| Abstract|| |
Context: HER2/neu testing in breast cancer is a mandate due to availability of trastuzumab, a monoclonal antibody targeted against this biomarker. Dual-color dual-hapten in situ hybridization (D-DISH) is a new test for assessment of HER2/neu gene overexpression on light microscopy. Aims: This was a validation study for D-DISH in our laboratory and was conducted to study the concordance between fluorescence in situ hybridization (FISH) and D-DISH for HER2/neu testing in breast cancer. Materials and Methods: In all, 150 cases of invasive breast carcinoma requested for FISH analysis were selected. Immunohistochemistry by Ventana PATHWAY anti-HER2/neu (4B5) antibody, FISH by ZytoLight SPEC ERBB2/CEN17 Dual Color Probe, and D-DISH using the Ventana INFORM HER2 Dual ISH DNA Probe Cocktail Assay was carried out. Statistical Analysis: Cohen's kappa coefficient was used to calculate concordance between FISH and D-DISH assays. The ratios and average number of signals were compared with Lin's concordance correlation coefficient. Results: About 93.1% of the cases showed concordance between FISH and D-DISH results. Cohen's kappa correlation coefficient was 0.836, indicating almost perfect level of agreement. Lin's concordance correlation coefficient (ρc) showed moderate strength of agreement for HER2/chromosome 17 ratios between FISH and D-DISH assays (ρc 0.9452). As per the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2018 updated guidelines, four of the cases that were nonamplified on FISH showed low-level amplification on D-DISH due to counting errors caused by faint signals or background dust. Genomic heterogeneity and larger red chromosome 17 signals on D-DISH led to discordance of the six cases amplified by FISH. D-DISH failure rate was 3.33%. Conclusion: Overall, D-DISH showed good concordance with FISH but needs expertise for reporting.
Keywords: Concordance, dual-color dual-hapten in situ hybridization, fluorescence in situ hybridization, HER2, invasive breast carcinoma, validation
|How to cite this article:|
Gajaria PK, Tambe S, Pai T, Patil A, Desai SB, Shet TM. Dual-color dual-hapten in situ hybridization (D-DISH) – Comparison with fluorescence in situ hybridization (FISH) for HER2/neu testing in breast cancer. Indian J Pathol Microbiol 2020;63:194-9
|How to cite this URL:|
Gajaria PK, Tambe S, Pai T, Patil A, Desai SB, Shet TM. Dual-color dual-hapten in situ hybridization (D-DISH) – Comparison with fluorescence in situ hybridization (FISH) for HER2/neu testing in breast cancer. Indian J Pathol Microbiol [serial online] 2020 [cited 2021 May 16];63:194-9. Available from: https://www.ijpmonline.org/text.asp?2020/63/2/194/282719
| Introduction|| |
Human epidermal growth factor receptor 2 (HER2/neu), a proto-oncogene, located on the long of chromosome 17 (17q12), was first implicated in the pathogenesis of breast cancer by Slamon et al. in 1987. With the advent of targeted therapy in the form of trastuzumab for these subsets of aggressive tumors, it has become important to correctly identify cases with HER2/neu amplification who would derive benefit from this therapy.
Various tests have been used for identification of this overexpression, ranging from the older techniques such as Southern or Western Blotting, enzyme-linked immunosorbent assay (ELISA) to the recently used immunohistochemistry (IHC), in situ hybridization (ISH) techniques, real-time polymerase chain reaction (PCR), digital PCR, and quasi-targeted proteomics, to name a few.,,
IHC and fluorescence in situ hybridization (FISH) are the most commonly used techniques for the detection of the same. Dual-color dual-hapten in situ hybridization (D-DISH) is a new kid on the block that uses nonfluorescent probes which produce bright-field microscopy signals for HER2/neu testing. Thus, it has an advantage of combining specificity of an ISH assay with convenience of light microscopic interpretation. We performed this study as a validation test for D-DISH testing in our laboratory and to study the concordance between FISH and D-DISH for HER2/neu testing in invasive breast carcinoma (IBC).
| Materials and Methods|| |
A total of 150 consecutive cases of IBC requested for HER2/neu FISH analysis were selected for the study. The same block was used for IHC, FISH, and D-DISH testing. The usual process of selection of block for FISH by a pathologist was used.
IHC for HER2/neu was done on formalin-fixed paraffin-embedded (FFPE) tissue with Ventana PATHWAY anti-HER2/neu antibody, clone 4B5 on a fully automated Ventana Benchmark XT platform. Antigen retrieval was performed on board using the Cell Conditioner 1 (CC1) solution for 9 min at 95°C and the antibody incubation time was 16 min at 37°C.
