Pericentromeric Probes Research Articles

New Data on Organization and Spatial Localization of B-Chromosomes in Cell Nuclei of the Yellow-Necked Mouse Apodemus flavicollis.

The gene composition, function and evolution of B-chromosomes (Bs) have been actively discussed in recent years. However, the additional genomic elements are still enigmatic. One of Bs mysteries is their spatial organization in the interphase nucleus. It is known that heterochromatic compartments are not randomly localized in a nucleus. The purpose of this work was to study the organization and three-dimensional spatial arrangement of Bs in the interphase nucleus. Using microdissection of Bs and autosome centromeric heterochromatic regions of the yellow-necked mouse (Apodemus flavicollis) we obtained DNA probes for further two-dimensional (2D)- and three-dimensional (3D)- fluorescence in situ hybridization (FISH) studies. Simultaneous in situ hybridization of obtained here B-specific DNA probes and autosomal C-positive pericentromeric region-specific probes further corroborated the previously stated hypothesis about the pseudoautosomal origin of the additional chromosomes of this species. Analysis of the spatial organization of the Bs demonstrated the peripheral location of B-specific chromatin within the interphase nucleus and feasible contact with the nuclear envelope (similarly to pericentromeric regions of autosomes and sex chromosomes). It is assumed that such interaction is essential for the regulation of nuclear architecture. It also points out that Bs may follow the same mechanism as sex chromosomes to avoid a meiotic checkpoint.

Tandem Repeat-Based Probes Support the Loop Model of Pericentromere Packing

Constitutive heterochromatin is the most mysterious part of the eukaryotic genome. It forms vital chromosome regions such as the centromeric and the pericentromeric ones. The main component of heterochromatic regions are tandem repeats (TR), and their specific organization complicates assembly, annotation, and mapping of these regions. Unannotated and unmapped TR arrays are still present in database contigs. In this study, we used a set of TR in the genomes of the pig (Sus scrofa) and the Chinese hamster (Cricetulus griseus) identified with the help of bioinformatics techniques and determined the specificity of the designed probes. The signal of the 4 pig TR probes in spermatogenic cells was often ring-shaped, especially in primary spermatocytes. The rings were located in the regions relatively weakly stained with DAPI. The unique assembly of the centromeric region was traced using the hamster meiotic chromosomes. The probe specific to chromosome 5 was used. Two signals, arranged as rings, were seen at the pachytene stage, similar to those in the pig spermatogenic cells. In the spermatogenic cells of both pig and hamster, the rings appeared on the chromosomes with pericentromeric TR probes. Our observations support the loop model of the centromeric region, the size of the loops being about 50 kb.

Small Supernumerary Marker Chromosome May Provide Information on Dosage-insensitive Pericentric Regions in Human.

Background: Cytogenetically visible chromosomal imbalances in humans are deleterious and adverse in the majority of the cases. However, healthy persons living with chromosomal imbalances in the range of several megabasepairs (Mbps) in size, like carriers of small Supernumerary Marker Chromosomes (sSMCs) exist.Materials & Methods: The identification of healthy sSMC carriers with euchromatic centromere-near (ECN) imbalances led to the following proposal: ECN-regions do not contain any dosage sensitive genes. Due to own previous work, dosage-insensitive pericentric ECN-regions were already determined with an accuracy of 0.3 and 5 Mbp. Based on this data we established 43 new pericentromeric probe sets spanning about 3-5 Mbp of each euchromatic human chromosome arm starting from the known insensitive regions towards distal. Such so called pericentromeric-critical region fluorescence in situ hybridization (PeCR-FISH) probe sets were applied exemplarily and successful here in 15 sSMC cases as available from the Else Kröner-Fresenius-sSMC-cellbank <http://ssmc-tl.com/ekf-cellbank.html>.Conclusion: Most of the involved sSMC breakpoints could be characterized as a higher resolution than before. An unexpected result was that in 5/15 cases cryptic mosaicism was characterized. The latter is also to be considered to have potentially an influence on the clinical outcome in these so-called discontinuous sSMCs. Overall, the suitability of PeCR-FISH to characterize sSMCs was proven; the potential of this probe set to further delineate sizes of dosage insensitive pericentric regions is obvious but dependent on suited cases. Furthermore, discontinuous sSMCs can be identified by this approach and this new subtype of sSMC needs to be studied in more detail in future.

