Rapid HER2 cytologic fluorescence in situ hybridization for breast cancer using noncontact alternating current electric field mixing.

BACKGROUND: Human epidermal growth factor receptor 2-in situ hybridization (HER2-ISH) is widely approved for diagnostic, prognostic biomarker testing of formalin-fixed paraffin-embedded tissue blocks. However, cytologic ISH analysis has a potential advantage in tumor samples such as pleural effusion and ascites that are difficult to obtain the histological specimens. Our aim was to evaluate the clinical reliability of a novel rapid cytologic HER2 fluorescence ISH protocol (rapid-CytoFISH). MATERIALS AND METHODS: Using a new device, we applied a high-voltage/frequency, noncontact alternating current electric field to tissue imprints and needle rinses, which mixed the probe within microdroplets as the voltage was switched on and off (AC mixing). Cytologic samples (n = 143) were collected from patients with immunohistochemically identified HER2 breast cancers. The specimens were then tested using standard dual-color ISH using formalin-fixed paraffin-embedded tissue (FFPE-tissue DISH) for HER2-targeted therapies, CytoFISH, and rapid-CytoFISH (completed within 4 h). RESULTS: All 143 collected cytologic specimens (50 imprinted cytology specimens from resected tumors and 93 liquid-based cytology specimens from needle rinses) were suitable for FISH analysis. The HER2/chromosome enumeration probe (CEP) 17 ratios did not significantly differ between FFPE-tissue DISH and either CytoFISH protocol. Based on HER2 scoring criteria, we found 95.1% agreement between FFPE-tissue DISH and CytoFISH (Cohen's kappa coefficient = 0.771 and 95% confidence interval (CI): 0.614-0.927). CONCLUSION: CytoFISH could potentially serve as a clinical tool for prompt determination of HER2 status in breast cancer cytology. Rapid-CytoFISH with AC mixing will enable cancer diagnoses and HER2 status to be determined on the same day a patient comes to a clinic or hospital.

Development of a Novel One-Step Automated Rapid in situ Hybridization for Anaplastic Lymphoma Kinase Rearrangement Using Non-Contact Alternating-Current Electric-Field Mixing.

Echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (ALK) fusion gene rearrangement is a key driver mutation in non-small cell lung cancer (NSCLC). Although Break-Apart ALK fluorescence in situ hybridization (FISH) is a reliable diagnostic method for detecting ALK gene rearrangement, it is also costly and time-consuming to use as a routine screening test. Our aim was to evaluate the clinical utility of a novel one-step, automated, rapid FISH (Auto-RaFISH) method developed to facilitate hybridization. This method takes advantage of the non-contact mixing effect of an alternating-current electric field. Ten representative specimens from 85 patients diagnosed at multiple centers with primary lung cancer with identified ALK-FISH status were collected. The specimens were all tested using FISH, RaFISH, and Auto-RaFISH. With both RaFISH protocols, the ALK test was completed within 4.5 h, as compared to the 20 h needed for the standard protocol. We found 100% agreement between the standard FISH, RaFISH, and new Auto-RaFISH based on the ALK status, and all methods stained equally well. These findings suggest that Auto-RaFISH could potentially serve as an automated clinical tool for prompt determination of ALK status in NSCLC.

Practical application of non-contact alternating current electric field mixing for reagent-saving in situ hybridisation of HER2.

AIMS: Human epidermal growth factor receptor 2 (HER2)-targeted agents are effective against HER2-positive breast cancers. However, their lack of survival benefit in HER2-negative patients as well as their toxic effects and high cost highlight the need for accurate assessment of HER2 status. Our aim was to evaluate the clinical utility of a reagent-saving in situ hybridisation (Saving ISH) that facilitates hybridisation and saves HER2/chromosome enumeration probe by taking advantage of the non-contact mixing effect of an alternating current (AC) electric field. METHODS: With a new device, we apply a high-voltage, low-frequency AC electric field to the tissue sections, which mixes the probe within microdroplets as the voltage is switched on and off. Specimens (n=113) from patients with breast cancers identified immunohistochemically as HER2 0/1(+), (2+) or (3+) were used. The specimens were all tested using conventional dual ISH (DISH), DISH with an automated slide stainer (ASS) and Saving ISH (1:1-1:3 dilution). RESULTS: The Saving ISH with 1:2 probe dilution produced stable results with less non-specific staining while using smaller amounts of probe. The accuracy of HER2 status with Saving ISH was equal to standard. We found 96.4% agreement between DISH using ASS and Saving ISH (kappa coefficient=0.912). CONCLUSIONS: These results suggest reagent-saving HER2 ISH could be used as a clinical tool for accurate and stable HER2 assessment, even when reagent concentrations vary.

Reagent-saving immunohistochemistry for HER2 using non-contact alternating current electric field mixing.

