Probe Kit Research Articles

Molecular and Genetic Characterization of Fit, Elderly Patients Receiving Oral Fludarabine, Oral Cyclophosphamide and Intravenous Rituximab (OFOCIR) As Initial Treatment of Chronic Lymphocytic Leukemia (CLL)

BackgroundCytogenetic characterization by fluorescence in-situ hybridization (FISH) identifies subgroups of patients with chronic lymphocytic leukemia likely to have poor responses or short remission duration following standard frontline chemoimmunotherapy. Next-generation sequencing (NGS) has identified new molecular targets associated with refractory or poorly responsive disease (eg. Notch1, SF3B1 or BIRC3) independent of cytogenetic abnormalities. We have performed genetic and molecular characterization of fit, elderly patients enrolled on the Australasian Leukemia and Lymphoma Group (ALLG) CLL5 randomized clinical trial of oral fludarabine, cyclophosphamide and rituximab (ACTRN12608000404325).MethodsPre-treatment peripheral blood and bone marrow aspirate samples were obtained from patients enrolled on a phase II randomized clinical trial investigating oral fludarabine, oral cyclophosphamide and intravenous rituximab (poFCivR) tolerance in previously untreated fit elderly patients with CLL (ALLG CLL5 study). Fitness was defined as Cumulative Illness Rating Scale (CIRS) score of ²6. Bone marrow aspirate samples were analysed for CLL-associated genomic changes with a Vysis CLL FISH probe kit (Abbott, Des Moines, IL) and ranked according to Dohner hierarchical classification. DNA was extracted from peripheral blood lymphocytes and we performed targeted exome sequencing of genes including TP53, ATM, NOTCH1, SF3B1, BIRC3, MYD88 and FBXW7 using a TruSeq Custom Amplicon Design Panel on a MiSeq DNA sequencer as per manufacturerÕs protocol (Illumina, San Diego, CA). Gene mutations were confirmed by Sanger sequencing. Data was analyzed using Illumina proprietary software, annotated using ANNOVAR software and compared to COSMIC and other genomic mutation databases.ResultsOf 116 analyzable patients enrolled on the clinical trial, 78 pts had available FISH results and 76 patients DNA sequencing. The ORR and CR for all patients on study were 96% and 56% respectively. There was no significant difference in ORR between cytogenetic risk groups (Table 1); however, only 1 of 9 patients with ATM deletion achieved CR (11%, p=0.0095). We identified 8 pts with TP53 mutations, only one patient (12.5%) achieved a CR (p=0.0084). CR rate for patients with mutations in ATM (n=9, CR 44%), NOTCH1 (n=10, CR 60%), SF3B1 (n=11, CR 91%), BIRC3 (n=2, CR 0%), XPO1 (n=6, CR 33%), myd88 (n=5, 100%) were not significantly different to patients without the respective mutations. Of 14 pts with normal FISH, 10 pts (71%) had molecular abnormalities identified by NGS (Figure 1). Median follow-up of patients is 20 months, with 91% patients alive at last follow up. At the time of analysis, there was no significant difference in progression free survival (PFS) between different FISH cytogenetic risk groups (Figure 1). Multivariable analysis identified patients with TP53 mutations (HR 4.3, p=0.04) and XPO1 mutations (HR 3.2, p=0.035) as independently associated with shorter PFS. Our analysis was limited by the small subgroups of patients with individual molecular mutations and currently relatively short follow-up of this study.ConclusionsMolecular characterization by DNA sequencing increases the yield of pre-treatment genetic alterations discovered in CLL patients. In this randomized clinical trial of elderly patients requiring first line therapy of CLL, we identified a high proportion of genomic alterations. Identification of genomic mutations may help further risk stratify CLL patients undergoing chemoimmunotherapy.Table 1NCR (n)CR (%)ORR (n)ORR (%)All patients116655611196FISH17p deletion191052178911q deletion9111889Trisomy 1215960149313q deletion3318553297No abnormal16138116100Not Done24148124100NGSAll Available7644587295mutationsTP538113787.5ATM94449100NOTCH110660990SF3B11110911091BIRC32002100XPO16233583Myd88551005100 [Display omitted] [Display omitted] DisclosuresBadoux:Roche: Honoraria. Mulligan:Roche, Abbvie: Consultancy, Honoraria. Kuss:Roche: Research Funding; Sanofi: Research Funding.