Noninvasive Detection of HER2 Amplification with Plasma DNA Digital PCR

Digital PCR is a highly accurate method of determining DNA concentration. We adapted digital PCR to determine the presence of oncogenic amplification through noninvasive analysis of circulating free plasma DNA and exemplify this approach by developing a plasma DNA digital PCR assay for HER2 copy number. The reference gene for copy number assessment was assessed experimentally and bioinformatically. Chromosome 17 pericentromeric probes were shown to be suboptimal, and EFTUD2 at chromosome position 17q21.31 was selected for analysis. Digital PCR assay parameters were determined on plasma samples from a development cohort of 65 patients and assessed in an independent validation cohort of plasma samples from 58 patients with metastatic breast cancer. The sequential probability ratio test was used to assign the plasma DNA digital PCR test as being HER2-positive or -negative in the validation cohort. In the development cohort, the HER2:EFTUD2 plasma DNA copy number ratio had a receiver operator area under the curve (AUC) = 0.92 [95% confidence interval (CI), 0.86-0.99, P = 0.0003]. In the independent validation cohort, 64% (7 of 11) of patients with HER2-amplified cancers were classified as plasma digital PCR HER2-positive and 94% (44 of 47) of patients with HER2-nonamplified cancers were classified as digital PCR HER2-negative, with a positive and negative predictive value of 70% and 92%, respectively. Analysis of plasma DNA with digital PCR has the potential to screen for the acquisition of HER2 amplification in metastatic breast cancer. This approach could potentially be adapted to the analysis of any locus amplified in cancer.

Design and validation of a pericentromeric BAC clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes

BackgroundSmall supernumerary marker chromosomes (sSMCs) are additional, structurally abnormal chromosomes, generally smaller than chromosome 20 of the same metaphase spread. Due to their small size, they are difficult to characterize by conventional cytogenetics alone. In regard to their clinical effects, sSMCs are a heterogeneous group: in particular, sSMCs containing pericentromeric euchromatin are likely to be associated with abnormal outcomes, although exceptions have been reported. To improve characterization of the genetic content of sSMCs, several approaches might be applied based on different molecular and molecular-cytogenetic assays, e.g., fluorescent in situ hybridization (FISH), array-based comparative genomic hybridization (array CGH), and multiplex ligation-dependent probe amplification (MLPA).To provide a complementary tool for the characterization of sSMCs, we constructed and validated a new, FISH-based, pericentromeric Bacterial Artificial Chromosome (BAC) clone set that with a high resolution spans the most proximal euchromatic sequences of all human chromosome arms, excluding the acrocentric short arms.ResultsBy FISH analysis, we assayed 561 pericentromeric BAC probes and excluded 75 that showed a wrong chromosomal localization. The remaining 486 probes were used to establish 43 BAC-based pericentromeric panels. Each panel consists of a core, which with a high resolution covers the most proximal euchromatic ~0.7 Mb (on average) of each chromosome arm and generally bridges the heterochromatin/euchromatin junction, as well as clones located proximally and distally to the core. The pericentromeric clone set was subsequently validated by the characterization of 19 sSMCs. Using the core probes, we could rapidly distinguish between heterochromatic (1/19) and euchromatic (11/19) sSMCs, and estimate the euchromatic DNA content, which ranged from approximately 0.13 to more than 10 Mb. The characterization was not completed for seven sSMCs due to a lack of information about the covered region in the reference sequence (1/19) or sample insufficiency (6/19).ConclusionsOur results demonstrate that this pericentromeric clone set is useful as an alternative tool for sSMC characterization, primarily in cases of very small SMCs that contain either heterochromatin exclusively or a tiny amount of euchromatic sequence, and also in cases of low-level or cryptic mosaicism. The resulting data will foster knowledge of human proximal euchromatic regions involved in chromosomal imbalances, thereby improving genotype–phenotype correlations.

