Multi-lineage interrogation of the performance characteristics of a split-signal fluorescence in situ hybridization probe for anaplastic lymphoma kinase gene rearrangements: a study of 101 cases characterized by immunohistomorphology on fixed archival tissue.

BACKGROUND: Fluorescence in situ hybridization (FISH) can identify chromosomal translocations on fixed archival tissue, but studies cross-validating the utility of FISH on lesions of different cell lineages that harbor similar translocations (e.g. those involving anaplastic lymphoma kinase [ALK]) have not been published. AIM: Our objective was to define the diagnostic utility, performance characteristics, and limitations of a commercially available, split-signal, FISH probe for ALK gene rearrangements on fixed, archived tissue from lesions of diverse cell lineage. STUDY DESIGN: The sensitivity, specificity, and positive and negative predictive values of the Vysis ALK FISH probe were compared with those of the ALK-1 antibody (Dako) in a series of 101 cases, comprising 43 hematolymphoid neoplasms, 4 reactive lymphoid controls, 50 non-hematolymphoid (including neuroectodermal, epithelial, myofibroblastic, and germ cell) lesions, and 4 early-trimester aborted fetuses that served as neuroblastic controls. METHODS: The study involved a predominantly (72%) Singaporean Chinese population aged between 9 months and 88 years (excluding the aborted fetal controls). All cases were reviewed both histologically and immunohistochemically with a wide panel of antibodies using the standard protocols in order to diagnose them according to the latest WHO classification systems. A positive cut-off value was determined, both by comparison with diagnostic categories with and without ALK translocations, as well as with negative controls. RESULTS: The ALK FISH probe suffered a 33% non-informative rate, but in informative cases it showed 94% concordance with the ALK-1 immunostain. A minimum cut-off value of 5 in 200 informative cells was adopted to make a positive call in each case. Of the ALK-1 immunoreactive lesions, nine lymphomas were concordantly ALK translocation-positive but one vesical inflammatory myofibroblastic tumor was discordantly FISH-negative. Among the ALK-1-immunonegative lesions, one case each of anaplastic lymphoma and pulmonary mycobacterial spindle cell pseudotumor were discordantly ALK FISH-positive, while a case each of intestinal myeloblastic tumor and ganglioglioma showed initial--but not reproducible--positive FISH readings. The remaining cases were concordantly negative. DISCUSSION: The discrepancies between ALK FISH results and well-established immunomorphological parameters indicate that interpretation is not always straightforward. Notably, the derivation of threshold cut-off values for positive calls on FISH assays has seldom been addressed in the literature, and has raised issues in interpreting cases with borderline positivity in this study. The factors that may influence such cut-off values are extensively reviewed. CONCLUSIONS: We propose the term 'conditional threshold positivity' to encourage the adoption of different cut-off values for making positive calls in lesions of different origin.

Detection of ALK gene rearrangements in formalin-fixed, paraffin-embedded tissue using a fluorescence in situ hybridization (FISH) probe: a search for optimum conditions of tissue archiving and preparation for FISH.

BACKGROUND: It is widely known that the efficiency of fluorescence in situ hybridization (FISH) probes applied to formalin-fixed, paraffin-embedded tissues is affected by the conditions under which the tissues are fixed and embedded. However, relatively few studies address exactly how tissue archiving conditions affect the performance of FISH probes. We report our experience based on use of an ALK FISH probe, during the validation of its diagnostic utility. METHODS: We applied the probe to 77 formalin-fixed, paraffin-embedded tissue blocks archived from 1991 through to 2000, and studied the interrelationship between the archival age (which ranged up to 10 years), type and condition of tissue, duration required for optimum hydrolysis, and obtainability of hybridization signals. RESULTS: We found that as archival age and tissue collagen content increased, not only did hydrolysis times have to be prolonged in order to yield interpretable hybridization signals, but also the likelihood of blocks becoming non-signaling increased. The most striking positive correlations were seen between the archival age of signaling lymphoid blocks and their requisite hydrolysis times. CONCLUSIONS: The difficulty in applying FISH on archival tissue increases with its archival age and collagen content, and may necessitate changes in laboratory protocol accordingly.

Evaluation of HER-2/neu oncogene status in breast tumors on tissue microarrays.

The amplification and/or overexpression of the HER-2/neu oncogene and its encoded receptor protein are increasingly used for prognostication and prediction of therapeutic response to Herceptin in breast cancer. However, large-scale examination of archival tumor blocks by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) is prohibitively laborious and technically challenging. The tissue microarray (TMA) technique enables hundreds of tumors to be studied simultaneously in a single experiment. To evaluate the HER-2/neu status of a selection of the breast tumors in our tumor bank, we constructed a TMA from 97 breast tumors, with a single 0.6-mm core per specimen. HER-2/neu gene amplification by FISH was found in 20 of the 87 interpretable cases (23%): in 14 of 14 IHC 3+ cases (100%), 5 of 8 IHC 2+ cases (62.5%) and 1 of 65 IHC 0/1+ cases (1.5%). Three of the 67 cases with no evidence of HER-2/neu gene amplification by FISH were moderately positive (2+) by IHC. A close relationship was observed between these 2 assays as applied to the TMA (95.4% concordance: 95% CI, -2.2% to 6.8%; P <0.0001), and both HER-2/neu gene amplification and protein overexpression were strongly associated with tumor grade, estrogen receptor status, and progesterone receptor status. Gene amplification was found in most of the tumors with high-level overexpression (IHC 3+) and not in the unequivocal IHC-negative cases. Complementary analysis by IHC and FISH are, however, recommended for tumors graded as 2+ by IHC, the group with the most result discrepancy. Hum Pathol 34:362-368.

Reliability of tissue microarrays in detecting protein expression and gene amplification in breast cancer.

Tissue microarrays allow high throughput molecular profiling of diagnostic or predictive markers in cancer specimens and rapid validation of novel potential candidates identified from genomic and proteomic analyses in a large number of tumor samples. To validate the use of tissue microarray technology for all the main biomarkers routinely used to decide breast cancer prognostication and postsurgical adjuvant therapy, we constructed a tissue microarray from 97 breast tumors, with a single 0.6 mm core per specimen. Immunostaining of tissue microarray sections and conventional full sections of each tumor were performed using well-characterized prognostic markers (estrogen receptor ER, progesterone receptor PR and c-erbB2). The full section versus tissue microarray concordance for these stains was 97% for ER, 98% for PR, and 97% for c-erbB2, respectively, with a strong statistical association (kappa value more than 0.90). Fluorescence in situ hybridization analysis for HER-2/neu gene amplification from the single-core tissue microarray was technically successful in about 90% (87/97) of the cases, with a concordance of 95% compared with parallel analyses with the full sections. The correlation with other pathological parameters was not significantly different between full-section and array-based results. It is concluded that the constructed tissue microarray with a single core per specimen ensures full biological representativeness to identify the associations between biomarkers and clinicopathological parameters, with no significant associated sampling bias.