191P - Experience of Alk Mutation Testing in 3351 Indian Patients of Nsclc

ABSTRACT Aim: In the last decade, we have witnessed a revolution in the molecular genetics of NSCLC (Non Small Cell Lung Cancer). These advances have led to the development of a multitude of prognostic and predictive biomarkers. Somatic mutations in EGFR (Epidermal Growth Factor Receptor) gene & ALK (Anaplastic Lymphoma Kinase) translocation are two “actionable” biomarkers that have passed clinical validation and are incorporated into current treatment paradigms. This abstract contributes towards understanding the epidemiology of ALK mutation in NSCLC patients in India. Methods: This was an observational study within the cohort of NSCLC patients. Pfizer Ltd supported this study as a part of Diagnostic Assistance Program. A total of 3351 patients of lung adenocarcinoma were tested from Jan 2013 to Mar 2014. These samples were collected from various locations across the country. ALK mutation was studied using the FDA approved Vysis ALK Gene Breakapart Probe Kit and the EGFR mutation testing (exons 18-21) was done using PCR followed by bidirectional Sanger's Sequencing. Results: Of the 3351 samples tested, 2146 (64.04%) were male & 1205 (35.96%) were females. Patients ranged in age from 25-90yrs with a median age of 57.94yrs. Biomarker testing for EGFR and ALK was performed on FFPE specimen of patients with confirmed adenocarcinoma histology. ALK testing was performed on 3351 samples and EGFR analysis on 3079. Of samples received for EGFR, 2653 (86.16%) were successfully analyzed & reported, 177 (5.75%) showed unsatisfactory results, and test could not be performed in 249 cases (8.09%). 2810 (83.86%) samples received for ALK testing by FISH could be successfully reported, 287 (8.56%) showed unsatisfactory results and 254 samples (7.58%) could not be tested. Common reasons for unsatisfactory results were presence of high degree of necrosis, scanty tumor or suboptimal fixation. Of the successfully reported cases, 28.19% were positive for presence of EGFR mutations. Mutations were observed in exon 18 (2.27%), exon 19 (72.99%), exon 20 (3.48%) and exon 21 (21.26%). 71 (2.53%) of the 2810 successfully reported cases were found Positive for ALK Gene Rearrangement. None of the samples showed a concomitant mutation. Conclusions: This study on 3351 adenocarcinoma enriched NSCLC patients of Indian origin, shows an ALK positivity of 2.53% & EGFR mutation positivity of 28.19%. Disclosure: All authors declare that he study was funded by Pfizer Ltd as a part of its Diagnostic Assistance Program. Other than that there is no disclosure to declare.

289P - Fish Testing of Her2 Ihc 1+ Early Breast Cancer with Unfavorable Prognostic Factors

ABSTRACT Aim: HER2-positive tumors are associated with a poor prognosis and a shortened disease-free and overall survival as well as with other unfavorable prognostic tumor characteristics (high histological grade, high proliferative index, negative or low estrogen receptor expression, etc.). HER2-positive tumors are also responsive to treatment with trastuzumab in reducing the risk of recurrence and improving survival. The aim of this study is to assess the incidence of HER2 gene amplification in selected tumors with adverse prognostic features which scored 1+ by immunohistochemistry (IHC). Methods: 75 women with infiltrating ductal carcinoma (IDC) and infiltrating lobular carcinoma (ILC) scoring 1+ by IHC were included. Forty-eight invasive breast carcinoma samples were selected according to unfavorable prognostic tumor characteristics and tested by FISH. HER2 amplification was evaluated using Vysis HER2/Cep17 probe (Path Vysion HER2 DNA Probe Kit®, Abbott Molecular, IL); ratio–based amplification was considered present when the HER2/Cep17 ratio was 2 or more and copy number-based amplification was considered present when the mean HER2 copy number was more than 6, in agreement with the ASCO/CAP/SIAPEC guidelines. Results: In 2013, 331 patients with invasive breast tumors were tested by IHC; 75 cases (23%) were scored 1+ of which 62 cases (19%) of IDC and 13 cases (4%) of ILC. Forty-eight invasive breast carcinoma samples (64%) were selected according to one or more unfavorable prognostic tumor characteristics; 22 out of 48 tumors (46%) showed high histological grade (G3); 27 cases (56%) had high proliferative index (Ki-67 ≥ 30%); 32 tumor samples (67%) were node-positive; and 29 cases (60%) showed vascular invasion. FISH was performed on 28 of the 1+ patients with adverse tumor characteristics and 6 IDC out of 48 (12.5%) showed HER2 amplification. Conclusions: Our preliminary retrospective data suggest that 6 patients out of 48 (12.5%) scoring 1+ by IHC show HER2 amplification, in agreement with the most recently published literature data. In order to not deny the benefit deriving from trastuzumab administration, in breast cancer patients showing IHC 1 + , it is advisable to test HER2 gene amplification by FISH. Disclosure: All authors have declared no conflicts of interest.

