Novel recurrent gene fusions between the androgen-regulated gene TMPRSS2 and the ETS family members ERG, ETV1, or ETV4 have been recently identified as a common molecular event in prostate cancer development. We comprehensively analyzed the frequency and risk of disease progression for the TMPRSS2 and ETS family genes rearrangements in a cohort of 96 American men surgically treated for clinically localized prostate cancer. Using three break apart (TMPRSS2, ERG, ETV4) and one fusion (TMPRSS:ETV1) fluorescence in situ hybridization (FISH) assays, we identified rearrangements in TMPRSS2, ERG, ETV1, and ETV4 in 65, 55, 2, and 2% of cases, respectively. Overall, 54 and 2% of cases demonstrated TMPRSS2:ERG and TMPRSS2:ETV1 fusions, respectively. As intronic loss of genomic DNA between TMPRSS2 and ERG has been identified as a mechanism of TMPRSS2:ERG fusion, our assays allowed us to detect deletion of the 3' end of TMPRSS2 and the 5' end of ERG in 41 and 39% of cases rearranged for respective genes. Prostate cancers demonstrating TMPRSS2 gene rearrangement were associated with high pathologic stage (P=0.04). Our results confirm that recurrent chromosomal aberrations in TMPRSS2 and/or ETS family members are found in about 70% of prostate cancers. Importantly, we define a novel approach to study these gene fusions and identified cases where TMPRSS2 was rearranged without rearrangement of ERG, ETV1 or ETV4 and cases with ETS family gene rearrangement without TMPRSS2 rearrangement, suggesting that novel 5' and 3' partners may be involved in gene fusions in prostate cancer.

The HER2 gene, amplified in 10 to 35% of invasive human breast carcinomas, has prognostic and therapeutic implications. Fluorescent in situ hybridization is one method currently used for assessing HER2 status, but fluorescent in situ hybridization involves the time-consuming step of manual signal enumeration. To address this issue, Vysis has developed an automated signal enumeration system, Vysis AutoVysion. A multicenter, blinded study was conducted on 39 formalin-fixed, paraffin-embedded invasive breast carcinoma specimens, including 20 HER2 nonamplified and 19 HER2 amplified (weakly to highly amplified), provided in duplicate to each study site for analysis. Calculation of the HER2/CEP17 ratio and the hands-on time of both manual and automated enumeration approaches were compared. Overall agreement of HER2 classification results (positive and negative) was 92.5% (196 of 212). The Vysis AutoVysion System requires manual enumeration for cases with scanner results within the ratio range of 1.5 to 3.0. When the data in this range are excluded, the agreement between manual and scanner results is 98.8% (169 of 171). The average Vysis AutoVysion System hands-on time per slide was 4.59 versus 7.47 minutes for manual signal enumeration (savings of 2.88 minutes/slide). These data suggest that the Vysis AutoVysion System can correctly classify specimens and may increase the overall efficiency of HER2 testing.

Recently, we identified recurrent gene fusions involving the 5' untranslated region of the androgen-regulated gene TMPRSS2 and the ETS (E26 transformation-specific) family genes ERG, ETV1 or ETV4 in most prostate cancers. Whereas TMPRSS2-ERG fusions are predominant, fewer TMPRSS2-ETV1 cases have been identified than expected on the basis of the frequency of high (outlier) expression of ETV1 (refs 3-13). Here we explore the mechanism of ETV1 outlier expression in human prostate tumours and prostate cancer cell lines. We identified previously unknown 5' fusion partners in prostate tumours with ETV1 outlier expression, including untranslated regions from a prostate-specific androgen-induced gene (SLC45A3) and an endogenous retroviral element (HERV-K_22q11.23), a prostate-specific androgen-repressed gene (C15orf21), and a strongly expressed housekeeping gene (HNRPA2B1). To study aberrant activation of ETV1, we identified two prostate cancer cell lines, LNCaP and MDA-PCa 2B, that had ETV1 outlier expression. Through distinct mechanisms, the entire ETV1 locus (7p21) is rearranged to a 1.5-megabase prostate-specific region at 14q13.3-14q21.1 in both LNCaP cells (cryptic insertion) and MDA-PCa 2B cells (balanced translocation). Because the common factor of these rearrangements is aberrant ETV1 overexpression, we recapitulated this event in vitro and in vivo, demonstrating that ETV1 overexpression in benign prostate cells and in the mouse prostate confers neoplastic phenotypes. Identification of distinct classes of ETS gene rearrangements demonstrates that dormant oncogenes can be activated in prostate cancer by juxtaposition to tissue-specific or ubiquitously active genomic loci. Subversion of active genomic regulatory elements may serve as a more generalized mechanism for carcinoma development. Furthermore, the identification of androgen-repressed and insensitive 5' fusion partners may have implications for the anti-androgen treatment of advanced prostate cancer.

