A clinical-molecular prognostic model to predict survival in patients with post polycythemia vera and post essential thrombocythemia myelofibrosis.

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms with variable risk of evolution into post-PV and post-ET myelofibrosis, from now on referred to as secondary myelofibrosis (SMF). No specific tools have been defined for risk stratification in SMF. To develop a prognostic model for predicting survival, we studied 685 JAK2, CALR, and MPL annotated patients with SMF. Median survival of the whole cohort was 9.3 years (95% CI: 8-not reached-NR-). Through penalized Cox regressions we identified negative predictors of survival and according to beta risk coefficients we assigned 2 points to hemoglobin level <11 g/dl, to circulating blasts ⩾3%, and to CALR-unmutated genotype, 1 point to platelet count <150 × 109/l and to constitutional symptoms, and 0.15 points to any year of age. Myelofibrosis Secondary to PV and ET-Prognostic Model (MYSEC-PM) allocated SMF patients into four risk categories with different survival (P<0.0001): low (median survival NR; 133 patients), intermediate-1 (9.3 years, 95% CI: 8.1-NR; 245 patients), intermediate-2 (4.4 years, 95% CI: 3.2-7.9; 126 patients), and high risk (2 years, 95% CI: 1.7-3.9; 75 patients). Finally, we found that the MYSEC-PM represents the most appropriate tool for SMF decision-making to be used in clinical and trial settings.

Extraordinary response to omalizumab in a child with severe chronic urticaria.

A case of immediate and definitive response to a single dose of omalizumab in a child with severe ciclosporin-resistant chronic urticaria is reported.

Microarray application in prenatal diagnosis: a position statement from the cytogenetics working group of the Italian Society of Human Genetics (SIGU), November 2011.

A precise guideline establishing chromosomal microarray analysis (CMA) applications and platforms in the prenatal setting does not exist. The controversial question is whether CMA technologies can or should soon replace standard karyotyping in prenatal diagnostic practice. A review of the recent literature and survey of the knowledge and experience of all members of the Italian Society of Human Genetics (SIGU) Committee were carried out in order to propose recommendations for the use of CMA in prenatal testing. The analysis of datasets reported in the medical literature showed a considerable 6.4% incidence of pathogenic copy number variations (CNVs) in the group of pregnancies with sonographically detected fetal abnormalities and normal karyotype. The reported CNVs are likely to have a relevant role in terms of nosology for the fetus and in the assessment of reproductive risk for the couple. Estimation of the frequency of copy number variations of uncertain significance (VOUS) varied depending on the different CMA platforms used, ranging from 0-4%, obtained using targeted arrays, to 9-12%, obtained using high-resolution whole genome single nucleotide polymorphism (SNP) arrays. CMA analysis can be considered a second-tier diagnostic test to be used after standard karyotyping in selected groups of pregnancies, namely those with single (apparently isolated) or multiple ultrasound fetal abnormalities, those with chromosomal rearrangements, even if apparently balanced, and those with supernumerary marker chromosomes.

Further clinical and molecular delineation of the 9q subtelomeric deletion syndrome supports a major contribution of EHMT1 haploinsufficiency to the core phenotype.

BACKGROUND: The 9q subtelomeric deletion syndrome (9qSTDS) is clinically characterised by moderate to severe mental retardation, childhood hypotonia and facial dysmorphisms. In addition, congenital heart defects, urogenital defects, epilepsy and behavioural problems are frequently observed. The syndrome can be either caused by a submicroscopic 9q34.3 deletion or by intragenic EHMT1 mutations leading to haploinsufficiency of the EHMT1 gene. So far it has not been established if and to what extent other genes in the 9q34.3 region contribute to the phenotype observed in deletion cases. This study reports the largest cohort of 9qSTDS cases so far. METHODS AND RESULTS: By a multiplex ligation dependent probe amplification (MLPA) approach, the authors identified and characterised 16 novel submicroscopic 9q deletions. Direct sequence analysis of the EHMT1 gene in 24 patients exhibiting the 9qSTD phenotype without such deletion identified six patients with an intragenic EHMT1 mutation. Five of these mutations predict a premature termination codon whereas one mutation gives rise to an amino acid substitution in a conserved domain of the protein. CONCLUSIONS: The data do not provide any evidence for phenotype-genotype correlations between size of the deletions or type of mutations and severity of clinical features. Therefore, the authors confirm the EHMT1 gene to be the major determinant of the 9qSTDS phenotype. Interestingly, five of six patients who had reached adulthood had developed severe psychiatric pathology, which may indicate that EHMT1 haploinsufficiency is associated with neurodegeneration in addition to neurodevelopmental defect.

