Chromogenic In Situ Hybridization Assay Research Articles

Abstract 433: Clinical validation of a Dickkopf-1 (DKK1) chromogenic in-situ hybridization (CISH) assay for manual and image-analysis assisted pathologist interpretation

Abstract Dickkopf-1 (DKK1) is a secreted modulator of Wnt signaling that is frequently overexpressed in tumors and associated with a poor prognosis. In this study we demonstrate an approach for clinically validating a RNAscope chromogenic in situ hybridization (CISH) assay for determining the level of DKK1 RNA in Gastric (G) and Gastroesophageal (GEJ) tumor tissues according to CLIA guidelines. This two-step process validated first the performance of the wet assay along with the ability of a pathologist to manually score the CISH signal according to a dot-based H-score paradigm, and second the ability of image analysis (IA) software (Flagship Biosciences) to unbiasedly and reproducibly quantify DKK1 staining in the same set of samples. The DKK1 CISH assay for manual scoring passed all pre-determined criteria of sensitivity, specificity, accuracy, and precision. 100% of the 40 evaluated G/GEJ tissues demonstrated acceptable staining in target tumor cells and absence in non-target cells. 100% of the evaluated tissues passed sensitivity with a broad dynamic range of signal expression across target cells, and negligible background staining. Reproducibility was measured by blinded pathology scoring of a serial subset of 12 cases, resulting in 11/12 (92%) with concordant DKK1 H-scores. Accuracy was assessed with a DKK1 qPCR assay on a 20-sample subset and a significant correlation with the H-score data was observed. The IA algorithm also passed all pre-determined criteria of sensitivity, specificity, accuracy, and precision. 36/40 samples (90%) passed analytical specificity with the IA algorithm correctly classifying staining on true cells belonging to the tumor. Failed samples were attributed to non-specific alkaline phosphatase activity, which in practice would be disregarded by the reviewing pathologist. 100% of samples passed the sensitivity criteria of appropriate cell identification and classification. IA precision compared H-scores across 3 days of staining, with 11/12 (92%) cases having concordant DKK1 H-scores and an ICC of 0.9009 (95% CI: 0.7117-0.9601). Digital H-scores were highly correlated to the validated manual H-scores of the 40-sample set (r = 0.72, p <0.0001). Taken together, these data demonstrate a clinically validated DKK1 RNAscope CISH laboratory-derived test (LDT) for manual and IA-assisted pathologist interpretation. The DKK1 RNAscope LDT is currently being applied as part of a phase 2 clinical study of DKN-01 in combination with tislelizumab to prospectively identify previously treated G/GEJ adenocarcinoma patients with elevated DKK1 tumor expression (NCT04363801). Citation Format: Charles Caldwell, Mike Kagey, Sofia Reitsma, Will Paces, Elizabeth Bueche, Vitria Adisetiyo, Roberto Gianani. Clinical validation of a Dickkopf-1 (DKK1) chromogenic in-situ hybridization (CISH) assay for manual and image-analysis assisted pathologist interpretation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 433.

Chromogenic Tissue-Based Methods for Detection of Gene Amplifications (or Rearrangements) Combined with Protein Overexpression in Clinical Samples.

Immunohistochemistry (IHC) is a well-established, tissue-based assay for the visualization of target proteins. For analysis of DNA targets, chromogenic in situ hybridization (CISH) applications have significant advantages over traditional fluorescence in situ hybridization (FISH). CISH slides can be analyzed using a regular light microscope, while FISH slides require the use of a specialized fluorescence microscope in a dark room. CISH slides allow observers to correlate the gene status (gene amplifications, gene rearrangements, and gene deletions) in the context of tissue morphology better than FISH slides. Recently, a combination of IHC and CISH assays (gene-protein assay, GPA) was developed to study the relationship between gene status and protein expression on the same tissue section. CISH and GPA applications can be optimized using an automated tissue slide processing system to generate reproducible results for a long and complex assay protocol. GPA applications are an ideal approach for tumor status and heterogeneity analyses for research and clinical investigations.

