Chromogenic In Situ Hybridization Method Research Articles

Evaluation of HER2 Gene Amplification using CISH in Patients with HER2 2+ (equivocal) Breast Carcinoma based on Immunohistochemistry in Imam Khomeini Cancer Institute from 2016 to 2018

Background: Human epidermal growth factor 2 (HER2) is known to be an important prognostic factor in breast cancer. Numerous studies have shown HER2+ breast cancers have reduced overall survival and recovery time, as well as the efficacy of anti-HER2 therapies along with chemotherapy in improving disease outcomes. For this reason, it is recommended that all patients with breast cancer should be evaluated for HER2 status. This study aimed to assess the HER2 gene amplification by the CISH method in evaluating the HER2 status in patients with immunohistochemistry (IHC) 2+ (equivocal) results. Methods: This retrospective cross-sectional study examined HER2 status based on the Chromogenic in situ hybridization (CISH) method in 280 breast carcinoma samples with an initial 2+ (equivocal) score in IHC. The relationship between HER2 amplification and hormone receptors (estrogen and progesterone) and Ki67 level was also investigated. Results: In sixty samples (21.4%), the HER2 gene was amplified based on the CISH method. The majority (215, 76.8%) of the samples were negative and 5 (1.8%) samples were indeterminate. No significant relationship was observed between HER2 amplification, estrogen receptor (p=0.143), and ki.67 protein level (p=0.977). There was a significant inverse relationship between HER2 amplification and progesterone receptor positivity (p=0.007). Conclusion: These results demonstrate that CISH is a helpful method to assess HER2 status in equivocal breast cancer and is positive (amplified) in about 21.4% of them.

Detection of High-Risk Human Papillomavirus by Chromogenic in Situ Hybridization Method in Liquid-Based Cervicovaginal Cytology Specimens with Atypical Squamous Cells of Undetermined Significance

Background : Cervical cancer is the second most common cancer after breast cancer in women worldwide. Owing to comprehensive cervical screening programs, a significant decrease has been observed in the incidence of invasive cervical cancers. Human Papillomavirus (HPV) is the underlying etiology in 99.7% of the cases of cervical cancer and is a major risk factor for the development of precancerous and cancerous cervical lesions. Chromogenic in situ hybridization method (CISH) is one of the methods used to detect high risk HPV in liquid -based smears. Aims : In this study, we conducted an archive search in the department of pathology and we used chromogenic in situ hybridization technique to investigate whether HPV DNA was present in liquid -based smears of 68 patients who had been found to have atypical squamous cells of undetermined significance (ASCUS) and whether a cervical intraepithelial lesion was present in the biopsy samples of patients who were positive for HPV DNA. Methods and Material :. We prepared additional samples from liquid -based preparations obtained by the ThinPrep method. The CISH procedure was performed on the Benchmark Automated Slide Stainer (Ventana) according to the manufacturer’s standard protocol. Results and Conclusions : We found HPV DNA in the samples of 10 patients by using INFORM HPV III high risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 68, and 70) (Ventana, Tucson, AZ) probe, with CISH method on each additional sample, and biopsy results of the respective patients, indicated low grade squamous intraepithelial lesion (LGSIL) in 8 patients and high grade squamous intraepithelial lesion (HGSIL) in 2 cases. Keywords: Human papillomavirus, chromogenic in situ hybridization, atypical squamous cells of undetermined significance DOI: 10.7176/JHMN/80-10 Publication date: September 30 th 2020

Predicting Effects of Clinicopathological Variables on Her2 Gene Amplification by Chromogenic in situ Hybridization (CISH) in IHC Her2 (2+) Breast Cancer Patients; A Study from Iran.

