RESUMEN
BACKGROUND: Recent studies reported abnormal alpha-synuclein deposition in biopsy-accessible sites of the peripheral nervous system in Parkinson's disease (PD). This has considerable implications for clinical diagnosis. Moreover, if deposition occurs early, it may enable tissue diagnosis of prodromal PD. OBJECTIVE: The aim of this study was to develop and test an automated bright-field immunohistochemical assay of cutaneous pathological alpha-synuclein deposition in patients with idiopathic rapid eye movement sleep behavior disorder, PD, and atypical parkinsonism and in control subjects. METHODS: For assay development, postmortem skin biopsies were taken from 28 patients with autopsy-confirmed Lewy body disease and 23 control subjects. Biopsies were stained for pathological alpha-synuclein in automated stainers using a novel dual-immunohistochemical assay for serine 129-phosphorylated alpha-synuclein and pan-neuronal marker protein gene product 9.5. After validation, single 3-mm punch skin biopsies were taken from the cervical 8 paravertebral area from 79 subjects (28 idiopathic rapid eye movement sleep behavior disorder, 20 PD, 10 atypical parkinsonism, and 21 control subjects). Raters blinded to clinical diagnosis assessed the biopsies. RESULTS: The immunohistochemistry assay differentiated alpha-synuclein pathology from nonpathological-appearing alpha-synuclein using combined phosphatase and protease treatments. Among autopsy samples, 26 of 28 Lewy body samples and none of the 23 controls were positive. Among living subjects, punch biopsies were positive in 23 (82%) subjects with idiopathic rapid eye movement sleep behavior disorder, 14 (70%) subjects with PD, 2 (20%) subjects with atypical parkinsonism, and none (0%) of the control subjects. After a 3-year follow-up, eight idiopathic rapid eye movement sleep behavior disorder subjects phenoconverted to defined neurodegenerative syndromes, in accordance with baseline biopsy results. CONCLUSION: Even with a single 3-mm punch biopsy, there is considerable promise for using pathological alpha-synuclein deposition in skin to diagnose both clinical and prodromal PD. © 2020 International Parkinson and Movement Disorder Society.
Asunto(s)
Enfermedad por Cuerpos de Lewy , Enfermedad de Parkinson , Trastorno de la Conducta del Sueño REM , Humanos , Piel , alfa-SinucleínaRESUMEN
The ability to simultaneously visualize the presence, abundance, location and functional state of many targets in cells and tissues has been described as a true next-generation approach in immunohistochemistry (IHC). A typical requirement for multiplex IHC (mIHC) is the use of different animal species for each primary (1°Ab) and secondary (2°Ab) antibody pair. Although 1°Abs from different species have been used with differently labeled species-specific 2°Abs, quite often the appropriate combination of antibodies is not available. More recently, sequential detection of multiple antigens using 1°Abs from the same species used a microwaving treatment between successive antigen detection cycles to elute previously bound 1°Ab/2°Ab complex and therefore to prevent the cross-reactivity of anti-species 2°Abs used in subsequent detection cycles. We present here a fully automated 1°Ab/2°Ab complex heat deactivation (HD) method on Ventana's BenchMark ULTRA slide stainer. This method is applied to detection using fluorophore-conjugated tyramide deposited on the tissue and takes advantage of the strong covalent bonding of the detection substrate to the tissue, preventing its elution in the HD process. The HD process was characterized for (1) effectiveness in preventing Ab cross-reactivity, (2) impact on the epitopes and (3) impact on the fluorophores. An automated 5-plex fluorescent IHC assay was further developed using the HD method and rabbit 1°Abs for CD3, CD8, CD20, CD68 and FoxP3 immune biomarkers in human tissue specimens. The fluorophores were carefully chosen and the narrow-band filters were designed to allow visualization of the staining under fluorescent microscope with minimal bleed through. The automated 5-plex fluorescent IHC assay achieved staining results comparable to the respective single-plex chromogenic IHC assays. This technology enables automated mIHC using unmodified 1°Abs from same species and the corresponding anti-species 2°Ab on a clinically established automated platform to ensure staining quality, reliability and reproducibility.
