Chromogenic In Situ Hybridization Research Articles

Evaluation of the prognostic/predictive value of HER2/TOP2A gene amplification and protein overexpression in patients with high-risk breast cancer treated with dose-dense sequential adjuvant chemotherapy.

e21059 Background: The prognostic/predictive value of HER2 and topoisomerase II alpha (TOP2A) gene status in patients with breast cancer treated with adjuvant dose-dense sequential chemotherapy is not well defined. Anthracyclines and taxanes are considered to be important drugs in this setting. Methods: Formalin-fixed tumor tissue samples were obtained from 354 patients participating in a phase III trial, in which 595 high-risk breast cancer patients were randomized to receive postoperative dose-dense sequential chemotherapy with epirubicin and CMF, with or without paclitaxel. HER2 and TOP2A gene amplification was evaluated by chromogen in situ hybridization (CISH) using ZYMED (InVitrogen) technology in 285 and 266 patients, respectively and protein expression was assessed by immunohistochemistry (IHC) in 297 and 248 patients, respectively. Results: Among the 52 tumors classified HER2-positive by IHC (3+, strong membranous staining in ≥30% of the tumor cells), 50 tumors (96%) were amplified by CISH. As for TOP2A, among the 153 tumors classified positive by IHC (2+ and 3+, moderate and strong nuclear staining in ≥5% of the tumor cells), only 13 (9%) of the tumors were amplified by CISH. 64% of the HER2-positive tumors were also TOP2A-positive when assessed by IHC, while only 21% of the HER2-amplified tumors were also TOP2A-amplified by CISH. Univariate Cox regression analysis did not show any association of HER2 and TOP2A gene amplification or IHC-positivity with OS or DFS, except for an increased risk for progression (HR=1.71, 95% CI: 1.07-2.73, Wald p=0.024) in HER2-positive patients by IHC. No benefit from paclitaxel treatment was detected for HER2 or TOP2A gene amplified or protein overexpressing tumors. Conclusions: HER2 and TOP2A gene amplification or protein overexpression do not appear to be predictive for response to paclitaxel in high-risk breast cancer patients treated with dose-dense sequential adjuvant chemotherapy. Evaluation of mRNA expression and gene amplification of HER2 and TOP2A by RT-PCR and FISH is in progress and the data will be presented at the meeting.

Comparison between HER2 extracellular domain in serum and HER2 overexpression in breast cancer tissue.

e11066 Background: Immunohistochemistry (IHC), fluorescent in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) are reliable ways to identify human epidermal growth factor receptor 2 (HER2) and HER2 amplification. Each technique requires access to tumor tissue and high-quality tissue sample, which may not be available in some patients. HER2 extracellular domain (ECD) may be shed to the serum. The importance of serum ECD in breast cancer (BC) is not yet determined. The first aim of this study was to explore the correlation between serum ECD and HER2 expression in breast cancer tissue. The second aim was to compare ECD levels with clinical and pathological features in early BC patients. Methods: This prospective trial included patients with stage I–III BC. Serum HER2 ECD levels were measured by ADVIA Centaur automated assays before surgical resection of the tumor. Tissue HER2 status was detected by IHC in all tumors. CISH test was utilized in HER2: +2 tumors by IHC. Serum ECD >15ng/ml w...

Frequent homozygous deletion of the LKB1/STK11 gene in non-small cell lung cancer

