Detection of numerical chromosomal abnormalities in malignant cells on body fluids by fluorescence in situ hybridization of interphase cell nuclei with chromosome-specific probes.

To detect numerical chromosomal abnormalities (NCA) in malignant cells on body fluids, Fluorescence in situ hybridization (FISH) technique was tested in clinical specimens from patients with metastatic disease. Directly labeled DNA probes specific for chromosomes 8, 12, X, and Y (Imagenetics, Naperville, IL) were used for in situ hybridization to interphase cell nuclei. Fifteen body fluids (BF) from various sites were studied. Based initially on the Papanicolaou-stained slides, there were seven malignant and eight benign samples. Blind analysis (200 cells/sample) showed that all benign samples had a normal number of chromosomes, whereas six of seven malignant samples showed different NCA comprising 5-60% of the cell population ranging from three to 10 chromosome signals per cell. We conclude that interphase cytogenetic cell analysis of BF by FISH is: (1) feasible and gives superior signals for detection of NCA, (2) helpful in detecting malignant cells, (3) relatively simple with a turnaround time of less than 24 hr. This method may have diagnostic and prognostic application in the study of the biologic behavior of malignant neoplasms.     

Numerical chromosomal aberrations in prostate cancer: correlation with morphology and cell kinetics

Eleven routinely processed radical prostatectomy specimens were studied for the presence of numerical chromosomal aberrations by means of in situ hybridization with nucleic acid probes specific for chromosomes 7, 10, 17, X, and Y. Cytogenetic information was correlated with morphology, tumour stage and volume as well as with cell kinetics, the latter being assessed by immunohistochemistry with antibodies raised against the proliferative cell nuclear antigen (PCNA) and against a formalin-resistant epitope of the Ki-67 antigen, MIB 1. In 5 of 11 cases, numerical aberrations of at least one chromosome were found. The cases with normal chromosome numbers were those with the smallest volumes of Gleason grade 4 and/or 5 tumour (mean 0.5 cm³) and represented tumours restricted to the prostate. Tumours with aberrations in the number of detected chromosomes showed advanced stages and large volumes of high-grade tumour (mean 12.5 cm³). All 4 tumours with positive surgical margins were recruited from a group with marked local heterogeneity in chromosome numbers. Immunostaining with MIB 1 and PCNA was most intense in areas of high-grade tumour and was positively correlated with the emergence of chromosomal aberrations. The data suggest that the appearance of numerical chromosomal aberrations in prostate cancer coincides with aggressive tumour behaviour and could be used as an additional prognostic marker.This work is part of E.K.'s doctoral thesis     

Photopolymerization shrinkage-stress reduction in polymer-based dental restoratives by surface modification of fillers

ObjectivesThis research explores the use of polymer brushes for surface treatment of fillers used in polymer-based dental restoratives with focus on shrinkage stress reduction. The influence of interfacial reactive groups on shrinkage stress is explored.MethodsOligomers of varying lengths and with varying number of reactive groups along the length were synthesized by modifying commercial oligomers. Surface of silica fillers (OX50) was treated with methylaminopropyltrimethoxysilane and this was further reacted with the synthesized oligomers to obtain a series of polymer brushes on the surface. Fillers modified with γ-methacryloxypropyltrimethoxysilane were used as a control. Filler surface treatment was confirmed using diffuse reflectance spectroscopy and thermogravimetric analysis. Fillers were added at 30 wt % to a resin made of BisGMA/TEGDMA and polymerization kinetics, shrinkage stress, volumetric shrinkage, flexural strength and modulus, viscosity were measured.ResultsComposites with polymer brush functionalized fillers showed up to a 30 % reduction in shrinkage stress as compared to the control, with no reduction in flexural strength and modulus. Shrinkage stress reduced with increasing length of the polymer brush and increased with increase in number of reactive groups along the length of the polymer brush.SignificanceThe interface between inorganic fillers and an organic polymer matrix has been utilized to reduce shrinkage stress in a composite with no compromise in mechanical properties. This study gives insights into the stress development mechanism at the interface.     

