Probe Kit Research Articles

The frequencies of human neutrophil alloantigens among the Japanese population

Human neutrophil antigens (HNAs) play an important role in a variety of clinical conditions including immune-mediated neutropenia, non-hemolytic transfusion reactions, and transfusion-related acute lung injury. The aim of this study was to investigate the frequency distribution of HNAs-1 to -5 among the Japanese population. We analyzed samples from 570 healthy Japanese by molecular and serologic techniques to estimate the gene frequencies of HNAs-1 to -5. DNA samples were obtained and typed for the HNA-1 (n = 523), -3 (n = 570), -4 (n = 570), and -5 (n = 508), by molecular techniques. The HNA-1 genotype was determined by using a commercial polymerase chain reaction-reverse sequence-specific oligonucleotide probes (PCR-rSSOP) kit. The HNA-3 to -5 genotypes were determined by the PCR-sequence specific primer (PCR-SSP), previously described, with a small modification. The HNA-2a phenotype was determined in 301 donors by granulocyte immunofluorescence test. In Japanese, the gene frequencies of HNA-1a, -1b, and -1c were 0.623, 0.377, and 0.000, respectively. The frequency of HNA-2a phenotype was 0.987, and the gene frequencies of HNA-3a and -3b were 0.654 and 0.346, respectively. HNA-4a and -4b were found at 1.000 and 0.000, respectively, and HNA-5a and -5b at 0.840 and 0.160, respectively. We describe, for the first time, the frequencies of all HNAs (HNA-1 to -5) among the Japanese population. This study will be helpful for the prediction of the risk of alloimmunization to HNA, especially to determine the risk of HNA alloantibody production by transfusion of HNA incompatible blood and feto-maternal incompatibility.

Application of fluorescencein situhybridization as a diagnostic tool in melanocytic lesions, using paraffin wax-embedded tissues and imprint-cytology specimens

Accurate histopathological diagnosis of certain melanocytic skin lesions as benign or malignant can be notoriously difficult. Recently, four-colour fluorescence in situ hybridization (FISH) has emerged as an important tool for classifying these lesions. To evaluate the sensitivity and specificity of a melanoma FISH probe kit for accurate diagnosis of melanocytic tumours, and to validate its use with imprint-cytology specimens from the cut surface of tumours. In total, 50 melanocytic skin lesions (31 malignant melanomas, 10 benign melanocytic naevi, and 9 histologically challenging benign melanocytic skin lesions) were evaluated. The samples comprise 47 tissue specimens embedded in paraffin wax, and three imprint-cytology specimens from the cut surface of melanomas. FISH was performed using four locus-specific identifier probes [Ras responsive element binding protein (RREB)1, myeloblastosis viral oncogene homologue (MYB), cyclin (CCN)D1 and centromere of chromosome (CEP)6], and results were compared with the clinical long-term follow-up and histopathological diagnosis data. The melanoma FISH probe distinguished between naevi and melanomas with a sensitivity of 100% and a specificity of 94.1%. The most sensitive criterion was a gain in 6p25 (RREB1), seen in 100% of cases, followed by CEP6-related MYB loss (48.1%), CCND1 gain (37%) and MYB gain (22.2%). More than three-quarters (77.8%) of melanomas were positive for two or more criteria. Positive FISH results were also obtained for the imprint-cytology specimens. FISH is a valuable diagnostic tool for differentiating between benign and malignant melanocytic lesions, providing a high degree of sensitivity and specificity. The probes displayed exceptional discriminative capacity in difficult or ambiguous lesions. To our knowledge, his is the first reported use of imprint-cytology specimens for FISH diagnosis.

Detection of Anaplastic Lymphoma Kinase (ALK) Gene Rearrangement in Non-Small Cell Lung Cancer and Related Issues in ALK Inhibitor Therapy

Anaplastic lymphoma kinase (ALK) encodes a receptor tyrosine kinase, and ALK gene rearrangement (ALK+) is implicated in the oncogenesis of non-small cell lung carcinomas (NSCLCs), especially adenocarcinomas. The ALK inhibitor crizotinib was approved in August 2011 by the US Food and Drug Administration (FDA) for treating late-stage NSCLCs that are ALK+, with a companion fluorescent in situ hybridization (FISH) test using the Vysis ALK Break Apart FISH Probe Kit. This review covers pertinent issues in ALK testing, including approaches to select target patients for the test, pros and cons of different detection methods, and mechanisms as well as monitoring of acquired crizotinib resistance in ALK+ NSCLCs.

Targeted therapies for the treatment of non-small cell lung cancer: Bull’s eye or missed target?