H3S10 Phosphorylation Marks Constitutive Heterochromatin During Interphase in Early Mouse Embryos Until the 4-Cell Stage

Phosphorylation of histone H3 at Ser10 (H3S10P) has been linked to a variety of cellular processes, such as chromosome condensation and gene activation/silencing. Remarkably, in mammalian somatic cells, H3S10P initiates in the pericentromeric heterochromatin during the late G2 phase, and phosphorylation spreads throughout the chromosomes arms in prophase, being maintained until the onset of anaphase when it gets dephosphorylated. Considerable studies have been carried out about H3S10P in different organisms; however, there is little information about this histone modification in mammalian embryos. We hypothesized that this epigenetic modification could also be a marker of pericentromeric heterochromatin in preimplantation embryos. We therefore followed the H3S10P distribution pattern in the G1/S and G2 phases through the entire preimplantation development in in vivo mouse embryos. We paid special attention to its localization relative to another pericentromeric heterochromatin marker, HP1β and performed immunoFISH using specific pericentromeric heterochromatin probes. Our results indicate that H3S10P presents a remarkable distribution pattern in preimplantation mouse embryos until the 4-cell stage and is a better marker of pericentromeric heterochromatin than HP1β. After the 8-cell stage, H3S10P kinetic is more similar to the somatic one, initiating during G2 in chromocenters and disappearing upon telophase. Based on these findings, we believe that H3S10P is a good marker of pericentromeric heterochromatin, especially in the late 1- and 2-cell stages as it labels both parental genomes and that it can be used to further investigate epigenetic regulation and heterochromatin mechanisms in early preimplantation embryos.

PD05-06: Determination of HER2 Status with Analysis of Plasma DNA by Digital PCR in Patients with Metastatic Breast Cancer.

Abstract Background. A small proportion of originally HER2 negative primary breast cancers relapse with HER2 amplified disease. Identification of these cancers is important as these patients may benefit from HER2 targeting therapies. A proportion of free plasma DNA originates from the cancer, and accurate determination of HER2 copy number in plasma DNA has the potential to non-invasively determine HER2 status in patients with metastatic breast cancer. Digital PCR can differentiate small increases in DNA concentration with a much higher degree of accuracy than conventional quantitative PCR, and here we assess the potential of plasma DNA digital PCR to determine HER2 status. Methods. We examined a cohort of 44 patients with metastatic breast cancer treated at the Royal Marsden Hospital who had received a median of 1.5 prior courses of chemotherapy for recurrent disease; 34 patients had ER positive and 9 HER2 positive (all 3+ positive HercepTest) disease as determined on biopsy of recurrent disease in 16 patients and original primary cancer in 28 patients. Following informed consent, plasma samples were taken, free plasma DNA was extracted and concentration determined with LINE1 quantitative PCR. We designed a custom HER2 copy number TaqMan MBG-probe (Applied Biosystems) and two chromosome 17 peri-centromeric control probes TUFMP1 and UBBP4, avoiding known SNPs and regions of normal copy number variation. Digital PCR was performed in 384 well format on a 7900HT Fast RT-PCR system, with copy number ratio determined using the Poisson distribution. Results. HER2 Digital PCR assay was initially tested on DNA from 9 HER2 amplified cell lines. HER2/UPPB4 ratio was increased in 9/9 HER2 amplified cell lines, and HER2/TUFMP1 in 7/9 due to co-amplification of the 17q peri-centromeric DNA in 2 cell lines. Dilution experiments with SKBR3 DNA in non-amplified DNA demonstrated sensitivity down to ∼1% levels. We selected HER2/UBBP4 digital PCR for examination of patient samples. Plasma DNA Digital PCR HER2/UBBP4 ratios differed substantially between HER2 non-amplified (mean 1.047, SD 0.149, median 1.046, range 0.70−1.264), and HER2 amplified cancers (median 2.231, range 1.138−7.89, p&amp;lt;0.001). Digital PCR HER2 ratios had a high diagnostic accuracy with ROC curve AUC 0.967 (95% CI 0.585−0.997). With a threshold Digital PCR HER2 ratio of 1.3 the test had 100% specificity and 89% sensitivity, with negative predictive value 97%. The single miscalled HER2 amplified patient had a low tumour burden. Results of a test set using sequential probability ratio test analysis will be presented at the conference. Conclusions: Plasma DNA Digital PCR accurately determined HER2 status in patients with metastatic breast cancer. Analyses of plasma DNA have the potential to replace biopsy sampling of metastatic disease to guided targeted therapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD05-06.