In bone marrow cells of 33 patients with myelodysplastic syndrome and acute myeloid leukemia, structural rearrangements of chromosome 7 were found with conventional G-banding: 8 with deletions 7q and 25 with translocations. In 29 of the patients, complex karyotypes were confirmed using multicolor fluorescence in situ hybridization (mFISH). Commercial probes (Abbot Molecular) were used for 7q22, 7q31, and 7q35, the regions most frequently deleted in myeloid malignancies. In three cases without deletions, high-resolution multicolor banding (mBAND) for chromosome 7 revealed other aberrations. Five groups of chromosomal rearrangements were established: (a) deletion 7q as a sole aberration (2 cases), (b) deletion 7q and complex karyotypes (6 cases), (c) combined translocations and deletions of 7q (17 cases), (d) combined translocation and deletion 7p (5 cases), and (e) translocation of chromosomes 7 without deletion 7p or 7q (3 cases). Deletions of all three FISH-screened regions were the most frequent, with heterogeneous breakpoints. The region 7p13.2 approximately p15.2 was most commonly deleted. Most of the deletions were cryptic, not detectable with conventional cytogenetics. Aberrations of chromosome 7 are associated with a very poor outcome; survival time in our cohort was short (median 7 months).

A substantial number of patients with early-stage colorectal cancer relapse from metastatic disease. Identification of these patients by genetic profiling of their primary tumours may allow more informed follow-up and tailored administration of adjuvant therapy. Primary tumours from 70 patients with early-stage and largely microsatellite-stable colorectal cancer were profiled using metaphase-based comparative genomic hybridization (CGH) and the aberrations confirmed independently in a subset of patients using microarray-based CGH. Of the 70 cancers, 61 were amenable to CGH, and follow-up data was available from 56 patients. Genomic aberrations were correlated with patients' survival using univariate, multivariate and Kaplan-Meier survival curves. Metastatic primary tumours exhibited more complex genomic aberrations than non-metastatic primary tumours. Loss of chromosome 4p was an independent prognostic factor in early-stage colorectal cancer using multivariate analysis (Hazard ratio, 9.6; 95% CI, 3.3-28; P = 0.0001). Loss of both chromosome arms 8p and 18q had a statistically significant negative effect on disease-free survival. Moreover, primary tumours with loss of both chromosomes 4 and 14q bestowed poorer prognosis than tumours with loss of any one of the two chromosomes (P<0.0001). Genetic profiling of primary tumours of patients with early-stage colorectal cancer is of significant value in identifying the subset of patients who may relapse with metastatic disease. Accordingly, the molecular genetic features of primary tumours should be considered in the mainstream management of patients with this specific stage of the disease.