LKB1/STK11 is a tumor suppressor and a negative regulator of mammalian target of rapamycin signaling. It is inactivated in 30% of lung cancer cell lines but only 5-15% of primary lung adenocarcinomas. There is evidence that homozygous deletion (HD) of chromosome 19p at the LKB locus contributes to the inactivation of the gene in primary human lung cancers. Here, we used several complementary genetic approaches to assess the LKB1 locus in primary non-small cell lung cancers (NSCLCs). We first analyzed 124 NSCLC cases for allelic imbalance using eight microsatellite markers on chromosome 19p, which revealed an overall rate of 65% (80 of 124) loss of heterozygosity (LOH). We next used chromogenic in situ hybridization (CISH) to directly examine the chromosomal status of the LKB1 locus. In all, 65 of 124 LOH tested samples were available for CISH and 58 of those (89%) showed either loss of one copy of chromosome 19p (LOH, 40 of 65 cases, 62%) or both copies (HD 18 of 65 cases, 28%). The occurrence of HD was significantly more frequent in Caucasian (35%) than in African-American patients (6%) (P=0.04). A total of 62 of 124 samples with LOH at one or both markers immediately flanking the LKB1 gene were further analyzed by directly sequencing the complete coding region, which identified 7 of 62 (11%) tumors with somatic mutations in the gene. Jointly, our data identified total inactivation of the LKB1 gene by either HD or LOH with somatic mutation in 39% of tested samples, whereas loss of chromosome 19p region by HD or LOH at the LKB1 region occured in 90% of NSCLC.

Abstract 2954: The transient receptor potential V6 (TRPV6) calcium channel in breast cancer cell lines and clinical breast cancer tissues

Abstract TRPV6 is an important calcium channel, which is highly selective for Ca2+ and has high basal activity. In prostate tumors TRPV6 over-expression is linked to enhanced invasiveness and proliferation. While some studies have demonstrated that TRPV6 is over-expressed in clinical breast cancer samples and studies from our laboratory have shown that TRPV6 mRNA is up-regulated in a sub-set of breast cancer cell lines, a link between TRPV6 expression and breast cancer aggressiveness has not been demonstrated. Furthermore, the mechanism for the over-expression of TRPV6 in breast cancer is unknown. To explore TRPV6 gene amplification as a possible mechanism for over-expression, we used a real-time PCR copy number assay and identified a sub-set of breast cancer cells lines that exhibited increased TRPV6 gene copy number. To assess the clinical relevance of this finding, we initially used a high throughout, clinically driven approach to examine TRPV6 gene amplification in breast tumors. Our findings showed that the mechanism of TRPV6 over-expression is due to, at least in part, gene amplification and that gene amplification may be pronounced in breast cancer subtypes with a poor prognosis. In clinical breast tumor samples, microarray based comparative genomic hybridization (aCGH) showed that the TRPV6 gene region (7q34) is gained in 0/16 invasive lobular carcinomas and in 11/35 high grade invasive ductal carcinomas. When stratified by estrogen receptor status, 7/13 estrogen receptor negative, high grade invasive ductal carcinomas harbored a gain in this region. To determine whether the copy number increase observed at the gene region 7q34 specifically corresponded with amplification of the TRPV6 gene, we performed chromogenic in situ hybridization (CISH) using a probe specific for the TRPV6 gene and a breast cancer tissue microarray. Our findings suggest that a sub-set of breast cancers may be characterized by TRPV6 gene amplification. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2954. doi:10.1158/1538-7445.AM2011-2954

A UK NEQAS ISH Multicenter Ring Study Using the Ventana HER2 Dual-Color ISH Assay

We performed a multicenter assessment of a new HER2 dual-color chromogenic in situ hybridization (CISH) test and herein report on concordance of CISH data with fluorescence in situ hybridization (FISH) data and intraobserver and interlaboratory scoring consistency. HER2 results were evaluated using duplicate cores from 30 breast cancers in 5 laboratories using the Ventana HER2 dual-color ISH assay (Ventana Medical Systems, Cambridgeshire, England) and in 1 central laboratory using a standard FISH assay. Overall 93.3% of cases were successfully analyzed by CISH across the 5 participating laboratories. There was excellent concordance (98.0% overall) for diagnosis of HER2 amplification by CISH compared with FISH. Intraobserver variability (7.7%) and intersite variability (9.1%) of absolute HER2/chromosome enumeration probe 17 ratios were tightly controlled across all participating laboratories. The Ventana HER2 dual-color ISH assay is robust and reproducible, shows good concordance with a standard FISH assay, and complies with requirements in national and international guidelines for performance of ISH-based diagnostic tests.

