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1.
BMC Cancer ; 18(1): 1266, 2018 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-30563489

RESUMO

BACKGROUND: Clinical assays for the assessment of the human epidermal growth factor receptor-2 (HER2) status in breast cancer include immunohistochemistry (IHC) and in situ hybridization (ISH), both of which have limitations. Recent studies have suggested that a more quantitative approach to the measurement of HER2 protein expression may improve specificity in selecting patients for HER-2 targeted therapy. In the current study, we have used HER2 expression in breast cancer cell lines and clinical samples as a model to explore the potential utility of a novel immunodetection technique, using streptavidin coated Phosphor Integrated Dot fluorescent nanoparticles (PID), which can be quantitatively measured using computer analysis. METHODS: The expression of HER2 protein in cell lines was evaluated with antibody-binding capacity using fluorescence-activated cell sorting (FACS) for comparison with PID measurements to test for correlations with existing quantitative protein analysis methodologies. Various other analytic validation tests were also performed, including accuracy, precision, sensitivity, robustness and reproducibility. A methods comparison study investigated correlations between PID versus IHC and ISH in clinical samples. Lastly, we measured HER2 protein expression using PID in the pretreatment biopsies from 34 HER2-positive carcinomas that had undergone neoadjuvant trastuzumab-based chemotherapy. RESULTS: In the analytic validation, PID HER2 measurements showed a strong linear correlation with FACS analysis in breast cell lines, and demonstrated significant correlations with all aspects of precision, sensitivity, robustness and reproducibility. PID also showed strong correlations with conventional HER2 testing methodologies (IHC and ISH). In the neoadjuvant study, patients with a pathologic complete response (pCR) had a significantly higher PID score compared with patients who did not achieve a pCR (p = 0.011), and was significantly correlated to residual cancer burden (RCB) class (p = 0.026, R2 = 0.9975). CONCLUSIONS: Analytic testing of PID showed that it may be a viable testing methodology that could offer advantages over other experimental or conventional biomarker diagnostic methodologies. Our data also suggests that PID quantitation of HER2 protein may offer an improvement over conventional HER2 testing in the selection of patients who will be the most likely to benefit from HER2-targeted therapy. Further studies with a larger cohort are warranted.


Assuntos
Neoplasias da Mama/genética , Citometria de Fluxo , Nanopartículas/química , Receptor ErbB-2/genética , Biomarcadores Tumorais/genética , Biópsia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hibridização in Situ Fluorescente , Terapia de Alvo Molecular , Nanopartículas/administração & dosagem , Terapia Neoadjuvante , Inclusão em Parafina , Trastuzumab/administração & dosagem
2.
PLoS One ; 19(5): e0303614, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38748758

RESUMO

HER2 expression in breast cancer is evaluated to select patients for anti-HER2 therapy. With the advent of newly approved HER2-targeted drugs for low HER2 expression breast cancer, more solid evidence on the whole spectrum of HER2 expression is needed. In this study, we quantitatively assessed HER2 expression from the whole core by combining high-intensity phosphor-integrated dot (PID) immunostaining and whole slide imaging (WSI) analysis. Two types of staining were performed using a 170-core tissue microarray of invasive breast cancer. First, HER2 was stained by immunohistochemistry (IHC), and IHC scores were determined by two practicing pathologists according to the ASCO/CAP HER2 guideline. Second, HER2 was stained with PID, and tentative PID scores were determined by quantitative analysis. The results show that PID can numerically classify HER2 expression status into scores 3+, 2+, 1+, and 0. The HER2 value quantified by PID strongly correlated with the 3, 3'-diaminobenzidine (DAB) IHC score determined by pathologists (R2 = 0.93). PID IHC score 1+ cases included both DAB IHC score 1+ and 0 cases, and low HER2 expression cases appeared to be often evaluated as DAB IHC score 0. Therefore, digital image analysis by PID and WSI can help stratify HER2 IHC. It may also help classify low HER2 expression.