3074 Background: Targeted drugs approved for advanced non-small cell lung cancer (NSCLC) include bevacizumab [(monoclonal antibody directed against vascular endothelial growth factor-A (VEGF-A)], erlotinib [(an epidermal growth factor receptor kinase (EGFR) inhibitor)] and crizotinib [(an anaplastic lymphoma kinase (ALK) inhibitor)]. The development programs for erlotinib and bevacizumab did not prospectively employ in vitro diagnostic tests to select patients (pts). Post-hoc exploratory analyses for bevacizumab based on VEGF-A levels have not identified subgroups with differential treatment effects: however for erlotinib, pts with activating EGFR mutations had greater improvement in overall response rates (ORR) and progression-free survival. Crizotinib is the first targeted therapy for NSCLC in which pts were selected using an analytically validated investigational test for ALK translocations. Crizotinib received accelerated approval on August 26, 2011 for the treatment of pts with locally advanced or metastatic ALK-positive NSCLC as detected by an FDA-approved test (Vysis ALK Break Apart FISH Probe Kit) that was approved by FDA on the same day. Methods: NDA file for crizotinib was examined. Results: Two single arm trials assessing efficacy of crizotinib monotherapy in ALK positive pts showed ORR of 61% and 50%. At FDA’s request, a cohort of 23 pts that tested negative for the ALK translocation evaluated showed ORR of 26%. Two additional pts had a single assessment and, if confirmed, the response rate would be 37% in this “marker negative” subgroup. Further exploration of this subgroup is a postmarketing commitment. Conclusions: Evaluation of target status (positive or negative) in all pts provides optimal information to guide clinical practice. As observed with crizotinib, responses may be observed in the “marker negative” subgroup. This unexpected finding may reflect the drug’s activity via alternate pathways, selection of a suboptimal biomarker for assessing ALK activation, or selection of a suboptimal cut-point for positive/negative. Accurate diagnostic tests will facilitate drug development with possible greater observed treatment effect and potentially smaller clinical trials.

Putting pharmacogenomics into practice

Putting pharmacogenomics into practice

Abstract 5098: Identification of copy number alterations associated with the progression and clinical outcomes of oral cavity squamous cell carcinoma

Abstract The molecular mechanisms contributing to the progression of oral cavity squamous cell carcinoma (OSCC) remain poorly understood. The primary treatment for advanced-stage OSCC is radical surgery with post-operative chemoradiation. However, the prognoses are usually discouraging. If we can better understand the characteristics of OSCCs, this may ultimately help clinicians to provide OSCC patients with more appropriate treatment. The advent of genome-wide profiling methods has opened up new possibility to catalogue chromosomal abnormal regions at high resolution. In the present study, 72 advanced-stage OSCCs were analyzed using two microarray platforms (26 cases with Affymetrix SNP 500K and 46 cases with Affymetrix SNP 6.0). Genomic copy number was determined by calculating the intensity ratio between the OSCC and a set of 270 normal DNAs from HapMap project. Segmented copy numbers for each tumor were inferred with the hidden markov model (HMM) using the Partek GS software. To identify regions for copy-number alterations (CNAs), GISTIC (genomic identification of significant targets in cancer) was applied to determine the significance. With this approach, 46 (14 gain and 32 loss) and 208 (48 gain and 160 loss) significant regions (FDR q-value < 0.25) were identified from SNP 500K and SNP 6.0 platform, respectively. Among them, 15 focal CNAs (8 gains (3q26.1, 7p11.2, 11q13.3, 13q22.1, 15q11.2, 17p13.3, 18p11.23-18p11.31, and 20p11.22) and 7 losses (1q21.1, 6p21.33, 7q31.3, 12q14.1, 2 focal regions in 14q11.2, 15q11.2)) are identical in these 2 platforms. Amplification of 11q13.3 containing a known oncogene CCND1 was confirmed by fluorescence in situ hybridization (FISH) with Vysis CCND1/CEP11 FISH probe kit. The clinicopathologic significances of these 15 CNAs were examined. Gain of 3q26.1 and 18p11.23-18p11.31, and loss of 15q11.2 were correlated significantly with lymph node metastasis. Furthermore, loss of two focal regions in 14q11.2 was significantly associated with disease-free and overall survival. In conclusion, CNAs associated with the progression and clinical outcomes of Taiwanese OSCC were identified by genomic approach using high-density SNP array. These results provide useful clues for future characterization of important oncogenes or tumor suppressor genes associated with the behaviors of the disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5098. doi:1538-7445.AM2012-5098

Dual color chromogenic in situ hybridization for determination of HER2 status in breast cancer: a large comparative study to current state of the art fluorescence in situ hybridization

