abnormal karyotype 47, XX+21. In order to investigate this result, FISH was performed, which identified the presence of three signals in 70% of the cells and two signals in 30% of bone marrow cells. GATA1 sequencing revealed the substitution of a single base (c.150delG) in exon 2. Seven months after the initial analysis, FISH and cytogenetic analyses of the stimulated/unstimulated peripheral blood cells and bone marrow cells were performed, revealing that each exhibited diploid signals, as observed in a normal karyotype.

Telomere shortening and telomere position effect in mild ring 17 syndrome.

BACKGROUND: Ring chromosome 17 syndrome is a rare disease that arises from the breakage and reunion of the short and long arms of chromosome 17. Usually this abnormality results in deletion of genetic material, which explains the clinical features of the syndrome. Moreover, similar phenotypic features have been observed in cases with complete or partial loss of the telomeric repeats and conservation of the euchromatic regions. We studied two different cases of ring 17 syndrome, firstly, to clarify, by analyzing gene expression analysis using real-time qPCR, the role of the telomere absence in relationship with the clinical symptoms, and secondly, to look for a new model of the mechanism of ring chromosome transmission in a rare case of familial mosaicism, through cytomolecular and quantitative fluorescence in-situ hybridization (Q-FISH) investigations. RESULTS: The results for the first case showed that the expression levels of genes selected, which were located close to the p and q ends of chromosome 17, were significantly downregulated in comparison with controls. Moreover, for the second case, we demonstrated that the telomeres were conserved, but were significantly shorter than those of age-matched controls; data from segregation analysis showed that the ring chromosome was transmitted only to the affected subjects of the family. CONCLUSIONS: Subtelomeric gene regulation is responsible for the phenotypic aspects of ring 17 syndrome; telomere shortening influences the phenotypic spectrum of this disease and strongly contributes to the familial transmission of the mosaic ring. Together, these results provide new insights into the genotype-phenotype relationships in mild ring 17 syndrome.

7T µMRI of mesenteric venous ischemia in a rat model: timing of the appearance of findings.

OBJECTIVES: The aim of this study is to analyze the chronological development of macroscopic, microscopic and magnetic resonance imaging (MRI) findings in a rat model of Superior Mesenteric Venous (SMV) ligation, and to evaluate the role of MRI in the diagnosis of mesenteric venous thrombosis. METHODS: Thirty adult Sprague-Dawley rats were used and divided in two different groups that underwent a different surgical model and a different monitoring of ischemic damage. Group I underwent macroscopical and histological observation; Group II underwent 7T µMRI evaluation and histological analysis. RESULTS: The first alterations occurred 30 min after SMV ligation and progressively worsened until the eighth hour. The morphological and MRI findings showed the same course. CONCLUSIONS: This study provides a systematic evaluation of early anatomopathological and MRI findings following the SMV ligation. MRI allows to identify the early pathological findings of venous mesenteric ischemia and allows to correlate those to the histopathological features. Our data suggest a relevant role of MRI in the diagnostic management of mesenteric venous thrombosis, allowing to non-invasively identify and characterize the histopathologic findings. So, thanks to these skills, its future application in early diagnosis of human mesenteric venous ischemia is supposable.

Array-CGH characterization and genotype-phenotype analysis in a patient with a ring chromosome 6.

BACKGROUND: Ring chromosome 6 is a rare constitutional abnormality that generally occurs de novo. The related phenotype may be highly variable ranging from an almost normal phenotype to severe malformations and mental retardation. These features are mainly present when genetic material at the end of the chromosome is lost. The severity of the phenotype seems to be related to the size of the deletion. About 25 cases have been described to date, but the vast majority reports only conventional cytogenetic investigations. CASE PRESENTATION: Here we present an accurate cyto-molecular characterization of a ring chromosome 6 in a 16-months-old Caucasian girl with mild motor developmental delay, cardiac defect, and facial anomalies. The cytogenetic investigations showed a karyotype 46,XX,r(6)(p25q27) and FISH analysis revealed the absence of the signals on both arms of the chromosome 6. These results were confirmed by means of array-CGH showing terminal deletions on 6p25.3 (1.3 Mb) and 6q26.27 (6.7 Mb). Our data were compared to current literature. CONCLUSIONS: Our report describes the case of a patient with a ring chromosome 6 abnormality completely characterized by array CGH which provided additional information for genotype-phenotype studies.

