Dual-color In Situ Hybridization Research Articles

Collision of Epstein\u2013Barr virus-positive and -negative gastric cancer, diagnosed by molecular analysis: a case report

BackgroundEpstein–Barr virus (EBV)-positive gastric carcinoma (GC) is defined by the proliferation of GC cells with EBV infection. The co-existence of EBV-positive and -negative components in a single GC is rare. We report a case of GC with the co-existence of EBV-positive and EBV-negative components, in which we performed—for the first time—various molecular analyses to elucidate their histogenesis.Case presentationAn 81-year-old man was diagnosed with GC based on the results of endoscopy and a pathological examination of the biopsy specimen. Systemic chemotherapy was performed, since lymph node and lung metastases were diagnosed based on computed tomography. Total gastrectomy and lymph node dissection were performed after chemotherapy, after confirming that the size of the metastatic lymph nodes had decreased and that the lung metastasis had disappeared. Grossly, a type 3 tumor was located in the middle posterior part of the stomach body. At the cut section, the tumor consisted of a white and solid part on the anal side of the tumor and a flat and elevated part on the oral side. Histologically, the former part consisted of GC with lymphoid stroma and the latter part was composed of poorly differentiated adenocarcinoma without prominent lymphocytic infiltration. The two histopathological components were clearly separated from each other. On EBV-encoded small RNA (EBER)-in situ hybridization (ISH), the part with the lymphoid stroma component was positive, while the other part was negative. Immunohistochemistry revealed that both components showed the overexpression of p53. Sequencing of TP53 using DNA extracted from the two components was conducted, and revealed different patterns. Targeted next generation sequencing revealed MYC amplification in the EBV-positive component of the tumor and HER2 amplification in the EBV-negative part. Immunohistochemistry revealed that the EBV-positive part was C-MYC( +)/HER2(−) and the EBV-negative part was C-MYC(−)/HER2( +). Correspondingly, chromogenic ISH and dual-color ISH showed amplification of C-MYC and no amplification of HER2 in the EBV-positive part, and no amplification of C-MYC and amplification of HER2 in the EBV-negative part.ConclusionWe presented a case of collision of two different GCs composed of EBER-ISH ( +)/C-MYC ( +) and EBER-ISH (−)/HER2 ( +) cells.

Correlation between HER2 Expression and Clinicopathological Features of Breast Cancer: A Cross- Sectional Study in Vietnam.

Background: HER2 is the target of the therapeutic agents which are used to treat HER2-positive breast cancer. Reports have shown that the HER2 oncogene expression and its association with clinicopathological factors remain unclear in breast cancer (BC) patients. This study aimed to determine the correlation between HER2 expression and clinicalpathological characteristics of breast cancer in Vietnamese women. Methods:Between June 2016 and August 2018, paraffin-embedded specimens from 237 patients with primary invasive breast carcinoma in Hue University Hospital and Hue Center Hospital, Hue city, Vietnam were examined for pathological features. The gene expression of HER2, ER, PR and Ki-67 were determined by immunohistochemistry (IHC). The gene amplification of Her2 was assessed by using Dual color in situ hybridization (DISH). Results:The most frequent histological type was invasive carcinoma of no special type (NST) with 77.35%, the highest percentage of patients with Grade II was detected (59.36%), tumor size > 2 cm accounted for 71.31% of cases, Lymph node metastases were available in 57.86% cases. Most patients were diagnosed at stage II (59.18%). The majority of patients were classified as moderate Nottingham prognostic index (54.9%). Estrogen receptor and Progesterone receptor were positive in 53.16% and 50.63%, respectively. 76.37% of cases were in high expression group of Ki-67 (≥14%). HER2 IHC 2+, 3+ were accounted for 28.69% and HER2 gene amplification was detected in 31% cases. HER2 gene amplification and/or overexpression was significantly associated with cell proliferation index Ki67. Furthermore, HER2 gene expression tended to be more frequently found in tumors with large tumor size, high grade, high stage and high Nottingham prognostic index and confirmed their prognostic independent role. Conclusions: Our data indicated that HER2 gene expression was significantly correlated with cell proliferation index Ki67, but not significantly associated with another clinicopathological factors in breast cancer of Vietnamese women.

HER2 expression and relevant clinicopathological features in esophageal squamous cell carcinoma in a Chinese population

