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Fluorescent in Situ Hybridization (FISH): "A type of IN SITU HYBRIDIZATION in which target sequences are stained with fluorescent dye so their location and size can be determined using fluorescence microscopy. This staining is sufficiently distinct that the hybridization signal can be seen both in metaphase spreads and in interphase nuclei." (Source: MeSH 2013)

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FISH Resources
Recent Publications: FISH

FISH Resources (2 links)

Recent Publications: FISH

Mizuno Y, Chinen Y, Tsukamoto T, et al.
A novel method of amplified fluorescent in situ hybridization for detection of chromosomal microdeletions in B cell lymphoma.
Int J Hematol. 2019; 109(5):593-602 [PubMed] Related Publications
Chromosomal microdeletions frequently cause loss of prognostically relevant tumor suppressor genes in hematologic malignancies; however, detection of minute deletions by conventional methods for chromosomal analysis, such as G-banding and fluorescence in situ hybridization (FISH), is difficult due to their low resolution. Here, we describe a new diagnostic modality that enables detection of chromosomal microdeletions, using CDKN2A gene deletion in B cell lymphomas (BCLs) as an example. In this method, which we refer to as amplified-FISH (AM-FISH), a 31-kb fluorescein isothiocyanate (FITC)-conjugated DNA probe encoding only CDKN2A was first hybridized with the chromosome, and then labeled with Alexa Fluor 488-conjugated anti-FITC secondary antibody to increase sensitivity. CDKN2A signals were equally identifiable by AM-FISH and conventional FISH in normal mononuclear blood cells. In contrast, when two BCL cell lines lacking CDKN2A were analyzed, CDKN2A signals were not detected by AM-FISH, whereas conventional FISH yielded false signals. Furthermore, AM-FISH detected CDKN2A deletions in two BCL patients with 9p21 microdeletions, which were not detected by conventional FISH. These results suggest that AM-FISH is a highly sensitive, specific, and simple method for diagnosis of chromosomal microdeletions.

Ridolfi M, Paudice M, Salvi S, et al.
Agar pre-embedding of small skin biopsies: real-life benefits and challenges in high throughput pathology laboratories.
J Clin Pathol. 2019; 72(6):448-451 [PubMed] Related Publications
Paraffin embedding of small, thin tissue samples requires specific expertise for optimal orientation before tissue sectioning. This study evaluates the real-life utility of the agar pre-embedding technique for small skin biopsies with regards to lengthening of work times, problems in orientation (re-embedding) and ancillary techniques (immunohistochemistry and in situ hybridisation) between two high work flow pathology laboratories, one of which routinely uses the agar pre-embedding technique and one which does not. The mean time required for pre-embedding in agar was 30.4 s, but time for paraffin embedding for agar pre-embedded samples was shorter than the traditional method (177 vs 296 s; p<0.005). The number of skin samples requiring re-embedding was significantly higher with the traditional embedding method (p<0.005). No problems in immunoreactivity were observed in all 1900 reactions performed with 17 different antibodies. Fluorescence in situ hybridisation analysis was optimised with a prolonged protease K incubation time (21 vs 18 min).

Giefing M, Siebert R
FISH and FICTION in Lymphoma Research.
Methods Mol Biol. 2019; 1956:249-267 [PubMed] Related Publications
Fluorescence in situ hybridization (FISH) is a powerful and robust technique allowing the visualization of target sequences like genes in interphase nuclei. It is widely used in routine diagnostics to identify cancer-specific aberrations including lymphoma-associated translocations or gene copy number changes in single tumor cells. By combining FISH with immunophenotyping-a technique called fluorescence immunophenotyping and interphase cytogenetic as a tool for investigation of neoplasia (FICTION)-it is moreover possible to identify a cell population of interest. Here we describe standard protocols for FISH and FICTION as used in our laboratories in diagnosis and research.

Lin T, Jin H, Gong L, et al.
Surveillance of non-muscle invasive bladder cancer using fluorescence in situ hybridization: Protocol for a systematic review and meta-analysis.
Medicine (Baltimore). 2019; 98(7):e14573 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: To evaluate the diagnostic effectiveness and predictive value of fluorescence in situ hybridization (FISH) in the surveillance of non-muscle invasive bladder cancer (NMIBC).
METHODS: PubMed/Medline, EMBASE, Web of Science, Ovid, Web of Knowledge, and Cochrane Library will be searched for studies related to the topic. The identification, inclusion, and exclusion flowcharts will be conducted according to preferred reporting items for systematic reviews and meta-analysis guidelines. The identified reports will be critically appraised according to the Newcastle-Ottawa scale, quality assessment of diagnostic accuracy studies-2 and standards for reporting of diagnostic accuracy 2015. Forest plots will be generated to display hazard ratios, sensitivities, and specificities. Pooled estimates with their 95% confidence intervals will be calculated using the bivariate model, the hierarchical summary receiver operating characteristic model and a fixed- or random-effects model.
RESULTS: This study will provide evidence and data to form a comprehensive understanding of the value of FISH in the surveillance of NMIBC.
CONCLUSION: The diagnostic efficacy of FISH will be affected by post-therapy factors. However, FISH still could facilitate the surveillance of NMIBC owing to its non-invasive feature. This study will improve the clinical decision-making and enlighten the future research of NMIBC.

