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BACKGROUND: Accurate estimation of cell viability is crucial in various applications such as cytotoxicity testing and routine cell culture on both industrial and laboratory scales. For this, the real-time monitoring of cell status would be beneficial. Conventional cell-based assays for cell viability have limitations in sensitivity and time-effectiveness. Analysis of cell-free DNA (cfDNA) in (culture) media is a good alternative as cfDNA release are a well-known phenomenon during cell death. RESULTS: We demonstrate a direct digital PCR (dPCR) method to estimate cell viability by analyzing cfDNA in media during induced cell death. After validating the duplex dPCR method for short and long amplicons of the SMAD4 and RPP30 loci, we determined that a media volume of 2 µL is feasible to measure the target DNA copy number with minimal negative effects on amplification. dPCR inhibition was evident with a higher media volume per reaction targeting long amplicons. Next, we applied our dPCR method using media cfDNA and other conventional methods to the monitoring of camptothecin (CPT)-induced cell death. Copy numbers increased significantly after 4 h of CPT treatment, showing a fold change of approximately 4-6 compared to the controls. Cell-based assays such as the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay and annexin V/7-AAD assay also indicated increased cell death at 4 h, but the trypan blue exclusion assay did not. SIGNIFICANCE: The developed media cfDNA direct dPCR method allows for efficient measurements of the degree of cell viability. Unlike other conventional cell-based assays, our method has advantages of no loss of cultured cells and the ability to implement online analysis. Accurate and sensitive media cfDNA analysis using dPCR can be adopted in various applications such as determining cytotoxicity levels in large-scale bioreactors or screening for effective anticancer drugs.
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Supervivencia Celular , Ácidos Nucleicos Libres de Células , Reacción en Cadena de la Polimerasa , Supervivencia Celular/efectos de los fármacos , Humanos , Reacción en Cadena de la Polimerasa/métodos , Camptotecina/farmacología , Medios de Cultivo/químicaRESUMEN
We investigated frequencies of HER2-low breast cancer (BC) (immunohistochemistry [IHC] 1+ or 2+ without gene amplification) before and after IHC conditions were modified in order to understand the impact of IHC staining conditions on frequencies of HER2-low BC. Primary BC cases diagnosed at the Yeungnam University Hospital (YUH, n = 728) or Keimyung University Dongsan Hospital (KUDH, n = 290) in 2022 were reviewed, and data on HER2 status and IHC conditions were collected (cohort 1). Both institutions used the 4B5 antibody for HER2 IHC but had different staining protocols. After modifications of the IHC conditions at both institutions, primary BC cases (YUH, n = 324 and KUDH, n = 135) diagnosed from April to July 2023 (cohort 2) were reviewed to assess any changes in the frequency of HER2 status. In cohort 1, of the 728 cases diagnosed at YUH, 556 (76.4%) were HER2-zero, 76 (10.4%) were HER2-low, and 96 (13.2%) were HER2-positive, and of the 290 cases diagnosed at KUDH, 135 (46.6%) were HER2-zero, 82 (28.3%) were HER2-low, and 73 (25.2%) were HER2-positive. Modifications in HER2 IHC staining conditions dramatically increased the frequencies of HER2-low BC in cohort 2 (YUH 38.9% and KUDH 49.6%), but they did not result in significant changes in the HER2-positive rates (YUH 15.4% and KUDH 25.2%) compared to cohort 1. In conclusion, minor modifications in HER2 IHC staining conditions significantly affected the frequency of HER2-low BC but had little impact on the HER2-positivity rate. Each pathology laboratory should verify IHC conditions using control slides (including 1+) to enable the accurate identification of HER2-low BC.
