RESUMEN
With the development of genomic technologies, the isolation of genomic DNA (gDNA) from clinical samples is increasingly required for clinical diagnostics and research studies. In this study, we explored the potential of utilizing various leftover blood samples obtained from routine clinical tests as a viable source of gDNA. Using an automated method with optimized pre-treatments, we obtained gDNA from seven types of clinical leftover blood, with average yields of gDNA ranging from 3.11 ± 0.45 to 22.45 ± 4.83 µg. Additionally, we investigated the impact of storage conditions on gDNA recovery, resulting in yields of 8.62-68.08 µg when extracting gDNA from EDTA leftover blood samples stored at 4 °C for up to 13 weeks or -80 °C for up to 78 weeks. Furthermore, we successfully obtained sequenceable gDNA from both Serum Separator Tube and EDTA Tube using a 96-well format extraction, with yields ranging from 0.61 to 71.29 µg and 3.94-215.98 µg, respectively. Our findings demonstrate the feasibility of using automated high-throughput platforms for gDNA extraction from various clinical leftover blood samples with the proper pre-treatments.
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ADN , Genoma , Ácido Edético , ADN/genética , Recolección de Muestras de Sangre , GenómicaRESUMEN
Multiplexed Imaging technologies are powerful techniques that enable ultrahigh-plex spatial phenotyping of whole tissue sections at single cell spatial resolution. Co-Detection by Indexing (CODEX) multiplexing can detect up to 100 proteins using cyclic detection of DNA conjugated antibodies applied to tissue sections. However, it is necessary to correlate multiplexed fluorescent (mIF) spatial images with Hematoxylin and Eosin (H&E) stained sections post analysis. To effectively correlate mIF spatial images with H&E morphology, an (H&E) staining protocol was developed that is directly applied to the CODEX Fusion flow-cell slide after analysis allowing for direct H&E correlation and annotation with mIF images.
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Colorantes , Proteínas , Eosina Amarillenta-(YS)/farmacología , Hematoxilina/farmacología , Coloración y Etiquetado , Colorantes/farmacologíaRESUMEN
OBJECTIVES: We studied the prevalence and prognostic significance of mismatch repair deficient (MMRD) and p53 aberrant ovarian clear cell carcinoma (CCO) and their association with other prognostic and theranostic biomarkers (p16, HER2, PD-L1). We also aimed to identify morphologic features to serve as screening tools for immunohistochemical testing for these biomarkers. METHODS: Tissue microarrays with 3-mm cores from 71 pure CCOs were immunostained with PMS2, MSH6, p53, p16, HER2, and PD-L1. Expression status was correlated with tumor recurrence/disease progression and survival. It was also correlated with morphologic features (tumor size, nuclear grade, tumor architecture, mitotic activity, presence of endometriosis, tumor budding, and tumor inflammation). RESULTS: p53 aberrant tumors were associated with shorter overall and recurrence-free survivals (P = .002 and P = .01, respectively). In multivariate analysis, p53 aberrant status and tumor stage were independently associated with recurrence/disease progression (hazard ratio [HR] = 3.31, P = .037 and HR = 1.465, P = .004, respectively). p53 aberrant status was associated with tumor budding (P = .037). MMRD, p16, HER2, and PD-L1 expression had no prognostic significance. HER2 and PD-L1 were expressed in 56% and 35% of tumors, respectively. MMRD was associated with tumor expression of PD-L1 (P > .05) but not with tumor inflammation. CONCLUSIONS: Aberrant p53 in CCO is infrequent but associated with poor prognosis independent of stage. Presence of tumor budding could be a screening tool for p53 testing. High prevalence of HER2 and PD-L1 expression indicates the eligibility of patients with CCO for ongoing clinical trials using these therapeutic targets.