Background & Objective:The her2 amplification plays an important role in breast cancer management. Therefore, there is a need for using supplementary molecular methods in IHC equivocal cases. Present study has been conducted to determine the effects of clinicopathological variables on her2 gene amplification by chromogenic in situ hybridization (CISH) in IHC Her2 (2+) breast cancer individuals.Methods:A cross-sectional study was conducted in Zaferanyeh Laboratory collaborated with Shahid Beheshti University of Medical Sciences (Tehran-Iran; 2015-2018). All pathological data related invasive breast cancer patients with equivocal IHC results were included. CISH method was performed as a supplementary technique. The associations between histopathologic variables, status of Ki-67 index, progesterone and estrogen receptors (PR & ER) with her2 amplification by CISH were investigated and analyzed. The level of significance was considered as P-value < 0.05.Results:Totally, 239 patients with mean age of 53.2 years were studied. CISH identified her2 gene amplification in 51 subjects (21.3%). The type of tumor (invasive ductal carcinoma), the tumor grade, and the value of Ki-67 index were directly correlated with her2 amplification. Significant negative associations were also observed between CISH results and ER and PR expression.Conclusion:As her2 gene amplification was identified in 21.3% of invasive breast cancer patients with equivocal IHC results, it is supposed that applying CISH method may consider as a potentially valuable supplementary method. Results have also shown that higher grades of tumor, invasive ductal carcinoma, absences of hormone receptors and high Ki-67 index significantly correlated with the her2 amplification.

Evaluating HER2 Gene Amplification Using Chromogenic In Situ Hybridization (CISH) Method In Comparison To Immunohistochemistry Method in Breast Carcinoma.

BACKGROUND:In patients with breast cancer, HER2 gene expression is of a great importance in reacting to Herceptin treatment. To evaluate this event, immunohistochemistry (IHC) has been done routinely on the basis of scoring it and so the patients were divided into 4 groups. Lately, as there have been disagreements about how to treat score 2 patients, chromogenic in situ hybridization (CISH) and florescence in situ hybridization (FISH) are introduced. Since CISH method is more convenient than FISH for gene amplification study, FISH has been substituted by CISH.AIM:The current study is conducted in order to investigate whether using CISH is a better method comparison to IHC method for determines HER2 expression in patients with breast cancer in.METHODS:In this cross-sectional descriptive analytical study, information of 44 female patients with invasive ductal breast cancer were gathered from Imam Reza and Omid Hospital in Mashhad. IHC staining was done for all patients in order to determine the level of HER2 expression, and after scoring them into 4 groups of 0, +1, +2 and +3, CISH staining was carried out for all 4 groups. At the end, results from both methods were statistically evaluated using SPSS software V.22.0.RESULTS:The average age of patients was 50.2 with the standard deviation of 10.96. Using IHC method was observed that 2.6% (1 patient), 26.3% (10 patients), 65.8% (25 patients) and 5.3% (2 patients) percentage of patients had scores of 0, +1, +2 and +3. On the other hand, CISH method showed 36 patients (90%) with no amplifications and 4 (10%) with sever amplifications. In a comparative study using Fisher’s exact test (p = 0.000), we found a significant relation between IHC method and CISH method indicating that all patients showing severe amplifications in CISH method, owned scores of +2 and +3 in IHC method.CONCLUSION:According to the present study and comparing the results with similar previous studies, it can be concluded that CISH method works highly effective in determining HER2 expression level in patients with breast cancer. This method is also able to determine the status of patients with score +2 in IHC for their treatment with herceptin.