BackgroundChromogenic in situ hybridization (CISH) is fast becoming a well established technique for easy and sensitive determination of HER2 gene status in breast cancer. However, for the chromogenic method to achieve status as a safe and reliable technique, the method needs to be validated against already known and validated FISH techniques.MethodsHere it is reported from a comparative study where HER2 gene status obtained by HER2 CISH pharmDx™ Kit was compared to HER2 gene status obtained by the FDA approved HER2 FISH pharmDx™ Kit and the PathVysion HER-2 DNA probe Kit. The study included 365 formalin fixed and paraffin-embedded invasive breast cancer tissue specimens collected consecutively at a US reference laboratory.ResultsThe data obtained revealed an overall HER2 status concordance of approximately 98% for comparisons of HER2 CISH pharmDx™ Kit to both HER2 FISH pharmDx™ Kit and PathVysion HER-2 DNA Probe Kit.ConclusionsThe concordance between results obtained using the recently FDA approved HER2 CISH pharmDx™ Kit with previously FDA approved FISH techniques for HER2 gene status determination indicate that the HER2 CISH pharmDx™ Kit is a reliable chromogenic alternative to fluorescence-based methods.

Relationship between serum HER2 level and histologic HER2 status in patients with advanced/recurrent gastric cancer.

24 Background: The HER2 extracellular domain can be quantitatively measured in terms of the serum HER2 level by using a CLIA. Increased serum HER2 levels have been reported to be found in 20-25% of metastatic breast cancer patients and correlated with HER2 protein overexpression, increased tumor volume, and worse prognosis. However, the prevalence of HER2 expression in the serum of gastric cancer (GC) patients is not known. Methods: This study retrospectively analyzed the tumor tissue and serum samples of 100 patients who had histologically confirmed advanced/recurrent stomach adenocarcinoma, had started chemotherapy at National Cancer Center Hospital East during 2007–2011, did not have any other active malignancies, and whose serum and tumor tissue samples had been collected and stored at the baseline. HER2 protein expression was determined by immunohistochemistry (IHC; I-VIEW Pathway 4B5 kit) with the GC scoring system (Hofmann, 2008), and gene amplification was determined by fluorescence in situ hybridization (FISH; PathVysion HER2 DNA probe kit), with HER2:CEP17 ≥ 2.0 seen in tumor tissue. Serum HER2 levels were measured using a CLIA (ADIVA Centaur XP). HER2 positivity was defined as IHC 2+ with FISH positive or IHC 3+. Results: The baseline characteristics were as follows: men, 66; median age, 66 years (range, 29–85); advanced/recurrent, 96/4; and intestinal/diffuse/mixed, 38/41/21. An IHC score of 0/1+/2+/3+ was noted in 41/32/6/21 patients, respectively. Gene amplification in FISH was noted in 28 cases. The number of HER2 positive patients was 21. The median serum HER2 level was 9.3 ng/mL (range, 5.0–332.4). With a cutoff level of 15 ng/mL (mean + 2SD of 241 healthy control women), the sensitivity of serum HER2 levels with respect to HER2 positivity was 52.4%; specificity, 93.7%; positive predictive value, 68.8%; negative predictive value, 84.1%; and accuracy, 85.0%. The area under the receiver operating characteristic curve was 0.894. Conclusions: Increased serum HER2 levels were noted in GC patients and were associated with HER2 overexpression in tumor tissue. Further investigations of serum HER2 in terms of the clinical significance are warranted.