Liu ZH, Wang K, Lin DY, et al.
Impact of the updated 2018 ASCO/CAP guidelines on HER2 FISH testing in invasive breast cancer: a retrospective study of HER2 fish results of 2233 cases.
Breast Cancer Res Treat. 2019; 175(1):51-57 [PubMed] Related Publications
OBJECTIVES: Human epidermal growth factor receptor 2 (HER2, ERBB2) is a valuable prognostic and predictive biomarker in breast cancer. Accurate assessment of HER2 status is essential in selecting the patients with invasive breast cancer who will likely response to HER2-targeted therapies. Some major modifications in the diagnostic recommendation for fluorescence in situ hybridization (FISH) have been made in the updated 2018 American Society of Clinical Oncology (ASCO)/College of American Pathologist (CAP) guideline. According to the revised guideline, concomitant IHC assays are required to arrive at the most accurate HER2 status designation after HER2 FISH equivocal results; however, little is known about its influence on the clinical practice of pathologist. The purpose of this study was to evaluate the impact of the revised 2018 ASCO/CAP guidelines on the HER2 status designation.
METHODS: We retrospectively reviewed the HER2 FISH testing results from 2233 cases of invasive breast cancer between January 2014 and December 2017. Concomitant immunohistochemistry (IHC) were performed on the same tissue blocks that were used for the FISH testing.
RESULTS: Compared to the 2013 guidelines, the HER2 status in 183 (8.2%) cases were re-defined when reassessed by the 2018 guidelines. Among these 183 cases, 175 equivocal cases according to the 2013 guideline were re-defined as HER2 negative (n = 173) or HER2 positive (n = 2). Eight previously classified as HER2 positive cases were converted to negative in the 2018 scheme, all of which were with HER2 IHC scores of 1+ or 2+. The number of cases in the negative category was 1705 according to the 2018 guidelines as opposed to 1524 by the 2013 guidelines.
CONCLUSIONS: The updated 2018 ASCO/CAP guidelines eliminated the FISH equivocal category, which can be attributed to reflex HER2 IHC, and partly ease the dilemma for clinical practice. Reflex IHC for FISH equivocal cases is of prime importance; furthermore, HER2 FISH results were converted from positivity to negativity based on the concomitant IHC results in a small percentage of cases. In all, implementation of the 2018 ASCO/CAP guidelines provides much clearer instructions and recommendations for the HER2 status designation, and thus reduces the risk of misdiagnosis.

Jin H, Lin T, Hao J, et al.
A comprehensive comparison of fluorescence in situ hybridization and cytology for the detection of upper urinary tract urothelial carcinoma: A systematic review and meta-analysis.
Medicine (Baltimore). 2018; 97(52):e13859 [PubMed] Free Access to Full Article Related Publications
OBJECTIVE: To compare the relative effectiveness of fluorescence in situ hybridization (FISH) and cytology in diagnosing upper urinary tract urothelial carcinoma (UUT-UC) and to evaluate the advantages and potential deficiencies of FISH analysis.
METHODS: We performed a complete systematic review based on studies from PubMed/Medline, Embase, Web of Science, Ovid, Web of Knowledge, and Cochrane Library. We identified 2031 patients with strict criteria in 14 individual studies between January 2005 to November 2017 in accordance to preferred reporting items for systematic reviews and meta-analysis (PRISMA) guidelines, we summarized the test performance using bivariate random effects models.
RESULTS: FISH was superior to cytology in terms of pooled sensitivities (84.0%, 95% confidence interval [CI] 74.4-90.5% vs 40.0%, 95% CI 33.6-46.7%). FISH and cytology were similar to each other in terms of pooled specificities, which were 89.5% (95% CI 85.3-92.6%) for FISH and 95.9% (95% CI 91.2-98.1%) for cytology.
CONCLUSION: We confirm the superiority of FISH over cytology in terms of sensitivity and find similar diagnostic outcomes between them based on systematic analysis. Therefore, we demonstrate that FISH is extremely sensitive while still very reliable with a relatively low error rate for diagnosing UUT-UC.

Freund JE, Liem EIML, Savci-Heijink CD, de Reijke TM
Fluorescence in situ hybridization in 1 mL of selective urine for the detection of upper tract urothelial carcinoma: a feasibility study.
Med Oncol. 2018; 36(1):10 [PubMed] Free Access to Full Article Related Publications
Kidney-sparing surgery of upper tract urothelial carcinoma (UTUC) requires a stringent follow-up with frequent ureteroscopies. Triage testing could reduce the number of follow-up ureteroscopies and hence minimize the invasiveness of follow-up. The use of urine-based markers for triage seems appealing but should be feasible with selective urine from outpatient cystoscopy to maximize the reduction of invasiveness. In this study, the feasibility of UroVysion

El-Menoufy MAM, Mourad ZI, Farahat NM
The prognostic impact of loss of chromosome 7 material detected by fluorescence in situ hybridization (FISH) in myeloid malignancies.
J Egypt Natl Canc Inst. 2018; 30(4):133-138 [PubMed] Related Publications
BACKGROUND: Monosomy 7 (-7) or deletion in its long arm [del(7q)] is among the most common chromosomal abnormalities in myeloid malignancies. There are prognostic variations between -7 and del(7q) in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).
AIM: To describe the clinical characteristics, response to treatment, and survival of patients with primary AML and MDS having -7 or del(7q) detected by fluorescence in situ hybridization (FISH).
PATIENTS AND METHODS: The study was conducted on 53 patients with primary AML and MDS. They were tested for chromosome 7 abnormality using FISH technique.
RESULTS: Thirty-one patients had chromosome 7 abnormality and 22 did not. Lower complete remission and higher death rates were observed in patients with -7 (47.6% and 62%, respectively) when compared to patients with del(7q) (70% and 40%, respectively) with no significant difference (p = 0.218 and 0.101, respectively). The median overall survival (OS) of patients with -7, del(7q) and normal chromosome 7 were 32.0, 43.0 and 50.0 months, respectively, with significant statistical difference (p = 0.001). This difference was evident between patients with -7 and those with normal chromosome 7 (p = 0.001), and less evident between patients with -7 and those with del(7q) (p = 0.021).
CONCLUSION: Chromosome 7 analysis has clear impact on the outcome of myeloid malignancies. The prognostic variations between -7 and del(7q) is attributed to multiple factors. Cases with del(7q) have better outcome than cases with -7. FISH provides a powerful tool for detecting and monitoring patients with chromosome 7 abnormalities.

Xu B, Shen J, Guo W, et al.
Impact of the 2018 ASCO/CAP HER2 guidelines update for HER2 testing by FISH in breast cancer.
Pathol Res Pract. 2019; 215(2):251-255 [PubMed] Related Publications
Recently, the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) updated the guidelines on HER2 testing for invasive breast cancer. Little is known about the impact of the guidelines update. We aimed to study the impact of the 2018 ASCO/CAP HER2 testing guidelines update. We compared the HER2 FISH results interpreted by 2013 and 2018 ASCO/CAP guidelines in 331 cases of invasive breast cancers. We also analyzed the pathological features and clinical outcomes of these cases. In comparing to the 2013 ASCO/CAP guidelines, the HER2 negative rate was increased significantly from 62.5% to 75.8%(P < 0.05), and 13.3% changed from equivocal to negative by the 2018 guidelines. Our findings indicate that the guidelines update significantly increased the rate of negative results. The reclassification of the equivocal results by the 2018 guidelines is the main reason for this change. Patients with HER2 equivocal results were associated with larger tumor size and higher Ki67 index than those with negative results, while clinical outcomes were similar between them.