A Broadband Sensor Interface IC for Miniaturized Dielectric Spectroscopy From MHz to GHz

This paper describes a broadband sensor interface IC as part of a miniaturized measurement platform for MHz-to-GHz dielectric spectroscopy. Developed in 0.35 μm 2P/4M RF CMOS, the IC measures frequency-dependent S 21 magnitude and phase of a microfluidic dielectric sensor fabricated in a thick gold-on-glass microfabrication process and loaded with a material-under-test (MUT). The IC architecture implements a broadband frequency response analysis (bFRA) method by first down-converting the sensor response signal from the RF excitation frequency to an intermediate frequency (IF) of 1 MHz using a low-noise amplifier (LNA) and active mixer, followed by down-converting the IF signal to dc using a coherent detector employing IF amplification stages with programmable gain, a passive mixer driven by in-phase (I) and quadrature-phase (Q) signals and an active-RC low-pass filter (LPF). The sensor interfaced with the IC is fully capable of differentiating among deionized (DI) water, phosphate buffered saline (PBS), ethanol and methanol in tests conducted at four different excitation frequencies of 50 MHz, 500 MHz, 1 GHz and 3 GHz. Further, dielectric readings of ethanol from the sensor interfaced with the IC at five excitation frequencies in the range of 50 MHz to 2 GHz are in excellent agreement (error <;1%) with those from using a vector network analyzer (VNA) as the sensor readout. A bulk-solution reference measurement by an Agilent 85070E dielectric probe kit interfaced with a VNA is also performed to verify proof-of-concept feasibility in conducting MHz-to-GHz dielectric spectroscopy with a miniaturized measurement platform using μL-sample volumes.

Morphology in conjunction with immunohistochemistry is sufficient for the diagnosis of mammary analogue secretory carcinoma.

The recently described mammary analogue secretory carcinoma (MASC) is a low-grade salivary gland malignancy that harbors the recurrent cytogenetic abnormality t(12;15) (p13;q25) ETV6-NTRK3. Confirmation of this is currently considered the gold standard for diagnosis. Some have postulated that morphology together with supporting immunohistochemistry is sufficient to diagnose MASC. In this study we retrospectively review a series of 19 MASCs diagnosed based on histology in conjunction with immunohistochemistry; subsequently we performed in situ hybridization using an ETV6 break-apart probe. Immunohistochemistry for S100 protein and mammaglobin as well as fluorescence in situ hybridization using the Vysis ETV6 Dual Color Break-Apart FISH Probe Kit were performed on all cases. The 19 cases were from 12 females and 7 males with ages ranging from 16 to 76 years (mean = 45 years). Sixteen cases were from the parotid gland, 1 case was from a periparotid lymph node and 2 cases were from the submandibular gland. All 19 cases demonstrated moderate to strong expression of S100 protein. Eighteen cases demonstrated strong, diffuse expression of mammaglobin, while one case had only rare tumor cells that strongly expressed mammaglobin. Eighteen of 19 cases (95 %) demonstrated the ETV6 rearrangement by fluorescence in situ hybridization. Given that morphology together with immunohistochemistry is highly correlated with the ETV6 gene rearrangement, we conclude that molecular confirmation is not required to diagnose MASC.

Correlation of HER2 gene status assessment by fluorescence in situ hybridization between histological sections and cytological specimens of breast cancer.

While HER2 gene detection in cytological specimens using fluorescence in situ hybridization (FISH) has been reported, the appropriate criteria for such specimens remain controversial. Fine needle aspiration (FNA) samples collected from surgically resected breast cancer specimens were rinsed in a cytopreservative solution containing fixative. Then, slides of the FNA samples were prepared by liquid-based cytology (LBC) (ThinPrep system, Hologic) according to the manufacturer's instructions, and a PathVision HER2 DNA probe kit (Abbott) was used for FISH staining. The results were evaluated using an automated MetaCyte imaging system (MetaSystems, Altlussheim, Germany). HER2 gene amplification was scored using the HER2/chromosome enumeration probe 17 (CEP17) signal count ratio as follows: amplified, >2.2; equivocal, 1.8-2.2; and unamplified, <1.8. The cytology results were compared with the histology results from concordant cases. Successful results were obtained in 98 of 100 cases, and results from the FNA specimens were in agreement with those from the histological sections in 97 of these 98 cases (accuracy rate, 99%; kappa, 0.962). FISH-based assessment of the HER2 gene status is consistent between histological sections and cytological specimens of breast cancer.

A PILOT STUDY OF HER-2/NEU GENE AMPLIFICATION ASSESSED BY FLUORESCENCE IN SITU HYBRIDIZATION (FISH) IN GASTRIC CANCER

Background: HER-2/neu is a predictive biomarker for treatment of gastric cancer using trastuzumab in combination with chemotherapy. This study aimed to evaluate the status of HER-2/neu gene amplification using fluorescence in situ hybridization (FISH) in gastric cancer. Patients and methods: thirty six gastric cancer patients were assessed HER-2/neu gene amplification by FISH using PathVysionTM HER-2 DNA Probe kit (including HER-2/neu probe and CEP-17 probe) with biopsy and surgical specimens. Results: The HER-2/neu gene amplification was observed in three cases (8.3%), the HER-2/neu gene amplification rate in Lauren’s intestinal-type and diffuse-type were 11.8% and 5.2%, respectively. Conclusion: We applied successfully FISH technique with gastric cancer tissue samples. This technique could be performed as routine test in gastric cancer in order to select patients that benefit from trastuzumab in combination with chemotherapy.

