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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

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Recent Publications: FISH

Zhang G, Lanigan CP, Goldblum JR, et al.
Automated Bright-Field Dual-Color In Situ Hybridization for MDM2: Interobserver Reproducibility and Correlation With Fluorescence In Situ Hybridization in a Series of Soft Tissue Consults.
Arch Pathol Lab Med. 2016; 140(10):1111-5 [PubMed] Related Publications
CONTEXT: -Atypical lipomatous tumors/well-differentiated liposarcomas contain alterations in the 12q13-15 region resulting in amplification of MDM2 and nearby genes. Identifying MDM2 amplification is a useful ancillary test, as the histologic mimics of atypical lipomatous tumors/well-differentiated liposarcomas have consistently shown a lack of MDM2 amplification.
OBJECTIVE: -To assess the interobserver reproducibility of a bright-field assay for MDM2 amplification (dual-color, dual-hapten in situ hybridization [DDISH]) among reviewers with varying degrees of experience with the assay and to assess the concordance of MDM2 DDISH with MDM2 fluorescence in situ hybridization (FISH).
DESIGN: -In total, 102 cases were assessed in parallel for MDM2 by FISH and DDISH. MDM2 amplification was defined as an MDM2 to chromosome 12 ratio of 2.0 or greater, whereas an MDM2 to chromosome 12 ratio of less than 2 was nonamplified. Fluorescence in situ hybridization was scored in the routine clinical laboratory and DDISH was evaluated by 3 different pathologists blinded to the final diagnosis and FISH results.
RESULTS: -Fluorescence in situ hybridization categorized 27 cases (26%) as MDM2 amplified and 75 cases (74%) as nonamplified; the consensus DDISH diagnosis was 98% concordant with FISH. Agreement between MDM2 DDISH by each reviewer and MDM2 FISH was highly concordant (99%, 98%, and 98%, respectively, for reviewers 1, 2 and 3). The κ agreement of the 3 reviewers scoring DDISH was excellent (κ = 0.949, 0.95, and 0.95, respectively, for reviewers 1, 2, and 3).
CONCLUSIONS: -This study highlights excellent concordance between DDISH and FISH in MDM2 copy number assessment. Moreover, excellent interobserver reproducibility of the DDISH assay was found among reviewers with varying levels of experience evaluating bright-field assays.

Akhter A, Mughal MK, Elyamany G, et al.
Multiplexed automated digital quantification of fusion transcripts: comparative study with fluorescent in-situ hybridization (FISH) technique in acute leukemia patients.
Diagn Pathol. 2016; 11(1):89 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: The World Health Organization (WHO) classification system defines recurrent chromosomal translocations as the sole diagnostic and prognostic criteria for acute leukemia (AL). These fusion transcripts are pivotal in the pathogenesis of AL. Clinical laboratories universally employ conventional karyotype/FISH to detect these chromosomal translocations, which is complex, labour intensive and lacks multiplexing capacity. Hence, it is imperative to explore and evaluate some newer automated, cost-efficient multiplexed technologies to accommodate the expanding genetic landscape in AL.
METHODS: "nCounter® Leukemia fusion gene expression assay" by NanoString was employed to detect various fusion transcripts in a large set samples (n = 94) utilizing RNA from formalin fixed paraffin embedded (FFPE) diagnostic bone marrow biopsy specimens. This series included AL patients with various recurrent translocations (n = 49), normal karyotype (n = 19), or complex karyotype (n = 21), as well as normal bone marrow samples (n = 5). Fusion gene expression data were compared with results obtained by conventional karyotype and FISH technology to determine sensitivity/specificity, as well as positive /negative predictive values.
RESULTS: Junction probes for PML/RARA; RUNX1-RUNX1T1; BCR/ABL1 showed 100 % sensitivity/specificity. A high degree of correlation was noted for MLL/AF4 (85 sensitivity/100 specificity) and TCF3-PBX1 (75 % sensitivity/100 % specificity) probes. CBFB-MYH11 fusion probes showed moderate sensitivity (57 %) but high specificity (100 %). ETV6/RUNX1 displayed discordance between fusion transcript assay and FISH results as well as rare non-specific binding in AL samples with normal or complex cytogenetics.
CONCLUSIONS: Our study presents preliminary data with high correlation between fusion transcript detection by a throughput automated multiplexed platform, compared to conventional karyotype/FISH technique for detection of chromosomal translocations in AL patients. Our preliminary observations, mandates further vast validation studies to explore automated molecular platforms in diagnostic pathology.

Li Y, Zhang R, Han Y, et al.
Comparison of the types of candidate reference samples for quality control of human epidermal growth factor receptor 2 status detection.
Diagn Pathol. 2016; 11(1):85 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Human epidermal growth factor receptor 2 (HER2) is as a target gene for trastuzumab in patients with breast cancer. Accurate determination of HER2 status and strict quality control are necessary to ensure reproducibility and accuracy of the techniques used for the determination of HER2 status.
METHODS: We used three different types of samples: formalin-fixed and paraffin-embedded (FFPE) samples prepared from cell lines, agarose gel samples using cell lines, and xenograft tumor samples. One cell line for FFPE or xenografts did not overexpress HER2, while the others showed different levels of HER2 overexpression. We compared the morphology, HER2 gene amplification status, and HER2 protein expression status of these samples with those of clinical specimens.
RESULTS: We successfully produced three kinds of samples for quality control. Cells from the cell line-sample sections were dispersed while those from the agarose gel-sample sections and xenograft tumor sample sections (prepared from the both cell lines) were concentrated in one area. The FISH results for all three kinds of samples were as expected. The IHC results of the cell line samples and xenograft tumor samples were as expected, but the staining level of the agarose gel samples, using HER2-overexpressed cell lines was weak which might be regarded as a false negative result.
CONCLUSIONS: Xenograft tumor samples might be used as an additional option for quality control in FISH and IHC. However, it might not replace the clinical specimen quality controls directly.