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Purpose: Notable effectiveness of trastuzumab deruxtecan (T-DXd) in patients with HER2-low advanced breast cancer (BC) has focused pathologists' attention. We studied the incidence and clinicopathologic characteristics of HER2-low BC, and the effects of immunohistochemistry (IHC) associated factors on HER2 IHC results. Materials and Methods: The Breast Pathology Study Group of the Korean Society of Pathologists conducted a nationwide study using real-world data on HER2 status generated between January 2022 and December 2022. Information on HER2 IHC protocols at each participating institution was also collected. Results: Total 11,416 patients from twenty-five institutions included in this study. Of these patients, 40.7% (range: 6.0%-76.3%) were classified as HER2-zero, 41.7% (range: 10.5%-69.1%) as HER2-low, and 17.5% (range: 6.7%-34.0%) as HER2-positive. HER2-low tumors were associated with positive ER and PR statuses (p<0.001 and p<0.001, respectively). Antigen retrieval times (≥ 36 min vs. < 36 min) and antibody incubation times (≥ 12 min vs. < 12 min) affected on the frequency of HER2 IHC 1+ BC at institutions using the PATHWAY HER2 (4B5) IHC assay and BenchMark XT or Ultra staining instruments. Furthermore, discordant results between core needle biopsy (CNB) and subsequent resection specimen HER2 statuses were observed in 24.1% (787/3259) of the patients. Conclusion: The overall incidence of HER2-low BC in South Korea concurs with those reported in previously published studies. Significant inter-institutional differences in HER2 IHC protocols were observed, and it may have impact on HER2-low status. Thus, we recommend standardizing HER2 IHC conditions to ensure precise patient selection for targeted therapy.
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Quantitative PCR (qPCR) is the gold standard for detecting nucleic acid sequences specific to the target pathogen. For COVID-19 diagnosis, several molecular assays have been developed. In this study, we present an optimization strategy for the measurement of SARS-CoV-2 RNA via multiplex qPCR and digital PCR (dPCR). Compared to qPCR, both droplet and chip-based dPCR, which are known to be more sensitive and accurate, showed a better resilience to suboptimal assay compositions and cycling conditions following the proposed optimizations. In particular, the formation of heterodimers among assays greatly interfered with qPCR results, but only minimally with dPCR results. In dPCR, existing heterodimers lowered the PCR efficiency, producing a dampened fluorescent signal in positive partitions. This can be corrected by adjusting the PCR cycling conditions, after which dPCR shows the capability of measuring the expected copy number. In addition, we present a process to improve the existing RdRp assay by correcting the primer sequences and matching the melting temperature, ultimately producing highly sensitive and robust assays. The results of this study can reduce the cost and time of SARS-CoV-2 diagnosis while increasing accuracy. Furthermore, our results suggest that dPCR is a reliable method for the accurate measurement of nucleic acid targets.
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COVID-19 , Ácidos Nucleicos , Humanos , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , SARS-CoV-2/genética , Prueba de COVID-19 , COVID-19/diagnósticoRESUMEN
Well-characterized reference materials support harmonization and accuracy when conducting nucleic acid-based tests (such as qPCR); digital PCR (dPCR) can measure the absolute concentration of a specific nucleic acid sequence in a background of non-target sequences, making it ideal for the characterization of nucleic acid-based reference materials. National Metrology Institutes are increasingly using dPCR to characterize and certify their reference materials, as it offers several advantages over indirect methods, such as UV-spectroscopy. While dPCR is gaining widespread adoption, it requires optimization and has certain limitations and considerations that users should be aware of when characterizing reference materials. This review highlights the technical considerations of dPCR, as well as its role when developing and characterizing nucleic acid-based reference materials.
