BRIEF REPORT |
Correspondence to: Puay-Hoon Tan, Dept. of Pathology, Singapore General Hospital, Outram Road, Singapore 169608. E-mail: gpttph@sgh.com.sg
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Summary |
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This study investigated if formalin fixation duration affects HER2/neu gene amplification detection by fluorescence in situ hybridization (FISH) in breast cancer. Tumor tissues from 35 cases were divided into three groups and subjected to two formalin fixation protocols per group (12 hr, 27 hr in the first; 2 hr, 17.5 hr in the second; 28.5 hr, 541 hr in the third) before FISH analysis. There was no significant difference in FISH signal detection between the two different fixation protocols in the first two groups. In the third, no signal was detected in 4/6 cases fixed for an extended duration.
(J Histochem Cytochem 50:16931696, 2002)
Key Words: FISH, HER2/neu, fixation, breast cancer, immunohistochemistry
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Introduction |
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The HER-2/neu gene (otherwise known as c-erbB2 gene), a proto-oncogene located on the long arm of chromosome 17, encodes a 185-kD transmembrane glycoprotein that belongs to the type I growth factor receptor family (
FISH is a powerful molecular cytogenetic method based on the hybridization of specific DNA sequences to the target genome and involving both histochemical and solid matrix hybridization techniques (
Paraffin-embedded breast carcinoma tissues from 35 women who underwent surgical resection of their tumors at the Singapore General Hospital were used for this study. Paraffinized sections were routinely stained with hematoxylineosin for histopathological diagnosis. Two sets of tumor tissues from each case were divided into three groups. They were fixed in 10% buffered formaldehyde for 12 ± 2.5 hr (range 918 hr, median 12 hr) and 27 ± 3.0 hr (range 2032 hr, median 27 hr) in the first group, 2 hr and 17.5 ± 1.5 hr (range 1620 hr, median 17.5 hr) in the second group, and 28.5 ± 2 hr (range 2630 hr, median 28.5 hr) and 541 hr ± 285 hr (range 1931010 hr, median 541 hr) in the third group. All cases in the first group represented the usual range of fixation durations for breast surgical specimens routinely handled in the laboratory. The second and third groups were used to test the effect of a shortened fixation duration (2 hr), and an extended fixation duration (median 541 hr) on FISH results compared with routinely fixed cases.
Three-µm sections were cut for FISH analysis and mounted on glass slides coated with silane (3-aminopropyltriethoxysilane; Sigma, St Louis MO). The sections were deparaffinized and pretreated to facilitate probe permeability using a Pretreatment Kit (Vysis; Downers Grove, IL) according to manufacturer's instructions with minor modifications. Briefly, slides were heated overnight (56C) and dewaxed in xylene, air-dried, and treated with 0.2 M HCl for 20 min at room temperature (RT). This was followed by incubation in 1 M sodium thiocyanate in 2 x SSC (0.5 M NaCl, 0.015 M sodium citrate) at 80C for 30 min, rinsed, and digested with protease (1.25 mg/ml) for 30 min at 37C. The slides were finally dehydrated in an ethanol series (70100%) and air-dried.
ISH was performed using the PathVysion HER2 DNA Probe Kit (Vysis) basically according to the manufacturer's recommendations (
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For HER2/neu IHC, 4-µm sections were mounted on glass slides coated with silane (3-aminopropyltriethoxysilane; Sigma), and dried overnight at 37C. The sections were deparaffinized in xylene and rehydrated via a series of graded alcohols. Endogenous peroxidase activity was blocked by incubating the sections in 12 ml of methanol containing 200 µl of 30% H2O2 for 10 min, followed by washing in running tapwater. Antigen unmasking was carried out by microwaving sections in citrate buffer for 10 min. Nonspecific binding sites were blocked by 10% normal swine serum for 10 min. Sections were then incubated with polyclonal anti Her2/neu antibody (Dako A0485; Carpinteria, CA) at 1:1000 dilution in a humid chamber for 30 min at RT. The primary antibody was rinsed off with Tris-buffered saline (TBS) at pH 7.4 and incubated with linking biotinylated antibody for 20 min, followed by incubation with peroxidase-conjugated streptavidin complex (Dako LSAB2 kit, K0675) for 20 min. Freshly prepared DAB solution (5 mg 3, 3'-diaminobenzidine tetrahydrochloride; Sigma D5637) was applied for 10 min after the tertiary layer was rinsed off with TBS. DAB was removed by rinsing with distilled water and slides were counterstained with hematoxylin, and mounted in depex. A breast tumor known to be reactive with HER2/neu antibody was used as positive control in each staining batch. IHC staining was classified according to the criteria recommended by the DAKO protocol: 0, no staining is observed or membrane staining is observed in <10% of tumor cells; 1+, faint/barely perceptible membrane staining is detected in >10% of tumor cells; 2+, weak to moderate complete membrane staining is observed in >10% of tumor cells; and 3+, strong complete membrane staining is observed in >10% of tumor cells.
Statistical analysis was performed using the GraphPad Prism statistical Package. The Student's paired t-test was performed to compare means and chi-squared test for comparing proportions. p<0.05 was considered as statistically significant.
There was no significant difference in detection of FISH signals for the 12-hr or 27-hr fixation protocols in the first group (p=0.476; Table 1; Fig 1A and Fig 1B), indicating that the range of fixation times used routinely in our surgical pathology laboratory did not affect FISH results. Of four amplified cases in this group, two cases revealed very high levels of gene amplification as shown by the HER2/neu/CEP17 ratio of 13.24, 10.6, and 14.65, 9.5 for the 12-hr and 27-hr fixation protocols, respectively. All tissues with 3+ IHC staining disclosed HER2/neu amplification (Fig 1C), whereas those with negative immunostaining (0 and 1+) showed no HER2/neu gene amplification (Table 1). No gene amplification was detected in the five immunopositive cases with 2+ IHC staining. In the second group there was also no significant difference in FISH results between the 2-hr and 17.5-hr fixation protocols (p=0.151; Table 2). In the third group signals were detected in all six cases in the 28.5-hr fixation protocol but only two cases showed signals in the extended-duration fixation protocol (cases 1 and 2; Table 3). In case 2, the fixation duration was approximately a week (the "shortest" period among those with protracted fixation). Case 1 showed high amplification, which likely accounted for the fact that despite prolonged formalin fixation for over 3 weeks, signals were observed, although diminished compared to those of the routinely fixed counterpart. When FISH signals were detected, there was a positive correlation between HER2/neu gene amplification by FISH and protein expression by IHC (p=0.0002) in all the groups.
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It appears from our study that a fixation period ranging from 2 hr to 1 week would not affect FISH results. Beyond 1 week however, no signals could be detected, with the exception of the highly amplified case 1 in group 3. Formalin fixation increases the complexity of cell structure and chromatin condensation, making it difficult for the probe to penetrate and interact with the target DNA (
The observation of a good concordance between FISH and HER2/neu IHC is in accord with those of other investigators (
In conclusion, reliable results for HER2/neu amplification are not compromised by the usual range of routine fixation periods of surgical breast specimens before processing and FISH analysis. Breast specimens fixed for a shortened period are also suitable for FISH analysis. However, a fixation period of more than a week appears to compromise the results obtained by FISH. Further study is necessary to determine if variable Her2/neu FISH results are also obtained on stored paraffin blocks.
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Acknowledgments |
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Supported by a grant from the SingHealth Cluster Research Fund No. BF006/2001.
Received for publication May 30, 2002; accepted August 10, 2002.
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