Assuntos
Neoplasias da Mama , Pontos Quânticos , Receptor ErbB-2 , Feminino , Humanos , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Imuno-Histoquímica/métodos , Invasividade Neoplásica , Receptor ErbB-2/análise , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Análise Serial de Tecidos/métodos
3.
Biochem Biophys Res Commun ; 426(3): 409-14, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-22959769

RESUMO

The detection of estrogen receptors (ERs) by immunohistochemistry (IHC) using 3,3'-diaminobenzidine (DAB) is slightly weak as a prognostic marker, but it is essential to the application of endocrine therapy, such as antiestrogen tamoxifen-based therapy. IHC using DAB is a poor quantitative method because horseradish peroxidase (HRP) activity depends on reaction time, temperature and substrate concentration. However, IHC using fluorescent material provides an effective method to quantitatively use IHC because the signal intensity is proportional to the intensity of the photon excitation energy. However, the high level of autofluorescence has impeded the development of quantitative IHC using fluorescence. We developed organic fluorescent material (tetramethylrhodamine)-assembled nanoparticles for IHC. Tissue autofluorescence is comparable to the fluorescence intensity of quantum dots, which are the most representative fluorescent nanoparticles. The fluorescent intensity of our novel nanoparticles was 10.2-fold greater than quantum dots, and they did not bind non-specifically to breast cancer tissues due to the polyethylene glycol chain that coated their surfaces. Therefore, the fluorescent intensity of our nanoparticles significantly exceeded autofluorescence, which produced a significantly higher signal-to-noise ratio on IHC-imaged cancer tissues than previous methods. Moreover, immunostaining data from our nanoparticle fluorescent IHC and IHC with DAB were compared in the same region of adjacent tissues sections to quantitatively examine the two methods. The results demonstrated that our nanoparticle staining analyzed a wide range of ER expression levels with higher accuracy and quantitative sensitivity than DAB staining. This enhancement in the diagnostic accuracy and sensitivity for ERs using our immunostaining method will improve the prediction of responses to therapies that target ERs and progesterone receptors that are induced by a downstream ER signal.


Assuntos
Neoplasias da Mama/diagnóstico , Corantes Fluorescentes/química , Imuno-Histoquímica/métodos , Nanopartículas/química , Receptores de Estrogênio/análise , Rodaminas/química , Coloração e Rotulagem/métodos , Feminino , Fluorescência , Humanos , Estreptavidina/química
4.
Mol Clin Oncol ; 10(3): 391-396, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30847180

RESUMO

One obstacle in diagnostic pathology is the harmonization of one drug-one diagnostic tests for programmed death ligand-1 (PD-L1). There are many challenges in accurate comparisons of diagnostic tests, such as differences in the titer of each antibody, detection system and dynamic range of visualization. Our previously developed digital immunostaining technique is highly sensitive and quantitative with the ability to quantify particles that bind in a one-to-one fashion with antibody in each cell. Determining the differences in the titer of each antibody with digital immunostaining may be beneficial for future harmonized analysis. To demonstrate the accuracy of digital immunostaining, the present study compared the number of particles with ELISA and nCounter data from five cell lines. NCI-H460 exhib-ited the highest level of PD-L1 protein, followed by A549, PC-3, NCI-H1299, and NCI-H446 cells. In addition, the PD-L1 mRNA values determined by nCounter corresponded with the order of the protein levels determined by ELISA. The present study revealed that digital immunostaining for PD-L1 was highly associated with ELISA and nCounter data. Among the four antibodies tested, the titer of all but SP142 coincided with ELISA and nCounter data. These results indicated that our digital immunostaining technique may be beneficial for future harmonized analysis.

5.
Cancers (Basel) ; 11(4)2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-31013810

RESUMO

In addition to genomic signaling, Estrogen receptor alpha (ERα) is associated with cell proliferation and survival through extranuclear signaling contributing to endocrine therapy (ET) resistance. However, the relationship between extranuclear ERα and ET resistance has not been extensively studied. We sought to measure extranuclear ERα expression by immunohistochemistry using phosphor-integrated dots (IHC-PIDs) and to assess its predictive value for ET resistance. After quantitative detection of ERα by IHC-PIDs in vitro, we developed "the nearest-neighbor method" to calculate the extranuclear ERα. Furthermore, tissue sections from 65 patients with HR+/HER2- BC were examined by IHC-PIDs, and the total ERα, nuclear ERα, extranuclear ERα PIDs score, and ratio of extranuclear-to-nuclear ERα (ENR) were measured using the novel method. We demonstrate that quantification of ERα using IHC-PIDs exhibited strong correlations to real-time qRT-PCR (r2 = 0.94) and flow cytometry (r2 = 0.98). High ERα ENR was significantly associated with poor overall survival (p = 0.048) and disease-free survival (DFS) (p = 0.007). Multivariate analysis revealed that the ERα ENR was an independent prognostic factor for DFS [hazard ratio, 3.8; 95% CI, 1.4-11.8; p = 0.006]. Our automated measurement has high accuracy to localize and assess extranuclear ERα. A high ERα ENR in HR+/HER2- BC indicates decreased likelihood of benefiting from ET.