MICS 대역과 ISM 대역에서 인체 전기적 상수를 갖는 준(準) 고체형 플랫 팬텀 제작

MICS 및 ISM 대역의 무선기기 설계 시에 인체 팬텀을 이용한 성능검증이 필수적으로 요구되고 있으나, 인체팬텀에 관한 연구 대부분이 휴대폰에 의한 생체 영향에 국한되어 있는 실정이다. 본 논문에서는 FCC에서 제안하고 있는 MICS 대역과 ISM 대역의 인체 팬텀에 대한 전기적 상수를 이용하여 준고체형 팬텀들을 제작하였다. 제작된 준 고체형 팬텀들은 각 대역에서 FCC에서 제시한 전기적 상수(MICS 대역에서 <TEX>$\varepsilon_r=56.7$</TEX>, <TEX>$\sigma=0.94$</TEX>, ISM 대역에서 <TEX>$\varepsilon_r=52.7$</TEX>, <TEX>$\sigma=1.95$</TEX>)들을 만족하였다. 또한, 제작된 준 고체형 팬텀의 재료들은 쉽게 구매 가능한 재료들로 구성되었으며, 팬텀 재료의 다양성을 위해서 각 대역에서 polyethylene과 TX-151을 이용한 방법과 글리세린을 이용한 두 가지 방법을 제시하였다. 제작된 준 고체형 팬텀은 평탄한 형태이며, 유전율 측정기로 제작후 1일 (24 시간)이 경과한 후 팬텀의 전기적 특성을 측정하였다. When wireless devises for MICS(Medical Implant Communication Service) or ISM(Industrial Scientific and Medical) bands are designed, it is necessary to verify the performance by using a human body flat phantom. However, most of studies on the phantom are limited to the biological effects of mobile-phone EMF. In this paper, semi-solid phantoms having the electric properties suggested by FCC at MICS and ISM bands are fabricated. The manufactured phantoms satisfy the electric properties(<TEX>$\varepsilon_r=56.7$</TEX> and <TEX>$\sigma=0.94$</TEX> at MICS band, <TEX>$\varepsilon_r=52.7$</TEX> and <TEX>$\sigma=1.95$</TEX> at ISM band) at each band. All the composing materials for phantoms are commercially available in domestic market. Two methods using both polyethylene powder and TX-151 and glycerin at each band are proposed for diverse purpose. The electrical properties of the fabricated phantoms are measured by a dielectric probe kit and network analyzer after the lapse of one day (24 hours).

P1-12-17: Fluorescent In Situ Hybridization Evaluation of HER2 Status in Tumors with Chromosome 17 Polysomy.

Abstract Introduction: According to the American Society of Clinical Oncology-College of American Pathologists recommendations for HER2 testing, a positive fluorescent in situ hybridization (FISH) result is defined as &amp;gt;6 HER2 gene copies/nucleus (for test systems with no internal control probe [single-color]) or as HER2 gene/chromosome 17 centromere (CEP17) ratio &amp;gt; 2.2 (for systems with an internal control probe [dual-color]). Although an increase in CEP17 copy number (average ≥ 3.0 copies/nucleus) is commonly considered to represent polysomy of chromosome 17, it can also be a result of gains of 17q with centromere involvement, or amplification of the centromeric region. The classification of HER2−positive tumors according to the HER2/CEP17 ratio may therefore misclassify a fraction of truly amplified cases as polysomic. We prospectively evaluated tumors with chromosome 17 polysomy but no HER2 amplification to assess HER2 status using the above two FISH classifications and immunohistochemistry (IHC). Materials and methods: Tumors were tested for gene amplification by FISH with probes to HER2/neu and CEP17 using the PathVysion HER-2 DNA Probe Kit (Vysis). Classification was based on the HER2/CEP17 ratio (amplified when &amp;gt; 2.2) and average HER2 gene copy number/nucleus (amplified when &amp;gt; 6 copies). Both polysomic and equivocal cases (HER2/CEP17 ratio 1.8 - 2.2) were further studied by IHC using the HercepTest (Dako) with 0–3 scoring system (overexpression when 3+). Results: From March 2010 to May 2011 we evaluated 31 primary breast cancers showing chromosome 17 polysomy. Median HER2/CEP17 ratio was 1.3 (range 0.5−1.9), median HER2 copy number was 5.4 (range 2.6−13.8), and median CEP17 copy number was 4.2 (range 3.2−8.0). Thirteen (42%) had an average HER2 gene copy number &amp;gt; 6/nucleus (median 6.8, range 6.1−13.8) and would therefore be considered as amplified if classified according to the absolute HER2 gene copy number. Nine (75%) of these were 3+ at IHC and the remaining 4 were 2+, whereas among the 18 cases with an average HER2 gene copy number &amp;lt; 6/nucleus, one was 2+, ten were 1+, and 7 scored 0. Twenty-nine cases showed negative HER2/CEP17 ratios (&amp;lt; 1.8) and three cases equivocal HER2/CEP17 ratios (between 1.8 and 2.2). Using HER2/CEP17 ratio as first assessment and IHC only in equivocal cases, only one of the 31 “polysomic” cases would have been classified as HER2−positive. However, 8 polysomic cases with HER2/CEP17 ratio &amp;lt; 1.8 showed 3+ immunostaining (all with average HER2 gene copy number &amp;gt; 6/nucleus), while 3 other cases had an average HER2 gene copy number &amp;gt; 6/nucleus with 2+ immunostaining. Conclusions: Our results show that both FISH evaluation criteria and IHC can modify the percentage of polysomic tumors classified as HER2−positive. However, the number of gene copies/nucleus appears more frequently associated with 3+ IHC than the HER2/CEP17 ratio. The predictive impact of the former method on response to anti-HER2 treatments warrants further investigation. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-17.

