Assessment of the HER2 status in breast cancer by fluorescence in situ hybridization: a technical review with interpretive guidelines

Diagnostic assays for HER2 in breast cancer provide important prognostic information and independently help guide management by identifying patients who are the most likely to benefit from Herceptin-targeted therapy. The biological events underlying HER2 -driven breast cancer that can be assessed in routine clinical specimens include the evaluation of gene amplification by fluorescence in situ hybridization (FISH), enhanced messenger RNA expression by real-time polymerase chain reaction, and the assessment of protein overexpression at the tumor cell membrane by immunohistochemistry (IHC). Immunohistochemistry and FISH methodologies have the advantage of being morphologically driven, allowing for correlations between HER2 expression and morphologic features. However, each has important advantages and disadvantages, which are discussed in detail. Although immunohistochemistry is familiar and readily accommodated in most surgical pathology laboratories, increasing demands for FISH testing in the clinical setting will require greater familiarity with the technical aspects of FISH assays and their interpretation by the greater laboratory community. In this review, we provide an overview of FISH testing for HER2 in breast cancer, with an emphasis on technical considerations, interpretive guidelines, scoring criteria, and quality control. The development of automated platforms for hybridization, image analysis for signal enumeration, and experience with FISH interpretation should broaden the availability of this technology for clinical diagnostic testing.     

Molecular diagnostic techniques for the clinical evaluation of gliomas.

Newly developed molecular techniques have been integrated into the routine assessment of gliomas in some laboratories. These tests serve to complement the subjective nature of morphologic analysis. Such strategies add useful information regarding pathogenicity, patient survival, and potential response to treatment. As we learn more about the molecular characteristics of these tumors, this information will provide the basis for the development of specific, targeted therapies. This review will describe the background, methods, clinical utility, and strengths and weaknesses of several molecular approaches, including fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), loss of heterozygosity (LOH)-testing, and nucleic acid sequencing, that are currently being employed in the diagnosis and evaluation of glial tumors.     

Characterization of sSMC by FISH and molecular techniques

Small supernumerary marker chromosome (sSMC) is a structurally altered additional chromosome that may not be explicitly clarified by conventional karyotyping alone. About one third of sSMC carriers have abnormal phenotypes and its clinical correlation is difficult, especially in prenatal studies. The present study was aimed at characterizing 19 sSMC identified in 15 patients with dysmorphic features with or without multiple congenital anomalies, conspicuous family history, short stature and/or ambiguous genitalia. All the sSMC were primarily identified by routine cytogenetics studies (performed with banding techniques) from peripheral blood except in one patient, where amniotic fluid was used. All sSMCs were further characterized by array-CGH (using 44?K oligonucleotide probe) and/or fluorescence in situ hybridization (FISH) using multicolor banding (MCB), centromere specific multicolor FISH (cenM-FISH), subcentromere-specific multicolor FISH (subcenM-FISH), micro-dissection and/or reverse FISH. This report demonstrates the worth of advanced molecular (cyto)genetic techniques in characterizing sSMC, their utility in genotype-phenotype correlation and risk of clinical presentation.     

Tissue microarray technology in the routine assessment of HER-2 status in invasive breast cancer: a prospective study of the use of immunohistochemistry and fluorescence in situ hybridization

Aims:  To compare tissue microarray (TMA) and whole-section (WS) techniques in the routine assessment of HER-2 status in invasive breast cancer by immunohistochemistry and fluorescence in situ hybridization (FISH).
Methods and results:  HER-2 status was assessed prospectively in 106 consecutive cases of excised high-grade and/or node-positive breast carcinoma using both WS- and TMA-based techniques. Whole sections were assessed by immunohistochemistry with FISH being performed on equivocal cases (scoring 2+ on Hercep^®Test) and randomly selected 3+ cases included for quality assurance. Five 0.6-mm cores from each tumour allowed accurate immunohistochemical and FISH testing in >95% of cases. Ninety-seven per cent concordance was achieved between WS and TMA approaches to FISH analysis, the only discrepancies being in cases that were borderline or near borderline by both techniques. TMA and WS approaches were comparable in terms of time for preparation and scoring.
Conclusions:  TMA technology is a robust method of assessing HER-2 status in invasive breast cancer. This is directly comparable to the current standard methodology using whole sections. The use of TMA technology offers several advantages over existing full-section methods in terms of cost, quality control, facilitation of future research and the facility to provide a high-throughput testing methodology.     

