Fluorescence In Situ Hybridization Assay Research Articles

Diagnostic Value of Fluorescence in Situ Hybridization Assay in Malignant Mesothelioma: A Meta-analysis

The diagnosis of malignant mesothelioma (MM) remains a clinical challenge and the fluorescence in situ hybridization (FISH) assay has been reported to be one promising tool. The present meta-analysis aimed to establish the overall diagnostic accuracy of FISH for diagnosing MM. After a systematic review of English language studies, the sensitivity, specificity and other measures of accuracy of FISH in the diagnosis of MM were pooled using random-effects models. Summary receiver operating characteristic curves were applied to summarize overall test performance. Nine studies met our inclusion criteria, the pooled sensitivity and specificity for FISH for diagnosing MM being 0.72 (95% CI 0.67-0.76) and 1.00 (95% CI 0.98-1.00), respectively. The positive likelihood ratio was 34.5 (95% CI 14.5-82.10), the negative likelihood ratio was 0.24 (95% CI 0.16-0.36), and the diagnostic odds ratio was 204.9 (95% CI 76.8-546.6), the area under the curve being 0.99. Our data suggest that the FISH assay is likely to be a useful diagnostic tool for confirming MM. However, considering the limited studies and patients included, further large scale studies are needed to confirm these findings.

Identification of a Novel, Recurrent SLC44A1‐PRKCA Fusion in Papillary Glioneuronal Tumor

Mixed neuronal-glial tumors are rare and challenging to subclassify. One recently recognized variant, papillary glioneuronal tumor (PGNT), is characterized by prominent pseudopapillary structures and glioneuronal elements. We identified a novel translocation, t(9;17)(q31;q24), as the sole karyotypic anomaly in two PGNTs. A fluorescence in situ hybridization (FISH)-based positional cloning strategy revealed SLC44A1, a member of the choline transporter-like protein family, and PRKCA, a protein kinase C family member of serine/threonine-specific protein kinases, as the 9q31 and 17q24 breakpoint candidate genes, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis using a forward primer from SLC44A1 exon 5 and a reverse primer from PRKCA exon 10 confirmed the presence of a SLC44A1-PRKCA fusion product in both tumors. Sequencing of each chimeric transcript uncovered an identical fusion cDNA junction occurring between SLC44A1 exon 15 and PRKCA exon 9. A dual-color breakpoint-spanning probe set custom-designed for interphase cell recognition of the translocation event identified the fusion in a third PGNT. These results suggest that the t(9;17)(q31;q24) with the resultant novel fusion oncogene SLC44A1-PRKCA is the defining molecular feature of PGNT that may be responsible for its pathogenesis. The FISH and RT-PCR assays developed in this study can serve as valuable diagnostic adjuncts for this rare disease entity.

Multiparametric molecular characterization of pulmonary sarcomatoid carcinoma reveals a nonrandom amplification of anaplastic lymphoma kinase (ALK) gene

BackgroundGenetic alterations for targeting therapy are largely unexplored issues in pulmonary sarcomatoid carcinoma (PSC), a life-threatening tumor subset. MethodsEGFR, HER2, KRAS, p53, CTNNB1, BRAF and PIK3CA mutations were assessed by direct sequencing, ALK, EGFR and HER2 gene status by fluorescence in situ hybridization (FISH), and ALK protein expression by immunohistochemistry (IHC) in 20 pleomorphic carcinomas (PLC), two pulmonary blastomas (PB) and one carcinosarcoma (CS). Surgical specimens and, in case of positivity, the corresponding preoperative biopsies were analyzed. Furthermore, 51 consecutive metastatic lung adenocarcinomas (MELAD) were used as controls for FISH and IHC assays of ALK gene. ResultsWhile no rearrangements of ALK were detected in PSC, relevant amplification was identified 5/23 (22%) surgical specimens and paired biopsies (four PLC and one PB, two females and three males, four current and one never smoker, aged 30–83 years). Considering tumor heterogeneity, the percentage of ALK amplified tumor cells ranged from 11% to 43%, with a mean gene copy gain (GCG±SD) of 6.9±0.8 and no signal differences between the epithelial (6.5±0.9) and the sarcoma-like components (6.8±0.9) of tumors. In the remaining 18 non-amplified PSC, the relevant value was 2.9±0.5 in 1–80% tumor cells (p<0.001). ALK amplification was closely associated with chromosome 7 (EGFR) or 17 (HER2) polysomy (p<0.001). Out of 51 MELAD, 10 were ALK-rearranged (p=0.026) and only one amplified (p=0.009). No amplified tumors, either PSC or MELAD, expressed the relevant protein by IHC, while the 10 ALK-rearranged MELAD were strongly positive. TP53, KRAS and CTNNB1 mutations accounted for 30%, 22%, and 4% of cases, respectively, with no significant relationship with ALK amplification. No mutations for EGFR, HER2, BRAF or PIK3CA gene were observed. ConclusionALK gene amplification is a nonrandom and clonally related event in a subset of PSC, but its biologic rationale deserves further investigation. KRAS mutation could represent a novel tool for therapy of such so deadly tumors with MEK inhibitors.