Abstract 5136: Development of an RNA hybridization technique for the in situ visualization of bacteria in cancer tissues

Abstract There is currently a great interest in understanding direct interactions between human-associated microbial populations and the tumor microenvironment. In this study, we aimed to develop a chromogenic in situ hybridization (CISH) assay for universal detection of bacteria in formalin fixed paraffin embedded (FFPE) cancer tissues. This was accomplished using the RNAscope assay (Advanced Cell Diagnostics) with a CISH probe set designed to target the most highly conserved region in the bacterial 16S ribosomal RNA (rRNA) gene. Optimization of the 16S rRNA CISH assay was conducted using FFPE mouse colon, Mycobacterium tuberculosis infected rabbit lung, and Propionibacterium acnes infected mouse prostate tissues. Positive control (peptidylprolyl isomerase B- PPIB) and negative control (maize –Zm) stains were also conducted on the tissues. We additionally developed immunohistochemistry (IHC) assays with antibodies against lipopolysaccharide (LPS) and lipoteichoic acid (LTA) to visualize gram negative and gram positive bacteria, respectively, as a means to verify the results of the CISH assay. As our laboratory has a specific interest in the interactions between prostate infections and prostate cancer, we used this technique to interrogate a series of 10 radical prostatectomy specimens that contained a very high degree of acute and chronic inflammation, and were suspicious for the presence of an infectious organism. Both CISH and IHC were conducted in FFPE radical prostatectomy samples. The 16S rRNA CISH assay detected bacteria in all of the control tissues. Interestingly, 3 of the 10 human radical prostatectomy samples that we examined showed bacterial 16S rRNA signal concentrated in areas with acute inflammation where the glandular lumens were filled with neutrophils. Two of the radical prostatectomy samples that showed positive bacterial 16S rRNA CISH signal also showed positive LPS staining indicative of the presence of gram negative bacteria. The remaining positive sample was suspicious for a gram positive intracellular organism. This study shows preliminary data supporting robust detection of bacteria in a subset of human prostate samples. We expect the frequency of bacterial infections of this nature present in radical prostatectomy specimens would be low, and that we were only able to identify these cases because we screened for samples with very high levels of inflammation. Detection of both bacterial RNA and protein in the tissues corroborates the strength and reliability of the 16S rRNA CISH. The use of these two methods concurrently can provide valuable information on bacterial presence in a range of clinical samples. In the future, we plan to use these methods to probe for bacterial presence and in understanding the spatial dynamics of the urinary microbiome. We expect that this method may also be of broader use in the study of many infection-associated cancers. Citation Format: Eva Shrestha, Karen S. Sfanos. Development of an RNA hybridization technique for the in situ visualization of bacteria in cancer tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5136.

Determination of HER-2/neu gene Status by Chromogenic in Situ Hybridisation Assay on Borderline (2+) Immunohistochemistry Cases in Patients with Invasive Breast Carcinoma: An Experimental Study on Preserved Tissue

HER-2 (also known as HER-2/neu) is a member of the epidermal growth factor (EGF) receptor family. The amplification of oncogene HER-2 is presented in 20 to 30% of breast cancers and results in an increase of the protein expression. HER-2 over expression has also been shown to correlate with poor prognosis. It is associated with poorly differentiated high grade tumors with lymph node involvement, greater risk of recurrence and relative resistance to some types of chemotherapy. The receptor is however a target for treatment with anti HER-2 antibody trastuzumab (Herceptin). Immunohistochemistry analysis for HER 2 scoring is subjective, requires trained personnel and expertise. One of the main concerns with IHC is that there is evidence of significant inter-observer variation in the assessment of staining, which can lead to misclassification of HER2 status. Chromogenic in situ hybridization (CISH) testing is sensitive and specific in detecting HER-2/neu gene amplification. Direct evaluation of gene amplification using CISH assay is a reliable method for routine diagnostic evaluation of HER2/neu status in breast cancer patients, especially in specimens showing 2+ IHC score. This observational experimental study was carried out in the Department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka, during the period of July 2014 to June 2015, The aim of this study was to assess HER-2 expression accurately in equivocal immunohistochemistry (2+) invasive breast cancer cases by Chromogenic in situ hybridization (CISH) and to associate the findings of CISH assay with the histologic prognostic and predictive factors (eg: tumor size, regional lymph node metastases, tumor grade and type. A total of 20 archival paraffin tissue blocks and IHC slides with IHC score 2+ for HER-2 were included in this study. All the slides were reviewed. CISH assay was done on section from archival paraffin block.CISH assay showed amplification of the HER-2 neu gene in 30% of cases. Majority (60%) was nonamplified. In two cases the results were unsatisfactory for interpretation. The differences were not statistically significant (p>0.05) among three groups (amplified, nonamplified and unsatisfactory) regarding the baseline characteristics (age and sex) with CISH. All the results of CISH assay in association with tumor size, tumor grade and lymphnode metastasis were not statistically significant. HER-2 / neu status is the one of the most important prognostic and predictive factor. It is a target for treatment with anti HER-2 antibody trastuzumab (Herceptin). Detection of HER-2/ neu status by IHC may sometimes be difficult and inaccurate, specially in IHC score 2+ cases. Based on the findings of the study, CISH can be considered a useful, simple and reproducible method for detecting HER-2/ neu gene amplification in cases with borderline (2+) immunohistochemistry score , and patients may be benefited from Trastuzumab therapy.