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Ácidos Nucleicos , Humanos , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
Methotrexate (MTX)-induced accelerated nodulosis (MIAN) reportedly occurs in patients with rheumatic arthritis receiving MTX therapy. However, it has also been reported in patients with other autoinflammatory conditions, such as systemic lupus erythematosus (SLE). A 38-year-old woman diagnosed with SLE presented with multiple movable, firm, flesh-colored nodules on both hands that had developed 3 years ago. She was taking oral medications, specifically hydroxychloroquine, azathioprine, and MTX. Histopathological examination revealed palisaded granulomatous inflammation, surrounded by histiocytes and lymphocytes, along the dermis to the subcutaneous fat layer. Fibrinoid degeneration was observed at the center of the granulomatous inflammation, and dermal mucin deposition was not observed. The patient was diagnosed with MIAN, and therefore discontinuation of MTX was recommended. Subsequently, the lesions almost completely disappeared with no signs of recurrence. MIAN exhibits clinicopathological features similar to those of rheumatoid nodules; therefore, it can be easily misdiagnosed. Herein, we report a case of MIAN in a patient with SLE to contribute to the accurate diagnosis and appropriate management.
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Spindle cell lipoma is a rare benign neoplasm that features a mixture of evenly aligned spindle cells, mature adipocytes, and ropey collagen. Most cases of spindle cell lipoma are found in the subcutaneous tissue, and intradermal spindle cell lipoma is rarely reported. We present a case of intradermal spindle cell lipoma in a 46-year-old female who presented with a 0.7-cm flesh-colored and dome-shaped nodule on the right temple that had developed 6 years ago. This mass was excised, and upon histopathologic examination, an unencapsulated lesion was located in the dermis, which consisted of bland spindle cells, scanty mature adipocytes, rare lipoblasts, and ropey collagen bundles with prominent basophilic myxoid stroma. Immunohistochemical staining showed diffuse positivity for CD34, negativity for the S-100 protein, and loss of retinoblastoma protein expression. Based on these features, intradermal low-fat spindle cell lipoma was diagnosed. No evidence of local recurrence was observed 4 months after excision. Intradermal low-fat spindle cell lipomas are extremely rare and can easily be mistaken for tumors that have similar clinical and histopathological findings. Herein, we report a globally rare case of an intradermal low-fat spindle cell lipoma.
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Superficial CD34-positive fibroblastic tumor (SCPFT) is a recently described disease entity characterized by marked nuclear pleomorphism, low mitotic count, and diffuse CD34 positivity. It is a rare, distinctive, low-grade fibroblastic neoplasm. To date, only 44 cases have been reported in the English-language literature. Herein, we report two cases of SCPFT involving a 48-year-old male and a 22-year-old male with superficial tumors on the right and left thighs, respectively. Excision was performed in both cases. Histologically, both tumors showed spindle-to-epithelioid cells arranged in fascicular or sheet-like patterns. Most cells displayed granular or eosinophilic glassy cytoplasm, marked nuclear pleomorphism, and a low mitotic rate. On immunohistochemical staining, tumor cells were diffusely positive for CD34 and negative for S100 protein, smooth muscle actin, and desmin. After wide excision, neither patient experienced recurrence or metastasis after 16 months and 11 months of clinical follow-up, respectively. To the best of our knowledge, these are the first two cases of SCPFT reported in Korea. We believe these case reports would contribute to the clinicopathological understanding of SCPFT and assist clinicians in differentiating this tumor from other superficial soft tissue neoplasms.
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PURPOSE: Oncotype DX (ODX) is a well-validated multigene assay that is increasingly used in Korean clinical practice. This study aimed to develop a clinicopathological prediction (CPP) model for the ODX recurrence scores (RSs). METHODS: A total of 297 patients (study group, n = 175; external validation group, n = 122) with estrogen receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative, T1-3N0-1M0 breast cancer, and available ODX test results were included in the study. Risk categorization as determined by ODX RSs concurred with the TAILORx study (low-risk, RS ≤ 25; high-risk, RS > 25). Univariate and multivariate logistic regression analyses were used to assess the relationships between clinicopathological variables and risk stratified by the ODX RSs. A CPP model was constructed based on regression coefficients (ß values) for clinicopathological variables significant by multivariate regression analysis. RESULTS: Progesterone receptor (PR) negativity, high Ki-67 index, and nuclear grade (NG) 3 independently predicted high-risk RS, and these variables were used to construct the CPP model. The C-index, which represented the discriminatory ability of our CPP model for predicting a high-risk RS, was 0.915 (95% confidence interval [CI], 0.859-0.971). When the CPP model was applied to the external validation group, the C-index was 0.926 (95% CI, 0.873-0.978). CONCLUSION: Our CPP model based on PR, Ki-67 index, and NG could aid in the selection of patients with breast cancer requiring an ODX test.