Abstract P3-10-24: Fibroblast Growth Factor Receptor 1 Amplification and Overexpression in Breast Cancer Tissue Microarrays Using Chromogenic In Situ Hybridization and Immunohistochemistry

Abstract Fibroblast growth factor receptors 1 (FGFR1) belongs to a subfamily of receptor tyrosine kinases (RTKs), which are involved in proliferation and apoptosis. FGFR1 amplification and mRNA overexpression occurs in ∼10% of breast cancers and has been associated with poor outcome. However, studies assessing FGFR1 amplification and protein overexpression in a large collection of breast tumors are lacking. In this prospective, we examined a series of primary breast cancers samples derived from the BCIRG trials assembled into 15 tissue microarrays. FGFR1 gene amplification was studied using chromogenic in situ hybridization (CISH) and evaluated with respect to association with level of protein expression and clinicopathological parameters. FGFR1 gene amplification was significantly associated with high protein levels as determined by immunohistochemistry (P<0.0001, 3294 pts with available matched CISH and IHC records). Overall, the incidence of FGFR1 amplification found was 9.7% (160 out of 1646 pts. with available clinicopathological records) without a statistically significant difference between Her2- (78 out 720 pts., 11%) and Her2+ (82 out of 926 pts., 9%) cancers. In both cohorts, the hormone receptor-positive (ER+/PR+) cancers showed statistically significant higher levels of FGFR1 amplification compared to hormone receptor-negative tumors. In the analysis of the association of FGFR1 and the presence of PIK3CA mutations, the incidence of FGFR1 amplificationwas greatly reduced in mutant vs. wt PI3KCA tumors. In these cohorts, a clear relationship between FGFR1 amplification status and clinical outcome was not detected. Data from this large study confirms recently reported incidences of FGFR1 amplification in breast cancer and shows for the first time an association between FGFR1 gene amplification and protein overexpression. Moreover, the lower incidence of FGFR1 amplification in PIK3CA mutated cancers suggests that these are largely exclusive molecular events that could benefit from different targeted therapies. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-24.

Prevalence and Prognostic Factors of EBV-Positive DLBCL of the Elderly In Peru

AbstractAbstract 4166 Background:EBV-positive diffuse large B-cell lymphoma (DLBCL) of the elderly is an entity recently described and included in the WHO classification of lymphomas. It usually affects patients older than 50 years with poor responses to chemotherapy and short survival. However, the majority of the cases are from Asian origin. In fact, in Western countries the incidence of EBV in patients with DLBCL is reported as <5%. The primary objective of this study is to establish the prevalence of EBV in patients with DLBCL and identify prognostic factors in these patients. Patients and methods:We investigated the EBV status by detection of EBV-encoded RNA (EBER) using a chromogenic in situ hybridization (CISH) technique in newly diagnosed patients with primarily nodal DLBCL, identified between January 2002 and December 2009. Clinical data were reviewed retrospectively and biopsies were analyzed for the presence of EBER by CISH and the immunohistochemical expression of BCL6, CD10 and MUM-1/IRF4 using standard procedures. Chi-square was used to compare the characteristics between EBER-positive and EBER-negative cases and to evaluate the association between complete response (CR) to chemotherapy and other clinical variables. Univariate survival estimates in patients who received chemotherapy were obtained using the Kaplan-Meier method. The multivariate survival analysis was performed using the Cox proportional-hazard regression test. Results:A total of 134 consecutive patients were eligible and were included in the comparative clinical analysis. In this cohort, the median age was 71 years (range 23–84) with a male-to-female ratio of 1.1 (71 male and 65 female cases) and a median overall survival (OS) of 47 months. 75% of the cases were 60 years or older, 62% had advanced clinical stage (III or IV), 63% has elevated LDH levels, 13% had involvement of 2 or more extranodal sites, 48% had an ECOG performance status of 2 or higher and 71% had a non-germinal center (NGC) immunohistochemical profile. Nineteen patients were positive for EBER but only 17 patients were included in the analysis because 2 patients were older than 50 years. When comparing EBER-positive and EBER-negative cases, there was an association between EBER expression and a worse outcome (p=0.003). EBER expression was not associated with gender, age, performance status, LDH levels, clinical stage, number of extranodal sites, B symptoms, immunohistochemical profile, overall response rate (ORR), CR rate, exposure to chemotherapy or IPI score. The only factors associated with CR were IPI score (p=0.047) and B symptoms (p=0.0004). Ninety nine patients received chemotherapy and were included in our survival analysis. In the univariate analysis, age over 60, performance status, LDH levels, number of extranodal sites, clinical stage, immunohistochemical profile and EBER expression were associated with OS. EBER-positive patients had a median OS of 12 months vs. 47 months in EBER-negative patients (p=0.045; Figure). In the multivariate analysis, performance status, LDH levels and EBER expression were independent factors for OS (p=0.02, 0.01 and 0.006, respectively). When evaluating EBER expression against the IPI score, both were independent prognostic factors for OS (p=0.002 and 0.03, respectively). [Display omitted] Conclusions:The prevalence of EBV-positive DLBCL of the elderly in Peru is the highest reported in the world (13%). DLBCL patients expressing EBER had a worse outcome in comparison to EBER-negative DLBCL patients. In the multivariate analysis, EBER expression in the tumoral cells was an independent prognostic factor for OS along with the IPI score. Disclosures:No relevant conflicts of interest to declare.