Novel, gross chromosomal alterations involving PTEN cooperate with allelic loss in prostate cancer

AbstractThere is increasing evidence that multiple chromosomal rearrangements occur in prostate cancer. PTEN loss is considered to be a key event in prostate carcinogenesis but the mechanisms of loss remain to be fully elucidated. We hypothesised that gross rearrangements may exist that cause disruption of the PTEN gene in the absence of genomic deletion. We therefore designed a novel fluorescence in situ hybridisation (FISH) assay with probes overlying regions 3′ and 5′ of PTEN and a third probe overlying the gene. We aimed to identify both genomic deletions and gross rearrangements of PTEN that would be overlooked by previously reported single-probe FISH assays. We proceeded to evaluate a tissue microarray with radical prostatectomy and trans-urethral resection of the prostate specimens from 187 patients. We identified PTEN genomic loss in 45/150 (30%) radical prostatectomy patients and 16/37 (43%) trans-urethral resection of the prostate patients. Importantly, our assay detected novel chromosomal alterations in the PTEN gene (characterised by splitting of FISH signals) in 13 tumours (6.9% of all prostate cancers; 21% of PTEN-lost cancers). All PTEN-rearranged tumours had genomic loss at the other allele and had no expression of PTEN by immunohistochemistry. PTEN-rearranged tumours were significantly more likely to have an underlying ERG rearrangement. Our assay differentiated loss of the probe overlying PTEN in isolation or in combination with either one of or both the probes overlying the 3′ and 5′ regions. This gave an indication of the size of genomic loss and we observed considerable inter-tumoural heterogeneity in the extent of genomic loss in PTEN-lost tumours. In summary, gross rearrangements of the PTEN locus occur in prostate cancer and can be detected by a ‘break-apart' FISH assay. This observation could explain the absence of PTEN protein expression in a subgroup of tumours previously classified as having heterozygous genomic loss using single-probe traditional FISH assays.

A Highly Specific and Discriminatory FISH Assay for Distinguishing Between Benign and Malignant Melanocytic Neoplasms

The diagnosis of certain melanocytic proliferations remains one of the most challenging areas in pathology. In recent times, fluorescence in situ hybridization (FISH) has emerged as a promising diagnostic aid to conventional microscopy. We previously showed that a 4-probe FISH assay targeting 6p25 (RREB1), 6q23 (MYB), Cep6 (centromere 6), and 11q13 (CCND1) could discriminate between histologically unequivocal melanomas and benign nevi with a sensitivity of 86.7% and specificity of 95.4%. However, the sensitivity of the assay is approximately 70% in melanomas with spitzoid morphology. Furthermore, differentiating true gains from tetraploidy may cause difficulties in interpretation by inexperienced examiners. Here we refine the current probe set to better target spitzoid melanomas and more easily distinguish cells with imbalanced copy number aberrations from tetraploid cells. Using FISH data from 3 training sets of 322 tumors, including 152 melanomas and 170 nevi, we identified 9p21, 6p25, 11q13, and 8q24 as a probe set with improved discriminatory power in differentiating melanomas from nevi. In a validation set of 51 melanomas and 51 nevi this probe set had a sensitivity of 94% and specificity of 98%, compared with the original probe set that had a sensitivity of 75% and specificity of 96% in the same validation cohort. We propose that by incorporating 9p21 into the 4-probe FISH assay, with a new diagnostic algorithm, this new probe set would have improved discriminatory power in melanocytic neoplasms and improved sensitivity for detecting spitzoid melanomas, as demonstrated by our previous studies.