6.
Sci Rep ; 7(1): 7509, 2017 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-28790306

RESUMO

The quantitative sensitivity and dynamic range of conventional immunohistochemistry (IHC) with 3,3'-diaminobenzidine (IHC-DAB) used in pathological diagnosis in hospitals are poor, because enzyme activity can affect the IHC-DAB chromogenic reaction. Although fluorescent IHC can effectively increase the quantitative sensitivity of conventional IHC, tissue autofluorescence interferes with the sensitivity. Here, we created new fluorescent nanoparticles called phosphor-integrated dots (PIDs). PIDs have 100-fold greater brightness and a more than 300-fold greater dynamic range than those of commercially available fluorescent nanoparticles, quantum dots, whose fluorescence intensity is comparable to tissue autofluorescence. Additionally, a newly developed image-processing method enabled the calculation of the PID particle number in the obtained image. To quantify the sensitivity of IHC using PIDs (IHC-PIDs), the IHC-PIDs method was compared with fluorescence-activated cell sorting (FACS), a method well suited for evaluating total protein amount, and the two values exhibited strong correlation (R = 0.94). We next applied IHC-PIDs to categorize the response to molecular target-based drug therapy in breast cancer patients. The results suggested that the PID particle number estimated by IHC-PIDs of breast cancer tissues obtained from biopsy before chemotherapy can provide a score for predicting the therapeutic effect of the human epidermal growth factor receptor 2-targeted drug trastuzumab.


Assuntos
Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/tratamento farmacológico , Diagnóstico por Imagem/métodos , Corantes Fluorescentes/química , Nanopartículas/química , Rodaminas/química , 3,3'-Diaminobenzidina/química , Anticorpos/química , Antineoplásicos Imunológicos/uso terapêutico , Biópsia , Biotina/química , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Diagnóstico por Imagem/instrumentação , Feminino , Fluorescência , Expressão Gênica , Humanos , Imidas/química , Imuno-Histoquímica/métodos , Pessoa de Meia-Idade , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Tamanho da Partícula , Perileno/análogos & derivados , Perileno/química , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Estreptavidina/química , Trastuzumab/uso terapêutico
7.
Astrobiology ; 5(2): 141-53, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15815165

RESUMO

Molecular genetic methods were used to analyze the remnants of microbial ecosystems contained within an ancient oceanic microbial habitat that was recovered from a continental drilled core of black shale approximately 100 million years in age. Bacterial ribosomal RNA genes were vertically amplified from the six different depths of a black shale core associated with a phosphate-rich stratum, defined as one of the mid-Cretaceous oceanic anoxic events (OAEs). Although the black shale core was recovered from a terrestrial coring effort, the recovered 16S rRNA gene sequences showed affinity to microbial communities previously seen in deep-sea sedimentary environments (i.e., the microbial assemblage was easily recognizable as a marine community). In particular, a number of 16S rRNA gene clones of oceanic sulfate-reducing bacteria within the delta-Proteobacteria predominated at the OAE layer. The recovered bacterial DNA signatures are consistent with the interpretation that the sequences are derived from the past microbial communities buried in either sea-bottom or subseafloor environments during the sedimentation process and, after ceasing growth, preserved until the present.


Assuntos
Bactérias/crescimento & desenvolvimento , Bactérias/genética , DNA Bacteriano , Fósseis , Genoma Bacteriano , Sedimentos Geológicos/microbiologia , Impressões Digitais de DNA , Dados de Sequência Molecular , Paleontologia , Filogenia , RNA Ribossômico 16S/análise
8.
J Phycol ; 47(5): 1164-76, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27020197