Quantification of EBV DNA In Whole Blood In Newly Diagnosed Patients with Diffuse Large B-Cell Lymphoma

AbstractAbstract 3102 Introduction:Epstein-Barr virus (EBV) is a well-known oncogenic virus associated with the development of several lymphoproliferative disorders. The quantification of EBV viral load in plasma and peripheral blood mononuclear cells has been reported as a useful biomarker for EBV-associated non-Hodgkin lymphoma (NHL). The role of EBV in diffuse large B-cell lymphoma (DLBCL) is unclear at this time. METHODS: In the present prospective study, using real-time quantitative polymerase chain reaction (RQ-PCR), whole blood specimens from patients with a new diagnosis of DLBCL were used to quantitatively determine EBV viral load. Consecutive patients who presented from January 1, 2009 to December 30, 2009 were selected for this study. The pathological samples from the patients showing elevated EBV DNA levels were then investigated for the presence of EBV-encoded RNA (EBER) using a chromogenic in situ hybridization (CISH) technique. Clinical data from these patients was collected and analyzed, and will be presented using descriptive statistics. Overall survival estimates were obtained using the Kaplan-Meier method. Results:Forty six patients with a new diagnosis of DLBCL were included in our study; from which five cases showed elevated levels of EBV DNA by RQ-PCR (10.8%). These five patients (100%) were positive for EBER expression within the tumoral cells by EBER CISH. One patient was evaluated at the end of treatment and blood EBV DNA level was undetectable. All EBV DNA-positive cases were male with a median age of 69 years (range 25–73 years); only one case was younger than 50 years. The median EBV DNA viral load was 59 copies/uL (range 0.225–43200 copies/uL). Three patients (60%) presented with advanced clinical stage (III or IV) and three patients (60%) had a primary extranodal site of involvement. Four patients (80%) received CHOP-based chemotherapy regimens. The three patients with advanced stage have deceased with a survival of <3 months. The two patients with early stage experienced a complete response with immunochemotherapy and are alive 6 and 9 months after diagnosis, respectively. The median overall survival is 4 months. Conclusions:These results, although preliminary, suggest that whole blood EBV DNA quantification might be useful at detecting patients with EBV-positive DLBCL. Further studies are needed to investigate the potential diagnostic and/or prognostic value of EBV DNA in DLBCL.CaseAgeSexStagePrimaryEBV VLIPITherapyResponseSurvivalOutcome125MIINodal57.31RCHOPCR6 mosAlive279MIVPeritoneum432004CHOPPD1 moDead384MIVNodal2724CHOPPD2 mosDead484MIVPalate594RT2 mosDead573MIIWaldeyer ring0.2252RCHOPCR9 mosAlive Disclosures:No relevant conflicts of interest to declare.