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Amidas/química , Anticuerpos/química , Colorantes Fluorescentes/química , Procesamiento de Imagen Asistido por Computador/métodos , Inmunohistoquímica/métodos , Amidas/metabolismo , Anticuerpos/metabolismo , Mama/química , Femenino , Colorantes Fluorescentes/metabolismo , Humanos , Neoplasias/química , Tonsila Palatina/química , Reproducibilidad de los ResultadosRESUMEN
The purpose of this study was to develop and analytically and functionally validate a new human papillomavirus (HPV) in-situ hybridization (ISH) assay and to determine whether the use of this assay combined with hemotoxylin and eosin (H&E) staining could potentially improve the diagnostic accuracy of interpreting cervical intraepithelial neoplasia (CIN) in human cervical tissue specimens. An automated HPV ISH assay was developed using probes that targeted the broad spectrum of HPV genotypes most commonly associated with CIN. In an exploratory study, tissue sections (n=118) were stained with H&E alone and H&E with HPV ISH and evaluated by 6 general surgical pathologists. Results were compared with diagnoses established by expert pathologists on H&E alone. The change in specificity (diagnosis of no-CIN) and sensitivity (diagnosis of CIN) using H&E plus HPV versus H&E alone was determined. The HPV ISH assay detected 21 HPV genotypes and demonstrated no cross-reactivity to Epstein-Barr virus, cytomegalovirus, herpes simplex virus (HSV)-1, HSV-2, or human placental DNA. The assay detected HPV in a range of 1 to 600 copies on CaSki, HeLa, and SiHa xenografts. Use of this assay with H&E staining improved the average diagnostic specificity of the surgical pathologists from 68.5% to 89.9% (P<0.001), with fewer false-positive CIN 1 results (122 vs. 39). The diagnostic sensitivity was similar for assessments made with H&E alone and those made with HPV plus H&E (93.1% vs. 93.6%). In conclusion, a new automated broad-spectrum HPV ISH assay combined with H&E-stained slides contributed to better ascertainment of CIN than H&E staining alone.
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Hibridación in Situ/métodos , Papillomaviridae/aislamiento & purificación , Displasia del Cuello del Útero/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Femenino , Humanos , Papillomaviridae/genética , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/virologíaRESUMEN
BACKGROUND: The human homolog of the mouse double minute 2 (MDM2) oncogene is amplified in about 20% of sarcomas. The measurement of the MDM2 amplification can aid in classification and may provide a predictive value for recently formulated therapies targeting MDM2. We have developed and validated an automated bright field dual-color in situ hybridization application to detect MDM2 gene amplification. DESIGN: A repeat-depleted MDM2 probe was constructed to target the MDM2 gene region at 12q15. A chromosome 12-specific probe (CHR12) was generated from a pα12H8 plasmid. The in situ hybridization assay was developed by using a dinitrophenyl-labeled MDM2 probe and a digoxigenin-labeled CHR12 probe on the Ventana Medical Systems' automated slide-staining platforms. The specificity of the MDM2 and CHR12 probes was shown on metaphase spreads and further validated against controls, including normal human tonsil and known MDM2-amplified samples. The assay performance was evaluated on a cohort of 100 formalin-fixed, paraffin-embedded specimens by using a conventional bright field microscope. RESULTS: Simultaneous hybridization and signal detection for MDM2 and CHR12 showed that both DNA targets were present in the same cells. One hundred soft tissue specimens were stained for MDM2 and CHR12. Although 26 of 29 lipomas were nonamplified and eusomic, MDM2 amplification was noted in 78% of atypical lipomatous tumors or well-differentiated liposarcomas. Five of 6 dedifferentiated liposarcoma cases were amplified for MDM2. MDM2 amplification was observed in 1 of 8 osteosarcomas; 3 showed CHR12 aneusomy. MDM2 amplification was present in 1 of 4 chondrosarcomas. Nine of 10 synovial sarcomas displayed no evidence of MDM2 amplification in most tumor cells. In pleomorphic sarcoma, not otherwise specified (pleomorphic malignant fibrous histiocytoma), MDM2 was amplified in 38% of cases, whereas 92% were aneusomic for CHR12. One alveolar rhabdomyosarcoma and 2 embryonal rhabdomyosarcomas displayed low-level aneusomy of CHR12 without net MDM2 amplifications. CONCLUSIONS: These results show that the use of the ISH MDM2 and CHR12 assay allows simultaneous analyses of the 2 DNA targets within the context of tissue morphology. This method combines the advantages of a fully automated protocol with bright field microscopy and has the potential for routine application in surgical pathology.