AIMS: Human epidermal growth factor receptor 2 (HER2)-targeted agents are an effective approach to treating patients with HER2-positive breast cancer. However, the lack of survival benefit in HER2-negative patients, as well as the toxic effects and high cost of the drugs, highlight the need for accurate and prompt assessment of HER2 status. Our aim was to evaluate the clinical utility of a novel reagent-saving immunohistochemistry method (AC-IHC) that saves HER2 antibody by taking advantage of the non-contact mixing effect in microdroplets subjected to an alternating current electric field. METHODS: Ninety-five specimens were used from patients diagnosed with primary breast cancers identified immunohistochemically as HER2 0/1+, 2+ or 3+ using ASCO/CAP guideline-certified standard IHC. The specimens were all tested using the conventional IHC method (1:50 antibody dilution) as well as AC-IHC (1:50 dilution) and reagent-saving AC-IHC (1:100 dilution). RESULTS: The reagent-saving AC-IHC produced stable results with less non-specific staining using smaller amounts of labelled antibody. Moreover, the staining and accuracy of HER2 status evaluated with the reagent-saving AC-IHC method was equal to that achieved with standard IHC. CONCLUSIONS: These results suggest reagent-saving AC-IHC could be used as a clinical tool for accurate and stable HER2 IHC, even when reagent concentrations vary.

Novel method for rapid fluorescence in-situ hybridization of ALK rearrangement using non-contact alternating current electric field mixing.

Echinoderm microtubule-associated protein-like 4 gene and anaplastic lymphoma kinase gene (EML4-ALK) rearrangement is a key driver mutation in non-small cell lung cancer (NSCLC). Although Break-Apart ALK fluorescence in situ hybridization (FISH) is a reliable diagnostic method for detecting ALK gene rearrangement, it is too costly and time-consuming for use as a routine screening test. Our aim was to evaluate the clinical utility of a novel rapid FISH (RaFISH) method developed to facilitate hybridization. RaFISH takes advantage of the non-contact mixing effect of an alternating current (AC) electric field. Eighty-five specimens were used from patients diagnosed with NSCLC identified immunohistochemically as ALK 0, (1/2+) or (3+). With RaFISH, the ALK test was completed within 4.5 h, as compared to 20 h needed for the standard FISH. Although RaFISH produced results more promptly, the staining and accuracy of the ALK evaluation with RaFISH was equal to the standard. We found 97.6% agreement between FISH and RaFISH based on the status of the ALK signals. These results suggest RaFISH could be used as a clinical tool to promptly determine ALK status.

Novel rapid-immunohistochemistry using an alternating current electric field for intraoperative diagnosis of sentinel lymph nodes in breast cancer.

Axillary lymph node status and pathological diagnosis of sentinel lymph nodes (SLNs) is a prognostic factor that influences management of postoperative therapy. Recent reports indicate that one-step nucleic acid amplification and hematoxylin and eosin (HE)-stained frozen sections are effective for intraoperative diagnosis of SLNs. In the present study, we report a rapid-immunohistochemical staining (R-IHC) method that enables intraoperative detection of SLN metastases within 16 min using an anti-cytokeratin antibody. This is the first report on SLN diagnosis using R-IHC in patients with breast cancer. We prospectively examined 160 dissected SLNs from 108 breast cancer patients who underwent surgery at our institute. The dissected SLNs were sectioned and conventionally stained with HE or immunohistochemically labeled with anti-cytokeratin antibody using R-IHC procedures. Intraoperative R-IHC analyses were completed within 16 min, after which diagnoses were made by two pathologists. The total time required for intraoperative diagnosis was about 20 min. In this study series, R-IHC detected four metastatic SLNs that were undetected using conventional HE staining (4/20, 20.0%). Compared with subsequent permanent diagnosis, R-IHC offered 95.2% sensitivity and 100% specificity. These findings indicate R-IHC is a clinically applicable technique that enables precise and quick intraoperative detection of micro- and macrometastasis in breast cancer.

Rapid Immunohistochemistry With Thyroid Transcription Factor-1 for Pulmonary Adenocarcinoma.

BACKGROUND: Intraoperative pathologic diagnosis of solitary pulmonary tumors to differentiate between metastatic and primary lung cancer is extremely important to determine the appropriate range of excision. Accurate intraoperative pathologic evaluation may be often difficult, however, and needs additional immunohistochemical (IHC) evaluation to support the diagnosis. Although conventional IHC is a powerful tool for diagnosis, its clinical use is limited intraoperatively because of time constraints. To address this issue, we developed a device that enables complete and rapid IHC (R-IHC) analyses within 20 minutes. We aimed to evaluate the discriminative ability of the R-IHC with anti-thyroid transcription factor-1 (TTF-1) antibody, which is a highly specific IHC marker for primary lung adenocarcinoma. METHODS: A total of 61 pulmonary tumors that were resected at our institute from May 2011 to September 2013 were retrospectively examined. The samples were sectioned, labeled with anti-TTF-1 antibody using the R-IHC method, and pathologically evaluated. The standard used for evaluation was conventional IHC with TTF-1. RESULTS: With the R-IHC procedure, analyses were completed within 20 minutes, with a diagnostic accuracy of 96.7% (59 of 61). Among the 47 primary lung adenocarcinomas, the R-IHC detected 31 (66%) tumors that were positive for TTF-1, with a positive predictive value of 100% (31 of 31). CONCLUSIONS: Our newly developed method of R-IHC with anti-TTF-1 antibody was useful for diagnosing and differentiation of solitary pulmonary tumors. This technology may prove to be an important supplement to standard intraoperative pathologic diagnosis in routine practice.