Keratoacanthoma (KA) is a benign keratinocytic neoplasm that usually presents as a solitary nodule on sunexposed areas, develops within 6-8 weeks and spontaneously regresses after 3-6 months. KAs share features such as infiltration and cytological atypia with squamous cell carcinomas (SCCs). Furthermore, there are reports of KAs that have metastasized, invoking the question of whether or not KA is a variant of SCC. To date no reported criteria are sensitive enough to discriminate reliably between KA and SCC, and consequently there is a clinical need for discriminating markers. We screened fresh frozen material from 132 KAs and 37 SCCs for gross chromosomal aberrations by using comparative genomic hybridization (CGH). Forty-nine KAs (37.1%) and 31 SCCs (83.7%) showed genomic aberrations, indicating a higher degree of chromosomal instability in SCCs. Gains of chromosomal material from 1p, 14q, 16q, 20q, and losses from 4p were seen significantly more frequently in SCCs compared with KAs (P-values 0.0033, 0.0198, 0.0301, 0.0017, and 0.0070), whereas loss from 9p was seen significantly more frequently in KAs (P-value 0.0434). The patterns of recurrent aberrations were also different in the two types of neoplasms, pointing to different genetic mechanisms involved in their developments.

This study investigated the effects of exposing human embryonic stem cells (hESC) to 4oC and 25oC for extended durations of 24h and 48h respectively. Cell survivability after low temperature exposure was assessed through the MTT assay. The results showed that hESC survivability after exposure to 25oC and 4oC for 24h was 77.3 ± 4.8 % and 64.4 ± 4.4 % respectively (significantly different, P < 0.05). The corresponding survival rates after 48h exposure to 25oC and 4oC was 71.0 ± 0.5 % and 69.0 ± 2.3 % respectively (not significantly different, P > 0.05). Spontaneous differentiation of hESC after low temperature exposure was assessed by morphological observations under bright-field and phase-contrast microscopy, and by immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. hESC colonies were assigned into 3 grades according to their degree of spontaneous differentiation: (1) Grade A which was completely or mostly undifferentiated, (2) Grade B which was partially differentiated, and (3) Grade C which was mostly differentiated. In all low temperature exposed groups, about 95% of colonies remain undifferentiated (Grade A), which was not significantly different (P > 0.05) from the unexposed control group maintained at 37oC. Additionally, normal karyotype was maintained in all low temperature-exposed groups, as assessed by fluorescence in situ hybridization (FISH) of metaphase spreads with telomere and centromere-specific PNA probes. Further analysis with m-FISH showed that chromosomal translocations were absent in all experimental groups. Hence, hESC possess relatively high-tolerance to extended durations of low temperature exposure, which could have useful implications for the salvage of hESC culture during infrequent occurrences of incubator break-down and power failure.

The Moloney murine leukemia virus-transformed suspension cell line WMP2 is derived from wild mice (Mus musculus) of the WMP/WMP strain. These mice carry nine pairs of metacentric Robertsonian translocation chromosomes. As the chromosomes of the wild-type mouse are all acrocentric, metaphase spreads of the WMP2 cells seam to be highly suited for physical gene mapping. Here we studied the WMP2 line using spectral karyotyping (SKY) combined with new established mouse specific multicolor banding (mcb) probes for the chromosomes X, 3, 4, 6 and 18. SKY revealed that the WMP2 cell line developed further four derivative chromosomes. After application of mcb five previously unrecognizable intrachromosomal rearrangements with 9 breakpoints were detected for the studied chromosomes.

During aging, telomeres are gradually shortened, eventually leading to cellular senescence. By T/C-FISH (telomere/centromere-FISH), we investigated human telomere length differences on single chromosome arms of 205 individuals in different age groups and sexes. For all chromosome arms, we found a linear correlation between telomere length and donor age. Generally, males had shorter telomeres and higher attrition rates. Every chromosome arm had its individual age-specific telomere length and erosion pattern, resulting in an unexpected heterogeneity in chromosome-specific regression lines. This differential erosion pattern, however, does not seem to be accidental, since we found a correlation between average telomere length of single chromosome arms in newborns and their annual attrition rate. Apart from the above-mentioned sex-specific discrepancies, chromosome arm-specific telomere lengths were strikingly similar in men and women. This implies a mechanism that arm specifically regulates the telomere length independent of gender, thus leading to interchromosomal telomere variations.