The protective effect of smilax glabra extract on advanced glycation end products-induced endothelial dysfunction in HUVECs via RAGE-ERK1/2-NF-κB pathway

Ethnopharmacological relevanceSmilax glabra Roxb. (SGR) is a traditional Chinese herb that has been used in folk for the treatment of diabetic vascular complications. Advanced glycation end products (AGEs)-induced endothelial dysfunction has been thought to be a major cause of diabetic vascular complications. The present study was conducted to investigate the protective effect of SGR extract on AGEs-induced endothelial dysfunction and its underlying mechanisms. Materials and methodsHuman umbilical vein endothelial cells (HUVECs) were exposed to 200μg/ml AGEs to induce endothelial dysfunction. Acridine orange/ethidium bromide (AO/EB) fluorescence assay and Annexin-V/PI double-staining were performed to determine endothelium apoptosis. Dihydroethidium (DHE) fluorescence probe, SOD and MDA kits were used to evaluate oxidative stress. The effect of SGR extract on AGEs-induced TGF-beta1 expression was determined by immunocytochemistry and western blotting. Attenuations of SGR extract on receptor for AGEs (RAGE) expression, extracellular regulated protein kinases (ERK1/2) activation and NF-κB phosphorylation were determined by immunofluorescence assay and western blotting. The blockade assays for RAGE and ERK1/2 were carried out using a specific RAGE-antibody (RAGE-Ab) or a selective ERK1/2 inhibitor PD98059 in immunofluorescence assay. ResultsThe pretreatment of SGR extract (0.125, 0.25 and 0.5mg crude drug/ml) significantly attenuated AGEs-induced endothelium apoptosis, and down-regulated TGF-beta1 protein expression in HUVECs. It was also well shown that SGR extract could down-regulate dose-dependently ROS over-generation, MDA content, TGF-beta1 expression, ERK1/2 and NF-κB activation whereas increase significantly SOD activity. Furthermore, the AGEs-induced ERK1/2 activation could be attenuated by the blockade of RAGE-Ab (5μg/ml) while the NF-κB activation was ameliorated by ERK1/2 inhibitor PD98059 (10μM). ConclusionThese results indicated that SGR extract could attenuate AGEs-induced endothelial dysfunction via RAGE-ERK1/2-NF-κB pathways. Our findings suggest that SGR extract may be beneficial for attenuating endothelial dysfunction in diabetic vascular complications.

Diagnostic and therapeutic issues for patients with advanced non‑small cell lung cancer harboring anaplastic lymphoma kinase rearrangement: European vs. US perspective (review).

The recent availability of crizotinib in clinical practice, for the treatment of patients with advanced non-small cell lung cancer (NSCLC) selected by the presence of anaplastic lymphoma kinase (ALK) rearrangement, has relevant implications for both the diagnostic phase and the treatment choices. In the United States, crizotinib was approved by the Food and Drug Administration (FDA) in 2011 for patients with ALK positivity detected by FDA-approved companion diagnostic test. As of January, 2014, the only FDA-approved diagnostic test is Vysis ALK Break-Apart FISH Probe Kit. In Europe, European Medicines Agency (EMA) approved crizotinib for ALK-positive patients in 2012, without specifying the type of test used for determining the positivity. FISH remains the reference technique for ALK determination, but, if fully validated, immunohistochemistry could challenge the current ALK screening practice. Given the robust evidence of activity of crizotinib in ALK-positive patients both pretreated and chemotherapy-naïve, and the favourable tolerability profile of the drug, many oncologists would prefer to administer the drug as early as possible. This is technically feasible in the United States, where crizotinib was approved well before the availability of the results of the randomized phase III trial comparing the drug with standard second-line chemotherapy, and the use of crizotinib in ALK-positive patients is not restricted to a specific line of treatment. On the contrary, in Europe, differently from the FDA decision, crizotinib cannot be used in chemotherapy-naïve patients. In both realities, a deeper knowledge of mechanisms of resistance, the role of repeated biopsies, the treatment strategy for patients experiencing disease progression with crizotinib, the choice of the best chemotherapy regimen are challenging topics for the management of ALK-positive patients in clinical practice.

P0097 ALK rearrangements in turkish patients with lung adenocarcinoma

<h3>Background</h3> We aimed to establish the frequency of ALK translocation in lung adenocarcinomas at a population level. <h3>Methods</h3> Between 2011 and 2013, 157 paraffin embedded tumour tissue samples were collected by our hospitals. Forty patients from this group were also scanned for EGFR mutations. FISH technique was used on paraffin embedded tissues, which were cut 4 microns thick. Vysis ALK Breakapart FISH probe kit was used to detect rearrangement of ALK gene. <h3>Findings</h3> Twenty patients (12.7%) were positive for ALK rearrangement. ALK-positive patients were younger than ALK-negative patients. <h3>Interpretation</h3> Our study is the first to investigate the ALK status of lung cancers in Turkish patients.