McHale T, Ohori NP, Cieply KM, et al.
Comparison of urinary cytology and fluorescence in situ hybridization in the detection of urothelial neoplasia: An analysis of discordant results.
Diagn Cytopathol. 2019; 47(4):282-288 [PubMed] Related Publications
BACKGROUND: We examine the performance of cytology and FISH in the detection of urothelial carcinoma (UC), and explore the reasons for discrepant results, and potential clinical implications.
METHODS: Urine samples from 89 patients were prospectively collected for simultaneous cytology and UroVysion FISH, and results correlated with concurrent biopsies and/or clinical or histologic follow-up data. Corresponding tissue biopsies, where available, were also evaluated by FISH.
RESULTS: Sensitivity and specificity of cytology and FISH for the detection of UC was 54.8% and 92% and 50% and 88%, respectively. Only one of seven false-positive urinary FISH results proved to be an "anticipatory positive" on extended follow-up. Five of eight (62.5%) high grade (HG) carcinomas with false-negative urinary FISH, were negative due to the absence/paucity of FISH-detectable changes in the tumor cells. In atypical cytology cases, the FISH result did not assist in identifying UC. There was no significant difference between an atypical cytology result and a positive FISH result, with respect to the identification of patients with UC.
CONCLUSIONS: We found urinary cytology to be more sensitivity and specific than FISH in the detection of UC, though the difference was not statistically significant. Up to 24% of HG UCs are FISH negative due to an absence of FISH-detectable abnormalities in the tumor cells. Paucity of neoplastic cells in the urine also contributes to false-negative FISH results in both HG and low grade tumors. Negative urinary FISH cannot be taken alone as indicating the absence of significant disease in patients with atypical cytology.

Meyer RG, Van Dyke DL
Analysis of Common Abnormalities Seen in Chronic Lymphocytic Leukemia Using Fluorescence In Situ Hybridization.
Methods Mol Biol. 2019; 1881:35-49 [PubMed] Related Publications
Since fluorescence in situ hybridization (FISH) was used to define a prognostic heierarchy for chronic lymphocytic leukemia (CLL) in 2000, the method has been employed widely in cytogenetics laboratories worldwide. This chapter describes techniques and trouble-shooting to maximize the efficiency of microscope slide preparation for FISH analysis in CLL.

Brajkovic S, Pelz B, Procopio MG, et al.
Microfluidics-based immunofluorescence for fast staining of ALK in lung adenocarcinoma.
Diagn Pathol. 2018; 13(1):79 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Anaplastic lymphoma kinase (ALK) is a key oncogenic driver in lung adenocarcinoma patients and its fusion proteins are routinely assessed. The microfluidic tissue processor (MTP) device is based on a chip-confined low-volume technology allowing for rapid immunohistochemistry/immunofluorescence (IHC/IF) stainings of formalin-fixed paraffin-embedded (FFPE) or frozen tissue samples.
METHODS: A novel ALK IF protocol was developed for the MTP device using the primary mouse anti-human ALK antibody clone 5A4. FFPE tumor whole sections from 14 resected lung adenocarcinoma patients documented to be ALK positive (ALK+) by automated chromogenic IHC and/or FISH were used. MTP-derived IF immunoreactivity was measured by computerized analysis of digitalized images on individual frames of tumor epithelia and surrounding stroma, using an ImageJ plug-in.
RESULTS: The 5A4 antibody yielded saturated immunoreactivity at an incubation time of 4 min on a titration curve ranging from 2 to 32 min. Total staining time on the MTP device was 18 min including secondary IgG Alexa Fluor 647. MTP-based ALK IF confirmed all 12 cases; with epithelial signal above stromal staining based on computerized pixel-based measurement. MTP-IF (mean intensity levels 458 to 1301) and chromogenic IHC (H-score 120 to 300) showed an equal range of variation of 2.8 and 2.5 folds, respectively, and a trend for direct correlation (p-value 0.051).
CONCLUSION: The newly developed protocol for immunofluorescent detection of ALK protein with the MTP device confirms chromogenic IHC results on FFPE lung adenocarcinoma specimens. MTP-based IF is fast and reliable. We foresee this study to be a first step opening the road for further realization of microfluidic-based assays for rapid simultaneous detection of targetable oncogenic and immune-system related markers in their topographical context to investigate tumour heterogeneity and micro-environmental interactions.

Ikeri NZ, Akinjo AO, Ajayi OO, Banjo AA
An audit of the diagnosis and reporting of soft tissue sarcomas at the Lagos University Teaching Hospital.
Niger J Clin Pract. 2018; 21(10):1330-1336 [PubMed] Related Publications
Background: : The effective management of patients with cancer is predicated on the right diagnoses and other relevant parameters included in the pathology report. This is particularly important in soft tissue pathology where arriving at the right diagnosis is often challenging. The aim of this study, therefore, was to perform an audit of sarcoma diagnosis and reporting in our institution.
Methods: Slides of soft tissue sarcomas diagnosed in our institution over a 5-year period were reviewed with specialist soft tissue pathologists. Ancillary immunohistochemistry and fluorescent in situ hybridization were performed where necessary. The contents of the reports were assessed using a diagnostic checklist developed by the Association of Directors of Anatomic and Surgical Pathology.
Results: Fifty-five of the 62 patients studied (88.7%) were correctly identified as sarcomas. However, the correct diagnoses were made in only 27 patients (43.6%). Kaposi sarcoma and dermatofibrosarcoma protuberans were the most recognized sarcomas, while leiomyosarcoma, myxofibrosarcoma, and malignant peripheral nerve sheath tumor were the least recognized sarcomas. The most reported parameters included the histologic type (100%) and size (89.7%), while the percentage of necrosis (0%) and the stage (0%) were the least reported parameters.
Conclusion: A pattern based approach is important for the accurate diagnosis of soft tissue sarcomas. Some essential prognostic parameters and information needed for management were not included in the histopathology reports. The adoption of a structured reporting format and multidisciplinary team meetings will help to ensure the inclusion of such important information in the pathology report.