Certain recurrent cytogenetic abnormalities are diagnostic of a specific neoplasm and may portend prognosis. As conventional cytogenetics may not reveal a neoplastic clone, and unfixed material for fluorescence in situ hybridization may be unavailable, performing fluorescence in situ hybridization on fixed tissues is diagnostically and prognostically valuable. Manual interpretation of fluorescence in situ hybridization signals may be difficult on paraffin-embedded tissue sections due to truncated nuclei. Therefore, we investigated the use of an automated image acquisition and analysis system (MetaSystems) for interpretation of fluorescence in situ hybridization signals in tissue sections from dual fusion translocation probes. Three probe sets were analyzed on archival specimens with a confirmed diagnosis of mantle cell lymphoma, follicular lymphoma or Burkitt lymphoma. 100% of mantle cell lymphomas (7/7) were positive for t(11;14), 91% of follicular lymphomas (10/11) for t(14;18) and 100% of Burkitt lymphomas (9/9) for t(8;14). Successful hybridization was achieved using various tissue fixatives and fluorescence in situ hybridization interpretation was blinded with respect to the underlying diagnosis. Based on these results, automated analysis of fluorescence in situ hybridization on fixed tissues is accurate and valuable in the evaluation of B-cell lymphoma, and may provide pertinent diagnostic and prognostic information.

Combined loss of heterozygosity (LOH) on 1p and 19q is reported in 50% to 90% of oligodendroglial tumors and has emerged as a strong and favorable prognostic factor. Fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) are the most widely used techniques. The aim of this study was to evaluate the reliability of FISH to predict LOH at 1p and 19q when performed on touch preparations from 40 oligodendroglial tumors, even if the majority of the nuclei showed chromosomal imbalance. PCR was used as the gold standard. The presence of none or one target signal was reported as FISH-LOH, whereas all other losses were defined as FISH-imbalance. The sum of nuclei with FISH-LOH and imbalance was calculated in each case (FISH-sum) and cut-off values were defined as the mean FISH-sum value in controls plus 3 standard deviations; 27.7% for 1p and 33.2% for 19q. These corresponded well with the optimal cut-off values for our data, calculated using the minimum error rate classification procedure (35.6% for 1p and 33.1% for 19q). Concurrent FISH and PCR results were encountered in 95% for 1p and 87.5% for 19q. FISH-sum was the best and simplest discriminating variable for correct classification of LOH status. Under these conditions, even a dominant population of nuclei showing FISH-imbalance represented an LOH status in the tumor cells. FISH on touch preparations is a quick and reliable method for 1p/19q testing, does not require normal DNA and can be easily performed in an immunohistochemistry unit.

Osteosarcoma (OS) is characterized by chromosomal instability and high copy number gene amplification. The breakage–fusion–bridge (BFB) cycle is a well-established mechanism of genome instability in tumors and in vitro models used to study the origins of complex chromosomal rearrangements and cancer genome amplification. To determine whether the BFB cycle could be increasing the de novo rate of formation of cytogenetic aberrations in OS, the frequency of anaphase bridge configurations and dicentric chromosomes in four OS cell lines was quantified. An increased level of anaphase bridges and dicentrics was observed in all the OS cell lines. There was also a strong association between the frequencies of anaphase bridges, dicentrics, centrosomal anomalies, and multipolar mitotic figures in all the OS cell lines, indicating a possible link in the mechanisms that led to the structural and numerical instabilities observed in OS. In summary, this study has provided strong support for the role of the BFB cycle in generating the extensive structural chromosome aberrations, as well as cell-to-cell cytogenetic variation observed in OS, thus conferring the genetic diversity for OS tumor progression.

Determination of HER2 status by fluorescence in situ hybridization (FISH) in breast carcinoma correlates well with response to targeted therapy and prognosis. However, manual time consuming methods and quantification aspects of the procedure may be challenging for some laboratories. We examined the feasibility of automating these components of the FISH assay using a tissue microarray (TMA-118 clinically annotated cases) and a series of 41 whole sections. An in situ hybridization automated staining workstation was used to automate a programmed overnight start, on line baking, deparaffinization, cell conditioning, protease digestion, and prehybridization buffer washing. Dual label probe/target codenaturation/hybridization and stringency washing were done off line. The HER2 and CEP17 spot counts were quantified, and the HER2/CEP17 ratio calculated, via an imaging workstation. Results were benchmarked against manual counts for whole sections, and bright field in situ hybridization [silver in situ hybridization (SISH)] for the TMA. Automated FISH results using whole sections correlated well with manual results: HER2/CEP17 ratio correlation coefficient r = 0.9154, r = 0.8380, P < 0.0001. Correlation between automated and manual TMA FISH results was also excellent, and disease-free survival was significantly shorter (P < 0.001) for the HER2 amplified cases. Automation of the laborious manual prehybridization and image quantification components of FISH using directly labeled probes is feasible. Operational gains and enhanced consistency are inherent in this automated approach to HER2 clinical FISH testing.