Comparison of different techniques for the detection of genetic risk-identifying chromosomal gains and losses in neuroblastoma

Neuroblastoma (NB) is a pediatric neoplasia that shows complex combinations of acquired genetic aberrations. The specific genes and the molecular mechanisms responsible for development and progression of NB remain poorly understood. Our main objective is to compare the results obtained with different techniques for the detection of genomic data in 20 patients with NB using the information obtained to select the appropriate technique in routine analysis for the therapeutic stratification. The genetic methods used in this study are multiprobe fluorescence in situ hybridization (FISH) assay, metaphasic comparative genomic hybridization (mCGH), array comparative genomic hybridization (aCGH), and the multiplex ligation-dependent probe amplification (MLPA). Genomic copy number abnormalities were used to group the cases in four categories: MYCN amplification cases; 11q deletion tumors; cases with partial chromosome gains or losses and samples with entire chromosome alterations. The data obtained from the multigenomic techniques showed a high degree of concordance and our findings support the hypothesis that NB consists of biologically distinct subgroups that differ by genetic characteristics of prognostic relevance. FISH will be essential for the mandatory study of MYCN status. The use of MLPA as routine technique is an advantage procedure for detecting the implication of the common genetic alterations in NB.     

Fluorescence In Situ Hybridization (FISH) in Diagnostic and Investigative Neuropathology

Over the last decade, fluorescence in situ hybridization (FISH) has emerged as a powerful clinical and research tool for the assessment of target DNA dosages within interphase nuclei. Detectable alterations include aneusomies, deletions, gene amplifications, and translocations, with primary advantages to the pathologist including its basis in morphology, its applicability to archival, formalin-fixed paraffin-embedded (FFPE) material, and its similarities to immunohistochemistry. Recent technical advances such as improved hybridization protocols, markedly expanded probe availability resulting from the human genome sequencing initiative, and the advent of high-throughput assays such as gene chip and tissue microarrays have greatly enhanced the applicability of FISH. In our lab, we currently utilize only a limited battery of DNA probes for routine diagnostic purposes, with determination of chromosome 1p and 19q dosage in oligodendroglial neoplasms representing the most common application. However, research applications are numerous and will likely translate into a growing list of clinically useful markers in the near future. In this review, we highlight the advantages and disadvantages of FISH and familiarize the reader with current applications in diagnostic and investigative neuropathology.     

Standardization of fluorescence in situ hybridization studies on chronic lymphocytic leukemia (CLL) blood and marrow cells by the CLL Research Consortium

Five laboratories in the Chronic Lymphocytic Leukemia (CLL) Research Consortium (CRC) investigated standardizing and pooling of fluorescence in situ hybridization (FISH) results as a collaborative research project. This investigation used fixed bone marrow and blood cells available from previous conventional cytogenetic or FISH studies in two pilot studies, a one-day workshop, and proficiency test. Multiple FISH probe strategies were used to detect 6q-, 11q-, +12, 13q-, 17p-, and IGH rearrangements. Ten specimens were studied by participants who used their own probes (pilot study 1). Of 312 FISH interpretations, 224 (72%) were true-negative, 74 (24%) true-positive, 6 (2%) false-negative, and 8 (3%) false-positive. In pilot study no. 2, each participant studied two specimens using identical FISH probe sets to control for variation due to probe sets and probe strategies. Of 80 FISH interpretations, no false interpretations were identified. At a subsequent workshop, discussions produced agreement on scoring criteria. The proficiency test that followed produced no false-negative results and 4% (3/68) false-positive interpretations. Interpretation disagreements among laboratories were primarily attributable to inadequate normal cutoffs, inconsistent scoring criteria, and the use of different FISH probe strategies. Collaborative organizations that use pooled FISH results may wish to impose more conservative empiric normal cutoff values or use an equivocal range between the normal cutoff and the abnormal reference range to eliminate false-positive interpretations. False-negative results will still occur, and would be expected in low-percentage positive cases; these would likely have less clinical significance than false positive results. Individual laboratories can help by closely following rigorous quality assurance guidelines to ensure accurate and consistent FISH studies in their clinical practice and research.     