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PURPOSE: The updated American Society of Clinical Oncology/College of American Pathologists guideline for estrogen receptor (ER) testing recommends that breast cancer with ER expression in 1-10% of tumor cells should be reported as ER-low positive (ERlow), although limited data are available on the overall benefits of endocrine therapy. We investigated the clinicopathological characteristics and clinical outcomes of ERlow breast cancer and to compare them with those of ER-negative (ERneg) and ER-high (> 10% of tumor cells, ERhigh) breast cancers. METHODS: Consecutive patients with invasive breast cancer who underwent curative surgery between November 2007 and December 2014 were included. Clinicopathological characteristics and disease-free survival (DFS) of ERlow tumors were compared with those of ERneg and ERhigh tumors. RESULTS: Of the 2,309 cases included, 46 (2%), 643 (27.8%), and 1,620 (70.2%) were ERlow, ERneg, and ERhigh, respectively. ERlow tumors were associated with no special type of histology (p = 0.011), advanced pT (p = 0.017), pN (p = 0.009) and anatomic stages (p < 0.001), high grade (p < 0.001), negative/low progesterone receptor (PR) status (p < 0.001), human epidermal growth factor receptor 2 positivity (p < 0.001), high Ki-67 (p < 0.001), and recurrence (p = 0.006) compared to ERhigh tumors. DFS was significantly dependent on ER status, and ERlow tumors showed poorer DFS than ERhigh tumors (p = 0.001), however, there was no significant survival difference between ERlow and ERneg tumors. Furthermore, DFS in ERhigh patients was affected by hormone therapy (p < 0.001), while it was not affected in ERlow patients. CONCLUSION: Patients with ERlow breast cancer have clinicopathological characteristics that differ from those with ERhigh tumors. Although this study was limited by the small sample size of the ERlow group, no benefit from hormone therapy was observed in the ERlow group compared with the ERhigh group.
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BACKGROUND: Acellular dermal matrix (ADM) in implant-based breast reconstruction can show various ultrasound (US) findings. However, there are limited reports on the US features of the ADM. The aims of this study were to evaluate US findings of the ADM in implant-based breast reconstruction and correlate them with histopathological findings. METHODS: Between January 2015 and August 2020, 250 women who underwent implant-based breast reconstruction with ADM and a breast US examination at 6 months to 1 year after reconstruction were retrospectively analyzed. Abnormal US findings were classified as type 1 (focal thickening with decreased echogenicity), 2 (diffusely hyperechoic), or 3 (bright echogenic spots). ADM biopsy was performed in 33 patients who underwent second stage or revisional surgeries. RESULTS: In total, 176 consecutive women with 207 US findings were analyzed. The US findings were normal in 52.2% of the women. The percentages of type 1, 2, and 3 patients were 13.5%, 11.1%, and 23. 2%, respectively. These patients had microscopic findings that showed patchy areas with chronic inflammatory infiltrates, dense collagen bundles without degenerative or inflammatory changes, and empty spaces or degenerated foci unaccompanied by inflammation. CONCLUSION: Knowing the various ADM presentations on US can help avoid unnecessary invasive procedures.