Chromogenic in situ Hybridization Compared with Real Time Quantitative Polymerase Chain Reaction to Evaluate HER2/neu Status in Breast Cancer

The assessment of human epidermal growth factor receptor 2 (HER2) status has become of great importance in the diagnosis of breast cancer. The aim of this study was to investigate the diagnostic value of quantitative Polymerase Chain Reaction (qPCR) and Chromogenic In Situ Hybridization (CISH) to assess HER2 status of biopsy specimens. To elucidate the status of HER2 gene amplification, biopsies of breast carcinoma from 120 patients with 2+ IHC status were analyzed by qPCR and CISH. The results of the two experiments were compared, and it was depicted that the concordance rate between CISH and qPCR assays was 88.1%.The quantification of HER2 gene with CISH and qPCR showed that there was a significant correlation (p value= 0.0001 and r= 0.808). The results of this research support the idea that qPCR is a precise and reproducible technique, which can be employed as a supplementary method to evaluate HER2 status.

Abstract 396: Analysis of MET-amplified solid tumors using chromogenic in situ hybridization (CISH)

Abstract MET amplification has been implicated in signaling pathways that promote cell proliferation, invasion, and survival. It has been identified as an oncogenic driver in various malignancies and is currently being investigated as a potential therapeutic target. To date, MET exon 14 skipping by sequencing and MET amplification by FISH have been found to have potential clinical utility in predicting those patients who may derive benefit from MET-targeted therapy. However, little research has been conducted on alternative technologies to FISH such as CISH, which does not require a dark room and can be interpreted by a board-certified pathologist. The purpose of this study is to report our experience with MET amplification across solid tumors using CISH. A retrospective analysis was done on 26,619 specimens analyzed for MET amplification by CISH at a CLIA-certified lab (Caris Life Sciences). The validated CISH assay, previously validated against a FISH assay, utilized a gene copy number > 5 to assess amplification. Concordance and correlative studies were done in MET-amplified, non-small cell lung cancer (NSCLC) specimens analyzed using a cMET IHC (SP44, 2+ or 3+ staining intensity in 50% or more tumor cell membrane) analyzing protein expression. Correlative studies involving co-existing aberrations, including PD-L1 (SP142, any intensity in at least 50% of tumor cells), in this MET-amplified, NSCLC cohort were also performed. MET amplification utilizing CISH was 0.7% (188/26,619) overall. MET-amplified solid tumors included carcinomas such as NSCLC (3.1%, 87/2767), gastric adenocarcinoma (3.8%, 11/293), esophageal and esophagogastric junction adenocarcinoma (3.3%, 11/338), and endometrial carcinoma (0.4%, 9/2020) along with non-carcinomas, including glioblastoma multiforme (1.0%, 5/510), uterine sarcoma (1.3%, 5/400), melanoma (0.4%, 2/538), and rare tumors such as placental-site trophoblastic tumor (100%, 1/1) and prostatic neuroendocrine tumor (100%, 1/1). A sub-analysis of MET-amplified, NSCLC specimens demonstrated co-occurring protein overexpression in 92.6% (75/81) of cases. These same MET-amplified, NSCLC specimens were found to have EGFR pathogenic/presumed pathogenic mutations (19.7%, 15/76), ALK rearrangements (2.5%, 2/80), and PD-L1 overexpression (27.5%, 14/51). ROS1 rearrangements were not detected in this NSCLC cohort (0%, 0/76). Our data suggest MET amplification detection utilizing CISH is a viable option for identifying MET-driven cancers. The presence of MET across various solid tumors contrasts with biomarkers like HER2, which are exclusive to carcinomas. A sub-analysis of our NSCLC population shows MET-amplified tumors contains a similar molecular distribution to the general NSCLC population. Future studies should incorporate MET CISH in clinical trials utilizing MET-targeted agents to determine its potential as a predictive test for evaluating who may derive the most benefit. Citation Format: David Arguello, Zoran Gatalica, Semir Vranic, Sandeep Reddy, Ari VanderWalde. Analysis of MET-amplified solid tumors using chromogenic in situ hybridization (CISH). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 396.