P3.05 A New Non-Toxic Hybridization Buffer Reduces the Fish Assay Time From 16-20 Hours to 3½ Hours Without Compromising the Quality

ABSTRACT Fluorescence in-situ hybridization (FISH) is a powerful technique for direct visualization and quantitative determination of gene amplifications, deletions and translocations in human cancer cells. It is recognized as a reliable and reproducible method, and FISH assays are often referred to as 'Gold Standard' for determining the patient status with predictive tests for HER2 and ALK. But FISH is also known to have characteristics which limit a more widespread use, including the long assay time (staining and scoring), requirement for a fluorescence microscope and complexity involved in handling small volumes and toxic reagents. Dako has addressed the toxicity and long hybridization time by introducing a new hybridization buffer. The new non-toxic hybridization buffer (IQFISH buffer), which allows for significant reduction in hybridization time, brings the total assay time for a FISH assay down from 16-20 hours to just 3½ hours without compromising the quality. The new Dako HER2 IQFISH pharmDx™ (assay time 3½ hours) has been compared with the traditional Dako HER2 FISH pharmDx™ Kit (assay time 16-20 hours) in a comparative study on 78 breast tissue specimens of human breast carcinoma ( Fig 1 ). Cross tabulation of HER2 status obtained by the two assays gave an overall agreement of 98.7% (95% CI: 94.2%-99.9%). Disagreement between the two assays was observed for one specimen that most likely was a heterogeneous tissue with a very small area of amplification. A similar comparative study on 79 gastric adenocarcinoma specimens showed an overall agreement of 98.7% (95% CI: 94.2%-99.9%). The disconcordant sample was a heterogeneous tissue with a very small area of amplification. A third concordance study on 80 breast cancer tissues compared TOP2A IQFISH pharmDx™ with the traditional TOP2A FISH pharmDx™ Kit ( Fig 2 ). A 100% overall agreement (95% CI: 96.9%-100%) was found. Conclusion: We have developed new non-toxic hybridization buffer that by reduction of the hybridization time from 16 to 2 hours allows for a ½ day FISH protocol. The IQFISH buffer has been validated for HER2 on breast and gastric cancer and for TOP2A on breast cancer. Both assays showed a very high agreement with the traditional 2 day FISH procedure. Download : Download full-size image Fig. 1 . Agreement between the new Dako HER2 IQFISH pharmDx™ and the traditional Dako HER2 FISH pharmDx™ Kit. Download : Download full-size image Fig. 2 . Agreement between the new Dako TOP2A IQFISH pharmDx™ and the traditional Dako TOP2A FISH pharmDx™ Kit.

Improved Method of Detecting the ERG Gene Rearrangement in Prostate Cancer Using Combined Dual-Color Chromogenic and Silver In Situ Hybridization

The recently detected TMPRSS2-ERG fusion gene was revealed as a recurrent and prevalent prostate cancer (PCa)-specific event, potentially qualifying it for clinical use. To detect this alteration, fluorescence in situ hybridization (FISH) is the method of choice. However, FISH has some disadvantages for widespread adoption in clinical practice. Subsequently, chromogenic in situ hybridization, which uses organic chromogens, and enzymatic metallography silver in situ hybridization have emerged as promising bright-field alternatives. Compared with chromogenic in situ hybridization, silver in situ hybridization signals are very distinct and superior with regard to signal clarity and resolution, but the method excludes multicolor protocols. Based on the ERG break-apart FISH assay, we established a dual-color ERG break-apart assay using combined chromogenic in situ hybridization and silver in situ hybridization (CS-ISH) and compared these results with those obtained by FISH. We assessed 178 PCa and 10 benign specimens for their ERG rearrangement status by applying dual-color FISH and CS-ISH ERG break-apart assays to consecutive sections. We observed a highly significant concordance (97.7%) between FISH- and CS-ISH-based results (Pearson's correlation coefficient = 0.955, P < 0.001). Our findings demonstrate that the ERG rearrangement status can reliably be assessed by CS-ISH. Further, the CS-ISH technique combines the accuracy and precision of FISH with the ease of bright-field microscopy. This tool allows a much broader spectrum of applications in which to study the biological role and clinical use of ERG rearrangements in PCa.