Bao Y, Tu X, Chang T, et al.
The role of fluorescence in situ hybridization to predict patient response to intravesical Bacillus Calmette-Guérin therapy for bladder cancer: A diagnostic meta-analysis and systematic review.
Medicine (Baltimore). 2018; 97(36):e12227 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: The aim of the study was to systematically review the relevant studies to assess the role of fluorescence in situ hybridization (FISH) test for predicting patient response to Bacillus Calmette-Guérin (BCG) therapy after transurethral resection of bladder tumor (TURBT).
METHODS: We searched PubMed, Embase, and the Cochrane Library from inception to July 5, 2018, and used Quality Assessment Tool for Diagnosis Accuracy Studies (QUADAS-2) to assess the quality. We pooled sensitivity, specificity, and area under curve (AUC) of baseline and post-BCG FISH test for predicting tumor recurrence. Hazard ratio (HR) with 95% confidence intervals (95% CIs) and a Fagan nomogram were applied to assess predictive accuracy of post-BCG FISH test.
RESULTS: A total of 6 studies with 442 participants for post-BCG test and 404 participants for baseline BCG test were included. The pooled analysis for post-BCG FISH test revealed the sensitivity of 0.54 (95% CI 0.38-0.69), specificity of 0.84 (95% CI: 0.72-0.91), and area under the curve (AUC) of 0.78 (95% CI: 0.74-0.81) for predicting tumor recurrence. Patients with positive post-BCG FISH test were more likely to recur during follow-up (HR 3.95, 95% CI 2.72-5.72). The Fagan nomogram revealed the "post-test" probability of tumor recurrence increased by 29% for patients with positive post-BCG FISH test. The baseline FISH test had a pooled sensitivity of 0.70 (95% CI 0.55-0.81), specificity of 0.41 (95% CI: 0.26-0.58), and AUC of 0.60 (95% CI: 0.56-0.64) for predicting recurrence.
CONCLUSION: The post-BCG FISH test can predict BCG failure with high specificity and patients with positive post-BCG FISH test were more likely to recur. However, the relatively low sensitivity of post-BCG FISH test and unsatisfactory performance of baseline FISH test may limit their mono-use.

Parihar M, Singh MK, Islam R, et al.
A triple-probe FISH screening strategy for risk-stratified therapy of acute lymphoblastic leukaemia in low-resource settings.
Pediatr Blood Cancer. 2018; 65(12):e27366 [PubMed] Free Access to Full Article Related Publications
Karyotyping along with a 3-probe fluorescence in situ hybridization (FISH) strategy was used to risk stratify therapy in 303 children with B-cell precursor acute lymphoblastic leukaemia. Of the 166 patients risk stratified, karyotype identified 91 (55%). FISH identified all karyotypes accurately, with the exception of hypodiploidy, and risk stratified an additional 75 patients. The frequency of ETV6-RUNX1 is lower and high hyperdiploidy, higher than reported in the west. An adapted 3-probe FISH strategy identified two patients with ETV6-ABL1 fusion who received imatinib. In limited-resource settings, a 3-probe FISH approach provides a practical approach for risk-stratified therapy in childhood ALL.

Linos K, Kozel JA, Hurley MY, Andea AA
Review of the medical literature and assessment of current utilization patterns regarding the use of two common fluorescence in situ hybridization assays in the diagnosis of dermatofibrosarcoma protuberans and clear cell sarcoma.
J Cutan Pathol. 2018; 45(12):905-913 [PubMed] Related Publications
BACKGROUND: Dermatofibrosarcoma protuberans (DFSP) is a tumor of intermediate malignancy, which in selected circumstances can pose difficulty in diagnosis. Clear cell sarcoma (CCS) is a very rare aggressive soft tissue sarcoma that can be difficult to distinguish histologically from melanoma.
METHODS: The current literature on t(17;22) COL1A1-PDGFB fluorescence in situ hybridization (FISH) assay in DFSP was reviewed. Also reviewed was the current literature on dual color break-apart EWSR1 FISH assay in CCS. Finally, the current utilization patterns of these tests was assessed in attendees of the American Society of Dermatopathology annual meeting (Chicago, 2016).
RESULTS: The literature indicates that (17;22) COL1A1-PDGFB FISH assay has limited value for classic DFSP, where the diagnosis can be established by routine morphology and immunohistochemistry. Given the high specificity of the EWSR1 FISH assay and significant complexity in the diagnosis of CCS, this ancillary study is helpful in distinguishing CCS from melanoma.
CONCLUSIONS: In attendees, t(17;22) COL1A1-PDGFB FISH testing for classic cases of DFSP is appropriately not being used by respondents. However, the literature sustains that it is useful in selected circumstances in which a definitive diagnosis is challenging. The majority of respondents are utilizing the EWSR1 FISH assay to distinguish CSS from melanoma as is supported by the literature.

Yang T, Li Y, Li J, et al.
Diagnostic Value Comparison of Urothelium Carcinoma Among Urine Exfoliated Cells Fluorescent In Situ Hybridization (FISH) Examination, Computerized Tomography (CT) Scan, and Urine Cytologic Examination.
Med Sci Monit. 2018; 24:5788-5792 [PubMed] Free Access to Full Article Related Publications
BACKGROUND This study aimed to compare the clinical effectiveness of urine exfoliated cells FISH examination, CT scan, and urine cytologic examination on the diagnosis of upper urinary tract urothelium carcinoma with hematuresis symptom. MATERIAL AND METHODS A total of 30 patients with suspicious upper urinary tract urothelium carcinoma between Aug 2010 and Aug 2011 were enrolled, including 23 males and 7 females. All the subjects received urine exfoliated cells FISH examination, CT scan, and urine cytologic examination. Twenty-one cases were diagnosed as urothelium carcinoma, including 14 cases of carcinoma of renal pelvis and 7 cases of carcinoma of ureter. There were 6 cases in stage Ta/T1, 12 cases in stage T2, and 3 cases in T3/T4. The other 9 cases consisted of 1 case of neuroendocrine carcinoma of the renal pelvis, 2 cases of nephrotuberculosis, and 6 cases of renal clear cell carcinoma. RESULTS The total sensitivity of FISH examination, CT scan, and urine cytologic examination on upper urinary tract urothelium carcinoma was 85.7%, 66.7%, and 28.6%, respectively (P<0.05). The tumor staging detection on Ta/T1, T2, and T3/T4 by FISH was 66.7%, 91.7%, 100%; by CT scan 33.3%, 75.0%, 100%; and by urine cytologic examination 0%, 25.0%, and 100%. Their diagnostic specificities were 88.9%, 77.8%, and 100%, respectively (P<0.05). CONCLUSIONS The diagnostic sensitivity on upper urinary tract urothelium carcinoma was highest in FISH examination, followed by CT scan and urine cytologic examination. FISH technique obviously improves the diagnosis of upper urinary tract urothelium carcinoma.

