FISH Analysis for the Detection of Lymphoma-Associated Chromosomal Abnormalities in Routine Paraffin-Embedded Tissue

Abstract

Over the last decade, fluorescence in situ hybridization (FISH) has become a firmly established technique in the diagnosis and assessment of lymphoid malignancies. However, this technique is not wide-ly used in the routine diagnostic evaluation of paraffin-embedded biopsies, most likely because of a perception that it is technically more demanding. There are also uncertainties regarding diagnostic thresholds and the way in which results should be interpreted. In this Review, we describe practical strategies for using FISH analysis to detect lymphoma-associated chromosomal abnormalities in routine paraffin-embedded lymphoma biopsies. Furthermore, we provide proposals on how FISH results should be interpreted (including how to calculate cutoff levels for FISH probes), recorded, and reported. An online appendix (available at http://jmd.amjpathol.org) details various simple, yet robust procedures for paraffin FISH analysis; it also provides additional information on the production of FISH probes, evaluating and reporting FISH results, sources for reagents and equipment, and troubleshooting. We hope that these suggestions will make FISH technology for the study of lymphoma biopsies more accessible to routine diagnostic and research laboratories.

Genomic and immunohistochemical profiles of enteropathy-associated T-cell lymphoma in Japan

“…Generation of FISH probes and the FISH technique were previously described. [19][20][21] BAC clones were labeled with Spectrum Green by nick translation (Abbott Laboratories, UK). Commercial probes were used with the Vysis paraffin pretreatment kit II (7J02-02, Vysis).…”

“…Nevertheless, histologic evidence of celiac disease in the adjacent mucosa of the lymphoma such as villous blunting/atrophy and crypt hyperplasia were not found. In seven cases (35%) the intraepithelial lymphocytes were seen not only in the vicinity of the (100) 5 (25) 2 (10) 7 (35) 10 (50) 19 (95) 4 (20) Abbreviations: L, large; M, medium; S, small.…”

PTEN losses exhibit heterogeneity in multifocal prostatic adenocarcinoma and are associated with higher Gleason grade

“…We have estimated the relative distance between the differentially colored probes of a signal pair within the nucleus as follows: (1) co-localized signals: distance between the 3 0 -ERG and 5 0 -ERG signals less than one signal diameter; (2) non-overlapping signals: distance between the 3 0 -ERG and 5 0 -ERG signals more than two times the estimated signal diameter. 55 Subsequently, the TMPRSS2-ERG rearrangement mechanisms were classified according to the pattern of interphase FISH signals. 56 Class N described the normal ERG locus; therefore, co-localization of the two ERG probe signals in close proximity to the TMPRSS2 signal.…”

“…On the basis of hybridization in control cores (benign prostatic epithelium, data not shown) and the tumor cohort, detection of the TMPRSS2-ERG rearrangement was considered to be present when a minimum of 10% of the counted cells contained an Esplit or a minimum of 20% of the cells contained an Edel in a given core. 55 A core was considered fusion-positive if any of its representative spots met the above cutoff values. The 10 (Esplit) and 20% (Edel) cutoff values were also reassessed by analyzing the percentage of fusionpositive nuclei in a given core, alongside the percentage of nuclei with normal signal for each core from the TMA.…”

TMPRSS2-ERG gene fusion in transition zone prostate cancer

“…Although cutoff levels in FISH analyses may vary for different probes and among different laboratories, we used the widely accepted definition of a proper cutoff as being the mean of false-positive findings in at least five negative control specimens plus three times the standard deviation. [7][8] Hence, we analyzed ERG rearrangement in normal prostate glands and established a cutoff level of 10% for our FISH analyses. In other words, if more than 90% of the evaluated nuclei in a TZ tumor focus showed no evidence of ERG rearrangement, then that TZ focus was considered to lack the TMPRSS2-ERG gene fusion.…”