Crizotinib and Testing for ALK

Crizotinib was recently approved by the US FDA for the treatment of advanced non-small cell lung cancer (NSCLC) harboring the ALK (anaplastic lymphoma kinase) gene rearrangement. To ensure identification of patients most likely to benefit, the FDA approved crizotinib concurrently with a companion diagnostic test-the Vysis ALK Break Apart FISH Probe Kit. This kit was used in 1 of the 2 pivotal trials leading to the FDA approval of crizotinib and has become the gold standard for detecting ALK rearrangement in NSCLC. Although ALK FISH is clinically validated, the assay can be technically challenging and costly. Therefore, other diagnostic modalities are being explored, including immunohistochemistry (IHC) and reverse transcriptase-polymerase chain reaction. This article provides an overview of the diagnostic assays available for detecting ALK rearrangement. Each assay, including ALK FISH, has its strengths and weaknesses. Recent work with commercially available ALK antibodies suggests that IHC-based tests may represent a reliable and cost-effective screening strategy; however, large multicenter studies comparing IHC with FISH are needed to validate ALK IHC. While ALK FISH remains the current standard for diagnosing ALK positivity, large-scale screening of patients with newly diagnosed advanced NSCLC, as recommended by NCCN, may require development and validation of alternative screening strategies, such as combination IHC and FISH.

Trastuzumab (Herceptin®) for Treatment of Refractory/Relapsed HER2 Positive Adult B-ALL: Results of a Phase II GRAALL Study

Abstract 1525Trastuzumab (rhu-mAb-HER2, Herceptin®, F. Hoffmann-La Roche, Basel, Switzerland) is the humanized equivalent of the murine 4D5 monoclonal antibody targeted against the HER2 cell-surface receptor. In combination with chemotherapy, trastuzumab has significantly improved the outcome of women with HER2+ breast cancer. We have previously shown that HER2 surface antigen is up-regulated in around one third of adult B-ALL and is associated with chemoresistance in these patients (Chevallier et al, Haematologica, 2004). We report here for the first time the results of a Phase II study evaluating the safety and efficacy of trastuzumab in refractory/relapsed HER2 + adult B-ALL patients.Prior to patient inclusion, HER2 positivity was assessed using multicolor flow cytometry with the phycoerythrin-conjugated HER2 Neu 24.7 antibody (BD) and a CD19+ CD45+low blast cell gating strategy. The mean fluorescence intensity (MFI) ratio was obtained by dividing the MFI of HER2 with that of its isotypic control. HER2 positivity threshold was defined by a ratio intensity (RI) >= 2. Also, HER2 oncogene amplification was assessed by FISH analysis using the HER2 DNA probe kit (Vysis, Downers Grove, IL, USA). Relapsed/refractory B-ALL patients aged >=18 years and with a HER2+ expression for at least 30% of the leukemic blast population in peripheral blood (PB) and/or bone marrow (BM) were included. Left ventricular ejection fraction has to be > 50%. All patients gave informed consent and the protocol was approved by the required regulatory authorities. The trial was registered at http://clinicaltrials.gov/ct no.NCT00724360. Trastuzumab was administered according to the approved schedule in breast cancer patients at 4 mg/kg intravenous loading dose followed by 2 mg/kg weekly. There was no corticosteroid premedication. Trastuzumab was provided by Roche.Out of 50 patients screened for the HER2 expression, 15 patients (30%) (male n=8; female n=7), with a median age of 62 years (range: 24–80), have been included in the study between November 2006 and July 2011. This was considered as a very high-risk population: 2 patients had refractory disease after 2 induction courses, 2 patients were in first untreated relapse while 11 patients had a refractory first relapse or were beyond first relapse. Median percentage of HER2+ leukemic blast population was 94% (range: 0–100) in PB and 100% (range: 31–100) in BM. Surprisingly, no HER2 gene amplification was detected in samples assayed by FISH. Normal and complex karyotype were detected in 7 and 3 patients respectively. Three patients had a Phi+ B-ALL, and 1 patient a monosomy 7 (unknown karyotype n=1).Currently, 3 patients are still receiving therapy. Considering the 12 other patients, the median number of trastuzumab infusions was 4 (1 month of treatment) (range: 2–20). No grade 3–4 toxicities were observed, with no cardiotoxic events. The overall response rate (CR or PR (decrease >=50% of blast population) or blast clearance in BM or PB) was 33%. No CR was observed. Two patients achieved partial response in BM (92% vs 12% after 9 infusions with loss of HER2 expression on blast population, total number of infusions (TNI) n=18); 25% vs 11% after 4 injections, TNI n=13) while blast clearance was observed in 2 other patients (96% vs 57% of blasts in BM after 8 infusions with loss of HER2 blast expression, TNI n=20; 20.5% vs 2% of blasts in PB after 3 infusions, TNI n=3).In all, we conclude that trastuzumab in monotherapy can allow for some responses in this very high-risk refractory/relapsed HER2+ adult B-ALL population. Combination of trastuzumab with chemotherapy or other therapeutic monoclonal antibodies should be tested in the future. An updated follow-up of the study will be provided during the meeting. Disclosures:No relevant conflicts of interest to declare.

