Multi-colour FISH in oesophageal adenocarcinoma –
predictors of prognosis independent from stage and grade
C.I. Geppert1, 8, P. Rümmele2, M. Sarbia4, R. Langer6, M. Feith3, L. Morrison7, E. Pestova5, R. Schneider-Stock1, 8, A. Hartmann1, 8, T.T. Rau1, 8
Supplemental methodologicalinformation:
TMA generation and staining procedure
Cylinders with a diameter of 0.65 mm were punched from the tumour area with a stainless steel tube and deposited in prepared TMA blocks. Each patient tumour specimen was represented by three punch samples on the TMA. Sections of the TMA blocks were cut to 3μm thickness and the sections placed onto SuperFrost®Plus coated slides (Menzel GmbH, Braunschweig, Germany) for in situ hybridization.
TMA sections were deparaffinised, rehydrated in a graded ethanol series and rinsed in distilled water. After that slides were placed in 0.01 M sodium citrate and heated for 40 minutes at 95°C in a water bath and digested in 0.1% pepsin (Sigma, Munich, Germany) and 0.01 M HCL for 10 minutes at 37°C. After washing in 2 × SSC (1 × SSC, standard saline citrate, is 150 mM sodium chloride and 15 mM sodium citrate, pH 7) and water, slides were dehydrated in a graded alcohol series and air-dried. Respective DNA probe sets (10μl each) were applied to the TMA. Sections were cover-slipped and the edges were sealed airtight with rubber cement (Fixogumm, Marabu GmbH & Co. KG Tamm, Germany). For co-denaturation of probe and target, DNA slides were placed for 5 minutes on a hotplate preheated to 73°C and afterwards transferred overnight to a warmed hybridization chamber at 37°C. After hybridization the rubber cement was removed and the slides were immersed successively in 4 × SSC plus 0.3% Igepal (Serva, Heidelberg, Germany), 2 × SSC and 1 × SSC for 10 minutes at 50°C. The slides were rinsed for one minute in water at room temperature and air-dried. After the completion of hybridization and post-hybridization washing, nuclei were counterstained by application of Vectashield mounting medium containing DAPI (4',6-diamidino-2-phenylindole) (Vector Laboratories, Burlingame, CA, USA), for nuclear boundary identification. The FISH-stained TMA slides were kept cool and dark at 4°C and were analysed by epifluorescence microscopy.
Statistics
Inter class correlation coefficient (kappa) highlight the correlation between morphological and molecular homogeneity during TMA punch selection. Each parameter showed high concordance between the three different punches in a test test of 10 tumours.
CDKN2A: interclass correlation in average measurement: κ=0.944, significance: p < 0.001
ERBB2: interclass correlation in average measurement: κ=0.944, significance: p = 0.001
ZNF217: interclass correlation in average measurement: κ=0.88, significance: p < 0.001
MYC: interclass correlation in average measurement: κ=0.74, significance: p =0.007
Additionally, student`s T-Test were calculated and no significant difference could be seen as well.
Thus, the results showed high homogeneity of each FISH-probe in the central tumour area.Counting 50 consecutive cells from one of the three samples of each tumour and each normal specimen could be relied upon to provide a representative result for each specimen.
Light spectra of the used FISH probes:
CDKN2A – Spectrum Red™, excitation 560nm (+ 20), emission 630nm (+ 30), filter: C-00431,
ERBB2 – Spectrum Green™, excitation 480nm (+ 15), emission 535nm (+ 20), filter: C-00431,
MYC – Spectrum Aqua™, excitation 436nm (+ 5), emission 480nm (+ 10), filter: C-00430,
ZNF217– Spectrum Gold™, excitation 538nm (+ 10), emission 575nm (+ 15), filter: C-00429,
DAPI, excitation 360nm (+ 20), emission 460nm (+ 25), filter: A-513678.
Slides were imaged with a fluorescence microscope (Axio-Imager Z.1, Zeiss, Göttingen, Germany) equipped with specific fluorescence filter sets for DAPI and LSI-probes.
Remarks about FISH evaluation especially facing nuclear truncation and trouble shooting
It is important to keep in mind that it is difficult to distinguish between high-grade dysplasia and EAC using FISH, which is possibly caused by the genetic similarity between high-grade dysplasia and EAC.(Brankley et al., 2006),(Reid, Haggitt, Rubin, & Rabinovitch, 1987),(Riegman et al., 2002)(Fritcher, Brankley, Kipp, & Voss, 2008). Pre-selection of region of interests like TMA punch biopsies from a certain area are therefore mandatory.