Comparison of MammaPrint and TargetPrint results with clinical parameters in German patients with early stage breast cancer

The 70-gene expression profile MammaPrint is a powerful prognostic indicator for disease outcome in breast cancer patients with improved prediction of recurrence risk compared to currently used guidelines. The microarray-based test TargetPrint further provides reliable, quantitative assessment of mRNA expression levels of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). This study was performed as a validation of MammaPrint and TargetPrint in an unselected German breast cancer population and was designed to determine the degree of concordance with currently applied clinical parameters. One hundred and forty cases of breast cancer stage I and II were classified as being low or high risk for distant metastasis using MammaPrint. Results were compared to current clinical risk classifications and adjuvant treatment management. Immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH)/chromogenic in situ hybridization (CISH) assessments of ER, PR and HER2 were further compared with gene expression read-outs using TargetPrint. Thirty-two percent of patients (19/59) with a poor prognosis-signature identified via MammaPrint did not receive adjuvant systemic treatment apart from endocrine therapy and were potentially undertreated; whereas 42% (35/77) of patients with a good prognosis-signature received chemotherapy and were potentially overtreated. Comparison of microarray receptor results with IHC and FISH/CISH were concordant in 97% for ER; 86% for PR; and 94% for HER2. In this German study population, MammaPrint would have resulted in altered treatment advice for adjuvant systemic therapy in 40% of patients. Furthermore, TargetPrint presented high concordance for ER, PR and Her2 with IHC and FISH/CISH analysis.

Clinicopathological Characteristics and Her-2/neu Status in Chinese Patients with Uterine Papillary Serous Carcinoma

Objective. To analyze clinico-pathological features of Chinese patients with UPSC, and investigate roles of Her-2/neu protein expression and gene amplification in UPSC prognosis. Methods. Thirty-six patients with UPSC treated in Cancer Hospital of Fudan University from 1996 to 2006 were analysed retrospectively. Chromogenic in situ hybridization (CISH) and immunohistochemistry (IHC) were performed to evaluate Her-2/neu gene amplification and protein expression respectively. Results. The median age was 63 years, and 61% (22/36) were late stages (stage III/IV). The 1-year, 3-year, and 5-year overall survival (OS) was 73.1%, 51.9% and 43.9%, respectively. Advanced stages (P = .0006) and deep myometrial invasion (P = .0138) were significantly associtated with a shorter OS. In 36 cases, 27.8% (10/36) showed 2+ staining and 8.3% (3/36) showed 3+ by IHC. Amplification of the Her-2/neu gene was observed in 11.1% (4/36) cases. The 5-year overall survival rate in Her-2/neu IHC 2 + ∼3+ and 0 ~ 1+ cases was 12.9% and 68.6% respectively. Her-2/neu protein expression 2 + ∼3+ was significantly associated with advanced surgical stage and worse overall survival (P = .03 and P = .0023, resp.). Conclusion. Chinese patients with UPSC showed characteristics of deep myometrial invasion, advanced stages and poor overall survival. Her-2/neu protein overexpression is associated with advanced stage and poor survival outcome.

Expression of human papillomavirus type 16/18 in human cervical carcinomas by the quantum dot fluorescent in-situ hybridization

To investigate fluorescence in situ hybridization labeled with quantum dots (QDs) for the detection of human papillomavirus 16/18 (HPV16/18) infection in cervical carcinoma patients. A total of 80 biopsy samples of squamous carcinoma of cervix were assayed for HPV 16/18 infection by using quantum dot labeled fluorescent in situ hybridization (QD-FISH) and chromogenic in situ hybridization (CISH) techniques, respectively. The results obtained by using two different methods were statistically analyzed. The positive rate for HPV16/18 by QD-FISH was 88.8% (71/80), higher than that (80.0%) by CISH, however, the result was statistically not significant (P=0.127). The positive detection rates for HPV16/18 by using both methods increased coincidentally with raising of the tumor grading stage. The sensitivity and specificity of HPV infection detectable by QD-FISH is higher than that by the CISH technique.