Modern Approach to Cervical Cancer Screening Program – Georgian Experience

1. Abstract 1.1.Objective:The study aimed to pilot the modern approach to cervical cancer screening program, which means the application of liquid based cytology and chromogenic in situ hybridization(CISH) for human papillomavirus (HPV) genotyping on atypical cervical smears. 1.2.Materials and Methods:1293 cervical cytology samples have been analyzed in country of Georgia. The samples had been collected and processed by the usage of materials and equipment provided by Hologic. Prepared smears were post-fixed in 96% ethanol and stained accordingly with Papanicolau protocol. The Bethesda 2001 system terminology was employed for reporting and diagnoses of cervical smears. Patients with diagnosed atypia were recalled for obtaining of material for HPV genotyping. This has been performed by usage of CISH method. 1.3.Results:The negative for intraepithelial lesion or malignancy (NILM) category was equal to 1156 cases (89.40%). Other categories in decreasing order were atypical squamous cells of undetermined significance (ASCUS) with 104 cases (8.04%), low grade squamous intraepithelial lesion (L-SIL) with 8 cases (0.62%), high grade squamous intraepithelial lesion (H-SIL) with 1 cases (0.08%), atypical squamous cells, cannot exclude high grade intraepithelial lesion (ASC-H) with 21 case (1.63%) and atypical glandular cells of undetermined significance (AGUS) with 3 case (0.23%). Cellularity was lower in liquid based cytology (LBC) as compared with conventional smears (CS). Also, nuclear overlap was significantly less observed compared to CS. The smear background was notably cleaner and cell morphology was better evaluated in LBC. In terms of Trichomonas and Candida detection, LBC was superior compared to CS. Doderlein lactobacilli were seen in significantly lesser amounts and were mainly situated in close vicinity to the squamous epithelial cells. Due to lack of pretreatment, the degree of inflammation was better assessed in CS. 1.4.Conclusion:Our experience shows that LBC is superior to CS in the evaluation of cell morphology and detection of certain microorganisms such as Trichomonas and Candida. The degree of inflammation is better assessed with CS. CISH is effective and easy for implementation method for HPV genotyping on cervical smears. There has been revealed 76.12% concordance in average between genotyping results and cytopathology findings of routine screening.

Relationship of Amplification and Expression of the C-MYC Gene with Survival among Gastric Cancer Patients.

During the past decades, the incidence and mortality rate of stomach cancer has demonstrated a great decrease in the world, but it is still one of the most common and fatal cancers especially among men worldwide, including Iran. The MYC proto-oncogene, which is located at 8q24.1, regulates 15% of genes and is activated in 20% of all human tumors. MYC amplification and overexpression of its protein product has been reported in 15-30% of gastric neoplasias. The aim of this investigation was to find the relative efficacy of CISH (chromogenic in situ hybridization) or IHC (immunohistochemistry) in diagnosis and prognosis of gastric cancer, as well as the relationship of amplification and expression of C-MYC gene with patient survival. In this cross-sectional study, 102 samples of gastric cancer were collected from patients who had undergone primary surgical resection at the Cancer Institute Hospital, Tehran University of Medical Sciences, from July 2009 to March 2014. All samples were randomly selected from those who were diagnosed with gastric adenocarcinomas. CISH and IHC methods were performed on all of them. Patients were classified into two groups. The first consisted of stage I and II cases, and the second of stage III and IV. Survival tests for both groups was carried out with referrnce to CISH test reults. Group II (stage III and IV) with CISH+ featured lower survival than those with CISH- (p=0.233), but group I (stage I and II) patients demonstrated no significant variation with CISH+ or CISH- (p=0.630). Kaplan-Meier for both groups was carried out with IHC test findings and showed similar results. This data revealed that both diffuse and intestinal types of gastric cancer occurred significantly more in men than women. Our data also showed that CISH+ patients (43%) were more frequent in comparison with IHC+ patients (14.7%). For planning treatment of gastric cancer patients, by focusing on expanding tumors, which is the greatest concern of the surgeons and patients, CISH is a better and more feasible test than IHC, in regard to sensitivity and specificity. Therefore, CISH can be used as a feasible test for tumor growth and prognosis in stage III and IV lesions. This study also indicated that C-MYC amplification in gastric cancer is correlated with survival in advanced stages.

Study of C-MYC amplification and expression in Iranian gastric cancer samples using CISH and IHC methods.

Background:Gastric cancer is the fourth most frequent malignancy and the second cause of cancer-related mortality worldwide. It has been suggested that in gastric carcinogenesis, the C-MYC gene has an important function. The objective of this study is to establish the preference of Chromogenic in situ hybridization (CISH) and Immunohistochemistry (IHC) in the diagnosis and prognosis of gastric cancer.Materials and Methods:Samples comprised of 50 randomly selected patients of whom 40 were male and 10 female. To evaluate the MYC copy number and its protein expression, CISH and IHC analyses were performed for 50 gastric adenocarcinomas, in Iran.Results:The location of the tumor in 64% of the patients was the fundus, and in 72% of patients, the tumors were of a diffuse type; 22 samples showed no amplification, and 28 samples were with amplification. MYC immunoreactivity was observed in 13 samples. Twelve samples showed both MYC amplification and MYC immunoreactivity. In addition, among the 28 CISH+ samples, 12 samples had positive signals for IHC and 16 samples had negative signals for IHC. A majority of the IHC-negative patients had no amplification, but only one patient with IHC positive had no amplification.Conclusion:Our conclusion was that for the management and treatment of gastric cancer, and for special attention of clinicians, for prognosis and tumor progression, the CISH was a better and more feasible test than IHC, in regard to the sensitivity and specificity.