U.S. Food and Drug Administration Approval: Crizotinib for Treatment of Advanced or Metastatic Non–Small Cell Lung Cancer That Is Anaplastic Lymphoma Kinase Positive

On August 26, 2011, the U.S. Food and Drug Administration (FDA) approved crizotinib (XALKORI Capsules, Pfizer Inc.) for treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) that is anaplastic lymphoma kinase (ALK) positive as detected by an FDA-approved test. The Vysis ALK Break-Apart FISH Probe Kit (Abbott Molecular, Inc.) was approved concurrently. In two multicenter, single-arm trials, patients with locally advanced or metastatic ALK-positive NSCLC previously treated with one or more systemic therapies received crizotinib orally at a dose of 250 mg twice daily. In 119 patients with ALK-positive NSCLC by local trial assay, the objective response rate (ORR) was 61% [95% confidence intervals (CI), 52%-70%] with a median response duration of 48 weeks. In 136 patients with ALK-positive NSCLC by the to-be-marketed test, the ORR was 50% (95% CI, 42%-59%) with a median response duration of 42 weeks. The most common adverse reactions (≥25%) were vision disorder, nausea, diarrhea, vomiting, edema, and constipation. Accelerated approval was granted on the basis of the high ORRs and durable responses. On November 20, 2013, crizotinib received full approval based on an improvement in progression-free survival in patients with metastatic ALK-positive NSCLC previously treated with one platinum-based chemotherapy regimen.

Cost Effectiveness of Crizotinib for Anaplastic Lymphoma Kinase–Positive, Non–Small-Cell Lung Cancer: Who Is Going to Blink at the Cost?

In 2014, lung cancer is the poster child for personalized medicine, and identifying targetable oncogenic drivers is now considered standard of care in stage IV disease. The anaplastic lymphoma kinase (ALK) gene has emerged as an important oncogenic driver in a small population of patients with adenocarcinoma. Crizotinib has received accelerated US Food and Drug Administration approval when used in conjunction with its companion diagnostic test to identify patients with the EML4-ALK gene rearrangement. No one who treats patients with lung cancer questions the exciting results obtained with crizotinib, or the need for a highly specific test. A recent phase III study compared crizotinib with standard chemotherapy in patients with locally advanced or metastatic ALK-positive lung cancer who had received one previous platinum-based regimen. Everything favored crizotinib: median progression-free survival (PFS), 7.7 versus 3.0 months; response rate, 65% versus 20%; and symptoms and quality of life were also substantially better. Overall survival was not improved, but 64% of the group receiving chemotherapy crossed over to crizotinib. Two main questions remain unanswered: Can we afford to screen everyone with lung cancer, given that only 3% to 5% of the population will be ALK positive, and can we afford to pay for crizotinib? In the article prompting this editorial, Djalalov et al present their results of a cost-effectiveness analysis of both EML4-ALK fusion testing and first-line crizotinib using a Markov model in newly diagnosed, previously untreated patients with stage IV non–small-cell lung cancer (NSCLC) and nonsquamous histology. The authors compared strategies for treatment: crizotinib for patients who test ALK positive versus standard chemotherapy for those who test negative. The incremental cost-effectiveness ratio (ICER) or change in costs divided by the change in benefits is commonly used in health economics to provide a practical approach to decision making regarding health interventions. The costs are described in monetary units, whereas the benefits or effects on health status are measured in quality-adjusted life years (QALYs) gained or lost. Using a relative efficacy estimate in the first-line setting that was derived from the second-line phase III trial, the author’s projected that first-line crizotinib gives 0.64 years (7.7 months) extra survival, or 0.38 QALYs, at an additional cost of $95,000 compared with standard of care, leading to ICERs of $148,000 per additional life year, or $250,000/ QALY gained. The authors concluded that EML4-ALK fusion testing and crizotinib treatment for patients positive for ALK is not costeffective (from a Canadian perspective) because of the low biomarker frequency in the population—and the high drug costs. Are their conclusions valid, what do they mean, and is crizotinib affordable? The model seems valid. The authors simulated the clinical trial of what happens if patients positive for ALK start with crizotinib while we await the results of the ongoing first-line phase III PROFILE 1014 trial (A Clinical Trial Testing the Efficacy of Crizotinib Versus Standard Chemotherapy Pemetrexed Plus Cisplatin or Carboplatin in Patients With ALK Positive Nonsquamous Cancer of the Lung). In this analysis, all patients receive crizotinib (or do not, if ALK negative), then cisplatin plus gemcitabine, then second-line pemetrexed, then thirdline erlotinib, regardless of epidermal growth factor receptor status. The authors transform the added 0.64 years of life to 0.38 qualityadjusted years by assuming that the quality of life is only 3% better for patients receiving crizotinib compared with regular chemotherapy. This seems ungenerous, given the rapid and often dramatic symptom improvement seen in practice, which should translate into greater quality time while receiving the drug. Their test strategy for ALK assessment is efficient—a critically important aspect, given the need to screen 100 patients to find approximately three who test positive. The only US Food and Drug Administration–approved test is the Vysis LSI ALK Break Apart FISH Probe Kit (Abbott Molecular, Des Plaines, IL), with near 100% accuracy but at a cost of more than $250/test. The correlation of results of ALK immunohistochemistry (IHC) and ALK fluorescent in situ hybridization (FISH) are excellent, but IHC has not been validated as a predictive marker for response to an ALK inhibitor in a large patient cohort. The US guidelines state that ALK IHC, if carefully validated, may be considered for screening patients with lung adenocarcinoma. The authors used ALK IHC (approximately $40) with 95% sensitivity as their baseline, with subsequent verification with ALK FISH if needed. Djalalov et al indicate that EML4-ALK fusion testing with crizotinib treatment for patients positive for ALK “is not cost-effective in the setting of high drug costs and a low biomarker JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 32 NUMBER 10 APRIL 1 2014

