Analysis of a giant marker chromosome in a well-differentiated liposarcoma using cytogenetics and fluorescence in situ hybridization.

Well-differentiated liposarcomas (LPS) are cytogenetically very complex, characterized by giant marker chromosomes, ring chromosomes, and telomeric associations. We report a case of well-differentiated LPS in which the only cytogenetic anomaly was an additional giant marker. In an attempt to identify the origin of this marker, centromeric probes (chosen on the basis of the morphology of the marker) to chromosomes 1,2,3,4,6,7,8,9,10,11,12,16,17, and X and a shared satellite probe for chromosomes 1,5, and 19, were used with fluorescence in situ hybridization (FISH). This was successful at eliminating certain chromosomes as candidates for centromeric trisomy but could not identify the origin of the marker. This case is unusual in that it does not conform to the typical cytogenetic pattern for well-differentiated LPS and is the first known example with an apparently normal diploid karyotype with only one additional change.     

A simplified protocol for fluorescence in situ hybridization with repetitive DNA probes and its use in clinical cytogenetics

A method for chromosome-specific staining and its use in clinical cytogenetics is described. This fluorescence in situ hybridization protocol for repetitive DNA probes results in yellow-green fluorescent signals on orange-red stained chromosomes. Special characteristics are its simplicity, the use of digoxigenin-11-dUTP for labeling, and the combination of high stringency criteria for hybridization and low stringency for washing. The method is particularly advantageous in cases with structurally abnormal extra chromosomes (ESACs), marker chromosomes of gonosomal origin, and chromosomal mosaicism. It may also facilitate the screening of cases for fragile X. The chromosome-specific staining can be done within 1 working-day.     

Molecular genetic study of benign pigmented neoplasms by fluorescence in situ hybridization

Twenty samples of benign pigmented neoplasms of the skin, including 9 intradermal nevi and 11 complex ones, were investigated. Fluorescence in situ hybridization was used to detect the copy number of the RREB1 (6p25), MYB (6q23), and CCND1 (11q13) genes. Analysis of the findings revealed no significant changes characteristic of melanoma in the nevi. However, the authors established that there was a direct correlation between the copy number of the MYB and RREB1 genes and that the amount of the MYB gene most frequently deviated from the normal values. In addition, a relationship was found between the number of MYB gene copies and the depth of the epidermal layer. In cases of an intradermal nevus, the copy numbers of the CCND1 and MYB genes were shown to vary more greatly than in cases of a complex nevus.     

染色体非平衡易位的分子细胞遗传学研究

目的 应用荧光原位杂交技术对１例染色体异常患者进行分析。方法 对经细胞遗传学提示的染色体结构异常的病例,选用１、１８号染色体控针池进行涂染。结果 证实患者由于染色体不平衡易位导致部分单体和部分三体。结论 １ｑ４２－ｑｔｅｒ、１８ｑ２２－ｑｔｅｒ可能与心脏的发育等胚过程有关。     

11q trisomy detected by fluorescence in situ hybridization

Takano T, Yamanouchi Y, Kawashima S, Date M, Hashira S, Kida M, Abe T, Nakahori Y, Nakagome Y. 11q trisomy detected by fluorescence in situ hybridization. Clin Genet 1993: 44: 324–328. © Munksgaard, 1993 A patient with psychomotor developmental delay, multiple minor anomalies, congenital heart disease and left inguinal hernia is reported. His karyotype was 45,X/46,X,+mar (3 : 37 cells), and the marker chromosome was identified as t(Y;11) (q12;q14?) using fluorescence in situ hybridization and fluorescent chromosome painting. He was diagnosed as mosaic for de novo 11q trisomy.     