MGMT and PTEN as potential prognostic markers in breast cancer

AimTo evaluate the prognostic importance of MGMT and PTEN concerning their correlation with other prognostic factors evaluated by immunohistochemistry (IHC) and the molecular phenotype of breast cancers. MethodsIHC for estrogen and progesterone receptors, HER2, Ki67, p53, p63, e-cadherin, EGFR, CK5, CK14, MGMT and PTEN was performed on 200 breast tumors. Basal-like and luminal breast carcinomas were defined by the IHC evaluation of these markers. Fluorescent in situ hybridization (FISH) was performed for PTEN and HER2 analysis using the Vysis PTEN and HER2 DNA probe kits (Abbott™). RT-PCR was performed to evaluate gene expressions of MGMT and PTEN in frozen tissue of 59/200 cases. Results147/200 cases were triple-negative (73.5%), 47/147 were basal-like carcinomas (31.9%). 53 cases (26.5%) were luminal-like type A or B. 56 (93.3%) and 46 samples (76.6%) expressed lower levels of MGMT and PTEN mRNA, respectively, compared with normal breast (p<0.001). There was a positive correlation between the IHC results and the RT-PCR values for MGMT and PTEN. Tumors with homozygotic deletion of PTEN expressed little or no mRNA or protein. Positive p53, high Ki67, and basal-like tumors expressed significant lower MGMT and PTEN. ConclusionsWe hypothesize that MGMT and PTEN expressions have prognostic significance in breast cancer. Also, based on their predictive value of response to therapy, evaluating MGMT and PTEN and learning to interpret their patterns of immunoexpression will undoubtedly lead to a greater understanding of breast cancer and its treatment.

Does a change from IHC to FISH enhance patient selection for HER2 therapy?

e11015 Background: Breast cancer is the most commonly diagnosed malignancy among women in the United States. Among various subtypes of breast cancer, tumors that overexpress the human epidermal growth factor receptor 2 (HER2) have an aggressive biology. The frequency of HER2 gene amplification is about 20% among breast cancer patients. The diagnostic tests to determine HER2 status in breast cancer include Immunohistochemisty (IHC), and Fluorescence In-Situ Hybridization (FISH). The interpretation of IHC is subjective and may depend on the pathologist. It is unknown whether the use of FISH has led to higher detection of HER2 overexpression. Methods: We conducted a retrospective review of patients diagnosed with breast cancer from 2000 to 2008. A total of 865 breast cancer patients were reviewed. One hundred and sixty five (19%) were diagnosed with HER2+ subtype. Our institution utilized IHC (HER2-pY 1248 antibody, Dako Corporation) from 2000 to 2003 and FISH (Path Vysion, HER-2 DNA Probe Kit) from 2004 to 2008. Results: The frequency of HER2 detection in breast cancer by IHC from 2000-2003 was 17%. The detection by FISH from 2004-2008 was 20%, an increase of 3%. Conclusions: We observed an increase in the diagnosis of HER2+ breast cancer with the introduction of FISH. This modest difference when applied to a large group of patients could translate into more patients being eligible for anti-HER2 therapy and may enhance accrual to clinical trials.

Human epidermal growth factor receptor 2 (HER2) positivity shown by fluorescence in-situ hybridization in patients with advanced gastric cancer: A multicenter study in Taiwan.