Nonrandom chromosome changes in Kaposi sarcoma: cytogenetic and FISH results in a new cell line (KS-IMM) and literature review.

The karyotype of a new tumorigenic Kaposi sarcoma (KS)-derived cell line, as defined by cytogenetic and fluorescence in situ hybridization (FISH) analysis is 49,XY,i(1)(q10),i(7)(p10),+i(7) (q10),+der(8)t(8;13)(p11;q11),-13,+del(14)(q22),+der(17)t(1;17)(p13;p13). Our aim was to point out some characteristics and recurrent chromosome changes probably playing a relevant role in the malignant progression of KS, by a comparison of the cytogenetic results obtained in the present study with data from the literature. The interpretation of the cytogenetic results is that KS development occurs by multiple steps: an initial reactive polyclonal cell proliferation is associated with chromosome instability; the cells in a later stage acquire clonal chromosome changes. If many chromosome changes are present, particularly 8q and 1q trisomy, 3p14-->pter deletion, 1p13, 13p14.3, 7q22, 8p11, 13q11, and 19q13 band rearrangements, KS acquires a neoplastic aggressive state.

Molecular cytogenetics, RFLP analysis and clinical characterization of a de novo trisomy 10p case.

A new case of a de novo trisomy 10cen-->10pter is described. The karyotype was exactly defined by high resolution banding and FISH analysis; the chromosome aberration was of maternal meiotic origin as demonstrated by RFLP analysis. Clinical data are reported and correlated with other trisomy 10p cases from the literature. A critical review of the literature was made to define the phenotype of trisomy 10p syndrome.

Cytogenetic and interphase FISH analyses of 73 basal cell and three squamous cell carcinomas: different findings in direct preparations and short-term cell cultures.

Cytogenetic analysis performed on 73 sporadic basal cell carcinomas (BCCs) and three squamous cell carcinomas (SCCs) showed different findings in direct preparations (24 hours) and in short-term cell cultures. Except for loss of the Y chromosome, not one of the other clonal (+6, +16, add(2)(q37), del(3)(q13), add(1)(p31), and near triploidy) or sporadic changes found in direct preparations was found in cell cultures and vice versa. Clonal trisomy 6 found in two BCC direct preparations and demonstrated by interphase fluorescence in situ hybridization in 8 other cases seems to be a nonrandom change in basal cell carcinoma. Immunohistochemistry showed that the cell type investigated was different in the two methods of analysis used: epithelial in direct preparations and fibroblastic in cell cultures. Thus, the results obtained in direct preparations indicate the BCC or SCC epithelial karyotype, whereas the aberrations found in cell cultures indicate the presence of chromosome instability in the fibroblastic stroma. The apparent lack of correspondence between direct and indirect preparations and the presence of clonal chromosome changes in both epithelial and stromal cells suggest tumor cell heterogeneity of BCC. The fibroblastic stroma seems to be implicated in the neoplastic process. This is not evident in SCC, in which clonal changes are present only in direct preparations. The chromosomal distribution of the breakpoints involved in structural changes in direct and cell culture preparations is random; together with those reported in the literature, the breakpoints found in BCC cultures show, however, a cluster to 1p36, 3q13, 9q22, 14p11, 15p11, and Xp11 bands. We did not find any significant correlations between BCC cytogenetic results and the clinical data (site, age, sex, recurrence). The incidence of cases of BCC (38%) and of SCC (100%) showing clonal chromosome changes agree with their benign and malignant nature, respectively. Finally, a significantly high incidence of constitutional inv(9) and dup(9)(q11q21) was found in the group of patients with BCC.

Cytokine production by a new undifferentiated human thyroid carcinoma cell line, FB-1.

A human anaplastic thyroid cancer cell line FB-1, derived from a 68-yr-old woman who underwent surgery for anaplastic thyroid cancer, has been established. The spindlelike cells have been proliferating stably for more than 2 yr. Karyotype analysis shows many abnormalities and many marker chromosomes have been observed. Heterotransplant of FB-1 cells into severe combined immunodeficient mice has resulted in rapidly growing tumors classified as anaplastic carcinomas, although 50% have shown areas with a trabecular pattern. FB-1 cells failed to express messenger RNA for thyroglobulin; TSH-receptor; thyroperoxidase, and placental angiogenic growth factor. Conversely, PAX8 and thyroid transcription factor 1, whose expression is thyroid specific, was kept in an FB-1 cell line at a level comparable with that observed in normal thyroid tissue. In addition, the present cell line expressed high levels of messenger RNA for high-mobility group proteins (Y) and -C. The in vitro study revealed that FB-1 cells are able to produce high levels of interleukin (IL)-8 and medium amount of IL-6, whereas no release of IL-1-alpha, IL-1-beta, and IL-4 was observed. No modulation of cell proliferation and DNA synthesis in FB-1 cells has been observed after the addition of exogenous IL-6.