According to clinical request, FISH testing was performed mostly on cases that showed equivocal results on IHC. FISH testing was performed manually using the ZytoLight SPEC ERBB2/CEN17 Dual Color Probe. It is a mixture of orange fluorochrome direct-labeled CEN17 (Centromere) probe specific for the alpha satellite centromeric region of chromosome 17 (D17Z1) and a green fluorochrome direct-labeled SPEC ERBB2 probe specific for the chromosomal region 17q12-q21.1 harboring the HER2/neu gene. Sections were cut at 2 μm thickness and spread on precoated charged slides. These were subject to pretreatment steps on day 1. Slides were deparaffinized in xylene which was preheated to 60°C (three washes for 10 min each), followed by two washes for 10 min each with xylene at room temperature. This was followed by two changes in 100% ethanol, each for 5 min. The slides were then incubated for 15 min in prewarmed heat pretreatment solution citric at 98°C, followed by two changes of 4 min each in 2× sodium citrate and sodium chloride solution. The sections were then subject to washing with distilled water. After incubation with pepsin solution for approximately 7 min at 37°C, the sections were passed through ascending grades of chilled ethanol. Once these pretreatment steps were completed and slides dried, 10 μL of probe was added on to the sections in dark. These were then covered with cover-slip using rubber cement and kept for denaturation at 75°C. The sections were then kept for overnight incubation in the hybridizer at 37°C. Day 2 had removal of cover-slip and post-hybridization washes to remove the unbound probe followed by counterstaining with 4',6-diamidino-2-phenylindole in dark. The slides were stored in refrigerator and in dark until reporting. FISH slides were evaluated on Olympus fluorescence microscope with the help of QCapture software.
D-DISH assay was carried out using the INFORM HER2 Dual ISH DNA Probe Cocktail Assay from Ventana Medical Systems. With this, HER2 gene gets detected by a dinitrophenyl (DNP)-labeled probe and visualized using ultraView silver in situ hybridization (SISH) DNP detection Kit. The chromosome 17 centromere is targeted with a digoxigenin (DIG)-labeled probe and detected using ultraView Red ISH DIG detection Kit. On light microscopy, HER2 appears as discrete black signals and chromosome 17 as red signals. The 2-μm-thick sections were loaded into the Ventana Benchmark XT machine. On board, a fully automated procedure was carried out as per the company recommended protocol with the following basic steps of deparaffinization followed by cell conditioning and protease digestion. Subsequently, the probe was applied followed by hybridization and application of the SISH Multimer. This was followed by application of silver chromogen, and then Red ISH Multimer and red chromogen. Finally, counterstain with hematoxylin was followed by clearing in xylene and mounting with dibutylphthalate polystyrene xylene. The slides were evaluated on Olympus CX21i light microscope using the 60× objective lens for better visualization and counting of the signals.
At least 40 nuclei were counted. IHC and FISH results were interpreted by one pathologist and D-DISH was evaluated independently by two other pathologists, who were blinded to the FISH and IHC results. Discrepant results for D-DISH were seen again and discussed to arrive at a consensus opinion. The results were interpreted based on the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) Clinical Practice Guideline Focused Update, 2018. An average of ≤1.4 chromosome 17 signals was considered as monosomy, while the average number of signals ≥3 was the criteria chosen for polysomy.
Cohen's kappa coefficient was used to calculate concordance between FISH and D-DISH assays using the Statistical Package for the Social Sciences (SPSS, Version 25) software. Lin's concordance correlation coefficient was calculated to compare the HER2/CEP 17 ratios, average HER2/neu signals, and average CEP 17 signals by both the tests using the MedCalc statistical software.
| Results|| |
FFPE from 150 IBC cases were analyzed by all three tests, IHC, FISH, and D-DISH. Most of the cases, 136 of 150 (90.7%), were equivocal (score 2+) on IHC, while 11 of 150 (7.3%) had positive (score 3+) staining, and 3 (2%) cases had been reported negative (score 0/1+).
FISH was uninterpretable in 3 (2%) cases. FISH was amplified in 54 cases (36%) and nonamplified in 93 of the 150 cases (62%) tested [Table 1] and [Figure 1]a, [Figure 1]b, [Figure 1]c. According to the ASCO/CAP 2018 guidelines, 53 of 150 (35.3%) belonged to Group 1 (HER2/CEP17 ratio ≥2.0 and average HER2 copy number ≥4.0 signals/cell) and 89 cases (59.3%) belonged to Group 5 (HER2/CEP17 ratio <2.0 with average HER2 copy number <4.0 signals/cell). Only 1 case (0.7%) belonged to Group 2 (HER2/CEP17 ratio ≥2.0 and average HER2 copy number <4.0 signals/cell), and in view of it showing monosomy, it was considered to be nonamplified after being evaluated by two observers, while none of the cases belonged to Group 3. Four cases (2.7%) fell into Group 4 (HER2/CEP17 ratio <2.0 and average HER2 copy number ≥4.0 and <6.0 signals/cell) [Table 2]. Of these, three were labeled nonamplified and one amplified after scoring by the three pathologists taking into consideration FISH and IHC profile. Nineteen cases (12.7%) showed monosomy of chromosome 17, while only 4 cases (2.7%) showed polysomy. Genomic heterogeneity was observed in five cases, with four of these showing that of the intermixed type, while only a single case revealed a cluster-type pattern.