Clinical experience with atypical spitzoid tumors in patients younger than age 18: Does fluorescence in situ hybridization predict lymph node metastasis?

8586 Background: Determining the malignant potential of atypical spitzoid melanocytic proliferations can be diagnostically challenging, and many patients are therefore managed as if they had melanoma. However, few studies focus specifically on atypical spitzoid tumors (AST). Further, cytogenetic analysis using fluorescence in situ hybridization (FISH) has been used to determine malignant potential. We reviewed our institutional experience to determine staging and clinical outcomes, and the correlation between FISH findings and regional nodal positivity in patients with AST. Methods: With IRB approval, we retrospectively reviewed all patients aged &lt;18 years treated for AST from 1981-2011 to determine staging variables, outcome, and the results of 4-probe FISH assay. A total of 44 patients with a median age of 11.5 years were identified. All pathology was re-reviewed by a single dermatopathologist. Staging was based on 2010 AJCC criteria. Correlations were determined using either the Pearson or Spearman correlation coefficient. Results: Median lesion thickness was 2.8 mm (range: 0.55-12) and eight (18%) lesions met spitzoid melanoma criteria. Median followup was 4.1 years for all patients (5.5 years for spitzoid melanomas). Thirty-nine patients (89%) underwent sentinel lymph node biopsies (SLNB) and 15 (38%) were positive. Lymph node (LN) positivity correlated with tumor thickness (p=0.002), stage (p=0.001), and mitotic rate (p=0.05). FISH was available for 17/39 patients who had SLNB; sensitivity and specificity for LN metastases were 50% and 54%, respectively. Of 15 patients with a positive SLNB, 12 underwent completion LN dissection, three of which were positive for 1, 2, and 3 additional nodes, respectively. There were no recurrences or disease-related deaths. Conclusions: Traditional indicators of thickness and stage predicted nodal involvement in pediatric patients with AST. However, FISH results did not predict nodal status and should not be used to guide management. With 100% disease specific survival and no recurrences, AST should be considered a separate entity from melanoma and complete excision may be sufficient therapy.

Prognosis significance of HER2/neu overexpression and amplification in patients with curatively resected gastric cancer.

e14539 Background: HER-2/neu targeted therapy has been successfully used in advanced gastric cancer, but the role of HER-2/neu inprognosis of gastric cancer is not yet clear. To investigate the correlation between HER-2/neu expression and amplification and their associations with clinicopathologic outcomes and prognosis in patients with curatively resected gastric cancer. Methods: We constructed tissue microarray blocks containing &gt;70% of gastric cancer tissue and matched adjacent normal gastric tissue for 229 patients. Expression of the HER-2/neu protein in these specimens was analyzed using immunohistochemical (IHC) staining. Amplification of HER-2/neu was also analyzed for the same samples using fluorescence in situ hybridization (FISH). Kaplan Myers curve and Cox proportional hazards model were used to assess the survival. Results: Of the 229 gastric cancer samples, 14.85% were positive for HER-2/neu protein expression that was closely correlated to the Lauren type, degree of differentiation, tumor size, lymph node metastasis and overall survival. Overall, 11.79% of the 229 gastric cancers were positive for amplification of HER-2/neu, which was also closely correlated to the Lauren type, degree of differentiation, tumor size and overall survival. Moreover, Cox proportional hazards model showed that positive expression of HER-2/neu, Lauren type, tumor size, vessel invasion, TNM stage were independent prognostic factor. Both IHC and FISH assays provide an important prognostic factor for gastric cancer with a concordance rate of 96.9%, but IHC was a stronger independent prognostic factor. Conclusions: Expression of the HER-2/neu protein, compared with its gene amplification, was an independent and more sensitive prognostic factor for curatively resected gastric cancer patients.