Cytogenetic and interphase cytogenetic analyses reveal chromosome instability but no clonal trisomy 8 in Dupuytren contracture.

The results of cytogenetic and FISH analysis performed in 26 cases of Dupuytren contracture are reported. Clonal or sporadic chromosome changes were found in 18 cases (69%). Clonal changes consisted of: +2, +16, -10, -Y, add(1)(p23), del(2)(q21), t(3;16)(p21;q24), add (3)(p24), del(18)(q21), t(Y;14)(p12;q24), +mar. The results differ from those obtained in normal palmar fascia used as control, in which -Y and +Y were the only clonal changes found in 2 of 11 analyzed cases (18%). No clonal trisomy 8 was found. FISH analysis performed in 11 cases (centromeric probe specific for chromosome 8) failed to show the presence of a cell population with +8. Clonal and sporadic structural changes were different from case to case and no clustering breakpoint was observed. The significance of the chromosome instability leading to clonal and sporadic chromosome changes not specific to Dupuytren contracture are discussed.

Age-related increase of baseline frequencies of sister chromatid exchanges, chromosome aberrations, and micronuclei in human lymphocytes.

Intra- and interindividual variations of baseline frequencies of cytogenetic end points in lymphocytes of human populations have been reported by various authors. Personal characteristics seem to account for a significant proportion of this variability. Several studies investigating the role of age as a confounding factor in cytogenetic biomonitoring found an age-related increase of micronucleus (MN) frequency, whereas contradictory results were reported for chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs). We have quantitatively evaluated the effect of age on SCE, CA, and MN through the analysis of a population sample that included data from several biomonitoring studies performed over the last few decades in 12 Italian laboratories. The large size of the data set, i.e., more than 2000 tests for each end point, allowed us to estimate the independent effect of age, taking into account other covariates, such as sex, smoking habits, occupational exposure, and inter- and intralaboratory variability. A greater frequency of the mean standardized values by increasing of age was observed for all of the end points. A leveling off was evident in the last age classes in the trend of MN frequencies. Frequency ratios (FRs), which express the increase of the cytogenetic damage with respect to the first age classes, i.e., 1-19 years, were estimated using Poisson regression analysis after adjustment for the potential confounding factors and confirmed the increasing trend by age class for all three end points. The most dramatic increase was observed for MN, with a FR that approaches the value of 2 at the age class 50-59 (FR, 1.97; 95% confidence interval, 1.43-2.71) and remains substantially unchanged thereafter. The trend of FRs for CA is more homogeneous, with a constant rise even in the older classes, whereas the frequency of SCE increases with age to a lesser extent, reaching a plateau in the age class 40-49 and the maximum value of FR in the age class over 70 (FR, 1.14; 95% confidence interval, 1.07-1.23). In conclusion, our results point to an age-related increase of the chromosome damage in lymphocytes and emphasize the need to take into account the potential confounding effect of this variable in the design of biomonitoring studies based on chromosome damage.

Large T antigen coding sequences of two DNA tumor viruses, BK and SV40, and nonrandom chromosome changes in two glioblastoma cell lines.

The T antigen (TAg) coding sequences of two DNA tumor viruses, BKV and SV40, were detected by Polymerase Chain Reaction (PCR) amplification followed by Southern-blot hybridization in two human glioblastoma multiforme derived cell lines. RT-PCR analysis indicated that these two TAg coding sequences were expressed in both tumor cell lines carrying the viral early region DNAs. Moreover, analytical polyacrylamide gel electrophoresis (PAGE) and DNA sequence analyses showed that the amplified PCR products are indistinguishable from the TAg coding sequences of BKV and SV40 wildtype strains. Cytogenetic study performed in the two cell lines showed unbalanced changes, mainly gains of chromosomes 3p, 5, 6, 7, and 19 and losses of chromosomes 3, 3q, 16, 9p22-->pter, 18, and 20. Excess of chromosomes 6 and 7 are common to the two cell lines. The putative role of the TAg of the two DNA tumor viruses in transformation and karyotype changes is discussed.