|Figure 1: (a) A case amplified by both FISH (ratio 9.8) and (b) D-DISH (ratio 10) at 1000× magnification. (c) A case nonamplified by both FISH (ratio 1.66) and (d) D-DISH (ratio 1.53) at 1000× magnification. (e) A case that is nonamplified by FISH (ratio 1.62) but (f) amplified by D-DISH (ratio 1.98, average HER2 signals 4.2) at 1000× magnification. (g) A case showing polysomy for chromosome 17 by both FISH and D-DISH (h) at 1000× magnification|
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|Table 2: Comparison between FISH and D-DISH according to groups as per ASCO/CAP 2018 guidelines|
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D-DISH was uninterpretable in five cases (3.3%). Fifty cases out of 150 (33.3%) showed amplification of HER2/neu gene, while 95 out of 150 (63.3%) were nonamplified [Table 1] and [Figure 1]b, [Figure 1]c, [Figure 1]d. Group 1 had 46 cases (30.7%), while 94 (62.7%) cases belonged to Group 5. The only case with Group 3 (HER2/CEP17 ratio <2.0 and average HER2 copy number ≥6.0 signals/cell) pattern was considered as amplified, even though it showed polysomy for chromosome 17 because of the fact that clusters of black signals were identified in most of the nuclei. Four cases showed Group 4 type of ratios and signals. These were interpreted similar to FISH with three nonamplified cases and one amplified case [Table 2]. Monosomy by D-DISH was observed in only 8 cases (5.3%), while 4 (2.7%) showed polysomy. Genomic heterogeneity was observed in the same cases as by FISH.
Comparison of FISH with D-DISH
Out of the 145 cases evaluable by D-DISH assay, 135 cases (93.1%) showed concordance between the respective FISH and D-DISH results [Table 3] and [Figure 1]e, [Figure 1]f. Cohen's kappa correlation coefficient for the same was 0.836, indicating almost perfect level of agreement between the two tests, which was statistically significant (P = 0.000). Lin's concordance correlation coefficient (ρc) showed a moderate strength of agreement in HER2/CEP 17 ratios between the FISH and D-DISH assays [ρc 0.9452, 95% confidence interval (CI) 0.9252–0.9599] [Graph 1]. The average number of HER2 signals by both tests showed a moderate level of agreement (ρc 0.9171, 95% CI 0.8905–0.9374). When average number of CEP 17 signals by both the tests was compared by Lin's concordance coefficient (ρc), it showed a poor strength of agreement (ρc 0.5452, 95% CI 0.4234–0.6476).
There was only fair level of agreement in the detection of monosomy and polysomy for chromosome 17 between both the tests (Cohen's kappa correlation coefficient 0.217, P = 0.01). Nineteen cases showed monosomy by FISH testing, but D-DISH could detect the same in only four of those cases and a single case showed polysomy with both the techniques [Figure 1]g, [Figure 1]h. On the other hand, four cases showed monosomy and three cases polysomy by D-DISH, which were not seen on FISH.
| Discussion|| |
Use of HER2/neu targeted therapy has resulted in better survival outcomes in patients with HER2-positive breast cancer. Monoclonal antibodies such as trastuzumab, pertuzumab and tyrosine kinase inhibitor lapatinib and neratinib are being currently used or are under clinical trials for the treatment of these patients. However, trastuzumab besides being expensive has some serious adverse effects associated with its use such as cardiomyopathy, pulmonary toxicity, and febrile neutropenia.,
Considering these aspects, it becomes very important to identify the population suitable for HER2 targeted therapy and to avoid unnecessary treatment and adverse events in patients who would not benefit from the same. This requires a reliable assay for identifying HER2/neu-positive tumors. IHC and FISH are commonly used in routine diagnosis. FISH is the gold standard especially for cases that show equivocal results on IHC. Many newer assays as mentioned above are being evaluated for their effectiveness in correctly diagnosing HER2/neu gene amplification.
D-DISH is one such technique that helps identify gene amplification under light microscopy as it uses chromogenic probes rather than fluorescent probes. It combines the two techniques of chromogen in situ hybridization (CISH) and SISH to target chromosome 17 centromere and HER2 gene, respectively. DIG-labeled probe targets the chromosome 17 centromere, which is then detected by Naphthol Fast Red ISH detection kit. Similarly, DNP probe targets the HER2 gene and is visualized using silver reagents. These probes are cohybridized during the assay, and therefore chromosome 17 appears as red signals and HER2 gene as black signals.