Satellited 4q identified in amniotic fluid cells

Extra material was identified on the distal long arm of a chromosome 4 in an amniotic fluid specimen sampled at 16.6 weeks of gestational age. There was no visible loss of material from chromosome 4, and no evidence for a balanced rearrangement. The primary counseling issue in this case was advanced maternal age. Ultrasound findings were normal, and family history was unremarkable. The identical 4qs chromosome was observed in cells from a paternal peripheral blood specimen and appeared to be an unbalanced rearrangement. This extra material was NOR positive in lymphocytes from the father, but was negative in the fetal amniocytes. Father's relatives were studied to verify the familial origin of this anomaly. In situ hybridization with both exon and intron sequences of ribosomal DNA demonstrated that ribosomal DNA is present at the terminus of the 4qs chromosome in the fetus, father, and paternal grandmother. This satellited 4q might have been derived from a translocation event that resulted in very little or no loss from the 4q and no specific phenotype. This derivative chromosome 4 has been inherited through at least 3 generations of phenotypically normal individuals. © 1995 Wiley-Liss, Inc.     

一种能精确检测人间期细胞核中21号染色体拷贝数的DNA探针

目的制备能精确检测人类间期细胞核中２１号染色体拷贝数的ＦＩＳＨ探针。方法利用万能引物ＰＣＲ法,从定位于人２１ｑ１１的ＹＡＣ克隆８８１Ｄ２分离制备ＤＮＡ探针,并用于与８例正常人和５例２１三体患者的外周血淋巴细胞中期相和间期核,及经细胞松弛素Ｂ（ｃｙｔｏｃｈａｌａｓｉｎＢ）诱导的双核细胞进行ＦＩＳＨ分析。结果该探针具有以下特点：（１）长度集中于３５０～７５０ｂｐ;（２）其靶序列位于２１号染色体长臂上且紧靠着丝粒;（３）特异性强;（４）杂交信号明亮,容易辨认;（５）对中期相及间期细胞核中２１号染色体的检出率分别高达９９．６０％和９８．４０％。结论制备的ＤＮＡ探针能精确检测人类间期细胞核中２１号染色体的拷贝数,且适用于细胞分裂时２１号染色体分离情况的研究     

荧光原位杂交技术在胎儿染色体异常产前诊断中的应用

目的探讨荧光原位杂交(FISH)技术在检测胎儿染色体异常的临床应用。方法选用13、21、18、X、Y特异性探针对1128例孕16～22周有产前诊断指征的妊娠妇女羊水间期细胞进行分析,并与同时进行的羊水细胞培养核型分析结果进行对照。结果被检1128例羊水间期细胞FISH检测均成功,其中检出正常核型1081例,数目异常核型20例,与常规细胞染色体核型分析结果一致,另外27例结构异常FISH技术未能检出。结论 FISH技术检测胎儿染色体数目异常具有快速、简便、准确性高、特异性强等优点,有较大的临床应用价值,并对产前细胞遗传学诊断有重要意义。     

核形正常的慢性淋巴细胞白血病13q14缺失的研究

目的研究核形正常的慢性淋巴细胞白血病（CLL）13q14缺失的情况。方法运用位于13q14的序列特异性DNA探针RB1、D13S319、D13S25和间期荧光原位杂交（I-FISH）技术对26例初发的CLL患者进行染色体13q14的检测。结果 26例B-CLL中12例（46.1%）有13q14缺失,阳性细胞率为25.0%～90.0%,其中RB1单独缺失0例,D13S319单独缺失3例（11.5%）,D13S25单独缺失3例（11.5%）;RB1、D13S319、D13S25同时缺失1例（3.9%）,D13S319、D13S25同时缺失有5例（19.2%）。结论 CLL患者13q14缺失区域是不恒定的,而I-FISH是研究13q14缺失准确而快速的方法。     

Cytogenetic discrepancy between cultured amniocytes and uncultured amniocytes in mosaic 46,XX,dup(9)(q22.3q34.1)/46,XX at amniocentesis in a pregnancy with a favorable outcome