BackgroundDespite great progress in surgery and other treatments, the prognosis of patients with esophageal squamous cell carcinoma (ESCC) is still very poor. HER2 has strong therapeutic implications in certain cancers, such as breast cancer and gastric cancer. However, literature on the frequency of HER2 expression in ESCC is scarce. In the present study, HER2 protein expression, HER2 gene amplification and the relationship between HER2 status and clinicopathological characteristics were evaluated in a large cohort of Chinese ESCC patients.MethodsA total of 857 consecutive ESCC patients who received radical esophagectomy without neoadjuvant therapy between January 2014 and October 2015 were included in this retrospective study. HER2 protein expression was analyzed by immunohistochemistry (IHC), and its correlation with clinicopathological parameters was assessed. In addition, 65 cases, including 13 HER2 overexpression (3+) cases and 52 HER2 equivocal (2+) cases from the 857-case cohort, and another 104 ESCC cases, including 1 HER2 overexpression (3+) case, 3 HER2 equivocal (2+) cases and 100 HER2 negative (1+/0) cases, were selected to construct tissue microarrays (TMAs). Dual-color in situ hybridization (DISH) was performed on the TMAs to assess HER2 gene amplification and the relationship with clinicopathological parameters.ResultsWe found HER2 overexpression (3+) status in 1.5% (13/857) of cases and HER2 equivocal (2+) status in 6.1% (52/857) of cases. HER2 IHC expression was significantly associated with gender (P = 0.028). However, there were no significant correlations between HER2 IHC expression and age, tumor differentiation, pT stage, pN stage, pM stage and pTNM stage (P > 0.05). Regarding the 169 cases analyzed by DISH, 14 (of 14, 100%) HER2 overexpression (3+) cases, 10 (of 55, 18.2%) HER2 equivocal (2+) cases, and 0 (of 100, 0%) HER2 negative (1+/0) cases showed HER2 gene amplification. HER2 gene amplification was not significantly associated with clinicopathological characteristics such as age, gender, tumor differentiation, pT stage, pN stage, pM stage and pTNM stage (P > 0.05).ConclusionsApproximately 1.5% of the Chinese ESCC patients had HER2 overexpression based on IHC. IHC and DISH had a high concordance rate. These results provide valuable insight for the future treatment of ESCC.

Diagnostic Utility and Limitations of Immunohistochemistry of p16, CDK4, and MDM2 and Automated Dual-color In Situ Hybridization of MDM2 for the Diagnosis of Challenging Cases of Dedifferentiated Liposarcoma.

The diagnosis of dedifferentiated liposarcoma (DDLPS) is challenging when an atypical lipomatous tumor component is absent or obscure. To analyze the utility and limitations of ancillary techniques, we studied 11 cases of DDLPS in challenging conditions and 17 cases of nonlipogenic high-grade sarcomas with immunohistochemistry (IHC) for p16, CDK4, and MDM2 and automated dual-color in situ hybridization (DISH) for MDM2 amplification. All DDLPS specimens lacked clear lipogenic components and were immunoreactive for p16, CDK4, and MDM2. DISH analyses also revealed high-level amplification of MDM2 in all DDLPS. In contrast, among nonlipogenic sarcomas, p16, CDK4, and MDM2 were expressed in 8, 9, and 3 cases, respectively. MDM2 amplification was detected in 3 of 8 studied. The MDM2-amplified tumors were the same as the MDM2-immunoreactive tumors. After careful reevaluation of these 3 sarcomas, 2 were reclassified as DDLPS because small areas of lipogenic components were detected in the original specimens. The respective sensitivities and specificities of these markers were as follows: p16 IHC (100% and 60%), CDK4 IHC (100% and 53.3%), MDM2 IHC (100% and 93.3%), and MDM2 DISH (100% and 83.3%). The results of MDM2 IHC completely coincided with those of MDM2 DISH. The present study confirmed the substantial utility of MDM2 IHC and MDM2 DISH in the diagnosis of DDLPS, especially when lipogenic components were indistinct compared with IHC for p16 and CDK4. Furthermore, automated DISH was more practical than fluorescent in situ hybridization.

MDM2 dual-color in situ hybridization (DISH) aids the diagnosis of intimal sarcomas.

Intimal sarcoma is a rare malignant mesenchymal tumor arising from the intima of the great vessels and the heart, and is associated with poor outcomes. As clinico-radiological findings and pathological features are often non-specific, the diagnosis of intimal sarcoma is challenging. Recently, MDM2 amplification was reported to be a characteristic genetic event in this tumor. In the present study, we examined MDM2 status by immunohistochemistry, and by fluorescence and dual-color in situ hybridization (FISH and DISH) using intimal sarcoma (10 tumors), angiosarcoma (5), pulmonary sarcomatoid carcinoma (p-SC) (14) and chronic pulmonary thrombosis (CPT) (3) to investigate MDM2 amplification for the diagnosis of intimal sarcoma. MDM2 and CDK4 were immunopositive in all 10 intimal sarcoma tumors, and high-level amplification of MDM2 was detected in eight tumors by both FISH and DISH. The other two tumors had polysomy of chromosome 12 and overexpression of p53 protein. Although MDM2 aberrations were observed in three p-SCs (two with amplification and one with polysomy), angiosarcomas and CPTs lacked MDM2 amplification. Furthermore, there was high concordance between FISH and DISH. In conclusion, we found that MDM2 amplification strongly supports the diagnosis of intimal sarcoma, and MDM2 DISH was a concordant method and an acceptable alternative to FISH. As MDM2 amplification and p53 overexpression were mutually exclusive, disruption of the MDM2-p53 pathway may be an essential genetic event for this malignant tumor.