Potential role of bone marrow-derived cells in the turnover of mesothelium

Background: Bone marrow cell has been proposed as a source of new mesothelium, but supporting evidence is rare. This study examines the validity of this hypothesis by using green fluorescent protein (GFP) and Y-chromosome trackers to identify the presence of donor marrow cells in peritoneal membrane of bone marrow transplant recipient mice. Methods: Cross-gender and GFP-mismatched bone marrow transplantation was undertaken in 20 FVB mice. Five recipients were killed 2, 4, and 6 weeks and 6 months later. Peritoneal tissues were obtained for the detection of GFP and Y chromosome by immunohistochemical staining (IHC) and chromogenic in situ hybridization (CISH). Results: GFP+ cells could be found in the peritoneal membrane of bone marrow transplant recipients. However, the level of engraftment was low, accounting for 0.9%, 0.8%, 0.7%, and 2.2% of the total counted mesothelial cells in intestinal serosa at 2, 4, and 6 weeks and 6 months post-transplantation, respectively. The presence of donor marrow cells within mesothelium was again confirmed by the detection of Y-chromosome-containing cells. Moreover, Y-chromosome+ cells incorporated within the mesothelium were positively stained by anticytokeratin antibody. Conclusions: Donor marrow cells could attach to mesothelium and exhibit mesothelial marker cytokeratin in bone marrow transplant recipients. This finding suggests that bone marrow-derived cells might participate in the turnover of mesothelium.

Chromogenic in situ hybridization to detect EGFR gene copy number in cell blocks from fine-needle aspirates of non small cell lung carcinomas and lung metastases from colo-rectal cancer

BackgroundSeveral studies demonstrated that epidermal growth factor receptor (EGFR) gene copy number (GCN) correlates to the response to tyrosine kinase inhibitors in non small cell lung cancer (NSCLC) and to anti-EGFR monoclonal antibodies (MoAbs) in metastatic colorectal cancer (CRC). In the presence of lung nodules, cytology is often the only possible diagnostic approach. Chromogenic in situ hybridization (CISH) is an alternative technique to fluorescence in situ hybridization (FISH), but its feasibility in detecting EGFR GCN in cell blocks from fine-needle aspiration cytology (FNAC) of lung nodules has not yet been established.MethodsWe evaluated the feasibility of CISH on 33 FNAC from 20 primary NSCLC (5 squamous carcinomas, 8 large cell carcinomas and 7 adenocarcinomas) and 13 lung metastases from CRC.ResultsOf the 33 FNAC analyzed by CISH, 27 (82%) presented a balanced increase in EGFR gene and chromosome 7 number: 10 cases (30%) showed a low polysomy, 15 (45%) a high polysomy and 2 (6%) NSCLC were amplified. No significant differences between NSCLC and CRC lung metastases were found in relation to disomic or polysomic status. In addition, no correlation between EGFR GCN and EGFR immunohistochemical overexpression was found. Furthermore, we compared CISH results with those obtained by FISH on the same samples and we found 97% overall agreement between the two assays (k = 0.78, p < 0.0001). Two cases were amplified with both assays, whereas 1 case of NSCLC was amplified by FISH only. CISH sensitivity was 67%, the specificity and positive predictive value (PPV) was 100%, and the negative predictive value (NPV) was 97%.ConclusionsOur study shows that CISH is a valid method to detect EGFR GCN in cell blocks from FNAC of primary NSCLC or metastatic CRC to the lung.