SUMMARY: BACKGROUND: Secretory carcinoma (SC) of the breast is a rare and indolent tumor. Although originally described in children, it is now known to occur in adults of both sexes. Recently, the tumor was associated with the ETV6-NTRK3 gene translocation. CASE PRESENTATION: A 52-year-old male was diagnosed with secretory breast carcinoma and underwent a modified radical mastectomy. At 18 months the tumor recurred at the chest wall and the patient developed lung metastases. He was treated concurrently with radiation and chemotherapy without response. His tumor showed the ETV6-NTRK3 translocation as demonstrated by fluorescent in situ hybridization (FISH). CONCLUSION: SC is a rare slow-growing tumor best treated surgically. There are insufficient data to support the use of adjuvant radiation or chemotherapy. Its association with the ETV6-NTRK3 fusion gene gives some clues for the better understanding of this neoplasm and eventually, the development of specific therapies.

OBJECTIVE: To determine whether it is possible to stratify patients with superficial bladder cancer into low- and high-risk groups for tumour recurrence/progression based on the chromosomal pattern detected by fluorescence in situ hybridization (FISH) in one urine cytology specimen used for follow-up testing. PATIENTS AND METHODS: Voided urine samples from 47 consecutive patients with urinary tract neoplasms (13 with no history of urothelial malignancy and 34 under follow-up after complete transurethral resection of superficial urothelial carcinoma of the bladder) were evaluated by liquid-based cytology (ThinPrep(R), CYTYC Corp., Boxborough, MA, USA) and UroVysion FISH (Vysis-Abbott, Downers Grove, IL). RESULTS: Of the 34 patients under surveillance, the UroVysion test was negative in four, 17 had loss of 9p21 sequences either alone or combined with low-frequency trisomy/ies or tetrasomy/ies of chromosomes 3, 7 and 17 in single cells (low-risk FISH), and 13 also had complex aneusomies of the remaining chromosomes (high-risk FISH). One of the four FISH-negative neoplasms, four of the 17 low-risk FISH cases and five of the 11 informative high-risk FISH-positive patients developed recurrence. Progression occurred only in patients with high-risk FISH results, showing high-frequency complex chromosomal polysomies (four of 11). CONCLUSION: The results from this pilot study indicate that the UroVysion FISH test may help to individually assess the clinical behaviour of superficial bladder cancer, based on the chromosomal pattern of exfoliated tumour cells in follow-up urinary cytology. It might be of use to identify those patients likely to progress at earlier and curable stages of disease, and lengthen the surveillance period in those with persistent or recurrent low-risk disease.