Abstract CT137: Phase II study of trastuzumab and docetaxel therapy in patients with HER2-positive recurrent and/or metastatic salivary gland carcinoma

Abstract Objectives: Salivary gland cancer is a rare cancer that has no effective drug therapy available. It comprises salivary duct carcinoma (SDC) as a major histology, which resembles high-grade breast ductal carcinoma and often overexpresses HER2. The purpose of this study is to evaluate the efficacy and safety of trastuzumab and docetaxel therapy in patients (pts) with HER2-positive salivary gland carcinoma. Methods: We conducted a multicenter, single-arm Phase II study of trastuzumab and docetaxel for pts with HER2-positive recurrent and/or metastatic salivary gland carcinoma pursuant to the J-GCP. HER2 positivity was defined as immunohistochemistry (IHC) 3+ or IHC 2+/dual color in situ hybridization (DISH) +. Eligible pts were treated with trastuzumab 6 mg/kg (loading dose 8 mg/kg) and docetaxel 70 mg/m2 every 3 weeks up to 8 cycles. The primary endpoint was overall response rate (ORR) using RECIST v1.1 assessed by the blinded independent review committee (BIRC). Secondary endpoints included progression-free survival (PFS), overall survival (OS) and safety. This study required 16 pts, with ORR of 25% considered non-promising and 70% promising (two-sided alpha = 0.05; beta = 0.1) and with 2 withdrawal pts. Results: Sixteen pts were recruited in this trial. The median age was 59 (range 26-72), 3 pts were female, and all pts had performance status 0-1. All pts had carcinomas histologically compatible with SDCs by central pathology review of samples used for HER2 evaluation, 14 of which were readily confirmed as SDCs. All pts had HER2 IHC 3+ tumors. Two pts were previously untreated and had metastatic disease. The remaining 14 pts had recurrence after surgery that was followed by adjuvant chemoradiotherapy in 5 pts and by adjuvant radiotherapy in 6 pts. No pts were previously treated with chemotherapy alone. After excluding a pt without measurable lesions evaluated by BIRC, ORR by BIRC was 60% (9/15; 95% CI, 32.3~83.7) with one complete response. Median PFS was 8.5 months (95% CI, 6.0~12.7). Median OS was not reached at a median follow-up of 17.9 months in survivors. Common grade 3/4 adverse events (> 10%) were neutropenia (16 pts, 100%), leukopenia (15 pts, 93.8%), lymphopenia (3 pts, 18.8%), and febrile neutropenia (2 pts, 12.5%). Treatment-related death occurred in 1 pt (6.3%) due to hypoalbuminemia. Conclusions: This study demonstrated a promising efficacy with predictable toxicities. Trastuzumab and docetaxel therapy may be an effective treatment option in pts with HER2-positive recurrent and/or metastatic salivary gland carcinoma. Citation Format: Ichiro Kinoshita, Satoshi Kano, Yasushi Shimizu, Naomi Kiyota, Yuichiro Tada, Kei Ijichi, Tmoko Yamazaki, Akihiro Homma, Yoichi M. Ito, Kota Ono, Keiko Kobayashi, Toshiyuki Isoe, Yutaka Hatanaka, Hitoshi Tsuda, Shojiroh Morinaga, Yoshihiro Matsuno, Hirotoshi Dosaka-Akita. Phase II study of trastuzumab and docetaxel therapy in patients with HER2-positive recurrent and/or metastatic salivary gland carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT137.

Formalin fixation on HER-2 and PD-L1 expression in gastric cancer: A pilot analysis using the same surgical specimens with different fixation times

BACKGROUNDThe needs for human epidermal growth factor receptor 2 (HER-2) and/or programmed death-ligand 1 (PD-L1) evaluations in gastric cancer are dramatically increasing. Although the importance of standardization of sample fixation has been widely recognized, most of the evidence regarding the fixation duration or type of fixing solution are based on breast cancer.AIMTo investigate the real effects of fixation conditions on HER-2 testing or PD-L1 testing for gastric cancer using gastrectomy specimens.METHODSThirty-two patients who underwent gastrectomy for gastric cancer were enrolled. Their resected specimens were each divided into four pieces and fixed in four strictly controlled different durations (6 h, 24 h, and 48 h, and 1 wk) by 10% formalin (n = 22) or 10% neutral buffered formalin (NBF) (n = 10). Immunohistochemistry (IHC) of HER-2 and PD-1 was performed, and a pathology examination was conducted. In the HER-2-immunoreactive cases, all four specimens were subjected to dual-color in situ hybridization (DISH). Five cases were assessed as HER-2-positive by IHC and DISH. We used the cut-off values of 1%, 10%, and 50% to assess the IHC findings of PD-L1.RESULTSNo significant difference was observed in comparisons between the shorter fixation period groups (6 h, 24 h, and 48 h) and the prolonged fixation period (1 wk) group in the HER-2 and PD-L1 analyses. Although no significant difference was observed between 10% formalin and 10% NBF within 1 wk of fixation, the superiority of 10% NBF was confirmed in a long-term (> 3 mo) fixation in both the HER-2 and PD-L1 analyses.CONCLUSIONIn this small-numbered pilot study, prolonged fixation within 1 wk showed no inferiority in HER-2 or PD-L1 testing. However, a large-numbered prospective study is needed to obtain conclusive results.