Fluorescence In Situ Hybridization (FISH) Analysis of Melanocytic Nevi and Melanomas

A 4-color fluorescence in situ hybridization (FISH) assay, using probes to chromosomes 11q, 6p, 6q, and 6 cent, has recently been proposed as an ancillary tool for the diagnosis of melanoma. The authors report herein their experience with this assay. To determine the sensitivity and specificity of the assay for histopathologically unequivocal cases, they analyzed 50 melanocytic nevi, 50 primary melanomas, and 15 metastatic melanomas. Of 50 melanocytic nevi, 47 were FISH negative on initial readout (test sensitivity, 94%); 49 were FISH negative after correction for tetraploidy (test specificity, 98%). Of 50 primary melanomas, 41 were FISH positive (test sensitivity, 82%). Of 15 metastatic lesions, 13 were FISH positive (test sensitivity, 85%). Of the 9 FISH-negative melanomas, 6 metastasized. The tumors of the 5 patients who had survived thick primary melanoma for more than 5 years without recurrence were all FISH positive. Half of the patients whose primary melanoma was tested by FISH had undergone sentinel lymph node (SLN) biopsy. When the authors compared the FISH results of those 25 melanomas with the SLN status, no statistically significant correlation was found. These findings document limitations of the current FISH assay. A rare nevus may be FISH positive. Some primary metastasizing melanomas are FISH negative. Even metastatic melanomas can be FISH negative. Awareness of the limitations in test sensitivity and specificity of the FISH assay is important to avoid an erroneous diagnosis by overreliance on cytogenetic findings. Correlation with clinical and histopathological findings is paramount for accurate diagnosis.

Twenty-four chromosome FISH in human IVF embryos reveals patterns of post-zygotic chromosome segregation and nuclear organisation

Fluorescence in situ hybridisation (FISH) was first applied on in vitro fertilisation (IVF) embryos for the preimplantation genetic diagnosis of sex, then chromosome translocations and later for chromosome copy number (PGS). Because of the controversy surrounding PGS diagnostically, it has been replaced by array-based approaches; however, FISH remains a powerful tool for investigating mechanisms of both post-zygotic segregation error and nuclear organisation, especially if most or all of the chromosomes in the karyotype can be analysed. The purpose of this study was to develop and apply a 24 chromosome FISH assay to investigate chromosome-specific rates of gain and loss, nuclear organisation patterns and the veracity of the original PGS result in days 5-6 human embryos. Analysis of 17 embryos by this newly developed approach gave strong signals for all chromosomes; it revealed chromosome copy number for each human chromosome per cell for each embryo and the nuclear address of the (mostly centromeric) loci probed. As all embryos were surplus to IVF requirements for both transfer and freezing (and many had an abnormal PGS indication) expected high levels of chromosome abnormalities were seen and no single nucleus displayed a normal complement; all were mosaic. Certain patterns emerged, however, namely that chromosome loss was more common than gain and apparent mitotic non-disjunction. Moreover, the centromeric probes tended preferentially to occupy the nuclear centre. Where we had a prior day 3 biopsy PGS result, it was confirmed, in part, by 24 colour FISH in most but not all cases.

Abstract 662: Effect of tobacco smoking on the risks of childhood acute lymphocytic and myeloid leukemias, by phenotypic and cytogenetic subtype