OBJECTIVE: A variety of methods have been used to select and identify fetal cells from maternal blood. In this study, a commonly used 3-step selection method is compared with selection directly from whole blood. Identification of fetal origin by XY FISH of male cells was also evaluated. METHODS: Maternal blood was drawn either before invasive chorion villus sampling (pre-CVS) or after (post-CVS) from women carrying a male fetus. Fetal cells were isolated either by density gradient centrifugation succeeded by CD45/CD14 depletion and CD71-positive selection from CD45/CD14-negative cells, or by CD71-positive selection directly from whole blood. The true origin of fetal cells recovered by the two methods was established by two rounds of XY chromosome FISH in reverse colors, in some instances combined with anti-zeta (zeta) or anti-zeta/anti-gamma (gamma) antibody staining. RESULTS: In blood samples taken post-CVS and enriched by CD71 selection directly from whole blood, fetal cells were identified with a frequency that was almost four orders of magnitude higher than in post-CVS samples enriched by the 3-step method. In blood samples taken pre-CVS and enriched by the 3-step procedure, no fetal cells were identified by reverse color FISH in 371 ml of blood. In similar samples enriched by CD71 selection on whole blood, two fetal cells were identified in 27 ml of blood. Rehybridization with X and Y chromosome probes with reverse colors was necessary to exclude false Y chromosome signals. Not all fetal cells identified by the presence of a true Y chromosome signal stained with anti-zeta antibody. CONCLUSIONS: Selection of fetal NRBCs from maternal blood by CD71-positive selection directly from whole blood is superior to density gradient centrifugation succeeded by CD45/CD14 depletion and CD71 selection of CD45/CD14-negative cells. Combining two markers for fetal origin is recommended for unambiguously identifying a cell as fetal.

OBJECTIVE: To study chromosome analysis by comparative genomic hybridization (CGH) compared with the conventional technique in early amniocentesis. MATERIAL AND METHOD: Cross-sectional descriptive study design was performed in 32 singleton pregnant women with gestational age between 12-15 weeks. Transabdominal amniocentesis was carried out under ultrasound guidance. The amniotic fluid samples were simultaneously investigated using CGH and the conventional cytogenetics study as a gold standard. RESULTS: Amniocentesis were done for advanced maternal age in all cases. The mean maternal age was 35.8 years (35-42 years). The mean gestational age was 13.7 weeks (12-15 weeks). The chromosome analysis by CGH technique of uncultured amniocyte showed 17 normal female chromosomes (53.1%) and 15 normal male chromosomes (46.9%). This finding was the same as the conventional cytogenetics method. The mean duration of the CGH method was 6 days and that of the conventional cytogenetics method was 13.7 days (10-23 days). CONCLUSION: The CGH technique is a reliable technique for a rapid prenatal diagnosis of chromosome study in early gestation.

PURPOSE: Uveal melanoma is a highly malignant disease with a mortality rate of 50% at 10 to 15 years. Previous studies have shown that chromosomal changes are associated with decreased survival of the patient. However, in these studies the small number of tumors analyzed did not allow robust statistical analysis. In the present study, the independent numerical changes in chromosomes 1, 3, 6, and 8 on disease-free survival (DFS) was assessed in a large series of patients with uveal melanoma. METHODS: One hundred twenty tumors from patients with uveal melanoma were analyzed for numerical changes in chromosomes 1, 3, 6, and 8, with cytogenetic analysis, fluorescent in situ hybridization, and/or comparative genomic hybridization. Data were correlated with disease outcome in univariate and multivariate analyses, by Kaplan-Meier and Cox regression analyses. RESULTS: At a mean follow-up time of 45 months, 42 patients had died or had metastatic disease. In the univariate analysis, loss of chromosome 3, gain of 8q, largest tumor diameter, or the presence of epithelioid cells was associated with a decreased DFS. In the multivariate analysis, the effect of monosomy 3 on survival was largely modified by changes in 1p36. Regarding all chromosomal changes, only the concurrent loss of the short arm of chromosome 1 and all of chromosome 3 was an independent prognostic parameter for disease-free survival (P < 0.001). CONCLUSIONS: In uveal melanoma, concurrent loss of the short arm of chromosome 1 and all of chromosome 3 is an independent predictor of decreased DFS.

<p>Recurrent chromosome breakpoints in tumour cells may point to cancer genes, but not many have been molecularly characterised. We have used multicolour-banding fluorescence in situ hybridisation (mbanding-FISH) on breast tumour cell lines to identify regions of chromosome break created by inversions, duplications, insertions and translocations on chromosomes 1, 5, 8, 12 and 17. We delineate a total of 136 regions of break, some of them occurring with high frequency. We further describe two examples of dual-colour FISH characterisation of breakpoints, which target the 1p36 and 5p11-12 regions. Both breaks involve genes whose function is unknown to date. The mbanding-FISH strategy constitutes an efficient first step in the search for potential cancer genes.</p>