In situ analysis of FGFR2 mRNA and comparison with FGFR2 gene copy number by dual-color in situ hybridization in a large cohort of gastric cancer patients

BackgroundFibroblast growth factor receptor (FGFR2) has been proposed as a target in gastric cancer. However, appropriate methods to select patients for anti-FGFR2 therapies have not yet been established.MethodsWe used in situ techniques to investigate FGFR2 mRNA expression and gene amplification in a large cohort of 1036 Japanese gastric cancer patients. FGFR2 mRNA expression was determined by RNAscope. FGFR2 gene amplification was determined by dual-color in situ hybridization (DISH).ResultsWe successfully analyzed 578 and 718 samples by DISH and RNAscope, respectively; 2% (12/578) showed strong FGFR2 gene amplification (FGFR2:CEN10 >10); moderate FGFR2 gene amplification (FGFR2:CEN10 <10; ≥2) was detected in 8% (47/578); and high FGFR2 mRNA expression of score 4 (>10 dots/cell and >10% of positive cells with dot clusters under a 20× objective) was seen in 4% (29/718). For 468 samples, both mRNA and DISH data were available. FGFR2 mRNA expression levels were associated with gene amplification; FGFR2 mRNA levels were highest in the highly amplified samples (n = 12). All highly amplified samples showed very strong FGFR2 mRNA expression (dense clusters of the signal visible under a 1× objective). Patients with very strong FGFR2 mRNA expression showed more homogeneous FGFR2 mRNA expression compared to patients with lower FGFGR2 mRNA expression. Gastric cancer patients with tumors that had an FGFR2 mRNA expression score of 4 had shorter RFS compared with score 0–3 patients.ConclusionRNAscope and DISH are suitable methods to evaluate FGFR2 status in gastric cancer. Formalin-fixed paraffin-embedded (FFPE) tissue slides allowed evaluation of the intratumor heterogeneity of these FGFR2 biomarkers.

Abstract P1-03-08: Simultaneous analyses of HER2 gene and protein status can more precisely predict pathological complete response (pCR) to neoadjuvant trastuzumab with chemotherapy in primary HER2-positive breast cancer

Abstract Background: HER2-positive breast cancer is characterized by overexpression of HER2 protein and/or amplification of the HER2 gene, assessed by immunohistochemistry (IHC) and in situ hybridization (ISH), respectively. According to the 2013 ASCO/CAP guidelines on HER2 testing in breast cancer, both protein and gene status can be classified into three categories: positive, equivocal and negative. Gene-protein assay (GPA) is a newly developed technique, in which IHC and dual color ISH (DISH) are simultaneously performed on a single slide. HER2 GPA slides allow bright-field analyses of both protein and gene expression status of each cell. Breast cancer cells can be classified into nine types based on the combination of protein and gene expression with GPA. Table 1. Three by three classification of cancer cells by the HER2 GPA analysis HER2 protein 3+ (positive)2+ (equivocal)0, 1+ (negative)HER2 gene copy number≥6 (positive)ABC 4, 5 (equivocal)DEF &amp;lt;4 (negative)GHI Purpose: We aimed to elucidate the relationship between the results of HER2 GPA analysis and the therapeutic effects of trastuzumab-based treatment. Methods: Fifty three (53) patients with HER2-positive breast cancer, who underwent neoadjuvant trastuzumab with chemotherapy, were analyzed for HER2 status and clinical outcome. First, HER2 protein and gene status in a pre-therapeutic biopsy material from each patient were separately assessed according to the ASCO/CAP guidelines using HER2 GPA slides. Second, all cancer cells in five representative microscopic images of GPA slides were assessed to determine protein expression and to count the gene copy number at individual cell levels. Finally, we investigated the relationship between the mixture composition of the nine cell types and pathological complete response (pCR) of trastuzumab-treated breast cancer. Results: The GPA results were concordant with the results of IHC in 98% of samples, and with DISH results in 100%. Two hundred eighty nine (289) cancer cells per patient were assessed on average, ranging from 137 to 490 cells. The mean proportion of type A cancer cells was 64%, ranging from 0% to 98%. Patients who had 50% or more of type A cancer cells achieved pCR in 72% of cases (28/39), while patients with less than 50% of type A cancer cells achieved pCR in 7% of cases (1/14) (P&amp;lt;0.0001). The mean proportion of type C cancer cells was 7%, ranging from 0% to 60%. Patients who had 10% or more of type C cancer cells achieved pCR in 10% of cases (1/10), while patients with less than 10% of type C cancer cells achieved pCR in 65% of cases (28/43) (P=0.0016). Conclusion: HER2 GPA analyses can more precisely predict therapeutic effect of trastuzumab-based treatment in primary HER2-positive breast cancer. Our study suggests that the HER2 GPA approach would further improve precision therapy in HER2-positive breast cancer. Citation Format: Horii R, Nitta H, Ito Y, Iwase T, Ohno S, Akiyama F. Simultaneous analyses of HER2 gene and protein status can more precisely predict pathological complete response (pCR) to neoadjuvant trastuzumab with chemotherapy in primary HER2-positive breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-03-08.