RESUMO

Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler is a cosmopolitan coccolithophore occurring from tropical to subpolar waters and exhibiting variations in morphology of coccoliths possibly related to environmental conditions. We examined morphological characters of coccoliths and partial mitochondrial sequences of the cytochrome oxidase 1b (cox1b) through adenosine triphosphate synthase 4 (atp4) genes of 39 clonal E. huxleyi strains from the Atlantic and Pacific Oceans, Mediterranean Sea, and their adjacent seas. Based on the morphological study of culture strains by SEM, Type O, a new morphotype characterized by coccoliths with an open central area, was separated from existing morphotypes A, B, B/C, C, R, and var. corona, characterized by coccoliths with central area elements. Molecular phylogenetic studies revealed that E. huxleyi consists of at least two mitochondrial sequence groups with different temperature preferences/tolerances: a cool-water group occurring in subarctic North Atlantic and Pacific and a warm-water group occurring in the subtropical Atlantic and Pacific and in the Mediterranean Sea.

9.
Environ Microbiol ; 7(7): 1009-16, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15946297

RESUMO

Stable carbon and nitrogen isotopic compositions were determined for individual photosynthetic pigments isolated and purified from the saline meromictic Lake Kaiike, Japan, to investigate species-independent biogeochemical processes of photoautotrophs in the natural environment. In the anoxic monimolimnion and benthic microbial mats, the carbon isotopic compositions of BChls e and isorenieratene related to brown-coloured strains of green sulfur bacteria are substantially ( approximately 10 per thousand) depleted in (13)C relative to those found in the chemocline. In conjunction with 16S rDNA evidence reported previously, it strongly suggests that Pelodyctyon luteolum inhabited and photosynthesized in the anoxic monimolimnion and benthic microbial mats by using (13)C-depleted regenerated CO(2). By contrast, both Chl a and BChl a in the monimolimnion and microbial mats have similar isotopic compositions as they do in the chemocline, implying that the source organisms live only in the chemocline. In the chemocline, the nitrogen isotopic compositions of BChl e homologues ranges from -7.7 to-6.5 per thousand, whereas that of BChl a is -2.1 per thousand. These isotopic compositions suggest that green sulfur bacteria Chlorobium phaeovibrioides would conduct nitrogen fixation in the chemocline, whereas purple sulfur bacteria Halochromatium sp. and cyanobacteria Synechococcus sp. may assimilate nitrite.


Assuntos
Bactérias/metabolismo , Bacterioclorofilas/metabolismo , Isótopos de Carbono/metabolismo , Água Doce/microbiologia , Isótopos de Nitrogênio/metabolismo , Cloreto de Sódio , Bactérias/classificação , Chlorobium/metabolismo , Chromatiaceae/metabolismo , Ecossistema , Água Doce/química , Japão , Fotossíntese , Synechococcus/metabolismo
10.
Environ Microbiol ; 5(11): 1103-10, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14641590

RESUMO

We investigated the distribution of chloropigments in a small meromictic lake, Lake Kaiike, south-west Japan. In the water-column, concentrations of Chl a related to cyanobacteria, BChl a related to purple sulphur bacteria, and three types of BChl e homologues (BChls e1, e2 and e3) related to brown-coloured green sulphur bacteria, were maximal at the redox boundary. Below the redox boundary, absolute concentrations of Chl a and BChl a gradually decreased with depth, whereas BChls e remained rather constant. Suspended particulate matter (SPM) at the deeper region of the anoxic water-column was enriched in highly alkylated BChl e homologues compared with SPM at the redox boundary. The shift in the relative content of highly alkylated BChl e homologues beneath the boundary was associated with community related adaptation of brown-coloured green sulphur bacteria to changes in light quality/quantity, resulting from the optical absorption and reflectance of SPMs in the overlying water-column. Benthic microbial mats were characterized by high abundances of BChls e, in which highly alkylated homologues were substantially abundant. This suggests that the BChls e in the microbial mat may be derived from the low-light adapted brown-coloured green sulphur bacteria forming the bacterial mat.


Assuntos
Água Doce/química , Sedimentos Geológicos/química , Pigmentos Biológicos/análise , Pigmentos Biológicos/isolamento & purificação , Microbiologia da Água , Bacterioclorofila A/análise , Bacterioclorofila A/isolamento & purificação , Bacterioclorofilas/análise , Bacterioclorofilas/isolamento & purificação , Clorofila/análise , Clorofila/isolamento & purificação , Clorofila A , Cromatografia Líquida de Alta Pressão , Sulfeto de Hidrogênio/análise , Japão , Oxigênio/análise , Análise Espectral
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