Chromogenic in situ hybridization is a reliable assay for detection of ALK rearrangements in adenocarcinomas of the lung

Reliable detection of anaplastic lymphoma kinase (ALK) rearrangements is a prerequisite for personalized treatment of lung cancer patients, as ALK rearrangements represent a predictive biomarker for the therapy with specific tyrosine kinase inhibitors. Currently, fluorescent in situ hybridization (FISH) is considered to be the standard method for assessing formalin-fixed and paraffin-embedded tissue for ALK inversions and translocations. However, FISH requires a specialized equipment, the signals fade rapidly and it is difficult to detect overall morphology and tumor heterogeneity. Chromogenic in situ hybridization (CISH) has been successfully introduced as an alternative test for the detection of several genetic aberrations. This study validates a newly developed ALK CISH assay by comparing FISH and CISH signal patterns in lung cancer samples with and without ALK rearrangements. One hundred adenocarcinomas of the lung were included in this study, among them 17 with known ALK rearrangement. FISH and CISH were carried out and evaluated according to the manufacturers' recommendations. For both assays, tumors were considered positive if ≥15% of tumor cells showed either isolated 3′ signals or break-apart patterns or a combination of both. A subset of tumors was exemplarily examined by using a novel EML4 (echinoderm microtubule-associated protein-like 4) CISH probe. Red, green and fusion CISH signals were clearcut and different signal patterns were easily recognized. The percentage of aberrant tumor cells was statistically highly correlated (P<0.001) between FISH and CISH. On the basis of 86 samples that were evaluable by ALK CISH, we found a 100% sensitivity and 100% specificity of this assay. Furthermore, EML4 rearrangements could be recognized by CISH. CISH is a highly reliable, sensitive and specific method for the detection of ALK gene rearrangements in pulmonary adenocarcinomas. Our results suggest that CISH might serve as a suitable alternative to FISH, which is the current gold standard.

Abstract 39: Clinical assessment of PTEN mutation in FFPE tissue: comparison of Sanger sequencing, immunohistochemistry and chromogenic in situ hybridization methods.