Comparison of HER2 Dual-Color and Fluorescence In Situ Hybridization in Breast Cancer

Human epidermal growth factor receptor 2 (HER2; ERBB2 gene) is of prognostic and predictive significance in breast carcinoma. Both fluorescence in situ hybridization (FISH) and dual-color in situ hybridization (DISH) methods are available. DISH and FISH are highly concordant in validation studies, but differences may be more prevalent in the equivocal range. Our goal was to compare FISH and DISH on a cohort enriched for equivocal cases, with respect to HER2 determination. The cohort was enriched for equivocal (2+) cases. DISH and FISH were evaluated using standard protocols and the results compared with respect to HER2 status, HER2 copy number, and HER2/chromosome 17 (Chr17) ratio. In total, 109 cases were identified. The agreement rate of DISH with FISH was 74%. The mean ± SD HER2/Chr17 ratio by DISH was 1.63 ± 0.08 vs 1.59 ± 0.26 by FISH (P = .45). The mean ± SD HER2 copy number by DISH was 4.56 ± 0.45 vs 4.75 ± 1.08 by FISH (P = .004). Individual signals were more easily resolved using FISH in cases with higher copy numbers. In our cohort enriched for equivocal cases, the numerical values of HER2 copy number were significantly lower using DISH, resulting in discordances. Although DISH is a valid method, variations with FISH may be expected in high-equivocal cases and in quality assurance activities.

Significant intratumoral heterogeneity of human epidermal growth factor receptor 2 status in gastric cancer: A comparative study of immunohistochemistry, FISH, and dual-color in situ hybridization.

The assessment of human epidermal growth factor receptor 2 (HER2) status is crucial for selecting patients with gastric cancer who may benefit from HER2‐targeted therapy. Accurate assessment using biopsy specimens is important for patients with advanced‐stage cancer. Intratumoral heterogeneity of HER2, however, is a major challenge in HER2 testing. Here, we aimed to examine whether assessment of HER2 status could be accurately carried out with currently used methods, namely, immunohistochemistry (IHC), FISH, and dual‐color in situ hybridization (DISH). Human epidermal growth factor receptor 2 status was evaluated in 108 biopsy tissues from patients with gastric adenocarcinoma and 70 matched surgical specimens by IHC, FISH, and DISH; HER2 amplification was detected in 11 (10.2%) out of 108 biopsy specimens. The IHC and FISH results were well correlated, and FISH and DISH results were consistent for all cases. The overall concordance rate of HER2 status between biopsy tissues and surgical specimens was 91.4%. All six discordant cases were false negative on biopsy; of these cases, five showed HER2 heterogeneity on surgical resection. Assessment of the HER2 status of biopsy tissues could predict the status of the whole tumor; however, a proportion of these cases may be discordant because of intratumoral heterogeneity.

Establishment and characterization of patient-derived tumor xenograft using gastroscopic biopsies in gastric cancer.

68 Background: The patient-derived tumor xenograft (PDTX) model has become the most realistic model for preclinical studies. PDTX models of gastric cancer using surgical tissues are reported occasionally; however, the PDTX model using gastroscopic biopsy, which is presented in this study, is very rare. Methods: A total of 185 fresh gastroscopic biopsies of gastric cancer were subcutaneously transplanted into NOD/SCID (Nonobese Diabetic/Severe Combined Immunodeficiency) mice. The characteristics of PDTX were identified by hematoxylin and eosin (H&amp;E) staining, immunohistochemistry (IHC), dual-color in-situ hybridization (DISH), and targeted next-generation sequencing. Chemosensitivity of PDTX model was tested using chemotherapy regimens based on patients. Results: Sixty-three PDTX models were successfully established (34.1%, 63/185) and passaged to maintain tumors in vivo, and the mean latency period of xenografts was 65.86±32.84 days (11-160 days). Biopsies of prior chemotherapy had a higher transplantation rate (52.1%, 37/71) than biopsies after chemotherapy (21.9%, 25/114; P&lt;0.01). No differences were found between the latency period of xenografts and characteristics of patients. The pathological and molecular features of PDTX as well as chemosensitivity were highly consistent with those of primary tumors of patients. The genetic characteristics were stable during passaging of PDTX models. Conclusions: PDTX models using gastroscopic biopsies in gastric cancer were demonstrated for the first time, and the biological characteristics of the PDTX models were highly consistent with patients, which provided the best preclinical study platform for gastric cancer. [Table: see text]

Abstract 921: Fibroblast growth factor receptor 1 (FGFR1) protein expression and gene copy number in small cell lung cancer