Comparison of HER2 status in breast cancer patients using CISH and immunohistochemical protocols

The evaluation of human epidermal growth factor receptor-2 (HER2) status in invasive breast carcinoma has been proven to be of prognostic value for the patients. HER2 overexpression is found in about 20% of the breast cancer cases and is thought to be associated with a poor clinical outcome. The test can be performed by different molecular methods, including in situ hybridization or immunohistochemistry. We evaluated the accuracy of our immu-nohistochemical protocol for HER2 breast cancer testing in comparison to the chromogenic in situ hybridization method (CISH). A total of 19 cases were analyzed. Using the HercepTest from Dako, three cases were initially evaluated as 3+ strong positive (with concomitant hormone receptor positivity). Fifteen cases were evaluated as 2+ weak positive and one triple negative case as 1+ negative. The results from CISH confirmed HER2 gene amplification in all three 3+ positive cases, whereas 46.7% of the 2+ cases showed gene amplification. The triple negative 1+ case did not reveal HER2 gene amplification. These results indicate that our immunohistochemical protocol has satisfactory sensitivity for detecting HER2 3+ positive cases and can reliably be used to select the HER2 negative cases. Nevertheless, CISH protocol should be used to clarify all ambiguous cases.

Chromosomal Aberrations Detected by Chromogenic In Situ Hybridization in Abdominal Wall Endometriosis After Cesarean Section

The goal of this study is to evaluate the chromosomal loss in abdominal wall endometriosis by chromogenic in situ hybridization (CISH). Twenty-four cases of abdominal wall endometriosis that developed after cesarean section at the Korea University Medical Center between January 1997 and December 2006 were selected. CISH was performed in the sections of tissue microarray block using the Zymed CISH centromeric probes for chromosomes 3, 7, 8, 9, 10, 11, 17, and 18. Monosomy was defined when the percentage of the nuclei with a single dot was more than mean+3 SD of the respective probe in normal control endometrium. CISH study was possible in more than half of the endometriosis samples, except for chromosome 9, and was most successful for chromosome 17. The frequency of monosomy was high for chromosomes 9 (75.0%) and 17 (73.9%), moderate for chromosomes 10 (57.1%) and 18 (56.3%), and low for chromosomes 3 (12.5%), 7 (22.2%), 8 (10.5%), and 11 (10.5%). Monosomy for >2 and 3 chromosomes occurred in 66.7% and 42.9% of the cases, respectively. It is concluded that CISH method may be considered a useful laboratory technique in detecting chromosomal loss, and multiple chromosomal loss is involved in the formation of ectopic endometrium in abdominal wall endometriosis.

Outcome and evaluation of 1p and 19q deletion by chromogenic in situ hybridization (CISH) in patients with diagnosis of oligodendroglioma (OD) and oligoastrocitoma (OA).