e14576 Background: Based on the results of ToGA (Trastuzumab for Gastric Cancer) study, trastuzumab in combination with chemotherapy can be considered as a new standard option for patients with human epidermal growth factor receptor 2 (HER2)-positive advanced gastric or gastro-esophageal junction (GEJ) cancer (Lancet 2010;376:687-97). In ToGA study, overall HER2-positivity rate was 22.1%; however, exceptionally low HER2-positivity rate was observed in some countries, including Taiwan (only 3%; ASCO-GI 2008:A11). The underlying factors contributing to low HER2-positivity rate in Taiwan remain obscured. Re-evaluation of HER2-positivity for Taiwanese GC patients in a larger sample size is highly warranted. Methods: We tested HER2-positivity by gene amplification using fluorescence in-situ hybridization (FISH) in formalin-fixed paraffin-embedded gastric or GEJ cancer samples. The testing was centralized to a single laboratory (Quest, UK). The PathVysion HER2 DNA probe Kit (Vysis, Abbott Molecular Inc., Germany) was used, which is FDA approved to detect the HER2 gene amplification. Results of hybridization on enumeration of 20 interphase nuclei from tumor cells are reported as the ratio of average HER2 copy number to that of CEP17, a specific region of the Chromosome 17’s centromere. Specimens with amplification showed a HER2 and CEP17 signal ratio of >/= 2.0. If the final ratio is calculated by the primary observer as near/at the cut-off point (1.8-2.2), an additional 20 cells is counted. The second observer will count an additional 20 cells. Results: Between Sep. 2007 and Nov. 2010, 180 patients (M:109, F:71) with gastric or GEJ adenocarcinoma from 8 medical centers in Taiwan were enrolled and evaluated by FISH testing. Among them, 47 patients (M:34, F:13) showed a HER2/CEP17 signal ratio of >/= 2.0, the overall HER2-positivity for Taiwanese GC patients was 26.1% (19.7%-32.5%, 95% CI). Conclusions: More than a quarter of Taiwanese advanced GC patients showed HER2-positivity by FISH, comparable with that of ToGA study. Novel HER2-targeted agents are justified for future active investigation in Taiwanese GC patients.

Comparison of GP and PathVysion FISH assays in measuring HER2 gene status in breast cancer

：Objective To evaluateclinical application of Jin Pujia GP HER2 probe kit in testing HER2 gene status of breastcancer through comparing it with PathVysion HER2 probe kit. Methods HER2 gene status weredetected from 108 cases with invasive ductal breast cancer using GP and PathVysion HER2probe kits by FISH. HER2 gene expression levels were measured by GP and PathVysion HER2probe kits, and the sensitivity, the specificity and the accuracy of GP HER2 probe kitwere evaluated. Results HER2 gene amplification positive rates detected by GP HER2 probekit and PathVysion HER2 probe kit were 25.0%(27/108) and 26.9% (29/108), respectively. Ascompared with PathVysion HER2 probe kit, the sensitivity, the specificity and the accuracyof the GP HER2 kit were 89. 7% (26/29), 98.7% (78/79)and 96. 3% ( 104/108), respectively,whereas the PPV and NPV were 96. 3% (26/27) and 96. 3% (78/81), respectively. The GP HER2probe kit had a sensitivity of 93.3% ( 14/15), a specificity of 100%(93/93) and anaccuracy of 99. 1% (107/108) for detecting polysomy 17. Conclusion GP HER2 probe kit hashigh sensitivity and specificity for detecting HER2 gene status in breast cancer patients,and it has clinical application value.

Identification of the Coexisting HER2 Gene Amplification and Novel Mutations in the HER2 Protein-Overexpressed Mucinous Epithelial Ovarian Cancer

Although overexpression, amplification, and somatic mutation of the HER2 gene have been noted in various types of human cancers, we report here for the first time that novel mutations and amplification of the HER2 gene occurred concomitantly in mucinous epithelial ovarian cancer (EOC). Twenty-seven tissue microarray samples from EOC patients were analyzed by immunohistochemistry (IHC) with Dako c-erb-B2 antibody and subsequently were examined by fluorescence in situ hybridization (FISH) with the Abbott PathVysion HER2 DNA Probe Kit. HER2 gene, exon 18-24, encoding a tyrosine kinase domain, was analyzed by polymerase chain reaction (PCR) and direct sequencing. The FISH-IHC paired results confirmed 19 concordant negative results and 3 concordant positive results. Moreover, all 4 HER2-amplified cases were of the mucinous type, whereas the remaining 23 HER2-nonamplified cases were of the nonmucinous type. The 4 mucinous EOC cases with HER2 gene amplification were selected and further analyzed for HER2 gene mutations. Data revealed that somatic mutations were present in 2 cases (R970W and E971G), but absent in the other 2 cases. HER2 protein overexpression correlated significantly with HER2 gene amplification in EOC (P = 0.027). It is surprising that all 4 cases of mucinous EOC showed HER2 gene amplification confirmed by FISH testing. However, we suppose that increasing the number of cases would possibly modify the results. This study also showed that both HER2 gene amplification and somatic mutations are not mutually exclusive in mucinous EOC. Further studies are warranted to investigate the potential role of anti-HER2 therapy.