Liu X, Zhang Z, Zhang B, et al.
Circulating tumor cells detection in neuroblastoma patients by EpCAM-independent enrichment and immunostaining-fluorescence in situ hybridization.
EBioMedicine. 2018; 35:244-250 [PubMed] Free Access to Full Article Related Publications
Detecting circulating tumor cells (CTCs) has proven valuable for evaluating the prognosis of cancer patients and for studying the mechanisms of treatment resistance. Owing to the lack of universal and specific tumor markers for neuroblastoma (NB), in this prospective study, we adopted an EpCAM-independent method to detect CTCs in the peripheral blood of NB patients. We used an EpCAM-independent assay to delete leukocytes and to enrich the CTCs. CTCs were identified by immunostaining of CD45, DAPI and DNA fluorescence in situ hybridization (FISH) of the centromere of chromosome 8 probe (CEP8). Cells that were DAPI+/CD45-/CEP8 ≥ 3 were considered CTCs. We collected peripheral blood from 28 NB patients as well as clinical and follow-up data. The number of CTCs among the different risk groups were significantly different (p = .0208, Kruskal-Wallis test). Patients with metastasis had more CTCs than those without metastasis (p < .0001, Mann-Whitney test). Patients with ≥3 CTCs per 4 ml of peripheral blood had an increased likelihood of having metastasis (sensitivity, 88.89%; specificity, 78.59%), and patients with ≥10 CTCs per 4 ml of peripheral blood had poorer overall survival. The EpCAM-independent assay along with immunostaining-FISH (i-FISH) described here can detect CTCs in patients with NB at a high sensitivity and may have clinical value for prognosis evaluation and diagnosing metastasis when imaging data are ambiguous.

Lateef F, Jamal S, Nasir S
Her-2/neu Oncogene Amplification by Fluorescence In Situ Hybridization and Protein Overexpression on Immunohistochemistry in Breast Cancer.
J Coll Physicians Surg Pak. 2018; 28(8):581-585 [PubMed] Related Publications
OBJECTIVE: To investigate the concordance and discordance between the test results of Her-2/neu by immunohisto-chemistry (IHC) and flourescence In Situ hybridization (FISH) in breast cancer cases.
STUDY DESIGN: Descriptive cross-sectional study.
PLACE AND DURATION OF STUDY: Department of Histopathology, Dr. Ziauddin Hospital, Karachi, from 2011 to 2016.
METHODOLOGY: Forty-three specimens of invasive ductal carcinoma of breast were evaluated for grade and Her-2/neu status using IHC and FISH methods. Concordance and discordance between their results was determined.
RESULTS: There is 100% concordance between FISH and IHC in cases scoring 0, 1+ (negative) and 3+ (positive) immunostaining. Tumour cases scoring 2+ immunostaining showed amplification in 69.2% cases. All grade-I tumours were non-amplified on FISH, while most of the grade-III tumours showed Her-2/neu amplification on FISH. There is significant association of Her-2/neu IHC with tumour grade and FISH (p<0.05). A fairly high proportion i.e. 69.7% of cases showed Her-2/neu gene amplification. There was high concordance between Her-2/neu testing on IHC and FISH, (Kappa co-efficient 0.466, p <0.001).
CONCLUSION: Her-2/neu amplification increases with increasing grade of breast cancer. A high proportion of Her-2/neu gene amplified cases indicates aggressive disease in that area and need for FISH testing on large scale, which is the gold standard for equivocal cases on immunohistochemistry.

Darriet F, Bernioles P, Loukil A, et al.
Fluorescence in situ hybridization microscopic detection of Bacilli Calmette Guérin mycobacteria in aortic lesions: A case report.
Medicine (Baltimore). 2018; 97(30):e11321 [PubMed] Free Access to Full Article Related Publications
RATIONALE: To improve the diagnosis of life-threatening Bacilli Calmette Guérin (BCG) arterial aneurysm in patients treated by intravesical instillation of BCG vaccine as adjunctive therapy for non-muscular bladder carcinoma, is a life-threatening condition. Its diagnosis remains cumbersome.
PATIENT CONCERNS: One patient with a history of intravesical BCG installation presented with aortic aneurysm with routine microscopic examination after Ziehl-Neelsen staining remaining negative.
DIAGNOSES: We used fluorescence in situ hybridization (FISH) to target the Mycobacterium tuberculosis complex rpob gene in a fresh aortic specimen. FISH yielded fluorescent mycobacteria in aortic lesions; mycobacteria were further confirmed as Mycobacterium bovis BCG mycobacteria by polymerase chain reaction (PCR) sequencing.
INTERVENTIONS: The patient benefited from an antituberculous treatment combining rifampicin, isoniazid, and ethambunol.
OUTCOME: A 9-month follow-up indicated a favorable outcome.
LESSONS: This case report teaches that FISH targeting the M tuberculosis complex rpoB gene should be incorporated in the laboratory investigation of aortic aneurysm in patients with a history of bladder carcinoma.