e21025 Background: In OD and OA, the 1p and 19q deletion has prognostic value in survival. It is also a predictive factor for response to chemotherapy. Fluorescence in situ hybridization (FISH) is the standard method for its evaluation. CISH could be an alternative that has already been validated in other neoplasias. In OD and OA, this combined deletion is present in about 50% of patients when analyzed with FISH. Methods: Patients resected at Clinica Reina Fabiola from january 2006 to january 2010 and diagnosed of OD and OA were propectivelly included. Paraffin-embebed tumor tissue was analyzed for 1p19q deletions by CISH. The results were correlated to the histology (OD and OA) and grade (II and III) of the tumors. Results: The demographic features of the patients from the present study coincide with literature. The 1p and 19q deletion was found in 3 of the 24 patients analyzed (13%). The combined deletion was only found in those with grade II OD. No combined deletion was found in patients diagnosed of grade III OD and grade II and III OA. In the subgroup of patients with grade II OD, the combined deletion was observed in 3 of 11 patients (27%). The 3 patients in which the deletion in both chromosomes was observed, received treatment with chemotherapy and radiotherapy, all of them with complete response. 5 years DFS was 90%-median follow up 36,8 (CI: 30,5-42,98) Conclusions: The detection of the combined deletion with CISH technique was inferior (13%) than the literature. We cannot demonstrate that CISH is a reliable method for the detection of the 1p and 19q deletion. The possible reasons of this difference could be attributed to the number of patients of the study, to deviations in the procedures of the test or to the fact that the CISH method is not coincident with FISH. This prospectivelly monoinstitutional results are also different to our previous report, and may be due to different pathological evaluation.

Detection of ALK Gene Rearrangement in Non-small Cell Lung Cancer: A Comparison of Fluorescence In Situ Hybridization and Chromogenic In Situ Hybridization with Correlation of ALK Protein Expression

Accurate determination of ALK rearrangement is important in lung cancer patients, especially in determining their eligibility for crizotinib therapy. Fluorescence in situ hybridization (FISH) has been regarded as the gold standard method for detecting ALK rearrangement. However, FISH requires a fluorescence microscope, and the signals are labile and rapidly fade over time. This study evaluates the concordance between ALK gene rearrangement in non-small cell lung cancer assessed by ALK FISH and a newly developed ALK chromogenic in situ hybridization (CISH) and correlates the results with ALK protein expression assessed by immunohistochemistry. A total of 465 formalin-fixed, paraffin-embedded non-small cell lung cancer samples were analyzed by ALK FISH (PathVysion, Vysis, Abbott) and ALK CISH. For comparison, all specimens were stained by immunohistochemistry (clone 5A4, Novocastra) and interobserver reproducibility was assessed. We found that agreement between the pathologists on the CISH-determined ALK status was achieved in 449 patients (96.6%), and ALK rearrangement was identified in 18 patients (4.0%) in CISH method. Among these cases, 443 cases (95.3%) had results matching the corresponding FISH results: 17 rearranged, 425 wild types, and 1 discordant case. There was high concordance in the assessment of ALK gene rearrangement between FISH and CISH techniques (κ = 0.92) and between observers (κ = 0.97). In addition, there was high concordance in the ALK gene status and ALK protein expression between CISH and IHC tests (κ = 0.82). CISH is a highly reproducible and practical method to detect ALK gene rearrangement and correlated well with ALK protein expression. Here, we present a diagnostic algorithm (Chung's SNUBH ALK protocol) to detect lung cancer with ALK rearrangements using IHC, FISH and CISH. Because CISH allows a concurrent analysis of histological features of the tumors and gene rearrangement, it appears to be a useful method in determining ALK gene rearrangement.

Visualization of FISH Probes by Dual-Color Chromogenic In Situ Hybridization

The overall purpose of the study was to demonstrate applicability of the DAKO dual-color chromogenic in situ hybridization (CISH) assay (DAKO Denmark, Glostrup) with respect to 4 fluorescence in situ hybridization (FISH) probes: MYC (c-MYC), EGFR, ERBB2 (HER2), and TOP2A. The study showed that the dual-color CISH assay can convert Texas red and fluorescein isothiocyanate (FITC) signals into chromogenic signals with an almost complete 1:1 conversion ratio. Agreement studies between the FISH assays for HER2 and TOP2A and the corresponding CISH conversion assays showed 100% concordance (kappa values of 1.0) between the CISH and FISH methods for HER2 and TOP2A status. The correlations of the gene copy number to centromere-17 ratios were similarly high, with a correlation coefficient (r) for HER2 and TOP2A of more than 0.95. Owing to the relatively small number of specimens in this study, it is important that the data are confirmed in a larger study.