Trisomy 18 is a consistent cytogenetic feature in pilomatricoma

Pilomatricoma, also known as ‘calcifying epithelioma of Malherbe’, is a common skin adnexal tumor that mimics hair growth. Its proliferating cells seem distinctly programmed to undergo terminal differentiation and death. We report the first cytogenetic investigations of pilomatricoma. Trisomy 18 was shown, in an index case, by G-banded karyotyping. This aberration was corroborated by interphase fluorescence in situ hybridization, using a chromosome 18 pericentromeric probe, in the basaloid epithelial component of 7 of 11 pilomatricomas, including the index case. Trisomy 18 was present in a small subset of cells, suggesting a role in pilomatricoma progression, rather than in tumor initiation. We conclude that trisomy 18 is a consistent feature in pilomatricoma, suggesting that genes carried on this chromosome, such as that for the antiapoptotic oncoprotein BCL2, may have a role in the growth and differentiation of this benign self-limited tumor.

Abstract 810: Amplification of 8q21 in breast cancer is independent of MYC and associated with poor patient outcome

Abstract Introduction Copy number gains involving the long arm of chromosome 8 including high-level amplifications at 8q21 and 8q24 have been reported in up to 60% of breast cancers. While the role of the MYC gene is well established, the significance of the 8q21 amplicon is less clear. The aim of our study was to analyze 8q21 amplification in a large set of breast cancers with clinical follow up data. Experimental procedures The breast cancer tissue microarray used for this study contains a total of 2.197 tumors and has been described in detail. A fluorescence in situ hybridization (FISH) probe was generated from genomic clone RZPDB737E022003D (imaGenes GmbH, Germany) that maps to the centre of the 8q21 amplicon as previously described in the SK-BR-3 breast cancer cell line. The probe contains the entire TMEM70 gene (transmembrane protein 70) and locates 4 kb downstream from TCEB1 (transcription elongation factor B, polypeptide 1), amplification of which was recently described in prostate cancer. A commercially available pericentromeric probe for chromosome 8 was used as reference (CEP 8Z2 SpectrumOrange, Vysis Inc.). A tumor was considered amplified if the ratio of 8q21/centromere 8 was ≥2.0. Ratios &amp;gt;1.0 and &amp;lt;2.0 had been considered as gains and a ratio ≤1.0 as normal. Results A total of 1458 (66.4%) of arrayed cancer samples were assessable by FISH. Copy number alterations of the 8q21 locus were found in 157 (10.7%) interpretable breast cancers, including amplification in 50 (3.4%) tumors and gains in 107 (7.3%). Amplification at 8q21 was associated to high tumor grade (p=0.042). There was no association between aberrations of the 8q21 locus and tumor stage, presence of lymph node metastases or estrogen receptor status (p&amp;gt;0.05 each). Data on MYC amplification were available from a previous study. A combined analysis of MYC and 8q21 identified 90 tumors with amplifications of MYC and/or 8q21. Of these, 28 tumors were amplified for 8q21 only and 54 for MYC only. Co-amplification of both genes was found in the remaining eight tumors. 8q21 amplification was strongly linked to adverse prognosis (hazard ratio 2.18, confidence interval 1.36 to 3.46, p=0.001). There was no impact of 8q21 gains on patient survival (p=0.48). However, a multivariate Cox analysis did not reveal an independent prognostic value of 8q21 amplification (p=0.23). Conclusions In summary, amplification of 8q21 occurs in a subgroup of breast carcinomas with poor prognosis. Copy number changes of 8q21 are independent from MYC and represent a separate amplicon in this chromosomal region. TMEM70 and the close TCEB1 gene in the centre of the 8q21 amplicon are the most likely possible candidate driver genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 810.