Vetro C, Haferlach T, Jeromin S, et al.
Identification of prognostic parameters in CLL with no abnormalities detected by chromosome banding and FISH analyses.
Br J Haematol. 2018; 183(1):47-59 [PubMed] Related Publications
Chronic Lymphocytic Leukaemia (CLL) is a heterogeneous disease with a clinical course dependent on cytogenetic features. However, in 15-20% of cases both chromosome banding and fluorescence in situ hybridisation analyses do not show any kind of abnormality. With the aim to identify dependable molecular prognostic factors in this subgroup, we performed a comprehensive analysis on 171 patients including genomic arrays (comparative genomic hybridisation and single nucleotide polymorphism), immunoglobulin heavy chain variable region genes (IGHV) status, flow cytometry and targeted sequencing. Genomic arrays detected 73 aberrations in 39 patients (23%). Most frequently, patients had 1 aberration (25/171; 15%), while 14 patients (8%) had at least 2 aberrations. IGHV status was unmutated in 53/171 (31%) patients. SF3B1 was the most frequently mutated gene (26/171 patients; 15%), followed by NOTCH1 (15/171; 9%). At univariate analysis, an adverse impact on time to treatment (TTT) was evident for SF3B1 mutations, higher white blood cell count, higher CLL cells percentage by flow cytometry, CD38 positivity, IGHV unmutated status and at least 2 genomic array abnormalities. Of these, SF3B1 mutations, CLL cells percentage, IGHV unmutated status and number of genomic array aberrations maintained their impact in multivariate analysis. In conclusion, by integrating genomic and molecular data, we identified patients at higher risk for treatment need.

Shlomi D, Peled N, Schwarz YA, et al.
Non-invasive early detection of malignant pulmonary nodules by FISH-based sputum test.
Cancer Genet. 2018; 226-227:1-10 [PubMed] Related Publications
BACKGROUND: Early detection decreases lung cancer mortality. The Target-FISH Lung Cancer Detection (LCD) Test is a non-invasive test designed to detect chromosomal changes (deletion or amplification) via Fluorescence in situ Hybridization (FISH) in sputum specimens from persons suspected of having lung cancer. We evaluated the performance of the LCD test in patients with highly suspicious pulmonary nodules who were scheduled for a biopsy procedure.
METHODS: Induced sputum was collected from patients who were scheduled for biopsy of a solitary pulmonary nodule (0.8-3 cm) in one of 6 tertiary medical centers in the US and Israel. The lung cancer detection (LCD) Test combined sputum cytology and Target-FISH analysis on the same target cells and the results were compared to the pathology. Participants with non-surgical negative biopsy results were followed for 2 years to determine their final diagnosis.
RESULTS: Of the 173 participants who were evaluated, 112 were available for analysis. Overall, the LCD test had a sensitivity of 85.5% (95% CI, 76.1-92.3), specificity of 69% (95% CI, 49.2-84.7) and an accuracy of 81.3% (95% CI, 72.8-88). The positive and negative predictive values (PPV, NPV) were 88.8% and 62.5%, respectively. The LCD test was positive in 9 of 11 lung cancer patients who had an initial negative biopsy.
CONCLUSIONS: In a cohort of patients with highly suspicious lung nodules, the LCD test is a non-invasive option with good sensitivity and a high positive predictive value. A positive LCD test reinforces the need to aggressively pursue a definitive diagnosis of suspicious nodules.

Kojima T, Nishiyama H, Ozono S, et al.
Clinical evaluation of two consecutive UroVysion fluorescence in situ hybridization tests to detect intravesical recurrence of bladder cancer: a prospective blinded comparative study in Japan.
Int J Clin Oncol. 2018; 23(6):1140-1147 [PubMed] Related Publications
BACKGROUND: We evaluated the use of UroVysion fluorescence in situ hybridization tests to detect the intravesical recurrence of bladder cancer during follow-up after a transurethral resection of bladder tumor (TURBT).
METHODS: In this prospective, blinded, comparative study, 486 patients treated by TURBT within the prior 2 years were registered at 12 centers. Urine cytology and UroVysion tests were performed once or twice at a central testing laboratory. For the patients with no suspicious findings of bladder cancer in the first analysis, the same examination set was repeated 3 months later as the second analysis. Totals of 468 and 399 patients were eligible for the first and second analyses, respectively. We determined the sensitivity and specificity of two consecutive UroVysion tests.
RESULTS: Bladder cancers were identified in 44 patients at the first analysis. The UroVysion test had 50.0% (95% CI 35.2-64.8%) sensitivity and 72.4% (68.3-76.8%). Urine cytology had 4.5% (0.0-10.7%) sensitivity and 99.8% (99.3-100.0%) specificity. The concordant rate of the first and second UroVysion test results was 72% (kappa coefficient 0.157). Interestingly, the patients with two consecutive positive UroVysion test results had the highest cancer detection rate (14.8%), which is greater than those of the patients with a positive result in either (7.2%) or neither (1.2%) of the two tests at the 3-month follow-up.
CONCLUSIONS: The UroVysion test provided higher sensitivity than urine cytology to detect bladder cancer during post-TURBT follow-up. Two consecutive UroVysion tests might be a better indicator to predict intravesical recurrence.

Cantú ES, Dong H, Forsyth DR, et al.
Discrepant Cytogenetic and Interphase Fluorescence In Situ Hybridization (I-FISH) Results from Bone Marrow Specimens of Patients with Hematologic Neoplasms.
Ann Clin Lab Sci. 2018; 48(3):264-272 [PubMed] Related Publications
Conventional cytogenetic and routine I-FISH (interphase fluorescence in-situ hybridization) studies periodically present discrepant results on the same sample calling into question their validity. Generally it is expected that these tests confirm each other, otherwise there is concern that they may represent laboratory error. We present data showing that these discrepant results are rarely due to laboratory error, and that M-FISH (metaphase fluorescence in-situ hybridization) can usually reconcile them by identifying the nature of these differences. This report includes 32 bone marrow (BM) samples from patients with hematologic neoplasms that showed incongruent cytogenetic/I-FISH results. M-FISH was selectively applied for further clarification of these discrepancies when deemed necessary. This study evaluated BM samples in our laboratory (Integrated Oncology, Phoenix, AZ) that represented 5 major categories of hematologic disorders (MDS/AML, MPN, NHL, CLL, & PCN). Five general categories of these cases were identified: 1) laboratory error (clerical), 2) limited resolution of testing methods, 3) cellular response to culture/preparative conditions, 4) cytogenetic bi-clonality and 5) failed hybridizations due to cover-slipping. Our results suggest that the majority of discrepant results are related to the intrinsic nature of the malignant cells (and how they respond to their growth environment) evaluated by these two testing methods.