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ABSTRACT: To investigate the correlations between ultrasonographic morphological characteristics quantitatively assessed using a deep learning-based computer-aided diagnostic system (DL-CAD) and histopathologic features of breast cancer.This retrospective study included 282 women with invasive breast cancer (<5âcm; mean age, 54.4 [range, 29-85] years) who underwent surgery between February 2016 and April 2017. The morphological characteristics of breast cancer on B-mode ultrasonography were analyzed using DL-CAD, and quantitative scores (0-1) were obtained. Associations between quantitative scores and tumor histologic type, grade, size, subtype, and lymph node status were compared.Two-hundred and thirty-six (83.7%) tumors were invasive ductal carcinoma, 18 (6.4%) invasive lobular carcinoma, and 28 (9.9%) micropapillary, apocrine, and mucinous. The mean size was 1.8â±â1.0 (standard deviation)âcm, and 108 (38.3%) cases were node positive. Irregular shape score was associated with tumor size (Pâ<â.001), lymph nodes status (Pâ=â.001), and estrogen receptor status (Pâ=â.016). Not-circumscribed margin (Pâ<â.001) and hypoechogenicity (Pâ=â.003) scores correlated with tumor size, and non-parallel orientation score correlated with histologic grade (Pâ=â.024). Luminal A tumors exhibited more irregular features (Pâ=â.048) with no parallel orientation (Pâ=â.002), whereas triple-negative breast cancer showed a rounder/more oval and parallel orientation.Quantitative morphological characteristics of breast cancers determined using DL-CAD correlated with histopathologic features and could provide useful information about breast cancer phenotypes.
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Neoplasias de la Mama/diagnóstico por imagen , Carcinoma Ductal de Mama/diagnóstico por imagen , Aprendizaje Profundo , Ultrasonografía/métodos , Adulto , Anciano , Anciano de 80 o más Años , Computadores , Femenino , Humanos , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
AIM: The Singapore nomogram was developed to predict recurrence risk of phyllodes tumours (PTs) of the breast based on histological features of stromal atypia, stromal mitoses, stromal overgrowth and surgical margin status. We aimed to validate the utility of the Singapore nomogram in a Korean PT cohort. METHODS: One hundred and twenty-four patients with Korean PT who underwent surgical resection between 1996 and 2015 were included in this study. Pathology reports and slides were reviewed to obtain histopathologic features and acquire Singapore nomogram scores. The probability of concordance between predicted and observed survivals by means of the Singapore nomogram was evaluated using a concordance index (C-index). RESULTS: Of the 124 cases, 57 (46%) were diagnosed as benign, 50 (40.3%) as borderline and 17 (13.7%) as malignant. Recurrences occurred in 25 (20.2%) patients. Univariate analysis showed PTs with higher stromal mitotic counts, marked stromal cellularity, stromal overgrowth, positive surgical margin, marked stromal atypia or a malignant grade presented higher risks of recurrence. Multivariate analysis showed stromal mitoses and surgical margin status independently predicted recurrence-free survival. Patients with high nomogram scores were at greater risk of recurrence (HR=1.05, 95% CI: 1.02 to 1.07, p<0.001) with a C-index of 0.762. CONCLUSION: The Singapore nomogram provided a useful means of predicting PT outcomes in a Korean PT cohort.
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Neoplasias de la Mama/diagnóstico , Tumor Filoide/diagnóstico , Adolescente , Adulto , Mama/patología , Neoplasias de la Mama/patología , Estudios de Cohortes , Femenino , Humanos , Márgenes de Escisión , Persona de Mediana Edad , Análisis Multivariante , Recurrencia Local de Neoplasia , Nomogramas , Tumor Filoide/patología , Pronóstico , República de Corea , Riesgo , Adulto JovenRESUMEN
Nucleic acid tests to detect the SARS-CoV-2 virus have been performed worldwide since the beginning of the COVID-19 pandemic. For the quality assessment of testing laboratories and the performance evaluation of molecular diagnosis products, reference materials (RMs) are required. In this work, we report the production of a lentiviral SARS-CoV-2 RM containing approximately 12 kilobases of its genome including common diagnostics targets such as RdRp, N, E, and S genes. The RM was measured with multiple assays using two different digital PCR platforms. To measure the homogeneity and stability of the lentiviral SARS-CoV-2 RM, reverse transcription droplet digital PCR (RT-ddPCR) was used with in-house duplex assays. The copy number concentration of each target gene in the extracted RNA solution was then converted to that of the RM solution. Their copy number values are measured to be from 1.5 × 105 to 2.0 × 105 copies/mL. The RM has a between-bottle homogeneity of 4.80-8.23% and is stable at 4 °C for 1 week and at -70 °C for 6 months. The lentiviral SARS-CoV-2 RM closely mimics real samples that undergo identical pre-analytical processes for SARS-CoV-2 molecular testing. By offering accurate reference values for the absolute copy number of viral target genes, the developed RM can be used to improve the reliability of SARS-CoV-2 molecular testing.