Primary Cutaneous T-Cell Lymphomas Do not Show Specific NAV3 Gene Deletion or Translocation

The mapping of a balanced t(12;18)(q21;q21.2) translocation in a Sézary syndrome (SS) case led Karenko et al. to identify NAV3 gene (12q21-22) deletion by interphase fluorescence in situ hybridization (FISH) in 15/21 patients with mycosis fungoides (MF) or SS. To determine whether the NAV3 deletion is the result of a specific gene breakpoint, we used FISH with dual-color split or break-apart bacterial artificial chromosome (BAC) probes covering the NAV3 locus. A total of 31 samples (18 skin, 11 blood, 1 lymph node, and 1 spleen) from 24 patients with advanced MF/SS (18 with large-cell transformation) were studied. Chromosome 12 imbalances were analyzed by comparative genomic hybridization (CGH) array with a 3K BAC probes in 24 samples from 22 patients. Both normal FISH and CGH array patterns were observed in 22 samples from 18 patients. In 6 patients, abnormal patterns were observed with an abnormal number of chromosome 12 set in 5 of them. Chromosome 12 structural abnormalities were seen in four of these six patients. An imbalanced FISH pattern between NAV3 and pericentromeric control probes was seen in three patients in accordance with CGH array data (one with a pericentromeric deletion and two with a large 12q deletion including NAV3). No NAV3 specific breakpoint or partial deletion was detected.

Assessment of chromosomal gains as compared to DNA content changes is more useful to detect dysplasia in Barrett's esophagus brush cytology specimens

Abnormal DNA ploidy status has been suggested as a prognostic factor for Barrett's esophagus progression into esophageal adenocarcinoma (EAC). The aim of the study was to compare image cytometry DNA analysis (ICDA) and fluorescent in situ hybridization (FISH) in the assessment of DNA ploidy status in Barrett's esophagus (BE), and to determine the value of these abnormalities as an adjunct to conventional cytology in detection of dysplasia and EAC. Brush cytology specimens of 90 BE patients were examined using ICDA and FISH with peri-centromeric probes for chromosomes 7 and 17. The results of ICDA and FISH were compared with each other, and with dysplasia grade or EAC as determined by histology and cytology. FISH and ICDA detected abnormalities in 41% (37/90) and 22% (19/90) of the BE cases, respectively. Gains of chromosome 7 and/or 17 were present in 13% of nondysplasia cases, which further increased with dysplasia stage, while overall DNA content aneuploidy was detected predominantly in high grade dysplasia (HGD) and EAC. Using FISH results combined with cytology, we were able to identify IND/LGD (indefinite/ low grade dysplasia) with a sensitivity and specificity of 75 and 76%, respectively. FISH alone detected HGD/EAC with a high sensitivity and specificity of 85 and 84%, which was superior to that of cytology alone. Thus, FISH is more sensitive than ICDA to detect chromosomal abnormalities in BE brush cytology specimens. FISH detects chromosomal gains in early stages of BE and represents a valuable adjunct to conventional cytology to detect dysplasia or EAC.

Extra copies of chromosomes 16 and X in invasive breast carcinomas are related to aggressive phenotype and poor prognosis

Background: Breast cancer is a genetically complex disease, which involves the accumulation of various structural and numerical chromosomal aberrations. Aim: To assess the numerical status of chromosomes 16 and X...