In situ hybridization for different mRNA in GH-secreting and in inactive pituitary adenomas

In a series of 40 pituitary adenomas in acromegaly all tumors showed mRNA for GH by in situ hybridization (ISH). The signals were mostly very strong and found in more than 80% of adenoma cells by using frozen sections. In paraffin sections the number of positive cells and the intensity of signals are lower. Prolactin mRNA was found in 87% of adenomas. In 27% more than 80% of cells were marked. beta-HCGmRNA (with 90% hormology for LH-mRNA) was demonstrable in very sparse cells of 25% of adenomas. Comparing ISH with immunohistology (IH) we found a correlation between signals and hormone content in 100% of adenomas for GH, in 60% for Prolactin and in 10% for Gonadotropins. In 18% Prolactin mRNA but not the hormone was demonstrable and in 5% Prolactin was immunostained but no hybridization signals were detected. In a series of 40 clinically inactive adenomas sparse cells of three tumors expressed GHmRNA and two of these contained also the hormone, whereas in one adenoma GH but not GHmRNA was demonstrable. Prolactin mRNA was found in 8 adenomas. 7 of these also contained the hormone. In two cases Prolactin but not Prolactin mRNA was present. Beta-HCG(LH)mRNA and the respective hormones were shown in very sparse cells of 6 adenomas, whereas only beta-HCG(LH)-mRNA was found in 8 cases. The significance of the findings is discussed.     

Human growth hormone and prolactin secreting pituitary adenomas analyzed by in situ hybridization

Acidophilic pituitary adenomas commonly produce growth hormone (GH) or prolactin (PRL), according to studies employing immunohistochemical and ultrastructural methods. To examine this question, in situ hybridization with oligonucleotide probes was done on routinely processed tissues received in the pathology laboratory to analyze for the presence of GH and PRL messenger RNA (mRNA) in 4 normal pituitaries, 10 prolactinomas, and 16 GH-secreting adenomas. Most acidophilic cells in normal pituitaries expressed either GH or PRL hormone and the respective mRNAs, but GH mRNA and PRL hormone were also detected in some of the same cells. Patients with a clinical diagnosis of prolactinoma had cells with only PRL mRNA in their tumors, while most (14 of 16) patients with a clinical diagnosis of acromegaly or gigantism had both GH and PRL mRNAs in their tumors. The GH adenomas varied in these studies. In situ hybridization was helpful in characterizing the adenoma from a patient with acromegaly who had immunoreactive PRL, but no immunoreactive GH in the resected tumor; in situ hybridization analysis revealed mRNAs for both GH and PRL in the same tumor cells. Our findings indicate that pituitary adenomas from patients with acromegaly commonly express PRL mRNA. It is concluded that in situ hybridization provides new information about the clinical biology and the histopathologic classification of pituitary adenomas.     

X染色体异常的细胞分子遗传学研究

目的我们从细胞遗传学和分子遗传学方面研究X染色体异常,探讨这些异常与患者临床特征的关系。方法采集患者外周血进行培养,常规制片后G显带染色体核型分析和荧光原位杂交技术分析。结果筛选200余例遗传咨询者共有15例为X染色体异常,其中X染色体数目异常8例,X染色体结构异常6例,数目异常伴结构异常1例。结论体征、性腺发育、智力等都与X染色体异常有关系。     

Loss of 1p in recurrent meningiomas. a comparative study in successive recurrences by cytogenetics and fluorescence in situ hybridization

Deletion of 1p is associated with histological progression to meningiomas. Detection of this alteration may be a predicting factor for recurrences in this tumor. We present 8 meningiomas from four patients: the original tumor and the first recurrence in one patient, and the first and second recurrences in the other three were studied. We compared results of monosomy 22 and deletion of chromosome 1p with cytogenetic methods and fluorescence in situ hybridization (FISH) analysis obtained from slides of direct preparations, of cultured cells and slides of touch preparations. The cytogenetic study showed normal chromosome 22 and deletion on 1p32 in both samples of one patient; only monosomy 22 in both recurrences in another patient, and normal karyotypes with different non-clonal anomalies in the other tumors. However, with FISH analysis, monosomy 22 in both recurrences of three patients was demonstrated, as well as the loss of 1p in all tumors. These results were more evident in the analysis of direct and touch preparations than in those of cultured cells.     