Smaller nuclei that resembled lymphocytes, overlapping nuclei, or nuclei with unclear boundaries were excluded from evaluation, but were used to monitor specificity and quality of the FISH staining on the TMA. Whenever signals suited evaluation, probe signals were counted using the appropriate microscope filter as described above. Two signals separated by a gap of less than one signal diameter were considered to be split signals and counted as one.
To assess performance of this 4-colour FISH assay in solid FFPE tissue, we first addressed possible FISH specific disturbances by nuclear truncation or genetic heterogeneity of the tumour. In a pre-test high homogeneity of each FISH-probe was seen in the tumour centre allowing us to use TMA as an appropriate research tool. As a typical histological phenomenon, we addressed nuclear truncation due to tissue sectioning for its effect on FISH signals. Due to nuclear truncation, lower levels of FISH signals are observed, requiring lower cut-offs for normal and tumour cells to compensate. 3-6 µm slides of normal tissue with diploid cells showed fewer than 2 signals per cell in about 20-30% of the cells due to nuclear truncation, what is in line with literature.(Nielsen et al., 2012)(Rauser et al., 2007)
Beyond these obstacles consistent well adapted cut-off levels could be defined in statistical analysis. Similar to the report of Fritcher E.B. et al. who used FISH markers in cytological specimens, we confirmed that the probes for ERRB2, CDKN2A, MYC and ZNF217 can also be used in FFPE tissue specimens in EAC with high specificity and sensitivity. (Fritcher et al., 2008)
References:
Brankley, S. M., Wang, K. K., Harwood, A. R., Miller, D. V, Legator, M. S., Lutzke, L. S., … Halling, K. C. (2006). The development of a fluorescence in situ hybridization assay for the detection of dysplasia and adenocarcinoma in Barrett’s esophagus. The Journal of molecular diagnostics : JMD, 8(2), 260–7. doi:10.2353/jmoldx.2006.050118
Fritcher, E. B., Brankley, S., Kipp, B., & Voss, J. (2008). A comparison of conventional cytology, DNA ploidy analysis, and fluorescence in situ hybridization for the detection of dysplasia and adenocarcinoma in patients with. Human pathology, 39(8), 1128–1135. doi:10.1016/j.humpath.2008.02.003.A
Nielsen, K. V., Ejlertsen, B., Møller, S., Jensen, M.-B., Balslev, E., Müller, S., … Mouridsen, H. T. (2012). Lack of independent prognostic and predictive value of centromere 17 copy number changes in breast cancer patients with known HER2 and TOP2A status. Molecular oncology, 6(1), 88–97. doi:10.1016/j.molonc.2011.11.006
Rauser, S., Weis, R., Braselmann, H., Feith, M., Stein, H. J., Langer, R., … Walch, A. (2007). Significance of HER2 low-level copy gain in Barrett’s cancer: implications for fluorescence in situ hybridization testing in tissues. Clinical cancer research : an official journal of the American Association for Cancer Research, 13(17), 5115–23. doi:10.1158/1078-0432.CCR-07-0465
Reid, B. J., Haggitt, R. C., Rubin, C. E., & Rabinovitch, P. S. (1987). Barrett’s esophagus. Correlation between flow cytometry and histology in detection of patients at risk for adenocarcinoma. Gastroenterology, 93(1), 1–11.
Riegman, P. H. J., Burgart, L. J., Wang, K. K., Wink-Godschalk, J. C. J., Dinjens, W. N. M., Siersema, P. D., van Dekken, H. (2002). Allelic imbalance of 7q32.3-q36.1 during tumorigenesis in Barrett’s esophagus. Cancer research, 62(5), 1531–3.
Figure legends:
Supplemental Figure 1: ERBB2/cell and the ratio of ERBB2/ZNF217 are able to subdivide individuals with EAC according to their prognosis.
A) Kaplan-Meier survival analysis in patients stratified by ERBB2/cell: definition and cut-off: ERBB2 copy number, cut-off <43% cells with ERBB2 >2 signals/cell, Log Rank p=0.0412. The median survival was 43.0 months (≥43% cells with gain), black line, vs. greater than study duration (<43% cells with gain), grey line.
B) Kaplan-Meier survival analysis in patients stratified by the ratio of ERBB2/ZNF217: definition and cut-off: ratio of ERBB2/ZNF217, cut-off 1.5 (Log-Rank test p=0.054). The median survival was 27.25 (ratio ≥1.5), black line, vs. 65.2 months (ratio <1.5), grey line.