410 POSTER Identification and characterization of novel, orally active inhibitors of c-MET and Ron receptor tyrosine kinases

The mapping of a balanced t(12;18)(q21;q21.2) translocation in a Sézary syndrome (SS) case led Karenko et al. to identify NAV3 gene (12q21–22) deletion by interphase fluorescence in situ hybridization (FISH) in 15/21 patients with mycosis fungoides (MF) or SS. To determine whether the NAV3 deletion is the result of a specific gene breakpoint, we used FISH with dual-color split or break-apart bacterial artificial chromosome (BAC) probes covering the NAV3 locus. A total of 31 samples (18 skin, 11 blood, 1 lymph node, and 1 spleen) from 24 patients with advanced MF/SS (18 with large-cell transformation) were studied. Chromosome 12 imbalances were analyzed by comparative genomic hybridization (CGH) array with a 3K BAC probes in 24 samples from 22 patients. Both normal FISH and CGH array patterns were observed in 22 samples from 18 patients. In 6 patients, abnormal patterns were observed with an abnormal number of chromosome 12 set in 5 of them. Chromosome 12 structural abnormalities were seen in four of these six patients. An imbalanced FISH pattern between NAV3 and pericentromeric control probes was seen in three patients in accordance with CGH array data (one with a pericentromeric deletion and two with a large 12q deletion including NAV3). No NAV3 specific breakpoint or partial deletion was detected.

Correlation between histologic findings and cytogenetic abnormalities in bladder carcinoma: A FISH study

Objectives To investigate the chromosomal abnormalities present in bladder carcinoma using a fluorescence in situ hybridization assay and to correlate the genetic findings with the pathologic grade and stage. Methods Samples from 37 bladder carcinomas were obtained at cystectomy or transurethral resection. In all cases, a histologic evaluation and fluorescence in situ hybridization analysis were performed. Pericentromeric DNA probes for chromosomes 7, 8, 9, and 17, and locus-specific DNA-probes for the 9p21 and 9q34 bands, were used in the fluorescence in situ hybridization assay. Results Grade 1-2 tumors were characterized by the loss of genetic material on chromosome 9 in 35.3% of cases and either no detectable alterations or multiple aneusomy events in 47.1% and 17.6% of the tumors, respectively. Polysomy was the most frequent occurrence in grade 3 and pT1-T4 carcinomas. No significant difference was found between the loss of 9p21, 9q34, or chromosome 9 and the different tumor classifications. A statistically significant difference was found in the frequency of polysomy between grade 1-2 and grade 3 tumors and between pTa and pT1-T4 tumors for chromosomes 7, 8, 9, and 17, as well as chromosome bands 9p21 and 9q34 ( P <0.005). Conclusions These findings show that chromosome 9 losses do not correlate with the tumor grade or stage, but are the only aberrations found at a significant frequency in low-grade lesions. The results suggest that pT1 tumors are closely related to muscle-invasive tumors at the genetic level and show that polysomy of the chromosomes assessed correlates with high-grade and high-stage bladder carcinoma.

PTEN protein expression correlates with PTEN gene molecular changes but not with VEGF expression in astrocytomas.

PTEN gene (10q23) is a relevant tumor suppressor gene whose protein is a phosphatase involved in the control of angiogenesis of some tumors including astrocytomas. There are no studies correlating molecular changes of PTEN and the immunohistochemical expression of its protein (pPTEN) with the expression of vascular endothelial growth factor (VEGF) in astrocytomas. Fifty-six surgically resected brain gliomas, 10 grade 2, 16 grade 3, and 30 grade 4, were studied by a combined approach, consisting of (1) PCR analysis using four microsatellite markers against the PTEN gene region (10q23), (2) the FISH technique to test chromosome 10 using a pericentromeric probe, and (3) immunohistochemical evaluation of pPTEN and VEGF. Loss of heterozygosity (LOH) of PTEN was observed in 10% of fibrillary grade 2 astrocytomas and all gemistocytic ones. In high-grade tumors, LOH was more frequent in grade 4 than in grade 3 (> or =2 loci deleted, 83% and 56%, respectively). Monosomy for chromosome 10 was observed especially in high-grade tumors (6% of grade 3 and 50% of grade 4) and in 20% of grade 2 tumors, corresponding to gemistocytic astrocytomas. Results with both antibodies against PTEN were concordant: loss of cytoplasmic immunoreactivity was frequently observed according to homogeneous or heterogeneous patterns in 70% and 50% of grades 4 and 3, respectively, but not in grade 2. Immunonegativity of pPTEN was associated with PTEN gene deletion (> or =2 loci deleted) (P = 0.04) but not with monosomy. Cytoplasmic immunoreactivity against VEGF was observed in high-grade and in gemistocytic astrocytomas, but not in conventional grade 2 tumors. Tumor expression of pPTEN was not associated with immunoreactivity against VEGF when the same areas were considered. In conclusion, loss of PTEN expression is frequent in high-grade astrocytomas, but not in grade 2 tumors, and correlates with PTEN deletion and loss of chromosome 10. PTEN immunoreactivity does not correlate with VEGF expression in astrocytomas when similar areas are considered.