DNA in situ hybridization (interphase cytogenetics) versus comparative genomic hybridization (CGH) in human cancer: detection of numerical and structural chromosome aberrations

DNA in situ hybridization techniques for cytogenetic analyses of human solid cancers are nowadays widely used for diagnostic and research purposes. The advantage of this methodology is that it can be applied to cells in the interphase state, thereby circumventing the need for high-quality metaphase preparations for karyotypic evaluation. In situ hybridization (ISH) with chromosome specific (peri)centromeric DNA probes, also termed "interphase cytogenetics", can be used to detect numerical changes, whereas comparative genomic hybridization (CGH) discloses chromosomal gains and losses, i.e. amplifications and deletions. We wanted to compare both methods in human solid tumors, and for this goal we evaluated ISH and CGH within a set of 20 selected prostatic adenocarcinomas. Chromosomes 7 and 8 were chosen for this analysis, since these chromosomes are frequently altered in prostate cancer. ISH with chromosome 7 and 8 specific centromeric DNA probes was applied to standard, formalin-fixed and paraffin-embedded, histological sections for numerical chromosome analysis. CGH with DNA's, extracted from the same histologic area of the archival specimens, was used for screening of gains and losses of 7 and 8. ISH with centromeric probes distinguished a total of 26 numerical aberrations of chromosome 7 and/or 8 in the set of 20 neoplasms. In the same set CGH revealed a total of 35 losses and gains. CGH alterations of 7 and 8 were seen in twenty-two of the 26 chromosomes (85%) that showed aberrations in the ISH analysis. Concordance between ISH and CGH was seen in 11 (of 26; 42%) chromosomes. Eight chromosomes were involved in gains (5 x #7, 3 x #8), three in losses (3 x #8). This included both complete (3/11) and partial (8/11) CGH confirmation of the numerical alteration. Partial CGH confirmation was defined as loss or gain of a chromosome arm with involvement of the centromeric region. In the majority of these cases it concerned a whole chromosome arm, mostly the long arm. We conclude that generally a fair correlation was found between ISH and CGH in interphase preparations of a series of prostate cancers. However, when specified in detail, most of the numerical ISH aberrations were only partly represented in the CGH analysis. On the one hand, it suggests that CGH does not adequately discriminate numerical abnormalities. On the other hand, it likely implies that not all numerical changes, as detected by interphase cytogenetics, are truly involving the whole chromosome. A part of these discrepancies might be caused by structural mechanisms, most notably isochromosome formation.     

Analysis of chromosome 1p abnormalities in renal oncocytomas by loss of heterozygosity studies: correlation with conventional cytogenetics and fluorescence in situ hybridization

We previously showed by cytogenetics and fluorescence in situ hybridization (FISH) that the most common chromosomal abnormality in renal oncocytomas is loss of chromosome 1 or 1p. In the present study, we evaluated chromosome 1 by loss of heterozygosity (LOH) studies. DNA was extracted from paraffin sections. Three microsatellite markers were used: D1S508, D1S199, and D1S2734. The regions targeted by FISH probes and LOH markers were close to each other but not overlapping.Among 16 tumors evaluated by all 3 techniques, in 2 cases, LOH could not be interpreted. LOH was detected in at least 1 locus in 12 (86%) of 14 renal oncocytomas studied, with other loci being noninformative or not interpretable (1 case). In 2 cases, the LOH results were inconclusive.These results provide further evidence to support widespread abnormalities in chromosome 1p in renal oncocytoma. Determining whether such abnormalities are unique to renal oncocytomas or are also present in other tumors requires further studies.     