MET Copy number in triple negative breast cancers

Background Triple negative breast cancers (TNBC) generally show a very poor prognosis with a need for targeted therapy. Expression of MET is elevated in 15-20% of breast cancers. Overexpression is a prognostic marker of poor survival, is associated with TNBCs and high risk of metastatic progression. MET is rarely mutated in breast cancer, however limited studies have indicated that MET overexpression is due to elevated copy number. Targeted inhibition of MET may provide a much needed therapeutic opportunity for treating TNBC. Methods 89 TNBC cases were analysed for MET copy number using fluorescence in situ hybridisation [Vysis MET SpectrumRed FISH Probe Kit and CEP 7 (D7Z1) Spectrum-Green Probe, Abbott Molecular]. MET protein expression was analysed using immunohistochemistry (SP44 clone antibody, Roche). Results One case (1.5%) showed elevated copy number for both MET and the CEP7 enumeration probe (average MET = 9.2, average CEP7 = 7.8, ratio =1.2). This case was not associated with recurrence or poor survival. Forty-seven of 89 (53%) showed MET immunoreactivity. Discussion Compared with another study 1 reporting MET ampli-ficatopm in 14% of TNBC, we have found elevated MET copy number occurs at a low frequency (1.5%) within our cohort and does not account for MET overexpression. Further investigation of MET copy number in larger cohorts is warranted.

HER2 Testing in Gastroesophageal Cancer Paired Biopsy and Resection Specimens – A Multi- Institutional Immunohistochemistry and Fluorescence In-Situ Hybridization Correlation Study

Eligibility for treatment with trastuzumab (Herceptin) for metastatic gastroesophageal cancer (GE) requires demonstration of HER2 protein overexpression or HER2 gene amplification. However, in the US there are no guidelines for best practices of HER2 testing in GE. The goals of this multicenter study were to first evaluate whether the published 2007 ASCO/CAP guidelines established for breast cancer could be extrapolated to GE cancers; and second, to implement an inter-laboratory comparison of HER2 IHC and FISH performance in paired biopsy and tumor resection specimens. Fifty paired formalin-fixed paraffin-embedded (FFPE) biopsy and resection specimens of GE carcinomas were selected from the pathology archives of three institutions. HER2 FISH analysis was performed using the PathVysion DNA probe kit. Immunohistochemistry was performed on the Ventana Benchmark XT with Ventana’s Pathway anti- HER-2/neu (4B5) antibody. Scoring and interpretation of results were according to the ASCO/CAP guidelines for breast carcinoma. Two of the 50 paired cases, were excluded due to insufficient quantity of residual tumor in the biopsy specimen. 48 and 38 pairs were successfully evaluated by IHC and FISH, respectively. Overall, the agreement of the IHC and FISH scoring between institutions was good; Ƙ = 0.76 (IHC) and very good; Ƙ = 0.89 (FISH). The concordance between biopsies and resection specimens was 96% and 97% with IHC and FISH, respectively. We present data from an inter-laboratory collaborative study of the performance of HER2 IHC and FISH within GE tumors in paired biopsy and resection specimens. We found that the 2007 ASCO/CAP guidelines can be applied to GE cancer with good inter-observer reproducibility.