Savic S, Bubendorf L
Common Fluorescence In Situ Hybridization Applications in Cytology.
Arch Pathol Lab Med. 2016; 140(12):1323-1330 [PubMed] Related Publications
CONTEXT: - Fluorescence in situ hybridization (FISH) is a well-established method for detection of genomic aberrations in diagnostic, prognostic, and predictive marker testing.
OBJECTIVE: - To review common applications of FISH in cytology.
DATA SOURCES: - The published literature was reviewed.
CONCLUSIONS: - Cytology is particularly well suited for all kinds of FISH applications, which is highlighted in respiratory tract cytology with an increasing demand for predictive FISH testing in lung cancer. Fluorescence in situ hybridization is the gold standard for detection of predictive anaplastic lymphoma kinase gene (ALK) rearrangements, and the same evaluation criteria as in histology apply to cytology. Several other gene rearrangements, including ROS proto-oncogene 1 receptor tyrosine kinase (ROS1), are becoming clinically important and share the same underlining cytogenetic mechanisms with ALK. MET amplification is one of the most common mechanisms of acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and can be targeted by crizotinib. As genomic aberrations are a hallmark of malignant cells, FISH is a valuable objective ancillary diagnostic tool. In urinary tract cytology, atypical urothelial cells equivocal for malignancy are a common diagnostic dilemma and multitarget FISH can help clarify such cells. Diagnosis of malignant mesothelioma remains one of the most challenging fields in effusion cytology, and ancillary FISH is useful in establishing the diagnosis. Fluorescence in situ hybridization is a morphology-based technique, and the prerequisite for reliable FISH results is a targeted evaluation of the cells in question (eg, cancer or atypical cells). Cytopathologists and cytotechnicians should therefore be involved in molecular testing in order to select the best material and to provide their morphologic expertise.

Zhou AG, Liu Y, Cyr MS, et al.
Role of Tetrasomy for the Diagnosis of Urothelial Carcinoma Using UroVysion Fluorescent In Situ Hybridization.
Arch Pathol Lab Med. 2016; 140(6):552-9 [PubMed] Related Publications
CONTEXT: -UroVysion fluorescent in situ hybridization (FISH) is routinely used to detect urothelial carcinoma (UC). A positive threshold is defined as chromosome polysomy in 4 or more cells, which also includes tetrasomy, a natural product of cell division.
OBJECTIVES: -To evaluate tetrasomy for UC detection and explore the relation to the surgical diagnosis or patient history.
DESIGN: -The FISH was performed on 1532 urine samples from patients with cytology results and 4 or more years of follow-up. We created separate polysomy and tetrasomy categories and constructed receiver operating curves to determine appropriate thresholds using biopsy (n = 194) as the gold standard. Standard FISH and a novel assay integrating cytomorphology and FISH (Target-FISH) were compared. Matching tissue biopsies of urine samples with 10 or more tetrasomy cells were analyzed.
RESULTS: -No significant threshold was found for tetrasomy cells. Exclusion of tetrasomy from the polysomy category changed the threshold from 8.5 to 4.5 cells, increased specificity (59.2% to 78.9%), but reduced sensitivity (78.9% to 65.9%). In Target-FISH, the same approach yielded a specificity of 93.7% and sensitivity of 65.2%. Similarly, specificity improved significantly for low- and high-grade UC, but sensitivity decreased for low-grade UC. No evidence of UC was observed in 95% (52 of 55) of the patients referred for screening who had 10 or more tetrasomy cells by FISH. Matching biopsies for urines containing 10 or more tetrasomy cells showed few or no tetrasomy cells.
CONCLUSIONS: -Tetrasomy is a nonspecific finding frequently encountered in urine FISH and should be excluded from the polysomy classification. Target-FISH is an optimal approach, offering the ability to detect rare tetrasomy tumors.

Nabeshima K, Matsumoto S, Hamasaki M, et al.
Use of p16 FISH for differential diagnosis of mesothelioma in smear preparations.
Diagn Cytopathol. 2016; 44(9):774-80 [PubMed] Related Publications
Because most of malignant pleural mesothelioma (MPM) patients first present with pleural effusion, detection of mesothelioma cells on effusion smears is critical for early diagnosis. Recently, accumulating evidence indicating that the cytological diagnosis of MPM supported by ancillary techniques is as reliable as that based on histopathology has led to new guidelines for the cytopathologic diagnosis of MPM. Based on the guidelines, a combination of cytomorphological criteria and verification by ancillary techniques is required for the cytologic diagnosis of MPM. Detection of p16 homozygous deletion by fluorescence in situ hybridization (FISH) is the most reliable ancillary technique for differentiating MPM from reactive mesothelial cells (RMC) because of its relatively high sensitivity and extremely high specificity. We showed that the p16 deletion status of MPM cells in pleural effusions reflected that of the underlying invasive MPM tissues, indicating the usefulness of p16 FISH in effusion smear cytology for MPM diagnosis. Thus, for differentiating MPM from RMC, we propose to perform p16 FISH as often as possible. A positive p16 homozygous deletion supports the diagnosis of MPM. However, a negative result does not rule out the possibility of MPM. In such cases, a morphological assessment is critical. Therefore, we analyzed the morphological characteristics of p16 deletion-positive mesothelioma cells using a combination of virtual microscopy and p16 FISH, and identified three morphological characteristics useful for the differentiation, including cell-in-cell engulfment with or without hump formation, multinucleate cells, and larger berry-like cell aggregates. Diagn. Cytopathol. 2016;44:774-780. © 2016 Wiley Periodicals, Inc.