Lipid-rich histology in a basal-type immuno-profile breast carcinoma: a clinicopathological histochemical and immunohistochemical analysis of a case.

We describe the clinicopathological and morphological features of an unusual breast carcinoma classifiable as a lipid-rich variant of ductal invasive carcinoma, with a basal-type immunohistochemical profile. Basal-type breast cancers show no hormonal receptor expression, rarely over-express HER-2 but exhibit molecular high weight cytokeratins, EGFR and c-kit positivity. Special stains and histochemistry tests were used to elucidate the nature of vescicles in the neoplastic cells. Sudan IV was performed on formalin-fixed tissue. Commercially available antibodies tested were: ER, PgR, EGFR, HER2, c-kit, high molecular weight cytokeratins. Cytoplasmic lipids were highlighted as red-orange droplets on Sudan IV staining. As for immunohistochemistry, the tumor showed no reactivity to ER, PgR and HER2 (triple negative), and diffuse and strong positivity to high weight cytokeratins, EGFR and c-kit, such as a basal-type breast carcinoma. A basaloid phenotype in a lipid-rich carcinoma has not been previously reported.

Endorectal coil MRI in local staging of rectal cancer.

PURPOSE: The choice of the therapeutic strategies in patients affected with rectal cancer is strictly dependent by the tumor stage. So, in order to obtain an improvement in preoperative staging accuracy, new imaging modalities are now under investigation. The aim of this work is the evaluation of endorectal-coil MRI in the local staging of rectal cancer. MATERIAL AND METHODS: Fourty-three patients affected with histologically proven rectal cancer, have been evaluated by an high-field strength magnet (1.5 T). In 14/43 patients neoadjuvant pre-operative chemotherapy had been previously performed. In all cases axial SE T1w and FSE T2w sequences and coronal or sagittal FSE T2w sequences, with and without fat suppression, were performed. Basing upon the TNM staging system and the previously reported MRI signs the local extent of the tumor was evaluated, focusing about the rectal wall infiltration and the perirectal lymph nodes involvement. All the patients underwent surgery and a comparative evaluation of MRI and pathological staging was done. RESULTS: At MRI the tumor was detected in 38/43 patients. In evaluating wall infiltration the MRI results agreed with pathological results in 89% of patients and showed 92% accuracy in T1-T2 stage and 94% in T3. In evaluating perirectal lymph nodes metastases MRI showed 69% accuracy, 82% sensitivity and 55%specificity. DISCUSSION AND CONCLUSIONS: The poor accuracy of CT and body-coil MRI in evaluating wall involvement in patients with rectal cancer is mainly related to their inability to demonstrate the single layers of the rectal wall. So transrectal ultrasound is now the first choice modalitiy in local staging of rectal cancer. However transrectal ultrasound showed low sensitivity in detecting perirectal lymph nodes metastases and low accuracy in evaluating the patients previously undergone to neoadjuvant chemotherapy or radiotherapy. On the other hand the improvement of MRI sequences and the availability of the endorectal coils allowed to visualize the single layers of the rectal wall so making the endorectal-coil MRI a reliable imaging technique to stage rectal cancer. The results of our work demonstrate a good diagnostic accuracy of endorectal-coil MRI in local staging of rectal cancer, in particular the degree of rectal wall infiltration was well demonstrated, while the perirectal lymph nodes metastases were demonstrated with less accuracy. The long examination time, the costs and the movement-related artefacts are the main limits of MRI. In particular the movement-related artifacts sometime do not allow the visualization of the wall layers so lowering the diagnostic accuracy in demonstrating the tumor wall infiltration. In conclusion, even though endorectal coil MRI proved to be a reliable imaging technique in local staging of rectal cancer, at present we are not able to state what may be its real role in diagnostic evaluation of the patients with rectal cancer, in particular if compared to endorectal ultrasound. Further, comparative studies, based upon larger patients series are probably needed to draw a definitive conclusion.