Analysis of HER2 status in breast carcinoma by fully automated HER2 fluorescence in situ hybridization (FISH): comparison of two immunohistochemical tests and manual FISH

Easy and accurate HER2 testing is essential when considering the prognostic and predictive significance of HER2 in breast cancer. The use of a fully automated, quantitative FISH assay would be helpful to detect HER2 amplification in breast cancer tissue specimens with reduced inter-laboratory variability. We compared the concordance of HER2 status as assessed by an automated FISH staining system to manual FISH testing. Using 60 formalin-fixed paraffin-embedded breast carcinoma specimens, we assessed HER2 immunoexpression with two antibodies (DAKO HercepTest and CB11). In addition, HER2 status was evaluated with automated FISH using the Leica FISH System for BOND and a manual FISH using the Abbott PathVysion DNA Probe Kit. All but one specimen were successfully stained using both FISH methods. When the data were divided into two groups according to HER2/CEP17 ratio, positive and negative, the results from both the automated and manual FISH techniques were identical for all 59 evaluable specimens. The HER2 and CEP17 copy numbers and HER2/CEP17 ratio showed great agreement between both FISH methods. The automated FISH technique was interpretable with signal intensity similar to those of the manual FISH technique. In contrast with manual FISH, the automated FISH technique showed well-preserved architecture due to low membrane digestion. HER2 immunohistochemistry and FISH results showed substantial significant agreement (κ=1.0, p<0.001). HER2 status can be reliably determined using a fully automated HER2 FISH system with high concordance to the well-established manual FISH method. Because of stable signal intensity and high staining quality, the automated FISH technique may be more appropriate than manual FISH for routine applications.

Abstract P3-05-01: Accurate assessment of HER2 status in “triple-negative” breast cancer requires both IHC and FISH testing

Abstract Introduction: Triple-negative invasive breast carcinoma (TNBC) does not over-express estrogen, progesterone receptors and HER2/neu. Approximately 15% breast cancers are “triple negative” and have poor overall survival with no targeted therapeutic options. HER2 positive patients also have a poor prognosis which is markedly improved with anti-HER2 therapy. ASCO/CAP guidelines have made substantial improvement in biomarker assessment by standardizing pre-analytic, analytic and post-analytic variables. Since 2009, we have performed HER2 fluorescence in situ hybridization (FISH) analysis in addition to immunohistochemistry (IHC) on all triple negative patients in an effort to accurately assess HER2 status. Materials and Methods: Pathology database at our institute was queried for triple negative breast cancer cases 2009-2012. 175 cases were identified with ER(SP1; Ventana)&amp;lt; = 1%, PR(1E2; Ventana)&amp;lt; = 1% and HER2 IHC (HercepTest® DAKO 2009-2011; 4B5-Ventana- 2012)&amp;lt; = 1+. FISH for Her2 was also performed on these cases. HER2/CEP17&amp;gt; = 2 was classified as HER2 positive. FISH for HER2 amplification was performed using the FDA-approved HER2 DNA Probe Kit (Vysis, Inc.). The hybridization results were recorded and analyzed by BioView Duet system (Allegro Plus Automated Scanner) with HER2 application software (BV-HER2-AF). Fisher's exact test was performed to correlate HER2 amplification with clinico-pathological parameters. Results: 175 breast cancers were classified as “triple-negative” by IHC between 2009-2012. The patients’ age ranged from 25 to 93 years old (mean = 58); 109 were Caucasian (62%), 33 African American (19%), 9 Asian (5%) and 4 Hispanic (2%). The majority of the cases (152, 86%) were invasive ductal carcinoma. The rest include 9 cases of medullary carcinoma, 3 cases of metaplastic carcinoma, 3 cases of apocrine carcinoma, 2 cases of papillary carcinoma and 2 cases of invasive lobular carcinoma. 34 had regional lymph node (axillary) metastasis (19%) and 9 had distant metastasis (5%). 23 of the 175 “triple-negative” cases (13%) showed HER2 amplification by HER2 FISH analysis (HER2/CEP17&amp;gt; = 2). 14.9% (20/134) TNBC cases were FISH positive 2009-11 (HercepTest®, DAKO) and 7.3% (3/41) cases were FISH positive in 2012 (4B5, Ventana). 14 of the 23 patients were Caucasian (61%), 4 African American (17%), 2 Asian and 1 Hispanic. 20 of the 23 cases (87%) had invasive ductal carcinoma, of which 18 (78%) were poorly differentiated. 8 of the 23 cases (35%) had lymph node metastasis and 1 case had distant metastasis. No statistically significant correlation was observed between the HER2 gene amplification and clinicopathological factors analyzed, including race (P = 1), age (P = 1), tumor size (P = 0.6), pathologic stage (P = 0.6), lymph node status (P = 0.08), and Ki-67 index (P = 0.8 with 20% cutoff; P = 0.1 with 70% cutoff). Conclusion: In this cohort, 13% TNBC were re-classified as HER2 positive by performing HER2 FISH test in addition to immunohistochemistry analysis. Rate of FISH positivity in TNBC declined over the four year study period. No clinicopathological variables are correlated with HER2 FISH amplification. These data suggest that dual testing of HER2 should be considered routinely in TNBC for accurate classification to guide therapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-05-01.