Supernumerary ring chromosome 20 characterized by fluorescence in situ hybridization

We report on a boy with mild dysmorphic features and developmental delay, in whom karyotyping showed an additional minute ring chromosome in 60% of metaphases. Fluorescence in situ hybridization (FISH) with a centromere specific probe demonstrated that the ring chromosome contained the centromeric region of chromosome 20. The ring was highlighted completely using a chromosome 20 painting probe. A cosmid probe for 20p12-13 gave a positive signal and hybridization with an all-telomere probe showed one signal, suggesting a breakpoint in the 20p telomere. The results suggested that only a small part of 20q was involved in this ring. The ring was also detected in 18% of nuclei of a buccal smear. The phenotypic similarities of symptoms in the proband to patients with a (partial) trisomy 20p and the dissimilarities to symptoms in patients with (partial) trisomy 20q were in agreement with the FISH results.     

Characterization of a small supernumerary ring X chromosome by fluorescence in situ hybridization

We report on a male with mild learning disabilities who has a supernumerary marker chromosome. The marker chromosome was defined by fluorescence in situ hybridization as a ring X chromosome with breakpoints in the juxacentromeric region. Replication studies suggest that the ring X is late-replicating. However XIST, a gene in the X inactivation centre interval which is expressed exclusively from the inactive X chromosome, is not present on the marker, nor is it expressed in the patient's cells. These results are discussed with respect to karyotype-phenotype correlations and X inactivation. © 1993 Wiley-Liss, Inc.     

Numerical aberrations of chromosome 8 detected by conventional cytogenetics and fluorescence in situ hybridization in individuals from northern Brazil with gastric adenocarcinoma

Gastric cancer is the third most frequent type of neoplasia and the second most important cause of cancer-related death in the world. In northern Brazil, the state of Pará shows a high incidence of this disease and the capital ranks among cities with the highest incidence of stomach cancer in the world. To evaluate chromosomal aberrations implicated in gastric carcinogenesis, we analyzed 16 samples of gastric adenocarcinoma by fluorescence in situ hybridization using a chromosome 8 alpha-satellite probe and by direct chromosomal analysis techniques. All lesions were classified as at advanced stages according to the recommendations of the Union Internationale Contre le Cancer (UICC). Trisomy 8 was the main finding of this study, observed in all cases. There was no significant difference between chromosome 8 ploidy and localization, stage, or histological type of adenocarcinoma in our sample. The high incidence of alterations we found in chromosome 8 may be a regional characteristic, related to the high incidence of this neoplasm in Pará state and a strong influence of external factors, such as eating habits. This aberration may comprise a cytogenetic subgroup of this neoplasm. Additional investigations are necessary to confirm the involvement of chromosome 8 and to identify genes in this chromosome related to gastric carcinogenesis.     

Molecular and fluorescence in situ hybridization analysis of a 10;11 rearrangement in a case of infant acute monocytic leukemia

Fluorescence in situ hybridization (FISH) analysis in a case of infant acute monocytic leukemia M5 revealed a complex rearrangement between chromosomes 10 and 11, leading to the disruption of the MLL gene. Using two painting probes for chromosomes 10 and 11 and a specific probe for the MLL gene localized on 11q23, we observed a paracentric inversion of the 11q13-q23 fragment translocated to 10p12. Molecular analysis showed that AF10 localized on 10p12 was the fusion partner gene of MLL in this rearrangement (10;11). This report underlined the usefulness of FISH and molecular techniques in identifying complex rearrangements.     

Confirmation of a cryptic unbalanced translocation using whole chromosome fluorescence in situ hybridization

We report on a 7-year-old boy with minor anomalies, growth retardation, and developmental delay with an initial 46,XY,der(18)t(18;?)(q23;?) chromosome constitution. To determine the origin of the additional chromosome segment, several candidate regions were identified including 4q and 18q. Clinical comparison showed more similarities to individuals with partial dup(4q) than to those with a dup(18q). Whole chromosome fluorescence in situ hybridization (FISH) was used to demonstrate the correct origin of the translocated region, clarifying the karyotype as 46,XY,der(18)t(4;18) (q28.2;q22.2), thus generating information of clinical importance. This illustrates the use of whole chromosome FISH to identify chromosome regions that cannot be determined conclusively using standard cytogenetic banding techniques. © Wiley-Liss, Inc.     