Sugita S, Asanuma H, Hasegawa T
Diagnostic use of fluorescence in situ hybridization in expert review in a phase 2 study of trabectedin monotherapy in patients with advanced, translocation-related sarcoma.
Diagn Pathol. 2016; 11:37 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Fluorescence in situ hybridization (FISH) is one of the most powerful genetic analysis tools for pathological diagnoses. FISH can detect various genetic abnormalities including gene translocation that was specifically found in translocation-related sarcomas (TRSs). Here, we report the use of FISH in expert review in a phase 2 study of trabectedin monotherapy for patients with advanced TRS.
METHODS: TRS patients (n = 76) were enrolled in the trial at 12 study sites after pathological diagnoses were made, including morphological examination with or without evidence of translocation by genetic testing. Following histological reviews of the representative specimens at the study sites, we performed immunohistochemistry using the appropriate antibodies and FISH for genetic confirmation of the tumor types in the expert review.
RESULTS: Among the 76 TRS cases, no split signal for SS18 probe was detected by FISH in three synovial sarcoma cases that were diagnosed at the study sites. Malignant peripheral nerve sheath tumor (MPNST) was diagnosed in two cases and sarcomatoid carcinoma in one. One of the cases was a small round cell variant of MPNST. After excluding these three cases, we assessed the other 73. There were no split signals detected in 7 of the 73 cases by FISH analysis, due to decalcification and hyperfixation procedures. Excluding these seven cases, FISH detected translocations in 95 % (63/66) of the study cases with a high sensitivity.
CONCLUSIONS: The diagnosis of TRS by FISH was highly sensitive and enabled genetic confirmation of the pathological diagnoses. We strongly recommend FISH as a confirmatory diagnostic test for TRS, which would enable the selection of patients with TRS in whom trabectedin is expected to be effective. This study was done in part that registered with Japan Pharmaceutical Information Center, number JapicCTI-121850.

Gao Y, Zhu Y, Zhang Z, et al.
Clinical significance of pancreatic circulating tumor cells using combined negative enrichment and immunostaining-fluorescence in situ hybridization.
J Exp Clin Cancer Res. 2016; 35:66 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Circulating tumor cells (CTCs) hold great potential in both clinical application and basic research for the managements of cancer. However, it remains to be an enormous challenge to obtain efficient detection of pancreatic CTCs. New detection platforms for the detection of pancreatic CTCs are urgently required.
METHODS: In the present study, we applied a newly-developed platform integrated subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) to analyze clinical significance of pancreatic CTCs. Immunostaining of CK, CD45, DAPI and FISH with the centromere of chromosome 8 (CEP8) were utilized to identify CTCs. Cells with features of CK+/CD45-/DAPI+/CEP8 = 2, CK+/CD45-/DAPI+/CEP8 > 2, CK-/CD45-/DAPI+/CEP8 > 2 were defined as pancreatic CTCs. The Kaplan-Meier method and Cox proportional hazards model were used to analyze the relationship of CTC level and other clinicopathological factors with pancreatic cancer clinical outcomes.
RESULTS: CTC count in pancreatic cancer was higher than healthy individuals (median, 3 vs. 0 per 7.5 ml; P < 0.001). SE-iFISH platform yielded a sensitivity of 88% and specificity of 90% in pancreatic cancer at the cutoff value of 2 cells/7.5 ml. Pancreatic cancer patients with lower CTC count (<3/7.5 ml) had substantially better overall survival (OS) compared with these with higher CTC count (≥3/7.5 ml) (15.2 vs. 10.2 months, P = 0.023). Multivariate analysis indicated that higher CTC count was a strong indicator for worse OS (HR = 4.547, P = 0.016).
CONCLUSION: Our current data showed that CTCs could be detected in pancreatic cancer patients in various stages, whether localized, locally advanced and metastatic. Besides, CTCs have shown the potential implication in predicting prognosis of pancreatic cancer.

Jonjić N, Mustać E, Tomić S, et al.
Acta Clin Croat. 2015; 54(4):479-85 [PubMed] Related Publications
Accurate assessment of HER-2 status is essential for identifying patients who will benefit from HER-2 targeted therapy. The aim of the present study was to show results on the concordance between local and central laboratory testing results in HER-2 positive breast cancer patients. In cases with discordant findings, the immunohistochemical (IHC) and/or in situ hybridization (FISH/SISH) analysis was performed in central laboratories. A total of 104 out of 143 (72.72%) breast carcinoma cases were HER-2 positive (score 3+), while nearly 14% of tumors (20/43) showed weak (score 2+) and 12% (19/143) negative IHC staining (score 0 and 1+). After repeated IHC and ISH, 88% (126/143) were classified as HER-2 positive and 12% (17/143) as HER-2 negative cases. The results obtained are in agreement with many studies that confirmed similar discordance in HER-2 testing by IHC and/or FISH between local and central laboratory. Thus, our findings as well as those from other studies support the importance of regular quality assessment of the staining procedures performed and consistency of interpretation of HER-2 test results.

Ferrara G, De Vanna AC
Fluorescence In Situ Hybridization for Melanoma Diagnosis: A Review and a Reappraisal.
Am J Dermatopathol. 2016; 38(4):253-69 [PubMed] Related Publications
Although conventional histopathological examination is the undisputable mainstay for the diagnosis of melanocytic skin neoplasms, fluorescence in situ hybridization (FISH) has the potential to provide important information to morphologically challenging cases. The standard melanoma FISH test targeting RREB1 (6p25), MYB (6q23), CCND1 (11q13), and centromere 6 is an effective compromise between cost, technical complexity, and sensitivity. The authors use the standard FISH-positivity as a tie-breaker for challenging melanocytic neoplasms mainly in a non-Spitzoid morphologic context because the currently available test leaves several unresolved issues: namely, a relatively low diagnostic accuracy in morphologically ambiguous melanocytic neoplasms; a relatively low sensitivity and specificity in Spitzoid neoplasms; and the occurrence of false positives due to tetraploidy in Spitz nevi and in nevi with an atypical epithelioid component. Under investigation is currently a new melanoma probe cocktail targeting RREB1 (6p25), C-MYC (8q24), CDKN2A (9p21), and CCND1 (11q13). However, CDKN2A is a significant parameter only if lost in homozygosis, and this complicates the interpretation of the results. Furthermore, the new melanoma probe cocktail has been tested on cases of atypical Spitzoid proliferations with fatal outcomes which at present are too few to allow definite conclusions. The authors propose the implementation of a FISH algorithm (standard 4-probe test followed by either C-MYC or CDKN2A/centromere 9) to assist the histopathological diagnosis and minimize the technical problems. Nevertheless, because the diagnostic accuracy of the FISH technique is far from being absolute, the overall clinicopathological context must always guide the decision-making process about the management of morphobiologically ambiguous melanocytic proliferations.