External quality assurance of HER2 fluorescence in situ hybridisation testing: results of a UK NEQAS pilot scheme

Background and AIMS: Trastuzumab provides clinical benefit for advanced and early breast cancer patients whose tumours over-express or have gene amplification of the HER2 oncogene. The UK National External Quality Assessment Scheme (NEQAS) for immunohistochemical testing was established to assess and improve the quality of HER2 immunohistochemical testing. However, until recently, no provision was available for HER2 fluorescence in situ hybridisation (FISH) testing. A pilot scheme was set up to review the performance of FISH testing in clinical diagnostic laboratories. METHODS: FISH was performed in 6 reference and 31 participating laboratories using a cell line panel with known HER2 status. RESULTS: Using results from reference laboratories as a criterion for acceptable performance, 60% of all results returned by participants were appropriate and 78% either appropriate or acceptable. However, 22.4% of results returned were deemed inappropriate, including 13 cases (4.2%) where a misdiagnosis would have been made had these been clinical specimens. CONCLUSIONS: The results of three consecutive runs show that both reference laboratories and a proportion of routine clinical diagnostic (about 25%) centres can consistently achieve acceptable quality control of HER2 testing. Data from a significant proportion of participating laboratories show that further steps are required, including those taken via review of performance under schemes such as NEQAS, to improve quality of HER2 testing by FISH in the "real world".     

Automation of manual components and image quantification of direct dual label fluorescence in situ hybridization (FISH) for HER2 gene amplification: A feasibility study.

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

Subtelomeric rearrangements in the mentally retarded: a comparison of detection methods

In recent years, subtelomeric rearrangements, e.g., chromosome deletions or duplications too small to be detected by conventional cytogenetic analysis, have emerged as a significant cause of both idiopathic and familial mental retardation. As mental retardation is a common disorder, many patients need to be tested on a routine basis. In this review, we will discuss the different methods that have been applied in laboratories worldwide, including multiprobe fluorescence in situ hybridization (FISH), multiallelic marker analysis, multiplex amplifiable probe hybridization (MAPH), multiplex ligation-dependent probe amplification (MLPA), quantitative real-time PCR, comparative genomic hybridization (CGH), and multicolor FISH, including spectral karyotyping (SKY), subtelomeric combined binary ratio labeling FISH (S-COBRA FISH), multiplex FISH telomere integrity assay (M-TEL), telomeric multiplex FISH (TM-FISH), and primed in situ labeling (PRINS).     

Comparison of HLA-A antigen typing by serology with two polymerase chain reaction based DNA typing methods: implications for proficiency testing

Serology has been routinely used for class I HLA typing for the selection of donors for allotransplantation. However, serology is not adequate for the assignment of all class I specificities especially when testing non-Caucasians subjects and it is necessary to adopt new strategies for routine testing. At the present time the extent of incorrect serologic HLA-A assignments in clinical testing is not known. The polymerase chain reaction (PCR) based techniques have become useful standard clinical typing methods of HLA class II alleles but most laboratories still use serology for class I typing. In this report we have compared two PCR based techniques, PCR amplification with sequencespecific primers (PCR-SSP) and PCR amplification and subsequent hybridization with sequence-specific oligonucleotide probes (PCR-SSOP), for the assignment of HLA-A specificities in 56 blood samples from patients and families serologically typed for HLA-A. This side-by-side comparison of PCR methods showed 100% correlation between them. However, serology showed 7.1% misassignments and, in an additional panel of 19 cells where serology produced equivocal results, the PCR-SSP and SSOP methods identified the correct HLA-A specificity. Our results emphasize the need to complement routine serologic testing of HLA specificities with a small number of primers designed to test HLA-A34, A36, A43, A66, A74 and A80, that are not detected with high precision by serology. We concluded that the PCR-SSP and -SSOP methods can be used in routine HLA-A typing of patients and donors for transplantation with a greater precision than serology.     

Fluorescence in situ hybridization (FISH) in the microbiological diagnostic routine laboratory: a review

Early identification of microbial pathogens is essential for rational and conservative antibiotic use especially in the case of known regional resistance patterns. Here, we describe fluorescence in situ hybridization (FISH) as one of the rapid methods for easy identification of microbial pathogens, and its advantages and disadvantages for the diagnosis of pathogens in human infections in the laboratory diagnostic routine. Binding of short fluorescence-labeled DNA or nucleic acid-mimicking PNA probes to ribosomes of infectious agents with consecutive analysis by fluorescence microscopy allows identification of bacterial and eukaryotic pathogens at genus or species level. FISH analysis leads to immediate differentiation of infectious agents without delay due to the need for microbial culture. As a microscopic technique, FISH has the unique potential to provide information about spatial resolution, morphology and identification of key pathogens in mixed species samples. On-going automation and commercialization of the FISH procedure has led to significant shortening of the time-to-result and increased test reliability. FISH is a useful tool for the rapid initial identification of microbial pathogens, even from primary materials. Among the rapidly developing alternative techniques, FISH serves as a bridging technology between microscopy, microbial culture, biochemical identification and molecular diagnostic procedures.     