O-18 PARP1 expression in hormone estrogen receptor negative breast cancer: Preferential expression in basal-like and HER2-positive tumours

breast cancer series prepared as tissue microarray (n = 1858) using IHC (HercepTest, DakoCytomation) and chromogenic ISH (CISH; DuoCISH, DakoCytomation) in order to identify relationships with clinico-pathological variables and patient outcome. None of these cases have received anti-HER2 therapy. There was excellent overall concordance between HercepTest negative (scores 0/1+) and positive (3+) with CISH positive/negative (defined as HER2/Chr17 copy number ratio of P2; p < 0.001). Twelve percent of cases were identified as HER2 positive (those with 3+ HercepTest scores or 2+ with gene amplification). Of the 74 borderline HercepTest 2+ cases, 44 cases (59%) showed HER2 gene amplification. We identified that HercepTest 2+ non-amplified cases were not significantly different from those amplified 2+ or 3+ cases with respect to their clinical outcome (BCSS and DFS). The overall concordance between HercepTest and CISH analysis for HER2 status was excellent. All HercepTest 2+ cases identified were observed to have poor outcomes similar to those HercepTest 3+ cases regardless of gene amplification status. In the current clinical environment, cases exhibiting IHC 2+ with non-amplified HER2 gene status are not offered targeted HER2 therapy but do exhibit aggressive clinical behavioural characteristics and therefore optimal treatment strategies for these patients need to be determined.

“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 …

Detection of SYT and EWS Gene Rearrangements by Dual-Color Break-Apart CISH in Liquid-Based Cytology Samples of Synovial Sarcoma and Ewing Sarcoma/Primitive Neuroectodermal Tumor

To improve cytologic diagnostic accuracy for translocation-associated sarcomas, we explored dual-color break-apart (dc) chromogenic in situ hybridization (CISH) on liquid-based cytology (LBC) samples of 2 prototypic sarcomas: synovial sarcoma (SS) and Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET). LBC samples of 10 cases of SS and 9 cases of ES/PNET were subjected to dc-CISH using probes for the specifically rearranged genes in each tumor entity: SYT in SS and EWS in ES/PNET. Rearranged SYT was successfully detected in all SSs but not in any ES/PNETs. In contrast, EWS rearrangement was identified in all ES/PNETs but not in any SSs. These results were validated by dc-fluorescence in situ hybridization and reverse transcription-polymerase chain reaction. dc-CISH on LBC samples is a reliable modality to detect gene rearrangements in sarcomas. This system has a clear advantage over other methods, enabling simultaneous visualization of the genetic abnormality and well-preserved, nonoverlapping cytomorphologic features with clear background under bright-field microscope.

Activation of the EGFR/ERK pathway in high-grade mucoepidermoid carcinomas of the salivary glands

Background:Mucoepidermoid carcinoma (MEC) shows differences in biological behaviour depending mainly on its histological grade. High-grade tumours usually have an aggressive biological course and they require additional oncological treatment after surgery.Methods:In a series of 43 MECs of the salivary glands, we studied the epidermal growth factor receptor (EGFR) gene by using dual-colour chromogenic in situ hybridisation (CISH). Moreover, we assessed the protein expressions of the EGFR and the activated extracellular signal-regulated kinases (pERK1/2) by using immunohistochemistry. These results were correlated with the histological grade of the tumours and the outcome of the patients.Results:The CISH study demonstrated a high-EGFR gene copy number, with balanced chromosome 7 polysomy, in 8 out of 11 high-grade MECs (72.7%), whereas 27 low-grade and 15 intermediate-grade tumours had a normal EGFR gene copy number (P<0.001). The EGFR gene gains correlated with disease-free interval (P=0.003) and overall survival of the patients (P=0.019). The EGFR protein expression had a significant correlation with the histological grade of the tumours but not with the outcome of the patients. The pERK1/2 expression correlated with histological grade of tumours (P<0.001), disease-free interval (P=0.004) and overall survival (P=0.001).Conclusions:The EGFR/ERK pathway is activated in high-grade MECs with aggressive behaviour. Patients with these tumours who require oncological treatment in addition to surgery could benefit from EGFR and mitogen-activated protein kinase pathway inhibitors.