Acs B, Szekely N, Szasz AM, et al.
Reliability of immunocytochemistry and fluorescence in situ hybridization on fine-needle aspiration cytology samples of breast cancers: A comparative study.
Diagn Cytopathol. 2016; 44(6):466-71 [PubMed] Related Publications
INTRODUCTION: To characterize breast tumors and metastases, fine-needle aspiration cytology (FNAB) can be a favorable first choice. However, the diagnostic accuracy of ancillary tests applied to FNAB samples is yet to be validated.
PATIENTS AND METHODS: We examined 110 breast cancer patients' paired cytological and surgical resection specimens evaluated between 2005 and 2014. Comparison of ER and Her2 immunocytochemical (ICC) and immunohistochemical (IHC) staining and HER2 fluorescence in situ hybridization (FISH) was performed.
RESULTS: Significant difference (p < 0.001) and moderate correlation (κ = 0.446) were noted between results of 97 paired ICC an IHC reactions for ER expression. ICC for ER status had a sensitivity of 75%, specificity of 100%, positive predictive value (PPV) of 100%, and negative predictive value (NPV) of 38.2%. Significant difference (p = 0.012) and moderate correlation (κ = 0.541) were found between results of 77 paired ICC an IHC reactions for Her2 expression. The Her2 ICC had a sensitivity of 54%, specificity of 95.4%, PPV of 66.7%, and NPV of 92.6%. The results of FISH carried out on 23 paired samples of FNAB and surgical specimens indicated perfect correlation (κ = 1.000) and no significant difference (p = 1.000). FISH performed on FNAB has sensitivity, specificity, PPV, and NPV of 100%.
CONCLUSION: The correlation of ICC and IHC is moderate regarding ER and Her2 expression of the same tumor. FISH performed on FNAB samples is suitable to categorize primary and metastatic breast cancer in regard of HER2 gene amplification status and can be used as a predictive test in respect of therapies targeting HER2. Diagn. Cytopathol. 2016;44:466-471. © 2016 Wiley Periodicals, Inc.

Koczkodaj D, Popek S, Zmorzyński S, et al.
Detection of chromosomal changes in chronic lymphocytic leukemia using classical cytogenetic methods and FISH: application of rich mitogen mixtures for lymphocyte cultures.
J Investig Med. 2016; 64(4):894-8 [PubMed] Related Publications
One of the research methods of prognostic value in chronic lymphocytic leukemia (CLL) is cytogenetic analysis. This method requires the presence of appropriate B-cell mitogens in cultures in order to obtain a high mitotic index. The aim of our research was to determine the most effective methods of in vitro B-cell stimulation to maximize the number of metaphases from peripheral blood cells of patients with CLL for classical cytogenetic examination, and then to correlate the results with those obtained using fluorescence in situ hybridization (FISH). The study group involved 50 consecutive patients with CLL. Cell cultures were maintained with the basic composition of culture medium and addition of respective stimulators. We used the following stimulators: Pokeweed Mitogen (PWM), 12-O-tetradecanoylphorbol 13-acetate (TPA), ionophore, lipopolysaccharide (LPS), and CpG-oligonucleotide DSP30. We received the highest mitotic index when using the mixture of PWM+TPA+I+DSP30. With classical cytogenetic tests using banding techniques, numerical and structural aberrations of chromosomes were detected in 46 patients, and no change was found in only four patients. Test results clearly confirmed the legitimacy of using cell cultures enriched with the mixture of cell stimulators and combining classical cytogenetic techniques with the FISH technique in later patient diagnosing.

Lim TH, Lim AS, Thike AA, et al.
Implications of the Updated 2013 American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations on Human Epidermal Growth Factor Receptor 2 Gene Testing Using Immunohistochemistry and Fluorescence In Situ Hybridization for Breast Cancer.
Arch Pathol Lab Med. 2016; 140(2):140-7 [PubMed] Related Publications
CONTEXT: Human epidermal growth factor receptor 2 (HER2/neu) amplification is used as a predictive marker for trastuzumab treatment in breast cancer. Both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) testing algorithms have been based on the 2007 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. In late 2013, the guidelines were updated with new scoring criteria.
OBJECTIVE: To assess the impact of the revised ASCO/CAP recommendations on both IHC and FISH results by using the dual-color HER2/neu and centromeric FISH probes.
DESIGN: Retrospective analysis of 590 invasive carcinomas with concurrent IHC and dual-color HER2/neu and centromeric 17 (CEP17) FISH results, based on 2007 ASCO/CAP guidelines, was conducted from July 2011 to June 2013. With the revised guidelines, patients were recategorized and concordance rates between the 2 assays were recalculated.
RESULTS: Overall concordance rates for FISH and IHC decreased from 94.9% to 93.8% with reclassification. Negative FISH cases decreased from 79.1% to 69.3%. However, equivocal FISH cases were significantly increased from 0.7% to 9.5%, leading to more retesting. Both positive IHC and FISH cases were also noted to be increased, leading to more patients being eligible for trastuzumab treatment, especially those patients with concurrent HER2/neu and CEP17 polysomy. Approximately 1% of patients with initial FISH negative results were reclassified as having positive results when both the ratios and average copy number of HER2/neu were considered under the revised guidelines.
CONCLUSIONS: The revised 2013 ASCO/CAP guidelines can potentially lead to more patients being eligible for trastuzumab therapy but additional retesting is to be expected owing to an increased number of equivocal FISH cases.