In situ hybridization study of pro-opiomelanocortin (POMC) gene expression in human pituitary corticotrophs and their adenomas

Summary Pro-opiomelanocortin (POMC) mRNA was detected on paraffin sections by in situ hybridization (ISH) in corticotrophs of 12 nontumorous pituitaries, 11 functioning corticotroph, and 11 silent pituitary adenomas. ISH combined with immunocytochemistry for adrenocorticotrophic hormone (ACTH), a POMC-derived peptide, was also performed. ACTH immunoreactive cells of the anterior lobes and those invading the posterior lobe showed a high or moderate level of POMC mRNA that was not correlated with the intensity of ACTH immunoreactivity. Variable levels of POMC gene expression were present in Crooke's cells, corticotrophs suppressed by glucocorticoid excess. Most functioning corticotroph adenomas and silent subtype 1 adenomas had an intense hybridization signal and ACTH immunoreactivity. In silent subtype 2 and 3 adenomas, POMC mRNA had a diffuse low level or was absent; in these adenomas ACTH immunoreactivity was diffuse, restricted to some cells, or negative. The results indicate that POMC gene is expressed in both normal and suppressed nontumorous corticotrophs. Intense signals for POMC mRNA are found in most functioning corticotroph adenomas. The difference between POMC gene expression in silent 1 and silent 2 and 3 adenomas suggests that different mechanisms are responsible for the lack of endocrine activity.     

Nonrandom gain of chromosome 7 in central neurocytoma: A chromosomal analysis and fluorescence in situ hybridization study

Central neurocytoma is a benign, slow-growing neoplasm with favourable prognosis. Biomolecular analysis has failed to demonstrate significant alterations, and no cytogenetic alterations have been reported. In this study we demonstrate chromosome 7 gain in three of nine neurocytomas (33%). Traditional cytogenetic analysis performed in four of the nine cases identified trisomy 7 as the sole chromosomal abnormality in one case. Interphase cytogenetics utilizing fluorescent in situ hybridization (FISH) on cell suspensions from formalin-fixed paraffin-embedded tumour tissue performed in all nine cases detected trisomy 7 in two more cases and tetrasomy in another. Our results suggest that chromosome 7 gain is a feature of neuroectodermal tumorigenesis, possibly conferring growth advantage on the neoplastic cells. FISH on interphase nuclei is a valuable adjunct in the genetic evaluation of rare central nervous system neoplasms with low baseline proliferative activity.     

Chromosomal changes in prostate cancer: a fluorescence in situ hybridization study

The incidence of prostate cancer (PC) is increasing steadily with the aging population in Singapore. As the pattern of chromosomal aberrations in Asian men with PC is poorly understood, we investigated the numerical aberrations for chromosomes 7, 8, 11, and 17 by fluorescence in situ hybridization (FISH). FISH was performed on standard sections and tissue microarrays of 54 PC and 33 benign prostatic hyperplasia (BPH) specimens. Among the 54 PC specimens, FISH detected 44 cases as aneusomy and two as disomy and was unsuccessful for eight cases. Cytogenetic alterations of two or more chromosomes per tumor were detected in 33/46 (72%) PCs. The most frequent alteration was aneusomy of chromosome 8 detected in 34/46 (74%) cases followed by numerical aberrations in chromosome 7 (61%). Gain of 8q24, loss of chromosome 7, and gain of 11q13 were associated with higher Gleason score and were statistically significant. Gain of chromosome 7 was more common in locally advanced disease, while gain of chromosome 11q13 and chromosome 7 was more common in metastatic disease.