Abstract Background: Tobacco smoke contains several myelotropic carcinogens (i.e., benzene, 1,3 butadiene and formaldehyde) and is strongly suspected of damaging pluripotential hematopoietic stem cells. Objective: To examine the role of tobacco smoking in the etiology of childhood acute lymphocytic leukemia (ALL) and myeloid leukemia (AML), defined by phenotypic feature and type of chromosomal aberrations at diagnosis, such as translocation t(12:21) or high hyperdiploidy (51+ chromosomes). Method: Children (0-14 years old) newly diagnosed with ALL (n=839) and AML (n=145) were enrolled in a case-control study in California (1995-2008). Population-based controls (n=1226) were matched to cases on age, sex, Hispanic status, and maternal race. Parents provided information on exposure to tobacco smoking before and after their child's birth. Conventional karyotype and fluorescence in situ hybridization (FISH) data were abstracted from the medical reports. Additional FISH assays using pre-treatment bone marrow specimens were conducted to complete cytogenetic classification. Unconditional logistic regression analyses were used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for developing leukemia, using binary variable for ever/never smoked (ORbinary) and continuous variable for number of cigarettes smoked per day (ORcig). All models included the matching factors and household income. Results: In univariate analyses, elevated risks of B-cell ALL (n=722) were associated with pre-conception paternal smoking (“PCPS”) (ORcig= 1.02; 95% CI:1.00-1.03; p=0.04) and child's passive smoking (“CPS”) after birth (ORbinary=1.32; 95% CI:1.21-3.50; p=0.02). Analyses using mutually exclusive exposure variables (i.e., only “PCPS,” only “CPS,” and both “PCPS-CPS,” compared to no smoking) showed that the elevated risk of B-cell ALL was limited to children exposed to both “PCPS-CPS” (OR=1.50, 95% CI:1.10-2.04). The magnitude of this association increased for 133 B-cell ALL cases harboring chromosomal translocation t(12;21) (OR=2.37; 95% CI:1.41-4.01). Weaker associations were seen between “PCPS” and “CPS” and B-cell ALL harboring other chromosome translocations (n=82) or deletions (n=63). Tobacco smoking did not appear to be associated with hyperdiploid B-cell ALL (n=228) and T-cell ALL (n=70). Child's passive smoking conferred a 1.5-fold increased risk of AML (95% CI:0.98-2.26; p=0.06), with a higher risk observed for those with chromosome aberrations linked to a “favorable” prognosis (ORbinary=1.94; p=0.07; n=80). No excess risks were seen with maternal smoking during pregnancy, after accounting for other sources of smoking. Conclusions: Exposure to tobacco smoking is an important predictor of childhood ALL, and possibly AML. The observed risks contrast by phenotypic and cytogenetic subtype, providing insights in the etiology of the disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 662. doi:1538-7445.AM2012-662

Correlation Between Histologic Assessment and Fluorescence In Situ Hybridization Using MelanoSITE in Evaluation of Histologically Ambiguous Melanocytic Lesions

The 4-probe, multicolor, fluorescence in situ hybridization (FISH) panel targeting chromosomes 6 and 11 has shown promising sensitivity and specificity in distinguishing between benign nevi and malignant melanoma. Only a few studies have assessed the potential utility of FISH in classification of histologically ambiguous melanocytic lesions. In the United States, this assay is exclusively licensed to NeoGenomics Laboratories (Irvine, California), which provides the technical component and has developed an innovative service (MelanoSITE) allowing pathologists to interpret FISH results using a dedicated Web portal. Thus far, use of MelanoSITE as a diagnostic adjunct in the diagnosis of melanocytic lesions has not, to our knowledge, been reported in the literature. To analyze 1.5 years of experience with the MelanoSITE melanoma FISH assay in the evaluation of histologically ambiguous lesions in the context of second opinion and routine dermatopathology practice. A prospective histologic/FISH correlation study of 140 cases. Twenty-seven percent of abnormal FISH results were false-positive results because of tetraploidy. After correcting for known false-positive results, all lesions considered atypical nevi showed normal FISH signals. Abnormal FISH signals were reported in 30% of lesions considered histologically borderline and in 48% of lesions in which a diagnosis of melanoma was favored. Four-probe, multicolor FISH results for melanoma correlate with the microscopic assessments of histologically ambiguous lesions. Pathologists using MelanoSITE must be aware of the high rate of false-positive results from tetraploidy.

Same‐day prenatal diagnosis of common chromosomal aneuploidies using microfluidics‐fluorescence in situ hybridization

Rapid molecular prenatal diagnostic methods, such as fluorescence in situ hybridization (FISH), quantitative fluorescence-PCR, and multiplex ligation-dependent probe amplification, can detect common fetal aneuploidies within 24 to 48 h. However, specific diagnosis or aneuploidy exclusion should be ideally available within the same day as fetal sampling to alleviate parental anxiety. Microfluidic technologies integrate different steps into a microchip, saving time and costs. We have developed a cost-effective, same-day prenatal diagnostic FISH assay using microfluidics. Amniotic fluids (1-4 mL from 40 pregnant women at 15-22 weeks of gestation) were fixed with Carnoy's before loading into the microchannels of a microfluidic FISH-integrated nanostructured device. The glass slides were coated with nanostructured titanium dioxide to facilitate cell adhesion. Pretreatment and hybridization were performed within the microchannels. Fifty nuclei were counted by two independent analysts, and all results were validated with their respective karyotypes. Of the 40 samples, we found three cases of fetal aneuploidies (trisomies 13, 18, and 21), whereas the remaining 37 cases were normal. Results were concordant with their karyotypes and ready to be released within 3 h of sample receipt. Microfluidic FISH, using 20-fold less than the recommended amount of probe, is a cost-effective method to diagnose common fetal aneuploidies within the same day of fetal sampling.