Priya D, Kumar RV, Appaji L, et al.
Histological diversity and clinical characteristics of Ewing sarcoma family of tumors in children: A series from a tertiary care center in South India.
Indian J Cancer. 2015 Jul-Sep; 52(3):331-5 [PubMed] Related Publications
BACKGROUND: The Ewing sarcoma family of tumors (ESFT) are aggressive malignant tumors with small round cell morphology affecting mainly children and adolescents. The aim of this study is to classify the histological diversity and clinical characteristics of ESFT in children from a Tertiary Care Center in South India.
MATERIALS AND METHODS: This retrospective descriptive study includes 51 cases of ES in children aged below 15 years. Clinical details were collected from case files. Histomorphological features were reviewed and tumors were subtyped into classic, primitive neuroectodermal tumor (PNET) and atypical variants along with immunohistochemical markers, cytogenetics, and fluorescence in situ hybridization (FISH).
RESULTS: Fifty-three percent were female and 47% were male with mean age of 10 years. The most common site of involvement was skeletal involvement in 71%, followed by soft tissue involvement in 23%, and visceral involvement in 6%. Localized disease at presentation was seen in 44%, locally advanced disease in 28%, and metastatic disease in 28%. Recurrence was documented during follow-up in 18% of the cases. Histomorphologically, classic type was the most common (72%) followed by PNET (20%) category and atypical variant (8%). All cases were immunoreactive for CD99. Cytogenetic study in 12 cases showed translocation t(11;22) (q24;12) in 80% and variant translocations such as t(3;16), t(3;11) with nonspecific numerical abnormalities in 20%. FISH was carried out for documentation of four cases with atypical histomorphology.
CONCLUSION: ESFT had wide histological variation which required confirmation by ancillary studies.

Hwang HC, Pyott S, Rodriguez S, et al.
BAP1 Immunohistochemistry and p16 FISH in the Diagnosis of Sarcomatous and Desmoplastic Mesotheliomas.
Am J Surg Pathol. 2016; 40(5):714-8 [PubMed] Related Publications
The separation of sarcomatous and desmoplastic mesotheliomas from benign organizing pleuritis can be morphologically very difficult. Deletion of p16 (CDKN2A) by fluorescence in situ hybridization (FISH) testing appears to be a reliable marker of malignancy in mesothelial proliferations, and more recently it has been reported that, in this setting, loss of BAP1 by immunohistochemistry is only seen in malignant mesotheliomas. To determine how useful these tests are with sarcomatous and desmoplastic mesotheliomas, we examined 20 such tumors. Loss of BAP1 was seen in 3/20 (15%) and deletion of p16 by FISH was seen in 16/20 (80%) cases. Loss of one or the other marker was observed in 17/20 (85%). We also examined 13 sarcomatoid carcinomas, an important differential diagnosis of sarcomatoid mesotheliomas, and found that BAP1 was never lost, but p16 was deleted in 3/11 (27%). We conclude that: (1) BAP1 immunohistochemistry is relatively insensitive in the context of sarcomatous and desmoplastic mesotheliomas, but as a matter of time and cost efficiency may nonetheless be a useful first approach to the problem; (2) deletion of p16 by FISH is considerably more sensitive, but there remain a proportion of cases in which p16 is not deleted; (3) a small improvement in sensitivity can be achieved by using both markers; (4) in the context of a spindle cell malignant tumor in the pleura or peritoneum, which morphologically might be a metastatic sarcomatoid carcinoma or a mesothelioma, the finding of BAP1 loss favors mesothelioma, but p16 FISH cannot be used to separate sarcomatous mesotheliomas from sarcomatoid carcinomas.

Fan YS, Casas CE, Peng J, et al.
HER2 FISH classification of equivocal HER2 IHC breast cancers with use of the 2013 ASCO/CAP practice guideline.
Breast Cancer Res Treat. 2016; 155(3):457-62 [PubMed] Related Publications
The status of human epidermal growth factor receptor 2 (HER2, ERBB2) determines the eligibility of breast cancer patients to receive HER2-targeted therapy. The majority of HER2 testing in the U.S. is performed using a combination of immunohistochemistry (IHC) screening followed by fluorescence in situ hybridization (FISH) for IHC equivocal cases. In 2013, the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) updated the guideline for HER2 testing. This study evaluates the impact of the 2013 ASCO/CAP updated guideline on final HER2 FISH classification of breast cancers with an equivocal IHC result. For each case, we reported a FISH result according to the 2013 updated guideline and recorded a separated result using the 2007 guideline for investigational purpose. McNemar's test and Bowker's symmetry test were used to compare the classifications by the two guidelines. Among 172 HER2 IHC 2+ equivocal cases, use of the 2103 guideline changed classifications in 36 cases (21 %) when compared with the results expected by use of the 2007 guideline, and yielded a higher proportion of positive (28.5 vs. 23.3 %) and equivocal (16.3 vs. 4.1 %), and a lower proportion of negative (55.2 vs. 72.7 %) cases (p < 0.001). The major classification change with use of the updated guideline is from the HER2 FISH negative to equivocal in 26 cases (15 %). Our study has shown that implementation of the 2013 ASCO/CAP updated guideline has significant impact on HER2 classification for breast cancers with an equivocal HER2 IHC result and therefore increased the use of HER2-targeted therapy. Our data have also shown that reflex FISH is effective for final classification of the IHC equivocal cases and that polysomy 17 (CEP17 copy number ≥3/cell) is present in a significantly higher proportion of cases with an equivocal HER2 FISH classification.

Qu Y, Gu C, Wang H, et al.
Diagnosis of adults Xp11.2 translocation renal cell carcinoma by immunohistochemistry and FISH assays: clinicopathological data from ethnic Chinese population.
Sci Rep. 2016; 6:21677 [PubMed] Free Access to Full Article Related Publications
This study aimed to assess the utility of transcription factor E3 (TFE3) break-apart fluorescence in situ hybridization (FISH) assay in diagnosis of Xp11.2 translocation renal cell carcinoma (Xp11.2 RCC) and to compare the clinicopathological features between adult Xp11.2 RCC and non-Xp11.2 RCC. 76 pathologically suspected Xp11.2 RCCs were recruited from our institution. Both TFE3 immunohistochemistry (IHC) and TFE3 FISH assay were performed for the entire cohort. The progression-free survival (PFS) and overall survival (OS) curves were estimated using the Kaplan-Meier method. FISH analysis confirmed 30 Xp11.2 RCCs, including 28 cases with positive TFE3 immunostaining and 2 cases with negative immunostaining. The false-positive and false-negative rates were 6.7% (2/30) and 4.3% (2/46), respectively, for TFE3 IHC compared with FISH assay. Xp11.2 RCC was significantly associated with higher pathological stage and Fuhrman nuclear grade compared with non-Xp11.2 RCC (P < 0.05). The median PFS and OS for TFE3 FISH-positive group were 13.0 months (95% CI, 8.4-17.6 months) and 50.0 months (95% CI, 27.6-72.4 months), respectively, while the median PFS and OS had not been reached for TFE3 FISH-negative group. In conclusion, TFE3 break-apart FISH assay is a highly useful and standard diagnostic method for Xp11.2 RCC. Adult Xp11.2 RCC is clinically aggressive and often presents at advanced stage with poor prognosis.