The abnormalities of chromosome 8 in two hepatocellular carcinoma cell clones with the same genetic background and different metastatic potential.

Two hepatocellular carcinoma (HCC) cell clones named MHCC97-H and MHCC97-L with different metastatic potential have recently been established from the same parent cell line MHCC97 in our institute. The cytogenetic alterations of these two clones were investigated in this study to explore the possible clues to the mechanism involved in HCC metastasis. Their chromosomal aberrations were analyzed with comparative genomic hybridization (CGH), chromosome-specific painting, and two-color fluorescence in situ hybridization (FISH). The aberrations were found in a total of 17 chromosomes, and six kinds of the aberrations including gains of 1q, 7q, 8q, 20, and the losses of 8p23, 21q were found both in the two cell clones and their parent cell line MHCC97. Using modified CGH, with the DNA of MHCC97-L as control to test the MHCC97-H clone, the loss of 8p23 and the gain of 1q31-32, 8q21.3-22, 13q22, 17q22 were highlighted, and the most significant finding was on chromosome 8. Dual color FISH combining a pericentromeric probe and a BAC probe mapping at 8q23.1 was then performed to verify this result, and the signal ratios of the BAC to centromere were 1.43 in MHCC97-H and 1.45 in MHCC97-L, confirming the over-representations at 8q in both cells. Another interesting finding in the dual-color metaphase FISH was the intrachromosomal translocation of 8q to 8p (looked like an isochromosome 8) and non-reciprocal translocation of part of 8q to 4q, which was further clarified and proved by the FISH with whole chromosome 8 painting probe. The high copies amplification on 8q, the formation of isochromosome 8, non-reciprocal translocation of partial 8q to 4q, and loss of 8p occurred at the same time and are the characteristic chromosomal aberrations of the two cell clones. The chromosome 8p, especially 8p23, might harbor some novel genes related to the HCC metastasis.

Interphase fluorescence in-situ hybridization in the diagnosis of bladder cancer

Interphase FISH is a technique that uses fluorescent molecules to detect chromosomes or specific regions of DNA. It is a rapid and powerful technique for detection of cytogenetic abnormalities in malignant cells independent of their cell cycle status. Using variety of pericentromeric and locus-specific probes, numerical chromosomal changes (aneusomy) as well as loss or gain/amplification of specific genetic regions can be detected in clinical samples. Numerous studies have identified genetic alterations at the DNA level, occurring in the pathogenesis of variety of human neoplasms including bladder cancer, some of which can be used for detection, prognosis, and as intermediate endpoints for evaluating the response to therapy. Recently, sensitivity and specificity of a multicolor FISH assay consisting of four probes (3, 7, 17 and 9p21) was analyzed in several prospective and retrospective studies. The data suggest that this method applicable to voided urine specimens may allow safe extension of the interval between cystoscopies in routine surveillance of patients with transitional cell carcinoma of the bladder. FISH analysis of cells isolated from bladder washings or voided urine is also holding promise for monitoring of treatment outcome and predicting recurrence and progression of the disease. Therefore, this technique can be an important aid in the efforts to reduce mortality from transitional cell carcinoma of the bladder, since it increases our ability to prevent progression to incurable muscle invasive disease.