We evaluated the HER2/neu gene amplification status by D-DISH and compared the results of those with FISH test. In our study, 93.1% of the cases showed concordance between the respective FISH and D-DISH results with Cohen's kappa correlation coefficient of 0.836, which indicated an almost perfect level of agreement between the two tests. This was similar to most other studies reported in the literature which have showed concordance ranging from 85% to 96% [Table 4]. Some of the studies did not find good correlation between these two tests and showed only fair level of agreement. Most of these studies have used the 2007 or 2013 ASCO/CAP guidelines for interpretation.
In our study, HER2/CEP17 ratio showed moderate strength of agreement as measured by the concordance correlation coefficient. Kosa et al. observed near-perfect agreement for HER2/CEP17 ratios between the PathVsyion FISH and D-DISH tests performed by them. Gao et al. compared the ratios with Cohen's kappa correlation coefficient and reported substantial agreement between the two tests. By regression analysis, Mansfield et al. concluded that the HER2/CEP17 ratios were on an average 0.12 lower by D-DISH when compared with the ratios obtained by FISH and the same was also demonstrated by Tang et al. with the help of linear regression analysis., Tang et al. also demonstrated an excellent correlation between both the tests for HER2/CEP17 ratios and for HER2 copies per nucleus.
Poor agreement for average chromosome 17 copies per nucleus by D-DISH assay as reported by us was also made by few other observers., Although this did not affect the overall ratio and final result, it did lead to missing of monosomy and polysomy for chromosome 17 by D-DISH assay.
D-DISH is by far a reproducible test, as observed by few authors who looked for interobserver variability. Similarly, a positive correlation (Pearson's correlation coefficient 0.91) was also observed between cell blocks and the respective histological sections tested by D-DISH.
This is the first study from India wherein the performance of the D-DISH assay has been reported and compared with FISH. Also, this study has taken into account the 2018 ASCO/CAP guidelines while evaluating D-DISH, when compared with previous studies which were conducted prior to these guidelines. Hence, we attempted to evaluate the results as per the groups defined in the newer guidelines.
Since all steps of D-DISH can be performed on the same machine as used for automated IHC, expensive equipment such as hybridizer, shaking water bath, and incubators need not be purchased. Another advantage with the D-DISH technique is that the turnaround time is reduced significantly, D-DISH being a 12- to 14-h procedure when compared with FISH which takes 2 days. The most important benefit of D-DISH is that the tumor can be seen clearly akin to routine hematoxylin and eosin sections, giving the pathologist the ability of selecting invasive foci and hence counting the signals in the most appropriate areas. Slides can be archived without causing quenching of signals and can be scanned for ease of evaluation.
The chief problem encountered in this study resulting in false negative D-DISH is that the target signal for HER/2neu is smaller, while that of chromosome 17 is larger making interpretation difficult when there are overlapping nuclei with masking of some black signals. While in FISH, the pathologist can count red and green signals separately under the two separate wavelength filters avoiding erroneous count due to overlapping of signals. Also, blotchy red signals were a common problem encountered by us in D-DISH. This could probably explain the discrepancy in evaluating chromosome 17 abnormalities in our study by D-DISH. All the D-DISH amplified were false positives due to overinterpretation of background dirt as HER2/neu signals.
About five of our cases showed a black dusty background, and hence the test had to be repeated. Similarly, red background was seen in few of the cases; however, this did not interfere with the counting of the signals. Four of the cases had to be subject to repeat testing in view of nuclear bubbling leading to absence of signals. Overall, D-DISH is more sensitive to fixation issues, water conductivity and pH, and clean equipment, and thus in five of our cases the test did not work. With FISH, certain stringent pretreatment measures could be undertaken and the signals were countable in two of these cases. The remaining cases were outside blocks that had come for referral testing at our institute and we were unaware of the quality and time of fixation of the tissue.
Overall though D-DISH showed good concordance with FISH and proved to be a quick and cost-effective assay, it has interpretative errors which can reduce with experience. D-DISH could help ease out the tedious task of performing FISH technique for every specimen and only those with borderline results need to be rechecked with FISH as all of our discordant cases occurred with an average HER2/neu copy number of four to five signals per cell and HER2/CEP17 ratio of 1.9–2.2.
| Conclusion|| |
Though D-DISH can serve as a good replacement for HER2/neu FISH in diagnostic setup to reduce the pressure of time on molecular diagnostic laboratory, it has an error rate which requires vigilance for picking up the same.
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Conflicts of interest
There are no conflicts of interest.
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Tanuja M Shet
Department of Histopathology, 8th Floor, Annexe Building, Tata Memorial Centre, Dr E Borges Road, Parel, Mumbai - 400 012, Maharashtra
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4]