The Correlation Between Dual-color Chromogenic In Situ Hybridization and Fluorescence In Situ Hybridization in Assessing HER2 Gene Amplification in Breast Cancer

Fluorescence in situ hybridization (FISH) is regarded as the gold standard method for detecting HER2 gene amplification. Chromogenic in situ hybridization (CISH) is a promising alternative to FISH because CISH has the advantages of being a method evaluated by bright-field microscopy and the generated chromogenic signals are also stable. This study presents a dual color CISH for simultaneous detection of the HER2 gene and chromosome 17. The CISH method performs a chromogenic detection "on top" of the Food and Drug Administration (FDA)-approved HER2 FISH pharmDx method, where the fluorochrome-labeled probes are detected using enzyme-labeled antibodies and visualized by chromogenic enzymatic reactions. The HER2 status (amplified/not amplified and HER2 ratios) was evaluated by the CISH method and compared with results obtained by the FDA-approved FISH method. Of the 72 successfully investigated invasive breast carcinomas, both FISH and CISH detected HER2 amplification in 24 cases and nonamplification was detected in 47 cases. One case showed a discrepancy between FISH and CISH. The concordance between CISH and FISH was found to be almost perfect (98.6%). The correlation between the HER2 ratios obtained by the 2 methods showed excellent correlation (correlation coefficient 0.95). In conclusion, it is possible by dual-color CISH method to demonstrate HER2 genes and chromosome 17 genes, in the same tissue section and reliably assess HER2 status. The CISH method is a very promising alternative to the FISH method.

Chromogenic in situ hybridization detection of chromosome 7 and 17 abnormalities in renal cell carcinomas and comparison to flow cytometric DNA ploidy patterns

ABSTRACT Background and aim: The chromogenic in situ hybridization (CISH) is a new method in which chromosome aberration is determined by using a chromogenic reaction. We report the aberrations of chromosome 7 and 17 in three major subtypes of renal cell carcinomas (RCC), and compared these chromosomal changes with the flow cytometric DNA ploidy patterns. Methods: Aberrations of chromosomes 7 and 17 in 21 clear cell (c), 6 papillary (p) and 15 chromophobe (ch) RCCs were studied and compared with flow cytometric DNA ploidy. Results: Two of 14 chRCCs (14.3%) were monosomic for chromosome 7, but none of the cRCCs or pRCCs were monosomic. Twelve (85.7%) chRCCs showed monosomy for chromosome 17, but none of the pRCCs were monosomic. Three pRCCs (50%), 5 of 14 (35.7%) chRCCs and 2 (9.5%) cRCCs were polysomic for chromosome 7, whereas only one pRCC was polysomic. DNA aneuploidy was found in 2 pRCCs (33.3%), 2 cRCCs (9.5%), and 6 chRCCs (46.2%). Hypodiploidy was seen in one chRCC, and triploidy was seen in all types of RCCs. DNA aneuploidy was more frequent in cases with both chromosome 7 and 17 abnormalities (80%) than in cases with abnormality of one (38.5%) or neither (4.5%). Conclusion: The CISH method is useful in detecting chromosomal change in RCC, and the identification of chromosome 17 monosomy is helpful in diagnosis of chRCC.

Amplification of the epidermal growth factor receptor gene in glioblastoma: An analysis of the relationship between genotype and phenotype by CISH method

We examined epidermal growth factor receptor (EGFR) overexpression and EGFR gene amplification using immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) in 109 glioblastomas, including 98 primary glioblastomas and 11 secondary glioblastomas. EGFR overexpression and EGFR gene amplification were found in 33% and 24% of glioblastoma, respectively, and all of those cases were primary glioblastoma. Large ischemic necrosis was significantly more frequent in primary glioblastomas than in secondary glioblastomas (54% vs. 18%), but pseudopalisading necrosis was not (65% vs. 54%). EGFR gene amplification was detected significantly more frequently in cases with both types of necrosis. Although glioblastomas with EGFR gene amplification invariably exhibited EGFR overexpression at the level of the whole tumor, tumor cells with EGFR gene amplification did not always show EGFR overexpression at the level of individual tumor cells. Cases of "strong" EGFR overexpression on IHC could be regarded as having EGFR gene amplification, and cases without EGFR overexpression could not. Cases of "weak" EGFR overexpression should be tested with CISH to confirm the presence of EGFR gene amplification. We found that 54% of glioblastomas with EGFR gene amplification were composed of areas with and without EGFR gene amplification; however, there were no obvious differences in morphology between tumor cells with and without EGFR gene amplification. Although small cell architecture might be associated with EGFR gene amplification at the level of the whole tumor, it did not always suggest amplification of the EGFR gene at the level of individual tumor cells. In one case, it seemed to suggest that a clone with EGFR gene amplification may arise in pre-existing tumor tissue and extend into the surrounding area. In cases of overall EGFR amplification, CISH would be a useful tool to decide the tumor border in areas infiltrated by tumor cells.