INVESTIGATION OF THE CHARACTERISTICS OF BARIUM STRONTIUM TITANATE (BST) DIELECTRIC RESONATOR CERAMIC LOADED ON ARRAY ANTENNAS

We investigated a dielectric resonator ceramic microstrip patch antenna. The antenna was formed using barium strontium titanate (BST), which had a dielectric constant of 15. A new approach, i.e., the use of a high temperature dielectric probe kit, was used to determine the dielectric constant of BST. A computer simulation technology (CST) microwave studio was used to simulate the BST array antennas, taking into consideration the dielectric constant. We also measured the gain of the antennas loaded with two-, four- , and six-element arrays of the BST antenna and found that the gain of a six-element BST array antenna was enhanced by a gain of about 1.6dB over the four-element BST array antenna at 2.3GHz. The impedance bandwidths of these BST array antennas for voltage standing wave ratio (VSWR) < 2 were in the application ranges,

Abstract P4-08-05: Delineation of HER2 Gene Status in Korean Breast Cancers by Standardized Immunohistochemistry (IHC) and Silver In Situ Hybridization (SISH): Comparison of Local IHC Result with Central IHC and SISH Results

Abstract Background: Amplification of the human epidermal growth factor receptor 2 (HER2) gene and concomitant protein overexpression are present in 20-25% of breast cancers. HER2 overexpression and/or gene amplification is associated with a worse clinical outcome and an important predictive marker for sensitivity to anthracycline-based chemotherapy and HER2- targeted therapy. In Korea, the HER2 status has usually been assessed by immunohistochemistry (IHC) in the laboratories. To verify the preanalytic, analytic, and postanalytic factors of IHC in multi-institutions, we assessed the interlaboratory variability of HER2 IHC by comparing the local IHC with the central IHC and SISH results, as a first step toward building a nationwide quality assurance program. Methods: The Breast Pathology Study Group of Korean Society of Pathologists collected 1,198 breast carcinoma samples from 7 university-based hospitals and constructed 56 tissue microarray (TMA) blocks with triplicate of 1 mm cores. Local IHC results for HER2 were obtained from 1,130 patients. We performed IHC (PATHWAY anti-HER2/neu (4B5) rabbit monoclonal antibody, Ventana Medical Systems) and SISH (INFORM HER2 DNA Probe Kit, Ventana Medical Systems) for HER2 on the TMA sections using the automated Benchmark platform (Ventana Medical Systems). We interpreted the results according to the American Society of Clinical Oncology/College of American Pathologists guidelines. Results from local IHC were compared with central IHC and SISH. The concordant rates between central IHC and SISH results were also calculated. Results: The total percentage of cases in each category of local IHC score was: 0/1+, 70.4%; 2+, 8.8% (range among hospitals, 0-36%); and 3+, 20.8% (10.8-28.8%). The percentage of central IHC results (1,110 patients) in each category was: 0/1+, 79.7%; 2+, 3.6% (1.1-8.3%); and 3+, 16.7% (13.1-18.8%). SISH results were obtained from 1,033 patients: negative, 79.5%; equivocal, 0.2% (0-1.6%); and positive, 20.3% (19.2-22.6%). SISH amplifications in each local IHC category were observed as follows: 0/1+, 4.3% (0.6-10%); 2+, 18.8% (5.6-66.7%); and 3+, 71.2% (50-97.3%). When considering the central SISH as gold standard, 3% of cases (30/995) were false negative and 6.3% (63/995) were false positive in local IHC. In central lab, the concordance rates between IHC 3+ and SISH-positive, and IHC 0/1+ and SISH-negative were 95.5% and 98%, respectively. Conclusions: The results of central IHC and SISH markedly decreased the interlaboratory variability of HER2 status. The measurement of HER2 gene amplification by SISH was less affected by the preanalytic factors than the measurement of overexpression of HER2 protein by IHC. Because the rate of false positivity is higher than that of false negativity in the local IHC, the quality assurance program needs to be focused on decreasing not only the false negativity, but also the false positivity of HER2 IHC in local laboratories Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-05.

Analyses of dielectric properties of fertilizers (urea and diammonium phosphate) in aqueous solution at different temperatures in microwave frequency.

This paper reports experimental results which have been carried out for understanding the behavior of dielectrics in the form of mixer of fertilizers and water content at wide frequency range in different temperatures. A simple and rapid measurement (reflection method) was used in determining the dielectric response at microwave frequency. The dielectric constant and loss have been measured in frequency range from 1 to 40 GHz with different temperature according to environment. The measurement procedure has been performed by dielectric probe kit using 50 GHz Performance Network Analyzer (PNA). The samples were prepared by mixing different fertilizers in water according to the Department of Agriculture. The different fertilizers (urea and diammonium phosphate) were taken from the market. The experimental work shows the dielectric behavior of solution of fertilizers and water in temperatures. The amount of water and fertilizers content were taken according to the Department of Agriculture. Keywords: Dielectric constant, dielectric loss, reflection, dielectric probe kit.