Abstract PURPOSE: Small cell lung cancer (SCLC) comprises approximately 15% of all lung cancers, corresponding to &amp;gt;30,000 new cases per year in the United States. No major therapeutic progress has been achieved in SCLC in the past 30 years. Identification of new therapies in SCLC is urgently needed. Fibroblast growth factor receptor 1 (FGFR1) is a tyrosine kinase receptor implicated in the pathogenesis of several cancer types and is potentially an attractive target for cancer treatment. FGFR1 amplification and overexpression are known as a frequent event in lung squamous cell carcinoma. However, very little is known about FGFR1 protein expression and gene copy number in SCLC. One difficulty with research on SCLC is the limited availability of tumor specimens sufficient for molecular studies. METHODS: We investigated FGFR1 protein expression and gene copy number in a tissue microarray of resected SCLC tumors from 90 patients (67 men and 23 women) from Poland with limited stage. FGFR1 protein expression was assessed by immunohistochemistry (IHC) with H scores ranging from 0 (no expression) to 300 (strong expression in all tumor cells) using a FGFR1 antibody (clone EPR806Y, Origene) . FGFR1 gene copy number was evaluated by dual-color ISH assay using a cocktail of FGFR1 DNP probe (FGFR1 locus on 8p12) and chromosome 8 DIG probe (centromere 8), ultraView SISH DNP detection kit and ultraView Red ISH DIG detection kit (Tucson, AZ, US). Positive FGFR1 protein expression was defined as having an H-score ≥11. FGFR1 gene amplification was defined as FGFR1/CEN8 ratio ≥2.2, or average FGFR1 signals ≥4 per nucleus. RESULTS: FGFR1 protein expression was evaluable in 83 of the 90 patients and gene copy number was evaluable in 77 patients. FGFR1 protein expression was positive in 6 cases (7.2%) (mean H score=129, median 70, range: 40-285). FGFR1 gene amplification was observed in 6 cases (7.8 %) of evaluable SCLCs. There were 30%-90% tumor cells containing clusters in the amplified cases. Four of the 6 cases were positive for both FGFR1 protein expression and FGFR1 gene amplification. FGFR1 protein and gene copy number were not associated with patient age, sex, tumor size or overall survival (all p-values &amp;gt;0.15).FGFR1 gene copy number was significantly associated with protein expression (p=0.03, based on a t-test). CONCLUSIONS: A small subset of patients with SCLC is characterized by tumors with FGFR1 gene amplification and/or FGFR1 protein expression. Further studies are needed to determine whether or not this subset of patients would benefit from FGFR1-directed targeted therapy. Citation Format: Liping Zhang, Andrzej Badzio, Theresa Boyle, Xian Lu, Rafal Dziadziuszko, Jacek Jassem, Fred R. Hirsch. Fibroblast growth factor receptor 1 (FGFR1) protein expression and gene copy number in small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 921. doi:10.1158/1538-7445.AM2014-921

A novel gene-protein assay for evaluating HER2 status in gastric cancer: simultaneous analyses of HER2 protein overexpression and gene amplification reveal intratumoral heterogeneity.

Human epidermal growth factor receptor 2 (HER2) protein overexpression and gene amplification are important biomarkers for trastuzumab treatment in breast and gastric cancer patients. Gastric cancer presents high rates of tumor heterogeneity, which may influence the results of HER2 testing. A novel gene-protein assay (GPA) can allow the simultaneous analysis of HER2 protein and gene status on a single slide. Using the tissue microarray technique, the HER2 status of each of 875 gastric cancer cases was evaluated by immunohistochemistry (IHC), brightfield dual-color in situ hybridization (DISH), and GPA. Intratumoral phenotypic and genotypic heterogeneity were evaluated by comparing the HER2 statuses of two tissue cores from each case. There was excellent concordance between GPA and IHC (99.2%), as well as between GPA and DISH results (99.3%). HER2 positivity obtained by GPA was almost identical (99.8%) to the results obtained by IHC and DISH assays. Intratumoral phenotypic heterogeneity was more frequently observed in IHC 2+ cases (63.5%) compared with IHC 3+ cases (28.3%). Phenotypic heterogeneity (48.8%) was more frequently observed than genotypic heterogeneity (26.8%). Tumor heterogeneity was consistently observed from early to advanced stages. HER2-positive gastric cancers presented different levels of HER2 protein expression and gene amplification statuses within the same lesion in almost half the cases examined. Evaluating both phenotypic and genotypic heterogeneity may contribute to a deeper understanding and improved prediction of clinical outcome in gastric cancer patients treated with trastuzumab. This newly established GPA technology may also be useful for developing biomarkers for other molecularly targeted therapies.

Combined Assessment of Endothelial Growth Factor Receptor Dual Color In Situ Hybridization and Immunohistochemistry with Downstream Gene Mutations in Prediction of Response to the Anti-EGFR Therapy for Patients with Metastatic Colorectal Cancer

Biomarkers associated with anti-EGFR antibodies therapy have been investigated in metastatic colorectal cancer (CRC). We conducted this study to evaluate the clinical utility of a combined assessment of EGFR status and genomic mutations of the EGFR downstream signal pathway in predicting the efficacy of anti-EGFR antibody treatment. We collected formalin-fixed paraffin-embedded tumor tissues and evaluated the EGFR status by immunohistochemistry (IHC), dual color in situ hybridization (DISH) and genomic analyses of KRAS, BRAF, PIK3CA and NRAS by direct sequencing. A total of 129 patients were evaluated in our study. Among KRAS wild-type patients, EGFR DISH positivity was associated with a higher response rate than DISH negativity (56.3 vs. 21.1%, p = 0.011). A subgroup with EGFR DISH positivity plus IHC3+ and wild-type of EGFR downstream gene mutations achieved higher response rate and disease control rate. EGFR DISH positivity, KRAS codon 146 mutation and NRAS codon 61 mutation were prognostic factors in both progression-free survival and overall survival by multivariate analyses. Combined assessment of DISH plus IHC and EGFR downstream gene mutations was useful to predict the response to anti-EGFR antibodies treatment in metastatic colorectal cancer patients in our study.

Development of an easy method to test for HER2 in breast cancer using dual-color in situ hybridization.