Clonal chromosome changes in non-neoplastic ureters.

Cytogenetic analysis was performed on 23 samples from non-neoplastic ureters. Clonal chromosome abnormalities were found in eight. They were: loss of Y chromosome, as a single abnormality (five cases) or associated with trisomy 10 and 20 (one case) or with trisomy 2 (one case); and duplication of Y chromosome (one case). Different numerical and structural sporadic abnormalities were found in nine cases. Immunohistochemical analysis and direct observation using the inverted microscope showed that the cells were mainly of the fibroblastic type. FISH analysis with chromosome 7 alpha-satellite probes failed to detect the presence of trisomy 7 in three epithelial cases tested.

Chromosome changes in lymphocytes of patients with scleroderma.

The authors have analyzed cytogenetically 28 cultured lymphocytes from females with Diffuse Scleroderma and 28 female controls between 30 and 70 years of age. Recurrent chromosome abnormalities were +8, +X, -X, and the PCD(X) phenomenon. Triplo X cells were significatively more frequent in patients than in controls. The incidence of +X and PCD(X) was significatively higher in the patients between 30 and 50 years of age, while the frequency of -X cells was higher in controls than in patients. None of these chromosome changes was correlated with the presence of anticentromere antibodies (ACA) in the patients' serum. Random structural chromosome abnormalities were also observed in the patients, but no break point clustering was observed. The incidence of chromosome breaks was significatively higher in patients than in controls. These data suggest a general tendency of females with Scleroderma to develop X polisomies and +X and the PCD(X) phenomenon may be considered Scleroderma related in younger patients.

Cerebral germ cell tumor and XXY karyotype.

Metaphases from a cultured cerebral germ cell tumor (CGCT) in a boy with a 46,XY constitutional karyotype had 47 chromosomes with an additional X chromosome and a translocation (1;21)(q11;p11). CGCT appear to be nonrandomly associated with Klinefelter syndrome, and a supernumerary X chromosome and trisomy of the 1q21-->1qter region may be clonal abnormalities in these tumors. The predisposition of Klinefelter patients to develop CGCT may be due to the pathogenetic relevance of the extra X chromosome both as an acquired and a constitutional abnormality.

Chromosome changes in benign prostatic hyperplasia and their significance in the origin of prostatic carcinoma.

Cytogenetic studies of benign prostatic hyperplasia (BHP) are scarce. We analyzed primary cell cultures obtained from biopsies of prostatic tissues from 10 patients (mean age: 60.7 years) with histologic diagnosis of BHP to compare the eventual chromosome changes with those reported in prostatic adenocarcinoma. Clonal chromosome abnormalities were noted in five of the 10 cases, with loss of Y chromosome in all. In one case, a clonal t(1;20) was observed with a -Y clone. Different numerical and structural sporadic abnormalities were evident in eight. Chromosome 1 was the chromosome most frequently involved in sporadic rearrangements. We concluded that -Y is a frequent nonrandom chromosome abnormality in BHP in this sample of patients. Immunohistochemical studies showed that loss of Y occurs in fibroblasts and not in epithelial cells; therefore, this anomaly is not related to cancer development.

Functional disomy of Xp22-pter in three males carrying a portion of Xp translocated to Yq.

A number of Xp22;Yq11 translocations involving the transposition of Yq material to the distal short arm of the X chromosome have been described. The reciprocal product, i.e. the derivative Y chromosome resulting from the translocation of a portion of Xp to Yq, has never been recovered. We searched for this reciprocal product by performing dosage analysis of Xp22-pter loci in 9 individuals carrying a non-fluorescent Y chromosome. In three mentally retarded and dysmorphic patients, dosage analysis indicated the duplication of Xp22 loci. Use of the highly polymorphic probe CRI-S232 demonstrated the inheritance of paternal Xp-specific alleles in the probands. In situ hybridization, performed in one case, confirmed that 29CL pseudoautosomal sequences were present, in addition to Xpter and Ypter, in the telomeric portion of Yq. To our knowledge, these are the first cases in which the translocation of Xp material to Yq has been demonstrated. The X and Y breakpoints were mapped in the three patients by dosage and deletion analysis. The X breakpoint falls, in the three cases, in a region of Xp22 that is not recognized as sharing sequence similarities with the Y chromosome, thus suggesting that these translocations are not the result of a homologous recombination event.