Li Y, Ma G, Zhao P, et al.
Improvement of sensitive and specific detection of circulating tumor cells using negative enrichment and immunostaining-FISH.
Clin Chim Acta. 2018; 485:95-102 [PubMed] Related Publications
BACKGROUND: Circulating tumor cells (CTCs) provide an opportunity to obtain pivotal biological information required for the development of personalized medicine. However, the current assays of CTCs' detection face serious challenges regarding specificity and sensitivity.
METHODS: In this study, we developed a novel strategy that combined negative enrichment (NE), immunocytochemistry CD45 staining and fluorescence in situ hybridization (FISH) to identify, enumerate and characterize CTCs. CTCs were identified as DAPI+/CD45-/Chromosome multiploid. The assay was evaluated with different cancer cell lines including lung, breast, esophageal and gastric cancer. And then, the developed assay was applied in cancer patients to explore the possibility of clinical application and whether CTC number was related to clinicopathological factors.
RESULTS: The average recover rate of esophageal cancer cell line Eca-109 using negative enrichment was higher than 80% and the multiploid cells rate of four cancer cell lines were >96%, which demonstrate the NE-FISH platform is favorable for CTCs detection. CTCs count was significantly higher in lung cancer patients than healthy controls and benign lung disease with an area under ROC curve of 0.905 (95% confidence interval 0.866-0.944, P < .001). Using a cutoff value of 2 CTCs, the positive rate of detecting lung, gastric, breast and esophageal cancer patients were 71.33%, 86.21%, 76.77% and 78.35%, respectively. Besides, CTCs could be detected in stage I with the positive rate of 64.15% for lung cancer, 83.33% for gastric cancer, 78.95% for breast cancer and 68.18% for esophageal cancer, which may promote the early diagnose and influence the treatment decision for better management of those cancer in clinic.
CONCLUSIONS: Our study showed that CTCs could be detected in diverse cancers using the novel NE-FISH platform with high sensitivity and specificity. Therefore, analysis of CTCs with NE-FISH has a clear potential to improve the management of cancer patients in clinical use.

Zare S, Lin L, Alghamdi AG, et al.
Comparative Pathologic Analysis of Breast Cancers Classified as HER2/neu-Amplified by FISH Using a Standard HER2/CEP17 Dual Probe and an Alternative Chromosome 17 Control Probe.
Am J Surg Pathol. 2018; 42(9):1208-1215 [PubMed] Related Publications
At our institution, breast cancer cases that generate an equivocal HER2/neu (HER2) result by fluorescence in situ hybridization (FISH) using the dual HER2/chromosome enumeration probe (CEP17) are reflexed to an assay that utilizes an alternative control probe (lissencephaly gene1 [LIS1] [17p13.3]/retinoic acid receptor α [RARA] [17q21.2]). This study examines whether cancers that are classified as HER2-amplified with an alternate probe are clinicopathologically similar to those that are classified as such using the HER2/CEP17 probe. Reports for 1201 breast cancers were reviewed, and clinicopathologic findings were compared between HER2/CEP17-equivocal cases that became HER2-amplified using the alternate probe (group A: n=48), HER2-amplified cases using the HER2/CEP17 probe (group B: n=169), and HER2-nonamplified cases using the HER2/CEP17 probe (group C: n=910). Of 1201 cases tested using the HER2/CEP17 probe, 169 (14%) were HER2-amplified, 122 (10%) were equivocal, and 910 (76%) were nonamplified. Additional testing with the alternative probe on the 122 equivocal cases reclassified 48 (39%) of them to HER2-amplified, and such cases comprised 22% of all HER2-amplified tumors. A higher proportion of tumors with HER2 copy number between 5.0 and 5.9 became positive upon additional testing when compared with those with a priori HER2 copy numbers between 4.0 and 4.9 (P=0.0362). Group A cases, compared with group B cases, were more frequently positive for estrogen receptor (97.91% vs. 72.18%, P<0.0001) and progesterone receptor (85.41% vs. 59.17%, P=0.0009). Most group A cases (71%) were HER2 equivocal (score 2+) by immunohistochemistry, whereas most group B cases (60%) were positive (score 3+). Groups A and B showed no significant differences regarding patient age, lymph node status, tumor grade, histotype, and stage distribution. In summary, among our HER2-amplified cohort of breast cancers, alternative probe-detected cases were more frequently estrogen receptor and progesterone receptor positive than HER2/CEP17-detected cases, and were more frequently discordant with HER2 immunohistochemistry results. These findings raise the possibility of underlying biologic differences between these 2 groups, which warrants further study. However, the tumors were largely comparable regarding all other clinicopathologic variables. As it is unknown whether HER2-targeted therapy is truly beneficial in this subgroup of patients, future clinical trials should specifically evaluate this subset.

Gupta S, Mani NR, Carvajal-Hausdorf DE, et al.
Macrodissection prior to closed system RT-qPCR is not necessary for estrogen receptor and HER2 concordance with IHC/FISH in breast cancer.
Lab Invest. 2018; 98(8):1076-1083 [PubMed] Free Access to Full Article Related Publications
An on-demand, closed RT-qPCR, the GeneXpert (GX) system, has the potential to provide biomarker information in low-resourced settings and elsewhere. We used this system with a research use only version of the Breast Cancer STRAT4 cartridge that measures the mRNA expression levels of ERBB2, ESR1, PGR, and MKi67. Here we evaluated the impact of non-macrodissected (non m-d) versus macrodissected (m-d) samples using STRAT4 on formalin-fixed, paraffin-embedded (FFPE) core needle biopsies. Two cohorts were assessed: (1) 60 FFPE infiltrating ductal carcinoma (IDCA) cases and (2) 20 FFPE IDCA cases with ductal carcinoma in situ (DCIS) with a range of HER2 expression as determined by clinical immunohistochemistry and fluorescence in situ hybridization (IHC/FISH). We observed about half of the core needle biopsy area as invasive tumor in both IDCA (mean = 51.5%) and IDCA with DCIS (mean = 53.5%) cohorts, but also found the mRNA levels were independent of tumor area. We found excellent agreement of the mRNA transcript level between the paired samples, m-d versus non m-d, for ERBB2, ESR1, PGR, and MKi67 for both the IDCA and IDCA with DCIS cohorts. No significant difference (P > 0.99) was observed when we compared the mRNA transcript level between the paired samples m-d versus non m-d. In addition, we noted a significant concordance (P < 0.001) between RT-qPCR and IHC/FISH for HER2-positivity, ER-positivity, and PR-positivity, independent of specimen dissection. These data suggest that mRNA expression for ERBB2, ESR, and PGR is sufficiently low in surrounding tissue cells such that macrodissection is not required for assessment of key breast cancer mRNA markers and is independent of the amount of input tumor. This approach may be valuable in settings lacking pathology expertise or using specimen types, such as fine-needle aspirates, where it may be challenging to separate non-tumor from tumor tissue.