ObjectiveWe present our observation of cytogenetic discrepancy between cultured amniocytes and uncultured amniocytes in mosaic dup(9)(q22.3q34.1) at amniocentesis in a pregnancy with a favorable outcome.Case reportA 37-year-old, gravida 4, para 0, woman underwent amniocentesis at 18 weeks of gestation because of advanced maternal age. Amniocentesis revealed a karyotype of 46,XX, dup(9)(q22.3q34.1)[8]/46,XX[16]. Prenatal ultrasound findings were unremarkable. She was referred for genetic counseling, and repeat amniocentesis was performed at 21 weeks of gestation, which revealed a karyotype of 46,XX,dup(9)(q22.3q34.1)[7]/46,XX[25]. Simultaneous array comparative genomic hybridization (aCGH) on the DNA extracted from uncultured amniocytes revealed no genomic imbalance, or arr (1–22,X) × 2. Interphase fluorescence in situ hybridization (FISH) analysis on 105 uncultured amniocytes detected only one cell with the dup 9q signal with a mosaic dup 9q level of 1%, compared with 0% in normal control. At 37 weeks of gestation, a 2640-g female baby was delivered with no phenotypic abnormality. The cord blood had a karyotype of 46,XX,dup(9) (q22.3q34.1)[4]/46,XX[36], the umbilical cord had a karyotype of 46,XX,dup(9) (q22.3q34.1)[2]/46,XX[38], and the placenta had a karyotype of 46,XX. aCGH analysis on cord blood revealed no genomic imbalance. At age 2½ months, the baby was doing well, the peripheral blood of the baby had a karyotype of 46,XX,dup(9) (q22.3q34.1)[4]/46,XX[36], and interphase FISH analysis on buccal mucosal cells revealed no dup 9q signal in 100 buccal mucosal cells.ConclusionCytogenetic discrepancy may occur between cultured amniocytes and uncultured amniocytes in mosaic dup(9) (q22.3q34.1). Molecular cytogenetic analysis on uncultured amniocytes is useful for rapid distinguishing pseudomosaicism from true mosaicism under such a circumstance.     

First-trimester prenatal diagnosis performed on pregnant women with fetal ultrasound abnormalities: The reliability of interphase fluorescence in situ hybridization (FISH) on mesenchymal core for the main aneuploidies

Objectives

To examine the reliability of interphase FISH analysis of the main aneuploidies performed on mesenchymal core when prenatal diagnosis was performed on pregnant women with first-trimester fetal abnormalities on ultrasound.

Study design

386 first-trimester prenatal examinations were investigated from chorionic villus samplings for increased nuchal translucencies or other fetal ultrasound abnormalities. Interphase fluorescence in situ hybridization (FISH) for the main aneuploidies (trisomies 13, 18, 21 and gonosomal aneuploidies) was performed on the mesenchymal core of villi. Molecular cytogenetic results were always complemented by conventional cytogenetic results on long-term cultured villi (LTC-villi). Short-term cultured villi (STC-villi) preparations were retrospectively performed only when a chromosomal abnormality was observed with interphase FISH and/or LTC-villi.

Results

88 chromosomal abnormalities (88/386 = 22.8% of first-trimester diagnoses) which could discuss subsequent abortions were observed after LTC-villi preparations. All cases possibly detectable by interphase FISH were detected. Thus, 85 aneuploidies (85/386 = 22.0% of first-trimester diagnoses; 85/88 = 96.6% of chromosomal abnormalities) were detected by interphase FISH, allowing early abortion by curettage before week 14 amenorrhea. No discrepancy occurred between interphase FISH and LTC-villi results for the aneuploidies studied. Three false-negative results (3/386 = 0.77% of first-trimester diagnoses; 3/88 = 3.41% of chromosomal abnormalities) were observed with STC-villi.

Conclusion

We observed a high rate of false-negative results on cytotrophoblast cells. Conversely, interphase FISH of the main aneuploidies on the mesenchymal core provided rapid and reliable results, and therefore should be preferred to STC-villi in first-trimester prenatal diagnosis performed on pregnant women with fetal abnormalities on ultrasound.     

APPLICATION OF TWO-COLOR INTERPHASE FISH USING SEX PROBE IN ALLOGENEIC STEM CELL TRANSPLANTATION

Karyotype aryalysis for sex chromosome or some determined hallmark has been primarily undertaken for engraftment evaluation and minimal residual disease (MRD) monitoring after allogeneic stem cell transplantation (alloSCT)[1,2]. Some special fusion gene detection by RT-PCR has also been applied in this area. However due to some limitation of these techniques, we investigated the application and significance of two-color interphase fluorescence in situ hybridization (FISH) technique using…