Chromogenic In Situ Hybridization to Detect HER-2/neu Gene Amplification in Histological and ThinPrep®-Processed Breast Cancer Fine-Needle Aspirates: A Sensitive and Practical Method in the Trastuzumab Era

The increasing evidence of trastuzumab efficacy in breast cancer (BC) patients means that an accurate and reproducible evaluation of HER-2 statusis of paramount importance in histological and in cytological samples. Currently, the two main methods used to analyze HER-2 amplification or overexpression are fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Although the two methods are strongly correlated for histological tissue, the evaluation of tumor morphology through FISH may be difficult and fluorescence fades quickly. These limitations can be overcome by chromogenic in situ hybridization (CISH), which can visualize the amplification product along with morphological features. In view of this, in the present study, we analyzed the usefulness of CISH on formalin-fixed, paraffin-embedded (FFPE) BC specimens and investigated whether CISH can be a valid technique in the determination of HER-2 status for fine-needle aspirates (FNAs) processed by liquid-based cytology. The results we obtained in a retrospective series of 111 FFPE BC specimens demonstrated good concordance between CISH and IHC and between CISH and FISH. The former concordance was comparable with that observed between FISH and IHC. When CISH was applied to a prospective series of 53 FNAs, from surgically removed BC, our data showed evidence of a higher concordance of results between liquid-based cytology and the companion FFPE tissues using CISH rather than HercepTesttrade mark. Therefore, CISH analysis, which is avaluable and reproducible alternative to FISH for selecting breast cancer patients for trastuzumab therapy, can lower false-positive immunocytochemistry findings in ThinPrep-processed FNAs.

HER2 Positivity in Breast Carcinoma: A Comparison of Chromogenic In Situ Hybridization With Fluorescence In Situ Hybridization in Tissue Microarrays, With Targeted Evaluation of Intratumoral Heterogeneity by In Situ Hybridization

An accurate and reproducible assay method for determining HER2 status is crucial, as a positive HER2 gene status is an eligibility requirement for Herceptintrade mark therapy. Although immunohistochemical (IHC) assessment is both practical and inexpensive, a worrying trend of high false-positive rates has been reported. Fluorescence in situ hybridization (FISH) is the universally accepted gold standard for confirming IHC 2+ cases and ambiguous results but is costly and requires specialized equipment and technical expertise. Chromogenic in situ hybridization (CISH) amalgamates the practical advantages of IHC with the reproducibility of FISH, and high concordance between the CISH and FISH methods has been reported in conventional sections. Tissue microarrays (TMAs) allow high throughput of specimens, and HER2 status assessment in TMA cores using IHC and FISH has correlated well with scores in conventional sections. The authors used TMA technology to compare the efficacy of ZYMED(R) CISH with PathVysiontrade mark FISH in a cohort of 119 archival breast resection cases and investigated possible intratumoral heterogeneity in a "mini-array" of 21 HercepTest "equivocal"/2+ cases. Concordance between FISH and CISH in TMA sections was 99%. All prescored 2+ HercepTest cases were nonamplified. Four 3+ HercepTest cases were classed as potential false-positives. The authors suggest that confirmatory ISH should be performed on all positive HercepTest cases. CISH was easier to perform and quicker to enumerate than FISH. The authors conclude that CISH is a practical alternative to FISH as a confirmatory tool for HER2 gene amplification status. Intratumoral heterogeneity did not affect the patient's HER2 status.