Novel method for rapid in-situ hybridization of HER2 using non-contact alternating-current electric-field mixing.

Human epidermal growth factor receptor 2 (HER2)-targeted agents are an effective approach to treating HER2-positive breast cancer patients. However, the lack of survival benefit in HER2-negative patients as well as the toxic effects and high cost of the drugs highlight the need for accurate and prompt assessment of HER2 status. Our aim was to evaluate the clinical utility of a novel rapid dual in-situ hybridization (RISH) method developed to facilitate hybridization. The method takes advantage of the non-contact mixing effect of an alternating current (AC) electric field. One hundred sixty-three specimens were used from patients diagnosed with primary breast cancers identified immunohistochemically as HER2 0/1(+), (2+) or (3+). The specimens were all tested using conventional dual in-situ hybridization (DISH), DISH with an automated slide stainer, and RISH. With RISH the HER2 test was completed within 6 h, as compared to 20-22 h needed for the standard protocol. Although RISH produced results more promptly using smaller amounts of labeled antibody, the staining and accuracy of HER2 status evaluation with RISH was equal to or greater than with DISH. These results suggest RISH could be used as a clinical tool to promptly determine HER2 status.

Novel method for immunofluorescence staining of mammalian eggs using non-contact alternating-current electric-field mixing of microdroplets.

Recently, a new technique was developed for non-catalytically mixing microdroplets. In this method, an alternating-current (AC) electric field is used to promote the antigen-antibody reaction within the microdroplet. Previously, this technique has only been applied to histological examinations of flat structures, such as surgical specimens. In this study, we applied this technique for the first time to immunofluorescence staining of three-dimensional structures, specifically, mammalian eggs. We diluted an antibody against microtubules from 1:1,000 to 1:16,000, and compared the chromatic degree and extent of fading across dilutions. In addition, we varied the frequency of AC electric-field mixing from 5 Hz to 46 Hz and evaluated the effect on microtubule staining. Microtubules were more strongly stained after AC electric-field mixing for only 5 minutes, even when the concentration of primary antibody was 10 times lower than in conventional methods. AC electric-field mixing also alleviated microtubule fading. At all frequencies tested, AC electric-field mixing resulted in stronger microtubule staining than in controls. There was no clear difference in a microtubule staining between frequencies. These results suggest that the novel method could reduce antibody consumption and shorten immunofluorescence staining time.

Rapid immunohistochemistry based on alternating current electric field for intraoperative diagnosis of brain tumors.

Rapid immunohistochemistry (R-IHC) can contribute to the intraoperative diagnosis of central nervous system (CNS) tumors. We have recently developed a new IHC method based on an alternating current electric field to facilitate the antigen-antibody reaction. To ensure the requirement of R-IHC for intraoperative diagnosis, 183 cases of CNS tumors were reviewed regarding the accuracy rate of diagnosis without R-IHC. The diagnostic accuracy was 90.7 % (166/183 cases) [corrected] in which definitive diagnoses were not provided in 17 cases because of the failure of glioma grading and differential diagnosis of lymphoma and glioma. To establish the clinicopathological application, R-IHC for frozen specimens was compared with standard IHC for permanent specimens. 33 gliomas were analyzed, and the Ki-67/MIB-1 indices of frozen specimens by R-IHC were consistent with the grade and statistically correlated with those of permanent specimens. Thus, R-IHC provided supportive information to determine the grade of glioma. For discrimination between glioma and lymphoma, R-IHC was able to provide clear results of CD20 and Ki-67/MIB-1 in four frozen specimens of CNS lymphoma as well as standard IHC. We conclude that the R-IHC for frozen specimens can provide important information for intraoperative diagnosis of CNS tumors.

A novel immunohistochemical staining method allows ultrarapid detection of lymph node micrometastases while conserving antibody.

We developed a novel ultrarapid immunohistochemical staining method in which an AC electric field is used to facilitate detection of tumor cells. Frozen sections of non-small cell lung cancer in lymph nodes were fixed in acetone for 2 min, after which they were incubated for 2 min with an anti-pancytokeratin antibody cocktail and then with EnVision(TM) complex under an alternating current (AC) electric field. The sections were then incubated with a chromogen (3,3'diaminobenzidine) for 3 min and counterstained with hematoxylin. This method enabled detection of tumor cells in frozen sections in less than 15 min. In addition, we were able to reduce the amount of antibody used by more than 90% when the sections were incubated under the AC electric field for a longer period. This method could be a useful tool for frozen section diagnosis and research. Furthermore, with this method the cost of immunohistochemical staining can be reduced.