Although human epidermal growth factor receptor 2 (HER2) is a significant clinical biomarker for breast cancer, the HER2 testing involves a complicated evaluation process. We devised an easy method for HER2 gene testing and investigated its utility. HER2 testing was performed by immunohistochemistry (IHC) and dual-color in situ hybridization (DISH) on surgical specimens from 50 patients with invasive breast cancer. DISH was evaluated by two methods. One was the DISH count method in which the HER2 and CEP17 signals were counted and the HER2/CEP17 signal ratio was calculated. The other was the DISH-easy method in which pathologists only observed slices without counting the signals. The DISH-easy method was performed by two pathologists. We analyzed the correlations between the DISH-easy method and the DISH count method by each pathologist and calculated the inter-observer concordance rate of the DISH-easy method. The results from two pathologists using the DISH-easy method corresponded to the IHC results (P < 0.01) and the DISH count method results (P < 0.01). All cases determined to be negative using the DISH-easy method were also negative via the DISH count method with a corresponding rate of 100 % for both pathologist A (34/34) and B (32/32). Most of the results for the positive cases also corresponded; the correspondence rate of pathologist A was 100 % (8/8) and 89 % for B (8/9). The inter-observer concordance rate was 81 % (κ = 0.65). The DISH-easy method is simple and useful for HER2 testing regardless of proficiency of the evaluators.

Abstract P4-06-12: Simultaneous phenotypic and genotypic heterogeneity at the cellular level approached by HER2 gene-protein assay (GPA) in the invasive ductal carcinoma of the breast: A new approach for cancer pathobiology

Abstract Introduction: Trastuzumab therapy has been given to the HER2 positive breast cancer (BC) patients (IHC 3+ and/or amplified HER2 gene). Heterogeneity of HER2 expression has been a problem when the recurrence is considered. Recently, the combined HER2 IHC and dual color ISH (DISH) method has been introduced as a gene-protein assay (GPA) (Roche Diagnostics Inc.). This method enables us to analyze heterogeneous expression of HER2 protein and HER2 gene at the individual cell level on the same tissue sections. This study is aimed to elucidate the relevance of GPA in studying the gene-protein(GP) heterogeneity of HER2 status at the individual cells of BCs and further to pursue the cellular GP heterogeneity as a possible cause of stage IV post-therapeutic recurrence. Materials and Methods: Formalin-fixed, paraffin-embedded sections from 17 HER2 IHC 3+ cases (5 immediate post-operative cases and 12 recurrence cases) and 11 HER2 IHC 2+ cases (9 immediate post-operative cases and 2 recurrence cases) were analyzed with HER2 GPA using BenchMark ULTRA (Roche Diagnostics K.K.). All slides were scored for HER2 protein expression (0 to 3+) and the ratio of HER2/CEN17 of 100 cells from the following areas: one hot spot and two non-hot spots. HER2 protein and gene status of individual cells were grouped from A to F according to the following definition: Group A: HER2 IHC positive (3+), DISH positive (HER2/CEN17 ratio ≥2.0) Group B: HER2 IHC positive (3+), DISH negative (&amp;lt;2.0) Group C: HER2 IHC equivocal (2+), DISH positive (≥2.0) Group D: HER2 IHC equivocal (2+), DISH negative (&amp;lt;2.0) Group E: HER2 IHC negative (0 or 1+), DISH positive (≥2.0) Group F: HER2 IHC negative (0 or 1+), DISH negative (&amp;lt;2.0) Results: HER2 GPA technology clearly demonstrated the HER2 heterogeneity at the individual cell level of invasive BCs. The GP heterogeneity was apparent in the HER2 3+ and 2+ immediate post-operative cases, particularly more obvious with HER2 IHC 2+ cases. For IHC 3+ cases (table 1), groups A and C are prominent with the HER2 GP heterogeneity compared with the other groups. For IHC 2+ cases (table 2), the HER2 GP heterogeneity was more obvious with prominent groups D and F than others. Table 1: Distribution of groups of HER2 status by GPA Cases with HER2 IHC 3+CasesABCDEFPost-operative79.22.88.407.02.6Recurrent50.83.017.06.17.68.9Post-operative and recurrent cases showing hetelogeneity as individual cell level For the recurrent cases, the GP heterogeneity was similar but more pronounced comparing to immediate post-operative tumors in IHC 3+ cases, and GP heterogeneity in recurrent cases was more pronounced in IHC 2+ cases. Conclusions: HER2 GPA system is an appropriate and essential technology to study the GP heterogeneity at the individual tumor cell level of BCs. We hypothesize that the GPA system may possibly predict the recurrence of post-therapeutic stage IV tumors by the presence of more pronounced HER2 GP heterogeneity. Table 2: Distribution of groups of HER2 status by GPA Cases with HER2 IHC 2+CasesABCDEFPost-operative01.714.944.78.630.2Recurrent007.00.548.044.5 Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-06-12.

Comparison of dual-color in-situ hybridization and fluorescence in-situ hybridization in HER2 gene amplification in breast cancer

HER2 is a prognostic factor in breast cancer, and is predictive of the effects of HER2-targeted drugs. HER2 tests are essential in invasive and metastatic breast cancer. Dual-color in-situ hybridization (DISH) is a novel genetic test, and we investigated its utility in HER2 testing in breast cancer. Using DISH and two FISH methods (FISH method 1, FISH method 2) with representative slices of surgical specimens from 134 invasive breast cancer patients, we performed HER2 gene testing and compared the results for HER2 gene/CEP17 signal ratio and HER2 gene diagnosis. Of 134 patients, either the HER2 gene or the CEP17 signal could not be counted in 2 patients by DISH, in 1 patient by FISH method 1, and in 1 patient by FISH method 2. HER2 gene/CEP17 signal ratios were strongly correlated in DISH and FISH method 1 (R=0.85, P<0.05). Agreement of DISH and FISH method 1 for HER2 gene diagnosis was 98.5% for all patients, irrespective of gene amplification (κ=0.97). HER2 gene/CEP17 signal ratios were strongly correlated in DISH and FISH method 2 (R=0.87, P<0.05). Agreement of DISH and FISH method 2 for HER2 gene diagnosis was 94.1% for gene amplification patients, 98.4% for gene non-amplification patients, and 96.2% for all patients (κ=0.92). DISH is useful for HER2 gene testing in breast cancer, and is recommended as a new option for assessing HER2 status.