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei(1). The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis(2). Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene(3-6), indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application(7). Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)(8). The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)(9). Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers(8,10).

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

Researchers find discordance between standard human epidermal growth factor receptor 2 (HER2) testing and HER2 status reported on Oncotype DX

Researchers find discordance between standard human epidermal growth factor receptor 2 (HER2) testing and HER2 status reported on Onco<i>type</i> DX

Dimethyl formamide-free, urea-NaCl fluorescence in situ hybridization assay for Staphylococcus aureus

To test the feasibility of identifying Staphylococcus aureus with a fluorescence in situ hybridization (FISH) assay that uses a single hot-plate and urea-NaCl reagents. Slides spotted with S. aureus and treated with methanol and lysozyme were incubated with urea-NaCl reagents on a hot-plate with a precise temperature control and identified with specific DNA probes. Staphylococcus aureus was detected and differentiated from Staphylococcus epidermidis in 1 h with a novel FISH method that used a single hot-plate and in the absence of dimethyl formamide. A rapid hot-plate FISH assay with urea-NaCl and without toxic dimethyl formamide might be useful if FISH is run infrequently or where resources are limited.

Fluorescence in situ hybridization assay detects upper urinary tract transitional cell carcinoma in patients with asymptomatic hematuria and negative urine cytology

We evaluated the performance of a multiprobe FISH (fluorescence in situ hybridization) assay for noninvasive detection of upper urinary tract transitional cell carcinoma (UUT-TCC) in patients with asymptomatic hematuria and negative urine cytology. Voided urine samples from 285 patients with asymptomatic hematuria and negative urine cytology were prospectively analyzed by FISH technique. FISH assays were performed to detect chromosomal changes frequently associated with TCC, including aneuploidy of chromosomes 3, 7 and 17, and loss of the 9p21 locus. Eleven (3.9%) had a positive FISH result. Of the 11 patients, nine (81.8%) were found to have a TCC of the upper urinary tract, while no patients with negative FISH findings were found to have UUT-TCC. In this selected cohort, the sensitivity and specificity of FISH for the detection of UUT-TCC was 100% and 99.3%, respectively. Our preliminary data suggest that the clinical utility of FISH assay of chromosomes 3, 7, 9, and 17 as a noninvasive ancillary test for the diagnosis of UUT-TCC in a selected patient population with asymptomatic hematuria and negative urine cytology and by significant high sensitivity and specificity may be a reliable diagnostic approach for early detection of UUT-TCC patients. Further larger prospective and multicenter trials are needed to confirm our results.

Validation of Fluorescence In Situ Hybridization Using an Analyte-Specific Reagent for Detection of Abnormalities Involving the Mixed Lineage Leukemia Gene

Fluorescence in situ hybridization (FISH) is a molecular cytogenetic assay that is commonly used in laboratory medicine. Most FISH assays are not approved by the US Food and Drug Administration but instead are laboratory-developed tests that use analyte-specific reagents. Although several guidelines exist for validation of FISH assays, few specific examples of FISH test validations are available in the literature. To provide an example of how a FISH assay, using an analyte-specific reagent probe, may be validated in a clinical laboratory. We describe the approach used by an individual laboratory for validation of a FISH assay for mixed lineage leukemia (MLL) gene. Specific validation data are provided illustrating how initial assay performance characteristics in a FISH assay for MLL may be established. Protocols for initial validation of FISH assays may vary between laboratories. However, all laboratories must establish several defined performance specifications prior to implementation of FISH assays for clinical use. We describe an approach used for assessing performance specifications and validation of an analyte-specific reagent FISH assay using probes for MLL rearrangement in interphase nuclei.