Aneusomy of chromosomes 7, 8, and 17 and amplification of HER-2/neu and epidermal growth factor receptor in Gleason score 7 prostate carcinoma: A differential fluorescent in situ hybridization study of Gleason pattern 3 and 4 using tissue microarray

Recent evidence shows that the proportion of poorly differentiated prostate carcinoma (Gleason pattern [GP] 4/5) is a surrogate factor for biochemical failure after radical prostatectomy (RP). However, little is known about specific molecular and cytogenetic changes in this aggressive component of localized prostate cancer. We constructed a tissue microarray containing areas of GP 3 and 4 from formalin-fixed radical prostatectomy specimens of 39 patients with Gleason score 7 carcinoma (≥50% GP 4), known pathologic staging parameters (stage < T3b), and biochemical failure data (mean follow-up, 30 months; range, 5 to 74 months). Interphase fluorescent in situ hybridization (FISH) was performed on 5-μm microarray sections using pericentromeric probes to chromosomes 7, 8, and 17 and probes for the HER-2/neu and epidermal growth factor receptor (EGFR) genes. Low-level amplification of HER-2/neu was found in 26% of cases (3 to 5 signals per nucleus, corrected for chromosome 17 aneusomy). Aneusomy of chromosomes 7, 8, and 17 was identified in 21%, 15%, and 5% of cases, respectively. All aberrations occurred almost exclusively in GP 4 carcinoma (8 of 8 aneusomies 7, 2 of 2 trisomies 17, 9 of 10 HER-2/neu amplifications, and 5 of 6 aneusomies 8; P <.001). The presence of HER-2/neu amplification was associated with high tumor volume (>2.0 cm3, P = 0.004). Among patients with negative surgical margins, gain of chromosome 7 was associated with biochemical failure after RP (P =.004, log-rank). Amplification of the EGFR gene occurred in only 1 case (3%). Significant differences in HER-2/neu amplification and gain of chromosomes 7, 8, and 17 were detected between GP 4 prostate carcinoma and GP 3. The frequency of aberrations increased with tumor volume. Chromosome 7 abnormalities may play an important role in cancer progression in margin-negative patients. EGFR amplification was rare, suggesting that this oncogene is not altered at the gene copy number level. HUM PATHOL 32:1392-1397. Copyright © 2001 by W.B. Saunders Company

Interphase cytogenetics in oncocytic adenomas and carcinomas of the thyroid gland

Follicular neoplasms of oncocytic type in the thyroid gland frequently cause diagnostic problems and prognostic uncertainties. To identify numerical chromosomal aberrations of possible pathogenetic importance, we determined chromosome copy numbers in situ in interphase nuclei of 31 oncocytic adenomas and 25 oncocytic carcinomas. Archival formaldehyde-fixed, paraffin-embedded tumor samples and normal control thyroid tissues were arranged in arrays and analyzed by fluorescence in situ hybridization (FISH). We used pericentromeric or locus specific probes for chromosomes 1, 7, 8, 9, 11, 12, 17, 18, 22, and X as well as for the oncogenes Her2/neu, cyclin D1, N-myc, and c-myc. The average number of aneusomies per nucleus was significantly higher in carcinomas than in adenomas, and in both, monosomies were more frequent than polysomies. Loss of chromosome 22 was found in 8 of 21 (38%) carcinomas; in 5 cases, it was associated with chromosome 2 monosomy. Conversely, chromosome 2 aberrations were not found in adenomas. Monosomies for chromosome 8 and X were detected in most adenomas and carcinomas. The most common gains in adenomas and carcinomas were for chromosome 7 (13.8% and 32.0% of the cases, respectively), chromosome 12 (9.6% and 12.0%), and chromosome 17 (19.3% and 32.0%). None of the adenomas with trisomy 17 was associated with gains for chromosomes 7 and 12. None of the analyzed oncogenes was found to be amplified by FISH analysis. Our results indicate that numerical chromosomal aberrations in oncocytic follicular tumors of the thyroid gland are common findings and suggest that different patterns of aberrations may occur in these neoplasms. HUM PATHOL 31:854-859.

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.