Abstract P2-05-08: Determination of HER2 amplification by dual-color in situ hybridization before and after neoadjuvant chemotherapy

Abstract Background: HER2-targeted therapies such as trastuzumab neoadjuvant chemotherapy for patients with HER2-positive breast cancer can increase pathological complete response (pCR) better than non-HER2-targeted therapies. Previous studies have suggested that trastuzumab may convert HER2-positive (HER+) primary breast tumors to HER2 negative (HER−) after neoadjuvant chemotherapy. We compared the HER2 status in breast cancer patients before and after neoadjuvant treatment by using dual-color in situ hybridization (DISH). Methods: We retrospectively identified 46 patients from the Breast Cancer database at Tokai University Hospital, in whom HER2-positive primary breast cancer was diagnosed between 2000 and 2010, and who were treated with neoadjuvant chemotherapy or endocrine therapy with or without trastuzumab. Surgical specimens from patients achieving less than pCR were assessed to determine if there was enough residual tissue to evaluate the post-treatment HER2 status by DISH. HER2 status was defined as positive if DISH demonstrated a gene copy ratio of HER2:CEP17 &amp;gt;2.0. Paraffin tissue sections (4-μm thick) were mounted on glass slides (New Sliane III, Catalog No. 5126–25; MUTO PURE CHEMICALS, CO. LTD., Tokyo, Japan) and stained using the newly developed, fully automated HER2 Dual ISH assay on a BenchMark® XT slide stainer according to the recommended procedure (Ventana Medical Systems Inc., Tucson, AZ). Thereafter, the stained slides were rinsed with tap water containing neutral detergent and then rinsed again with distilled water. The slides were dried at room temperature for at least 60 min and cover-slipped with cover grass for SGC (MUTO PURE CHEMICALS). HER2 Dual ISH and H&amp;E images were obtained using an Olympus BX51 microscope. Results: A pCR was achieved in 9 of the 46 patients (19.6%). Specimens from core needle biopsy were not sufficient to assess the pretreatment HER2 status in 3 patients. In 9 patients, the post-treatment HER2 status could not be assessed because residual tumors were DCIS only in 4 patients and there were less than 20 invasive cells in 5 patients. Residual tumor was sufficient to assess the post-treatment HER2 status in 25 patients. In pretreatment specimens, of the 25 patients identified as HER2 + by immunohistochemical analysis, 3 patients were identified as HER2− by DISH. No post-treatment specimens were found to be HER2− by DISH among the tumors identified as HER2+ by DISH at pretreatment. Among the 3 pretreatment tumors identified as HER2− by DISH, 1 tumor was found to be HER2+ by DISH at post-treatment, and 2 showed a stable HER2 status. Conclusion: DISH revealed a stable HER2 status in pretreatment breast tumors and in residual tumors. However, we found a discrepancy in the HER2 status between the immunohistochemical analysis and DISH. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-05-08.

HER2 expression and its clinicopathological features in resectable gastric cancer

A recent randomized controlled trial (Trastuzumab for Gastric Cancer [ToGA] study) established standard scoring criteria of human epidermal growth factor receptor 2 (HER2) for gastric cancer and demonstrated the efficacy of trastuzumab for treating metastatic gastric cancer. The aim of the present study was to evaluate the frequency of HER2-positive cases by application of the standard criteria in patients with resectable gastric cancer and to examine the relationships between HER2 expression and prognosis, mucin phenotype, p53 status, and clinicopathological features. A total of 213 patients were included in this retrospective study. All tumor samples were examined for HER2 expression by immunohistochemistry (IHC), HER2 amplification by in situ hybridization, and mucin and p53 expression by staining for CD10, MUC2, MUC5AC, MUC6, and p53. HER2-positive tumors were identified in 25 patients (11.7%). HER2-positive cases were more frequently found in men, older patients, and in the intestinal histological type (P=0.0048, 0.0309, and <0.0001, respectively). Although no association was found between HER2 overexpression and mucin phenotype, the expression of CD10 and p53 was significantly correlated with HER2 positivity (P=0.0079 and 0.013). The overall survival of HER2-negative and -positive patients was not significantly different. However, in patients with stage III/IV, overall survival was worse in HER2-positive patients (P=0.0149). In a comparison between dual-color in situ hybridization (DISH) and fluorescence in situ hybridization (FISH), four IHC2+/3+ cases that were DISH-positive were judged as negative by FISH. Our study indicated that HER2 expression was less frequent in resectable gastric cancer than in metastatic gastric cancer. The impact of HER2 expression on survival was limited. DISH was superior to FISH for evaluating cases with limited HER2 expression.