Vargas AC, Selinger C, Satgunaseelan L, et al.
FISH analysis of selected soft tissue tumors: Diagnostic experience in a tertiary center.
Asia Pac J Clin Oncol. 2019; 15(1):38-47 [PubMed] Related Publications
AIM: Fluorescence in situ hybridization (FISH) is an important ancillary tool for the classification of bone/soft tissue (BST) tumors. The aim of this study was to evaluate the contribution of FISH to the final classification of common BST entities in the molecular pathology department of the Royal Prince Alfred Hospital (RPAH), which is one of the most important referral centers for the management of sarcomas in Australia.
METHODS: All routine diagnostic FISH tests performed on BST formalin-fixed paraffin embedded (FFPE) tissue specimens at the RPAH in a 5-year period (February, 2010-November, 2015) were reviewed. FISH analyses presented in this study include commercial break-apart probes (SS18, FUS, DDIT3, FUS, USP6, PDGFB, TFE3 and ALK) and a single enumeration (MDM2) probe.
RESULTS: There were 434 interpretable FISH assays on BST samples including MDM2 (n=180), SS18 (n=97), FUS (n=64), DDIT3 (n=37), USP6 (n=30), PDGFB (n=13), TFE3 (n=8) and ALK (n=5). Discrepancies between the histopathological diagnosis and the FISH results were seen in 12% of the cases. In this subset of discordant cases, FISH contributed to the re-classification of 7% of cases originally diagnosed as synovial sarcoma (SS18) and 6% of adipocytic neoplasms (MDM2) based on the presence or absence of the expected gene alteration.
CONCLUSION: Our study confirms that paraffin FISH is a sensitive and specific ancillary tool in the diagnosis of BST neoplasms when used in the appropriate clinicopathological context. These findings highlight the need for further ancillary molecular tools in the diagnosis and characterization of challenging cases.

Ginestet F, Lambros L, Le Flahec G, et al.
Evaluation of a Dual ALK/ROS1 Fluorescent In Situ Hybridization Test in Non-Small-cell Lung Cancer.
Clin Lung Cancer. 2018; 19(5):e647-e653 [PubMed] Related Publications
BACKGROUND: Several therapeutics targets have emerged to treat patients with non-small-cell lung carcinoma (NSCLC), with numerous biomarkers available to test for treatment choices. Minimum tumor wastage is necessary to permit the analysis of every potentially relevant target. Searching for targetable ALK and ROS1 rearrangements is now mandatory in NSCLC. In the present study, we evaluated the performance of a dual ALK/ROS1 fluorescent in situ hybridization (FISH) probe that concurrently analyzed the 2 oncogenes on a same FISH slide.
MATERIALS AND METHODS: We used the FlexISH ALK/ROS1 DistinguISH Probe (Zytovision, Bremerhaven, Germany) to analyze a set of 28 formalin-fixed paraffin-embedded NSCLC tumor samples enriched in tumors with ALK- and ROS1-rearranged status.
RESULTS: The dual ALK/ROS1 FISH probe test results were fully concordant with the results of previous single ALK and ROS1 FISH tests (15 ALK and 3 ROS1 rearrangements) without any false-positive results. Dual- and single-probe FISH test results were also concordant regarding the unusual ALK FISH patterns. These included 1 sample that had negative FISH results with diffuse single 5'-ALK signals and positive ALK immunohistochemistry findings in a patient with a response to crizotinib, 2 paired samples with high percentages of ALK FISH-rearranged nuclei despite negative ALK immunohistochemistry findings, and ALK FISH-positive samples from 2 patients lacking a response to crizotinib therapy despite concordant ALK FISH and immunohistochemistry-positive results.
CONCLUSION: The dual ALK/ROS1 FISH probe test is a valuable tool to search concurrently for both ALK and ROS1 rearrangements on a same FISH slide and could help to spare tumor tissue for other biomarkers tests.

Bagir EK, Acikalin A, Alsancak P, et al.
Prevalence of cytogenetic abnormalities in chronic lymphocytic leukemia in the southern part of Turkey.
Indian J Cancer. 2017 Jul-Sep; 54(3):572-575 [PubMed] Related Publications
BACKGROUND: Chronic lymphocytic leukemia (CLL) is the most common type of leukemia among adults in Western populations. CLL has a wide range of clinical presentations and varied outcomes. For CLL, cytogenetic assessment is essential for estimating prognoses and determining the treatment of choice. The fluorescence in situ hybridization (FISH) technique is widely used for genetic assessment due to its high sensitivity.
AIM: This study aimed to evaluate the frequencies of deletions of 13q14.3, 17p13.1, 11q22.3, and 13q34 and of trisomy 12 and to observe their effects on survival in 226 Turkish CLL patients using FISH analysis.
RESULT AND CONCLUSION: The frequencies of abnormalities were 65.4% for del 13q14.3, 39.8% for del 17p13.1, 19% for del 11q22.3 (del ATM), and 15.9% for trisomy 12. No patients had a 13q34.3 aberration. Our results are partially consistent with literature findings. However, certain conflicts with prior results were observed, particularly with respect to the high prevalence of 17p13.1 deletions and the enhanced survival of patients with such deletions. These inconsistencies may represent population-based differences in the genetic epidemiology of CLL.

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