Gain of chromosome 7 by chromogenic in situ hybridization (CISH) in chordomas is correlated to c-MET expression

Chordomas are low to intermediate grade malignancies that arise from remnants of embryonic notochord. They often recur after surgery and are highly resistant to conventional adjuvant therapies. Recently, the development of effective targeted molecular therapy has been investigated in chordomas that show receptors for tyrosine kinase (RTKs) activation. Expression of specific RTKs such as Epidermal Growth Factor Receptor (EGFR) and Mesenchymal-epithelial transition factor (c-MET) in chordomas may offer valuable therapeutic options. We investigated changes in copy number of chromosome 7 and correlated it with EGFR gene status and EGFR and c-MET protein expression in 22 chordoma samples. Chromosome 7 copy number was evaluated by chromogenic in situ hybridization (CISH) and protein expression of EGFR and c-MET by immunohistochemistry. Tumors mostly showed conventional histopathologic features and were found mainly in sacral (41%) and cranial sites (54.5%). Aneusomy of chromosome 7 was seen in 73% of the samples, 62% of primary tumors and in all recurrent chordomas. EGFR and c-MET were both expressed, but only c-MET protein expression was significantly correlated with chromosome 7 aneusomy (P ≤ 0.001). c-MET overexpression may represent an early chromosome 7 alteration that could play an important role during chordoma pathogenesis. c-MET overexpression shows promise as a molecular marker of response to targeted molecular therapy in the treatment of chordomas.

Molecular Testing in Lung Carcinoma

The development of molecular targeted therapies with small-molecule inhibitors of the epidermal growth factor receptor (EGFR) has resulted in new therapeutic options for patients with advanced stage lung cancer. Certain clinical characteristics have been associated with an increased likelihood of response to EGFR tyrosine kinase inhibitors (TKIs; women, never smokers, East Asian ethnicity, and adenocarcinoma histology). Furthermore, several molecular events can also affect the efficacy of these therapies. Activating mutations in EGFR exons 18 through 21 (insert deletions in exon 19 and the single L858R point mutation in exon 21 being the most common) seem to be more reliable predictors of response than clinical features and are associated with increased response and survival after TKI therapy.1–3 In contrast, K- ras mutations, insertion mutations or T790M point mutations in exon 20 of EGFR , and MET amplification are negative predictors of response.4–6 These observations have resulted in numerous retrospective studies that correlate patient response to the molecular profile of lung adenocarcinomas. Unfortunately, different methodological approaches have been used for EGFR assessment, including DNA mutational analysis, fluorescence in situ hybridization (FISH) or chromogenic in situ hybridization (CISH) for gene copy number changes, and immunohistochemical analysis for protein expression. Not surprisingly, reported results are inconsistent, reflecting the lack of standardization of the method and interpretation criteria. In general, EGFR FISH and DNA mutation analyses are the 2 most extensively studied methods for selection of candidates for …

Evaluation of HER2 gene amplification in invasive breast cancer using a dual-color chromogenic in situ hybridization (dual CISH)

Fluorescence in situ hybridization (FISH) assay is considered the 'gold standard' for evaluation of HER2/neu (HER2) gene status, however, it is difficult to recognize morphologic features of tumors using fluorescence microscopy. Thus, chromogenic in situ hybridization (CISH) has been proposed as an alternative method to evaluate HER2 gene amplification. Here, we examined the dual color CISH (dual CISH) method which provides information regarding the copy number of the HER2 gene and chromosome 17 centromere from a single slide. We examined 40 cases of invasive ductal carcinomas of the breast that were resected surgically. HER2 gene status was assessed with FISH (Abbott) and dual CISH (Dako). HER2 gene amplification status was classified according to the guidelines of the American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP). Comparison of the cut-off values for HER2/chromosome 17 centromere copy number ratio obtained by dual CISH and FISH showed that there was almost perfect agreement between two methods (Kappa coefficient 0.96). The results of the two commercial products were almost consistent for evaluation of HER2 gene counts on the sections. The current study proved that dual CISH is comparable with FISH for evaluating HER2 gene status.