Picanço-Albuquerque CG, Morais CL, Carvalho FL, et al.
In prostate cancer needle biopsies, detections of PTEN loss by fluorescence in situ hybridization (FISH) and by immunohistochemistry (IHC) are concordant and show consistent association with upgrading.
Virchows Arch. 2016; 468(5):607-17 [PubMed] Related Publications
The prognostic value of phosphatase and tensin homolog (PTEN) loss in prostate cancer has primarily been evaluated by either fluorescence in situ hybridization (FISH) or immunohistochemistry (IHC). Previously, we found that PTEN loss by IHC was associated with increased risk of upgrading from biopsy (Gleason 3 + 3) to prostatectomy (Gleason 7+). Now, using an evaluable subset of 111 patients with adjacent biopsy sections, we analyzed the association between PTEN deletion in cancer and the odds of upgrading by a highly sensitive and specific four-color FISH assay. We also compared the concordance of PTEN loss by IHC and PTEN deletion by FISH. PTEN deletion was found in 27 % (12/45) of upgraded cases compared with 11 % (7/66) of controls (P = 0.03). Cancers with PTEN deletions were more likely to be upgraded than those without deletions (adjusting for age odds ratio = 3.40, 95 % confidence interval 1.14-10.11). With respect to concordance, of 93 biopsies with PTEN protein detected by IHC, 89 (96 %) had no PTEN deletion by FISH, and of 18 biopsies without PTEN protein by IHC, 15 had homozygous or hemizygous PTEN deletion by FISH. Only 4 biopsies of the 93 (4 %) with PTEN protein intact had PTEN deletion by FISH. When the regions of uncertainty in these biopsies were systematically studied by FISH, intra-tumoral variation of PTEN deletion was found, which could account for variation in immunoreactivity. Thus, FISH provides a different approach to determining PTEN loss when IHC is uncertain. Both FISH and IHC are concordant, showing consistent positive associations between PTEN loss and upgrading.

Van Dyke DL, Werner L, Rassenti LZ, et al.
The Dohner fluorescence in situ hybridization prognostic classification of chronic lymphocytic leukaemia (CLL): the CLL Research Consortium experience.
Br J Haematol. 2016; 173(1):105-13 [PubMed] Free Access to Full Article Related Publications
This study revisited the Dohner prognostic hierarchy in a cohort of 1585 well-documented patients with chronic lymphocytic leukaemia. The duration of both time to first treatment (TTFT) and overall survival (OS) were significantly longer than observed previously, and this is at least partly due to improved therapeutic options. Deletion 13q remains the most favourable prognostic group with median TTFT and OS from fluorescence in situ hybridization (FISH) testing of 72 months and >12 years, respectively. Deletion 11q had the poorest median TTFT (22 months) and 17p deletion the poorest median OS (5 years). The percentages of abnormal nuclei were significantly associated with differential TTFT for the trisomy 12, 13q and 17p deletion cohorts but not for the 11q deletion cohort. From the date of the first FISH study, patients with >85% 13q deletion nuclei had a notably shorter TTFT (24 months). Patients with ≤20% 17p deletion nuclei had longer median TTFT and OS from the date of the first FISH study (44 months and 11 years), and were more likely to be IGHV mutated.

Parikh SA, Strati P, Tsang M, et al.
Should IGHV status and FISH testing be performed in all CLL patients at diagnosis? A systematic review and meta-analysis.
Blood. 2016; 127(14):1752-60 [PubMed] Related Publications
Since the first description of the natural history of chronic lymphocytic leukemia (CLL) by David Galton in 1966, the considerable heterogeneity in the disease course has been well recognized. The Rai and Binet staging systems described ∼40 years ago have proven to be robust prognostic tools. Over the past 2 decades, several novel biological, genetic, and molecular markers have been shown to be useful adjuncts to the Rai and Binet staging systems. In this systematic review, we examined the role of immunoglobulin heavy-chain variable region gene (IGHV) mutation status and genetic abnormalities determined by interphase fluorescence in situ hybridization (FISH) in patients with newly diagnosed CLL. The cumulative evidence presented in this systematic review is sufficient to recommend that FISH and IGHV be performed as standard clinical tests for all patients with newly diagnosed CLL in those countries with the resources to do so. In addition to clinical stage, these parameters could represent the minimal standard initial prognostic evaluation for patients with CLL. This approach will allow the application of powerful, recently developed prognostic indices (all of which are dependent on IGHV and FISH results) to all patients with newly diagnosed CLL.