Abstract Background: PTEN is a tumor suppressor that negatively regulates the PI3K signaling pathway by dephosphorylating PIP3, converting it to PIP2. PTEN loss of function results in activation of Akt and downstream signaling pathways, and has been associated with numerous cancers, most commonly endometrial cancer, glioblastoma, melanoma and prostate cancer. PTEN loss can be a result of multiple different aberrations which include mutations in the coding region leading to frame-shift or stop codons, genomic deletions as well as promoter methylation leading to loss of PTEN protein. Determination of PTEN status, both at the DNA and protein levels, may be important for clinical decision-making because loss of PTEN is associated with resistance to various targeted therapies, while inactivating mutations may confer sensitivity to therapeutics targeting PI3K pathway members. We have developed Sanger sequencing, immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) assays to assess PTEN mutation status in a set of FFPE patient specimens. Methods: Our Sanger sequencing assay for PTEN was developed to detect known hotspot mutations in exons 5-8, which occur in the majority of PTEN mutations reported in the COSMIC database. The IHC and CISH assays were developed using commercially available reagents. All assays were validated using a combination of plasmids, cell lines and FFPE tissues for specificity, sensitivity, reproducibility and concordance. Results: 22 patient FFPE tissue specimens from a variety of tumor types including colon, lung, pancreas, uterus and prostate were evaluated for PTEN mutation and expression status by both Sanger sequencing and IHC methods. Sequencing results were: 9 wild-type, 12 heterozygous mutant, and 1 hemizygous mutant. 7 of the 9 specimens reported as wild-type by sequencing exhibited normal protein expression levels by IHC, while 2 were PTEN negative by IHC, suggesting that mechanisms other than mutations in exons 5-8 contributed to the loss of expression of PTEN protein. These 2 specimens were further analyzed by CISH to determine copy number. Of the 13 specimens reported as mutant by sequencing, IHC protein expression results indicated that 6 were PTEN positive and 7 were PTEN negative, underscoring the observation that exon 5-8 mutation status alone is not sufficient to predict PTEN expression levels. Data on CISH analysis of the specimen that was hemizygous mutant by sequencing will be reported to confirm the copy loss. Conclusion: The data suggest that additional mutations outside the exon 5-8 region as well as promoter methylation may result in loss of PTEN protein, and that accurate clinical assessment of PTEN mutation and protein expression status in FFPE tissues requires complimentary methods including Sanger sequencing, IHC and CISH methodologies. Citation Format: Kellen Sakrison, Jennifer Wright, Eric Bruening, Stephane Wong, Cynthia Spittle, Sabita Sankar, Chad Galderisi. Clinical assessment of PTEN mutation in FFPE tissue: comparison of Sanger sequencing, immunohistochemistry and chromogenic in situ hybridization methods. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 39. doi:10.1158/1538-7445.AM2013-39

Evaluation of Myc Gene Amplification in Prostate Cancer Using a Dual Color Chromogenic in-Situ Hybridization (Dual CISH) Assay

Objetive: 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 fluorescence in situ hybridization (FISH) probes MYC-C. Methods: MYC gene amplification by FISH and Dako dual-color CISH Results: The study showed that the dual-color CISH assay can convert Texas red and fluorescein isothiocyanate (FITC) signals into chromogenic signals. The dual –color CISH assay was performed on 40 cases of prostate cancer. Amplification was identified in 12 of 40 (30%) tumors. No amplification was seen in 28 of 40 (70%) tumors. FISH data were available in total of 40 tumors. All tumors showed concordant results between dual-color CISH and FISH for classifying a tumor as MYC amplified or not amplified. Conclusions: We conclude that dual-color Dako CISH assay is an accurate method for determining MYC gene amplification with added advantages that make it a more practically useful method.

Detection of Tritrichomonas foetus and Pentatrichomonas hominis in intestinal tissue specimens of cats by chromogenic in situ hybridization

In this retrospective study 102 cats were analyzed for the presence of trichomonads in intestinal tissue sections using chromogenic in situ hybridization (CISH). Two intestinal trichomonad species are described in cats: Pentatrichomonas hominis and Tritrichomonas foetus. While P. hominis is considered a mere commensal, T. foetus has been found to be the causative agent of feline large-bowel diarrhea. For the detection of both agents within intestinal tissue CISH assays using three different probes were performed. In the first CISH run a probe specific for all relevant members of the order Trichomonadida (OT probe) was used. In a second CISH run all positive samples were further examined on three consecutive tissue sections using the OT probe, a probe specific for the family of Tritrichomonadidae (Tritri probe) and a newly designed probe specifically detecting P. hominis (Penta hom probe). In total, four of the 102 cats were found to be positive with the OT probe. Thereof, one cat gave a positive reaction with the P. hominis probe and three cats were positive with the T. foetus probe. All Trichomonas-positive cats were pure-bred and between 8 and 32 weeks of age. In one cat positive for T. foetus large amounts of parasites were found in the gut lumen and invading the intestinal mucosa. The species of the detected trichomonads were confirmed by polymerase chain reaction and nucleotide sequencing of a part of the 18S ribosomal RNA gene. In this study, the usefulness of CISH to detect intestinal trichomonads within feline tissue samples was shown. Additionally, the specific detection of P. hominis using CISH was established. Generally, it was shown that CISH is well suited for detection and differentiation of trichomonosis in retrospective studies using tissue samples.