Comparison of high resolution chromosome banding and fluorescence in situ hybridization (FISH) for the laboratory evaluation of Prader-Willi syndrome and angelman syndrome

The development of probes containing segments of DNA from chromosome region 15q11-q13 provides the opportunity to confirm the diagnosis of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) by fluorescence in situ hybridization (FISH). We have evaluated FISH studies and high resolution chromosome banding studies in 14 patients referred to confirm or rule out PWS and five patients referred to confirm or rule out AS. In four patients (three from the PWS category and 1 from the AS group) chromosome analysis suggested that a deletion was present but FISH failed to confirm the finding. In one AS group patient, FISH identified a deletion not detectable by high resolution banding. Review of the clinical findings in the discrepant cases suggested that the FISH results were correct and high resolution findings were erroneous. Studies with a chromosome 15 alpha satellite probe (D15Z) on both normal and abnormal individuals suggested that incorrect interpretation of chromosome banding may occasionally be attributable to alpha satellite polymorphism but other variation of 15q11-q13 chromosome bands also contributes to misinterpretation. We conclude that patients who have been reported to have a cytogenetic deletion of 15q11-q13 and who have clinical findings inconsistent with PWS and AS should be reevaluated by molecular genetic techniques. © 1994 Wiley-Liss, Inc.     

Microarray-based genomic profiling as a diagnostic tool in acute lymphoblastic leukemia

In acute lymphoblastic leukemia (ALL) specific genomic abnormalities provide important clinical information. In most routine clinical diagnostic laboratories conventional karyotyping, in conjunction with targeted screens using e.g., fluorescence in situ hybridization (FISH), is currently considered as the gold standard to detect such aberrations. Conventional karyotyping, however, is limited in its resolution and yield, thus hampering the genetic diagnosis of ALL. We explored whether microarray-based genomic profiling would be feasible as an alternative strategy in a routine clinical diagnostic setting. To this end, we compared conventional karyotypes with microarray-deduced copy number aberration (CNA) karyotypes in 60 ALL cases. Microarray-based genomic profiling resulted in a CNA detection rate of 90%, whereas for conventional karyotyping this was 61%. In addition, many small (< 5 Mb) genetic lesions were encountered, frequently harboring clinically relevant ALL-related genes such as CDKN2A/B, ETV6, PAX5, and IKZF1. From our data we conclude that microarray-based genomic profiling serves as a robust tool in the genetic diagnosis of ALL, outreaching conventional karyotyping in CNA detection both in terms of sensitivity and specificity. We also propose a practical workflow for a comprehensive and objective interpretation of CNAs obtained through microarray-based genomic profiling, thereby facilitating its application in a routine clinical diagnostic setting.     

Venous thromboembolism laboratory testing (factor V Leiden andfactor II c.*97G>A), 2018 update: a technical standard of the American College of Medical Genetics and Genomics (ACMG)

Factor V Leiden and factor II c.*97G>A (formerly referred to asprothrombin 20210G>A) are the two most common genetic variants associated withvenous thromboembolism (VTE). Testing for these variants is one of the most commonreferrals in clinical genetics laboratories. While the methodologies for testingthese two variants are relatively straightforward, the clinical implementation canbe complicated with regard to test indications, risk assessment of occurrence andrecurrence of VTE, and related genetic counseling. This document provides anoverview of VTE, information about the variants and their influence on risk,considerations before initiating genetic testing, and the clinical and analyticalsensitivity and specificity of the tests. Key information that should be included inthe laboratory report is also provided. Disease-specific statements are intended toaugment the general American College of Medical Genetics and Genomics (ACMG)technical standards for clinical genetics laboratories. Individual laboratories areresponsible for meeting the Clinical Laboratory Improvement Amendments(CLIA)/College of American Pathologists (CAP) quality assurance standards withrespect to appropriate sample documentation, assay validation, general proficiencytesting, and quality control measures. This 2018 edition of the ACMG technicalstandard updates and supersedes the 2005 edition on this topic. It is designed to bea checklist for genetic testing professionals who are already familiar with thedisease and the methods of analysis.     