HER2 amplification and overexpression are significantly correlated in mucinous epithelial ovarian cancer

HER2 gene amplification and protein over-expression are important factors in predicting clinical sensitivity to anti-HER2 therapies in breast, gastric or gastroesophageal junction cancer patients. The aim of this study was to evaluate the correlation between HER2 gene copy numbers and HER2 protein expressions in mucinous epithelial ovarian cancer (EOC). Of the 49 tissue microarray samples of mucinous EOC, we applied 2010 ToGA trial (Trastuzumab for Gastric Cancer) surgical specimen scoring criteria to analyze the HER2 protein expression by an immunohistochemistry (IHC) test with Dako (Carpenteria, CA), c-erb-B2 antibody, and the HER2 gene amplification by the fluorescence in situ hybridization (FISH) test with Abbott/Vysis PathVysion HER2 DNA Probe Kit (Abbott Molecular Inc., Des Plaines, IA). We achieved a high overall concordance of 97.56% between nonequivocal HER2 results by IHC and FISH tests. In addition, HER2 gene copies before chromosome-17 correction increased significantly in a stepwise order through the negative, equivocal and positive IHC result categories (P<.001), as did the HER2 gene copies after chromosome-17 correction (P<.001). On the other hand, HER2 IHC results correlated significantly with both chromosome-17-uncorrected HER2 gene copy numbers (ρ=0.630, P<.001) and chromosome-17 corrected HER2 gene copy numbers (ρ=0.558, P<.001). We concluded that both chromosome-17 corrected and uncorrected HER2 gene copies correlated significantly with HER2 IHC results. Tests for the HER2 gene copies per tumor cell either before or after correction of chromosome-17 can be applied as a potentially valuable tool to analyze the HER2 status in mucinous EOC.

One-Day FISH Approach for the High-Speed Determination of HER2 Gene Copy Status in Breast Carcinoma

Fluorescence in situ hybridization (FISH) is a commonly used method to detect chromosomal aberrations, for example, to assess human epidermal growth factor receptor 2 (HER2) gene status in breast carcinoma. The classical FISH approach requires overnight incubation for proper hybridization result. Tissue morphologic features are varying because of aggressive pretreatment and application of high temperatures. To eliminate some of the methodological problems, a new 1-day FISH method was recently introduced. The aim of our study was to evaluate the utility of the Instant Quality FISH with the conventional FISH kit from the same provider (Dako pharmDx) for determination of HER2 status. We performed in situ hybridization on the same 40 invasive breast carcinoma samples with both probe kits, and HER2/CEN17 and chromosome 17/cell nucleus ratios were calculated. FISH signal stability was also tested by the reassessment of the slides after 2 months storage. The accordance regarding HER2 gene amplification status between the 2 FISH kits tested was 100%. There was an excellent correlation between HER2/CEN17 ratios with a concordance correlation coefficient of 0.958 and correlation coefficient (R) of 0.959. The 1-day HER2 Instant Quality FISH diagnostic kit points with fast and stable reaction showing the same result in the diagnostic practice when compared with the conventional overnight FISH method.

Investigating Electromagnetic Properties of Yttria-Stabilized Zirconia (YSZ) for Wireless Gas Sensor Applications

The article describes the investigation of electromagnetic properties of YSZ for wireless gas sensor applications. In particular, the dielectric properties of YSZ were found with a view to implement on-chip antennas. Agilent Dielectric Probe Kit and network analyzer were used for dielectric constant and dielectric loss measurements. The dielectric properties were measured as a function of YSZ sintering temperature. The average dielectric constant (over frequency) of YSZ was seen to increase with an increase in sintering temperature. The average loss tangent (over frequency) was seen to decrease with an increase in sintering temperature. Substrates based on the dielectric ceramic materials such as YSZ, Al2O3, and Low Temperature Co-fired Ceramic are promising candidates for antenna miniaturization because of their high permittivity, low loss, light weight, low cost, and market availability.

Detection of copy number changes in genes associated with Parkinson's disease in Iranian patients

Parkinson's disease (PD) is the second most common neurodegenerative disorder, after Alzheimer's disease. Genomic rearrangements are common mutations reported in PD patients. In this study, we investigated the prevalence of genomic rearrangements in a total of 232 Iranian PD patients, out of which 102 were sporadic early-onset (age-at-onset ≤45 years) and 51 had a family history. We used multiplex ligation-dependent probe amplification (MLPA) method to detect exon dosage changes. Two new improved probe kits, SALSA P051 and P052, were used and altogether α-synuclein, parkin, UCHL1, PINK1, DJ-1, LRRK2, GCH1, ATP13A2, CAV1, CAV2, LPA and TNFRSF9 genes were analyzed. Exon or whole-gene rearrangements were identified in 14 (13.7%) sporadic early-onset PD patients in parkin, α-synuclein and PINK1. Of familial PD patients 46 cases from 18 families (35.3%) showed exon or whole-gene rearrangements in parkin, α-synuclein, PINK1, DJ-1, and ATP13A2 genes. All changes were verified by quantitative PCR (qPCR). Novel mutations and unusual clinical features are reported in this study. Mutations in parkin were the predominant genetic cause in both early-onset and familial PD groups. Also the mutations observed in family PD group are more in number and diversity than the sporadic early-onset PD group.