Tang SM, Soong IS, Luk MY, et al.
Comparison of fluorescence in-situ hybridisation with dual-colour in-situ hybridisation for assessment of HER2 gene amplification of breast cancer in Hong Kong.
Hong Kong Med J. 2016; 22(2):144-51 [PubMed] Related Publications
OBJECTIVES: To compare the PathVysion fluorescence in-situ hybridisation assay with the INFORM HER2 Dual in-situ hybridisation assay on 104 invasive breast cancers with a broad spectrum of immunohistochemistry scores.
METHODS: This case series involved consecutive patients diagnosed with invasive breast carcinoma with equivocal immunohistochemistry score and referred for further HER2 assessment from the departments of Surgery and/or Clinical Oncology of the two hospitals between January 2013 and February 2014. An additional 10 cases with negative HER2 immunohistochemistry and 11 cases with positive HER2 immunohistochemistry were further included.
RESULTS: The results of both fluorescence in-situ hybridisation and dual in-situ hybridisation were available in 99 of 104 cases, respectively. Student’st test showed no statistically significant difference in the mean number of HER2 count, CEP17 copies, or HER2/CEP17 ratio between that obtained by fluorescence in-situ hybridisation and that obtained by dual in-situ hybridisation. Pearson’s correlation of results for the two assays was strong for HER2/CEP17 signal ratio (R=0.963, P<0.001) and mean HER2 copies per nucleus (R=0.897, P<0.001). Overall agreement was 96.0% (95 out of 99 cases, ĸ0.882). Three of the four discordant cases were equivocal for either fluorescence in-situ hybridisation or dual in-situ hybridisation. The results of immunohistochemistry 0/1+ and 3+ cases showed 100% concordance between the two assays. The failure rate was 0.96% for fluorescence in-situ hybridisation and 3.85% for dual in-situ hybridisation. Cases that failed for fluorescence in-situ hybridisation were successful for dual in-situ hybridisation and vice versa.
CONCLUSIONS: Our study showed that dual in-situ hybridisation is a reliable and useful option for HER2 testing in breast cancer.

Sarova I, Brezinova J, Zemanova Z, et al.
Molecular cytogenetic analysis of dicentric chromosomes in acute myeloid leukemia.
Leuk Res. 2016; 43:51-7 [PubMed] Related Publications
Dicentric chromosomes (DCs) have been described in many hematological diseases, including acute myeloid leukemia (AML). They are markers of cancer and induce chromosomal instability, leading to the formation of other chromosomal aberrations and the clonal evolution of pathological cells. Our knowledge of the roles and behavior of human DCs is often derived from studies of induced DCs and cell lines. It is difficult to identify all the DCs in the karyotypes of patients because of the limitations of metaphase cytogenetic methods. The aim of this study was to revise the karyotypes of 20 AML patients in whom DCs were found with conventional G-banding or multicolor fluorescence in situ hybridization (mFISH) with (multi)centromeric probes and to characterize the DCs at the molecular cytogenetic level. FISH analyses confirmed 23 of the 29 expected DCs in 18 of 20 patients and identified 13 others that had not been detected cytogenetically. Fourteen DCs were altered by other chromosomal changes. In conclusion, karyotypes with DCs are usually very complex, and we have shown that they often contain more than one DC, which can be missed with conventional or mFISH methods. Our study indicates an association between number of DCs in karyotype and very short survival of patients.

Hwang HC, Gown AM
Evaluation of Human Epidermal Growth Factor Receptor 2 (HER2) Gene Status in Human Breast Cancer Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Specimens by Fluorescence In Situ Hybridization (FISH).
Methods Mol Biol. 2016; 1406:61-70 [PubMed] Related Publications
Current standard of care requires that HER2 gene testing be performed on all newly diagnosed invasive breast cancers in order to determine eligibility for anti-HER2 antibody therapy and should be performed in accordance with current ASCO-CAP guidelines (Hammond et al., J Clin Oncol 29(15):e458, 2011; Wolff et al., J Clin Oncol 31(31):3997-4013, 2013). Here we describe a HER2 FISH methodology to evaluate HER2 gene status in FFPE breast tumor specimens.

Min L, Shou C
In Situ Hybridization of Breast Cancer Markers.
Methods Mol Biol. 2016; 1406:53-9 [PubMed] Related Publications
In situ hybridization is an important technique in breast cancer research, which is widely applied in detection of specific nucleic acid sequences. Here, we describe the detailed protocol of fluorescence in situ hybridization and chromogenic in situ hybridization in detection of gene HER2/neu amplification in breast cancer tissues.

Pelosi G, Perrone F, Tamborini E, et al.
Doing more with less: fluorescence in situ hybridization and gene sequencing assays can be reliably performed on archival stained tumor tissue sections.
Virchows Arch. 2016; 468(4):451-61 [PubMed] Related Publications
Little is known about molecular testing on tumor tissue retrieved from stained sections, for which there may be a clinical need. We retrospectively analyzed 112 sections from 56 tumor patients using either fluorescence in situ hybridization (FISH) with different probes (19 sections from 17 patients) or Sanger or targeted next generation sequencing for detection of BRAF, EGFR, KRAS, C-KIT, and TP53 mutations (93 sections from 39 patients). Tumor tissue sections had been stained by hematoxylin and eosin (H&E) (42 sections) or by immunohistochemistry for cytoplasmic or nuclear/nuclear-cytoplasmic markers (70 sections) with a peroxidase (P-IHC, with 3,3'-diaminobenzidine as chromogen) or alkaline phosphatase label (AP-IHC, with Warp Red™ as chromogen). For FISH analysis, the concordance rate between the original diagnosis and that obtained on H&E- or P-IHC-stained tissue sections (AP-IHC was not on record for this set of patients) was 95% (18 out of 19 tumor sections). Only one tumor sample, diffusely positive for MLH1, did not yield any nuclear hybridization signal. For sequencing analysis, the concordance rate was 100% on negative P-IHC and positive AP-IHC-stained sections, regardless of the subcellular localization of the reaction product. Mutations were detected in only 52% of cases expressing nuclear/nuclear-cytoplasmic markers, regardless of the sequencing technology used (p = 0.0002). In conclusion, stained sections may be a valuable resource for FISH or sequencing analysis, but on cases expressing nuclear markers sequencing results need to be interpreted cautiously.

Wesoła M, Jeleń M
A Comparison of IHC and FISH Cytogenetic Methods in the Evaluation of HER2 Status in Breast Cancer.
Adv Clin Exp Med. 2015 Sep-Oct; 24(5):899-903 [PubMed] Related Publications
The HER2 gene is responsible for the formation of the HER2 receptor on the surface of epithelial cells. Increased numbers of this receptor are associated with a worse prognosis in cancer. Increased numbers of copies of the HER2 gene occur in about 20-30% of breast cancer patients, so determining HER2 receptor levels is important in the current diagnosis and treatment of breast cancer. One diagnostic technique is the immunohistochemical (IHC) method, which permits indirect measurement of overexpression of HER2 receptors, based on subjective determination of the intensity of the color reaction. Another technique is the use of fluorescent in situ hybridization (FISH), which permits the exact number of copies of the HER2 gene to be specified. Based on the results of FISH tests, patients can be qualified for treatment with antibodies that partially block HER2 receptors. This treatment causes inhibition of tumor growth signals. Determining the HER2 status in breast cancer with the FISH method allows the further progress of the disease to be predicted, the right treatment to be chosen and the response to the treatment to be foreseen. Because of the widespread use of the FISH and IHC methods, comparing the advantages and disadvantages of these two methods seems to be relevant.