Recommendations for reporting results of diagnostic genetic testing (biochemical,cytogenetic and molecular genetic)

Genetic test results can have considerable importance for patients, their parents and more remote family members. Clinical therapy and surveillance, reproductive decisions and genetic diagnostics in family members, including prenatal diagnosis, are based on these results. The genetic test report should therefore provide a clear, concise, accurate, fully interpretative and authoritative answer to the clinical question. The need for harmonizing reporting practice of genetic tests has been recognised by the External Quality Assessment (EQA), providers and laboratories. The ESHG Genetic Services Quality Committee has produced reporting guidelines for the genetic disciplines (biochemical, cytogenetic and molecular genetic). These guidelines give assistance on report content, including the interpretation of results. Selected examples of genetic test reports for all three disciplines are provided in an annexe.Diagnostic genetic testing is an extremely rapidly expanding area encompassing a broad range of laboratory investigations to analyse chromosomes (from classical karyotype to molecular cytogenetics), nucleic acids (DNA, RNA), proteins and metabolites used to detect heritable or somatic mutations, genotypes or phenotypes related to disease and health. Genetic testing requires particular consideration in that it is usually performed only once in a patient''s lifetime, and the results may have considerable importance for lifetime decisions not only for the individuals being tested but also for children and family. Interpreting and reporting variation in germline chromosomes, DNA sequences or their products is a heavy clinical responsibility for prediction of susceptibility to disease, patient diagnosis, prognosis, counselling, treatment or family planning. Providing a set of reporting frameworks that can be customised for different testing contexts but share some common principles could be beneficial to the practice of a number of laboratories, including non-OECD members and/or laboratories that do not participate in External Quality Assessments (EQA), and to laboratories with blurred boundaries between research and genetic testing services.Although several guidelines already exist for reporting the results of genetic testing,^1,2,3 these focus on molecular genetic testing and do not cover the other two branches of laboratory genetics, namely biochemical genetics and cytogenetics. Based on recent surveys of EQA results presented by some European EQA providers and the request from genetic laboratories for comprehensive reporting guidelines, it was considered that a unifying attempt to harmonise the reporting practice of genetic tests in Europe and neighbouring countries would be welcome.     

La CGH array : un bouleversement de la pratique hospitalière en cytogénétique

The advent of comparative genomic hybridization on DNA chips or CGH-arrays is changing drastically the present clinical practice in the detection and diagnosis of human chromosome abnormalities. Several studies have shown that, using classical and molecular cytogenetics techniques, a chromosome abnormality is observed in at least 15% of mentally-retarded patients with or without a congenital malformation (2–3% of the general population). Also, this technology contributes to the nosology and to our understanding of the molecular basis of numerous malignancies, improving therefore their diagnosis and opening up new avenues for drugs discovery. Today, still limited to some research laboratories, this novel technology is establishing itself as the method of choice to detect and diagnose human constitutional and acquired chromosome abnormalities observed in hospital-based laboratories. In the present review, we are explaining the basic principles of CGH-arrays, showing its main place in the armamentarium of cytogenetic techniques, stating its indications and limitations in the detection and the diagnosis of human chromosome abnormalities and describing the drastic changes induced by its application into hospital-based laboratories.     

Identification of Acinetobacter isolates in the A. calcoaceticus-A. baumannii complex by restriction analysis of the 16S-23S rRNA intergenic-spacer sequences.

Members of the genus Acinetobacter are reported to be involved in hospital-acquired infections with an increasing frequency. However, clinical laboratories still lack simple methods that allow complete identification of some pathogenic species, i.e., those corresponding to A. baumannii (DNA group or genospecies 2), unnamed genospecies 3 and 13, and two new genospecies that have recently been described. In fact, a complete discrimination between these species is possible only by DNA-DNA hybridization or ribotyping. Both of these techniques are complex and time-consuming and cannot be performed in most clinical laboratories. As a consequence, isolates belonging to these genospecies are often not differentiated and included, together with the environmental genospecies 1, in the A. calcoaceticus-A. baumannii complex. In this report, a simple and rapid method for the identification of the genospecies belonging to the A. calcoaceticus-A. baumannii complex is proposed. It is based on the combined digestion by the restriction endonuclease AluI and NdeII of the DNA fragments resulting from the amplification of the 16S-23S rRNA intergenic spacer sequences. The analysis of 36 strains characterized by DNA-DNA hybridization in previous studies showed that the restriction profiles obtained are highly reproducible and characteristic for each genospecies. Moreover, extending this study to 68 clinical strains, which were assigned to the A. calcoaceticus-A. baumannii complex by phenotypic tests, confirmed the existence of a panel of limited and well-conserved restriction patterns and allowed the identification of the strains tested.(ABSTRACT TRUNCATED AT 250 WORDS)