“CISH the FISH” forHER2: Our Laboratory Experience

We read with great interest the article by Hoff et al1 on the applicability of visualization of fluorescence in situ hybridization (FISH) probes by dual-color chromogenic in situ hybridization (CISH) and totally agree about the need to perform studies on a larger number of samples to confirm their data. In our laboratory, we have been using this technique for 1 year, and to date our sample is composed of 40 cases tested for HER2 /centromere 17 status by FISH and subsequent CISH conversion. In our sample, the concordance between FISH and CISH assays is 100%, in complete agreement with the results reported by Hoff et al.1 As a matter of fact, as shown in Figure 1, all pairs of ratio values were consistently above or …

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.

P16INK4A Immunohistochemistry is Superior to HPV In Situ Hybridization for the Detection of High-risk HPV in Atypical Squamous Metaplasia

In situ hybridization (ISH) assays for high-risk human papillomavirus (HR-HPV) and immunohistochemical (IHC) assays for surrogate markers such as p16 can be useful in detecting HR-HPV in cervical dysplasia, but the use of these markers in problematic cervical biopsies has not been well-established. We evaluated 3 chromogenic ISH assays (Ventana INFORM HPVII and HPVIII and DakoCytomation GenPoint) in conjunction with p16 IHC and HPV polymerase chain reaction in a study set consisting of 12 low-grade squamous intraepithelial lesions, 16 high-grade squamous intraepithelial lesions, and 30 benign cervix samples. A test set of 28 cases of atypical squamous metaplasia were also evaluated withVentana HPVIII ISH and p16 IHC. In the study set, the sensitivity of the DakoCytomation ISH assay (which detects HPV subtypes 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, and 68) was similar to the Ventana HPVII assay but less than that of the Ventana HPVIII ISH assay (both of which detect HPV subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66) and less than p16 IHC (55.6% vs. 53.6 vs. 69.2% vs. 82.1%). All HPV ISH assays exhibited 100% specificity. p16 reactivity consisted of 2 patterns: focal strong and diffuse strong. Because focal strong p16 reactivity was identified in benign squamous epithelium (6.7% cases) and dysplastic epithelium, it was considered an equivocal result and only diffuse strong reactivity was considered to be specific for the presence of HR-HPV. In the squamous intraepithelial lesions study set, the difference in sensitivity between Ventana HPVIII ISH and p16 was not statistically significant. However, in the atypical squamous metaplasia test set cases, p16 reactivity (focal strong and diffuse strong) was significantly more sensitive than Ventana HPVIII ISH in correlating with the presence of human papillomavirus as detected by polymerase chain reaction (83.3% vs. 33.3% P=0.004). Because focal strong p16 reactivity is less specific, cases with this staining pattern are considered atypical and require further evaluation by other means. Overall, p16 IHC is considered the best candidate for the initial assessment of cervical biopsies that are histologically indeterminate for dysplasia given its wide availability, comparative ease of interpretation, and high sensitivity and specificity.

Her2 Amplification

Detecting Her2 gene amplification has become routine in predicting therapeutic responsiveness in patients with breast carcinoma. Fluorescence in situ hybridization (FISH) is a common technique for detecting Her2 amplification, yet dark field fluorescence microscopy remains problematic for many pathologists. Thus, a technique such as chromogenic in situ hybridization (CISH), in which the more familiar light microscopy can be used, is appealing. Paraffin-embedded sections from 61 breast carcinomas were tested for Her2 amplification by immunohistochemistry (IHC) and CISH. FISH was used to confirm CISH results. Excellent correlation was found between IHC and CISH except in cases considered negative (1+ on the DAKO scale) by IHC. CISH detected low-level Her2 amplification in 4 of 9 of these cases. Amplification was subsequently confirmed by FISH in all but 1 case. When compared with FISH, CISH was more sensitive than IHC for detecting low levels of Her2 gene amplification. Moreover, excellent concordance was found between FISH and CISH, supporting the conclusion that the CISH assay for Her2 gene amplification provides an accurate, effective, and practical alternative to FISH.