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Cromosomas Humanos Par 12/genética , Amplificación de Genes , Hibridación Fluorescente in Situ/métodos , Proteínas Proto-Oncogénicas c-mdm2/genética , Sarcoma/genética , Sarcoma/patología , Aneuploidia , Animales , Cromosomas Humanos Par 12/metabolismo , Humanos , Hibridación in Situ , Ratones , Sondas de Oligonucleótidos/química , Sondas de Oligonucleótidos/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Sarcoma/metabolismoRESUMEN
With the advent of personalized medicine, anatomic pathology-based molecular assays, including in situ hybridization (ISH) and mRNA detection tests, are performed routinely in many laboratories and have increased in their clinical importance and complexity. These assays require appropriately fixed tissue samples that preserve both nucleic acid targets and histomorphology to ensure reliable test results for determining patient treatment options. However, all aspects of tissue processing, including time until tissue fixation, type of fixative, duration of fixation, post-fixation treatments, and sectioning of the sample, impact the staining results. ASCO/CAP has issued pre-analytical guidelines to standardize tissue processing for HER2 testing in breast carcinoma specimens: 10% neutral-buffered formalin (NBF) with a fixation time from at least 6 to 48h [1]. Often, this recommendation is not followed to the detriment of staining results [2]. In this paper, we used a human breast carcinoma cell line (MCF7) generated as xenograft tumors as a model system to analyze the effects of different pre-analytical conditions on ISH staining. We performed H&E, FISH and dual colorimetric HER2 ISH assays using specimens fixed across a range of times in six different commonly used fixatives. Additionally, we investigated the effects of varying tissue section thickness, which also impacted the quality of ISH staining. Finally, we evaluated the effects of three different decalcifying solutions on human breast specimens, typically a treatment that occurs post-fixation for evaluating metastases to bone. The results indicate that time and type of fixation treatment, as well as appropriate tissue thickness and post-fixation treatment, all contribute to the quality of ISH staining results. Our data support the ASCO/CAP recommendations for standardized tissue processing (at least 6h in formalin-based fixatives and 4µm section thickness) and indicate that certain fixatives and post-fixative treatments are detrimental to molecular staining results.
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Neoplasias de la Mama/química , Hibridación Fluorescente in Situ/métodos , Hibridación in Situ/métodos , Fijación del Tejido/métodos , Animales , Línea Celular Tumoral , Eosina Amarillenta-(YS) , Femenino , Fijadores , Haptenos , Hematoxilina , Humanos , Ratones , Receptor ErbB-2/análisis , Coloración y Etiquetado , Trasplante HeterólogoRESUMEN
Cancer diagnosis can be a complex process, which takes consideration of histopathological, clinical, immunophenotypic, and genetic features. Since non-random chromosomal translocations are specifically involved in the development of various cancers, the detection of these gene aberrations becomes increasingly important. In recent years, break-apart (or split-signal) fluorescence in situ hybridization (FISH) has emerged as an advantageous technique to detect gene translocations on tissue sections. However, FISH assays are technically challenging and require specialized fluorescence microscopes. Furthermore, the FISH signal is not stable for long term archiving due to photo bleaching. Our objective was to demonstrate the feasibility of brightfield break-apart in situ hybridization (ba-ISH) for anaplastic lymphoma kinase (ALK) and mucosa-associated lymphoid tissue translocation protein 1 (MALT1) genes as models. ALK or MALT1 break-apart probes were labeled with digoxigenin (DIG) or 2,4-dinitrophenyl (DNP) on both sides of a known gene breakpoint region and the hybridization sites were visualized with the combination of alkaline phosphatase (AP)-based blue and red detection. Therefore, normal genes are detected as purple dots by mixing blue and red colors while translocated genes are detected as isolated blue or red dots. Formalin-fixed, paraffin-embedded tonsil was used as control for the co-localized 5' and 3' probes. Gene translocations of ALK or MALT1 were detected as separate blue and red dots on ALCL and MALT lymphoma cases. Thus, ISH analyses of gene translocations can be conducted with a regular light microscope and the long term archiving of break-apart ISH slides can be achieved.