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Adenocarcinoma de Células Claras , Antígeno B7-H1 , Femenino , Humanos , Antígeno B7-H1/metabolismo , Proteína p53 Supresora de Tumor , Biomarcadores de Tumor/metabolismo , Reparación de la Incompatibilidad de ADN , Pronóstico , Progresión de la Enfermedad , InflamaciónRESUMEN
Whereas the cellular and molecular features of human inflammatory skin diseases are well characterized, their tissue context and systemic impact remain poorly understood. We thus profiled human psoriasis (PsO) as a prototypic immune-mediated condition with a high predilection for extracutaneous involvement. Spatial transcriptomics (ST) analyses of 25 healthy, active lesion, and clinically uninvolved skin biopsies and integration with public single-cell transcriptomics data revealed marked differences in immune microniches between healthy and inflamed skin. Tissue-scale cartography further identified core disease features across all active lesions, including the emergence of an inflamed suprabasal epidermal state and the presence of B lymphocytes in lesional skin. Both lesional and distal nonlesional samples were stratified by skin disease severity and not by the presence of systemic disease. This segregation was driven by macrophage-, fibroblast-, and lymphatic-enriched spatial regions with gene signatures associated with metabolic dysfunction. Together, these findings suggest that mild and severe forms of PsO have distinct molecular features and that severe PsO may profoundly alter the cellular and metabolic composition of distal unaffected skin sites. In addition, our study provides a valuable resource for the research community to study spatial gene organization of healthy and inflamed human skin.
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Ecosistema , Psoriasis , Humanos , Transcriptoma , Piel/patología , Psoriasis/genética , Gravedad del PacienteRESUMEN
Approximately 30% of early-stage lung adenocarcinoma patients present with disease progression after successful surgical resection. Despite efforts of mapping the genetic landscape, there has been limited success in discovering predictive biomarkers of disease outcomes. Here we performed a systematic multi-omic assessment of 143 tumors and matched tumor-adjacent, histologically-normal lung tissue with long-term patient follow-up. Through histologic, mutational, and transcriptomic profiling of tumor and adjacent-normal tissue, we identified an inflammatory gene signature in tumor-adjacent tissue as the strongest clinical predictor of disease progression. Single-cell transcriptomic analysis demonstrated the progression-associated inflammatory signature was expressed in both immune and non-immune cells, and cell type-specific profiling in monocytes further improved outcome predictions. Additional analyses of tumor-adjacent transcriptomic data from The Cancer Genome Atlas validated the association of the inflammatory signature with worse outcomes across cancers. Collectively, our study suggests that molecular profiling of tumor-adjacent tissue can identify patients at high risk for disease progression.
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Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Humanos , Adenocarcinoma del Pulmón/genética , Inflamación/genética , Neoplasias Pulmonares/genética , Pulmón , Progresión de la EnfermedadRESUMEN
The skin is an external organ that is most frequently exposed to radiation. High-dose radiation initiates and promotes skin cancer and acute radiation injury. It is important to investigate the influence of high-dose radiation exposure on the skin at the molecular level to understand acute radiation injury. To identify genes that are associated with injury caused by high-dose radiation exposure of the skin, we used microarray technology to examine the effect of irradiation on approximately 1000 genes in normal human epidermal keratinocytes at 3 h postirradiation with a cytotoxic dose of X-ray (5 Gy). We found that 16 and 59 genes were up- and down-regulated respectively in the keratinocytes. Several apoptosis-related genes, for example, BAK and TSC-22, and anti-proliferative genes, for example, BTG-1 and BTG-3, were up-regulated. We focused on ATF3 because ATF3 is induced most strongly by X-irradiation, and its function in keratinocytes is unknown. The induction of the ATF3 mRNA and protein in keratinocytes following X-ray was confirmed by RT-PCR and western blot analysis. ATF3 was also induced and accumulated within the nuclei of keratinocytes after X-ray irradiation in vivo and in vitro. Exogenous EYFP-ATF3 also accumulated within the nuclei of keratinocytes. In the transient expression assay, EYFP-ATF3, but not EYFP, induced apoptosis in keratinocytes. Taken together, these results suggest that ATF3 plays a role in apoptosis in keratinocytes and is associated with skin injury caused by ionizing radiation.