Hopley R, Gillespie A
Cervical FISH Testing for Triage and Support of Challenging Diagnoses: A Case Study of 2 Patients.
Lab Med. 2016; 47(1):52-6 [PubMed] Related Publications
PATIENTS: A 29-year-old Caucasian woman (patient 1) and a 23-year-old Caucasian woman (patient 2).Chief Complaints: Abnormal Pap tests with high risk HPV positivity.

Shigeto S, Matsuda K, Yamaguchi A, et al.
Rapid diagnosis of acute promyelocytic leukemia with the PML-RARA fusion gene using a combination of droplet-reverse transcription-polymerase chain reaction and instant-quality fluorescence in situ hybridization.
Clin Chim Acta. 2016; 453:38-41 [PubMed] Related Publications
BACKGROUND: Acute promyelocytic leukemia (APL) with the PML-RARA fusion gene can be effectively cured using molecular-targeted therapies, which require both detection and quantification of the PML-RARA fusion gene. Here, we developed a rapid assay for identifying and measuring the PML-RARA fusion gene in patients with APL using droplet-reverse transcription-polymerase chain reaction (droplet-RT-PCR) and instant quality-fluorescence in situ hybridization (IQ-FISH).
METHODS: RNA for droplet-RT-PCR and fixed-cell suspensions for IQ-FISH were prepared from five patients with APL and three controls. We evaluated the amplification efficiency and reaction time with droplet-RT-PCR and signal clarity and hybridization time with IQ-FISH.
RESULTS: The reaction using droplet-RT-PCR was completed in 26min. The PML-RARA fusion gene was detected in all samples from the five patients. IQ-FISH yielded clear signals after 1h of hybridization. There were no significant differences in signal clarity or positive signal ratios between IQ-FISH and conventional FISH.
CONCLUSIONS: Simultaneous droplet-RT-PCR and IQ-FISH, in addition to morphological examination of blood smears, can be used to diagnose patients as having APL within 4h based on molecular/cytogenetic results. Rapid diagnosis can allow effective therapies to be started promptly.

Mehrazma M, Kalantari E, Rezvani H, et al.
Chromogenic In Situ Hybridisation Test for Breast Cancer Patients with Equivocal IHC Results--a Study from Iran.
Asian Pac J Cancer Prev. 2015; 16(17):7695-700 [PubMed] Related Publications
BACKGROUND: HER2/neu overexpression on cell membranes of breast cancer cells is due to HER2/neu gene amplification and it is important to identify potential candidates for anti HER2 therapy with trastuzumab. IHC, FISH and CISH are standard FDA approved assays currently used to determine HER2 status in routine practice. The aim of this study was to determine HER2 gene amplification, using the CISH method in breast carcinoma samples which had IHC +2 reactions.
MATERIALS AND METHODS: This study was conducted from 2008- 2010 using 334 consecutive breast carcinoma samples referred from local laboratories to Mehr Hospital. CISH assays were performed for all cases, and IHC tests were also done for determining efficacy and accuracy of local labs. HER2 status in local IHC tests was compared with central IHC and CISH results.
RESULTS: Of 334 breast cancer patients, 16 were negative for HER2 IHC (0, +1), 201 cases were equivocal (+2), and 31 positive (+3). Of 334 referral cases, 88 were CISH positive (26.3%) and 246 were CISH negative (73.7%). Of 201 IHC +2 cases, HER2 gene amplification was observed in 42 cases (kappa: 0.42). A 29.9% concordance was found between local IHC and central IHC. Sensitivity and specificity of local IHC were 90% and 53.8%, respectively.
CONCLUSIONS: Low accuracy of IHC results in local labs was associated with the following factors: using former FDA-approved criteria for HER2 interpretation, utilizing non-validated kits, and lack of any quality assurance program. Therefore, following the new 2014 ASCO/CAP guideline and comprehensive quality assurance should be implemented to ensure accuracy of HER2 testing.

Zeng M, Yang Z, Hu X, et al.
Grb7 gene amplification and protein expression by FISH and IHC in ovarian cancer.
Int J Clin Exp Pathol. 2015; 8(9):11296-304 [PubMed] Free Access to Full Article Related Publications
OBJECTIVE: Overexpression of growth factor receptor-bound protein 7 (Grb7) has been found in numerous human cancers. The aim of this study was to evaluate the correlation between Grb7 gene amplification and protein expression in ovarian cancer (OC).
METHODS: We use Tissue Microarray (TMA) respectively to detect the gene amplification and protein expression of Grb7 in 90 cases OC and 10 control specimens of normal ovarian tissues by IHC and FISH.
RESULTS: The Grb7 protein expression by IHC analysis was observed in 52/90 (57.8%) OC with 3 cases (3.3%) scored 3(+) and 9 cases (10%) scored 2(+) Grb7 gene amplification by FISH analysis was successfully detectable in 6 specimens with a positive rate of 6.8% (6/88) in which immunostaining 3(+), 2(+) and negative (1(+)/0) expressions of Grb7 were 100.0% (3/3), 11.1% (1/9) and 2.6% (2/76), respectively. Our data exhibited that the IHC and FISH results had a good consistency between Grb7 gene amplification and Grb7 protein expression (Kappa = 0.651, P < 0.001). Both the results of IHC and FISH revealed that Grb7 did not seem to have a role in OC clinicopathology.
CONCLUSION: There is a close relationship between Grb7 gene amplification and GRB7 protein overexpression in human OC. IHC might have limited diagnostic value especially in these tumors and especially in characterizing genetically diverse borderline cases, FISH could be superior to IHC.

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