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Prueba de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Genoma Viral , ARN Viral/genética , Juego de Reactivos para Diagnóstico/normas , SARS-CoV-2/genética , COVID-19/virología , Prueba de Ácido Nucleico para COVID-19/normas , Proteínas de la Envoltura de Coronavirus/genética , Proteínas de la Envoltura de Coronavirus/metabolismo , Proteínas de la Nucleocápside de Coronavirus/genética , Proteínas de la Nucleocápside de Coronavirus/metabolismo , ARN Polimerasa Dependiente de ARN de Coronavirus/genética , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , Dosificación de Gen , Expresión Génica , Humanos , Células Jurkat , Lentivirus/genética , Lentivirus/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN Viral/metabolismo , ARN Viral/normas , Juego de Reactivos para Diagnóstico/provisión & distribución , Estándares de Referencia , Reproducibilidad de los Resultados , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Empaquetamiento del Genoma ViralRESUMEN
Nucleic acid analysis is used in many areas of life sciences such as medicine, food safety, and environmental monitoring. Accurate, reliable measurements of nucleic acids are crucial for maximum impact, yet users are often unaware of the global metrological infrastructure that exists to support these measurements. In this work, we describe international efforts to improve nucleic acid analysis, with a focus on the Nucleic Acid Analysis Working Group (NAWG) of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM). The NAWG is an international group dedicated to improving the global comparability of nucleic acid measurements; its primary focus is to support the development and maintenance of measurement capabilities and the dissemination of measurement services from its members: the National Metrology Institutes (NMIs) and Designated Institutes (DIs). These NMIs and DIs provide DNA and RNA measurement services developed in response to the needs of their stakeholders. The NAWG members have conducted cutting edge work over the last 20 years, demonstrating the ability to support the reliability, comparability, and traceability of nucleic acid measurement results in a variety of sectors.
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Ácidos Nucleicos/análisis , Ácidos Nucleicos/normas , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA quantities, measured by reverse transcription quantitative PCR (RT-qPCR), have been proposed to stratify clinical risk or determine analytical performance targets. We investigated reproducibility and how setting diagnostic cutoffs altered the clinical sensitivity of coronavirus disease 2019 (COVID-19) testing. METHODS: Quantitative SARS-CoV-2 RNA distributions [quantification cycle (Cq) and copies/mL] from more than 6000 patients from 3 clinical laboratories in United Kingdom, Belgium, and the Republic of Korea were analyzed. Impact of Cq cutoffs on clinical sensitivity was assessed. The June/July 2020 INSTAND external quality assessment scheme SARS-CoV-2 materials were used to estimate laboratory reported copies/mL and to estimate the variation in copies/mL for a given Cq. RESULTS: When the WHO-suggested Cq cutoff of 25 was applied, the clinical sensitivity dropped to about 16%. Clinical sensitivity also dropped to about 27% when a simulated limit of detection of 106 copies/mL was applied. The interlaboratory variation for a given Cq value was >1000 fold in copies/mL (99% CI). CONCLUSION: While RT-qPCR has been instrumental in the response to COVID-19, we recommend Cq (cycle threshold or crossing point) values not be used to set clinical cutoffs or diagnostic performance targets due to poor interlaboratory reproducibility; calibrated copy-based units (used elsewhere in virology) offer more reproducible alternatives. We also report a phenomenon where diagnostic performance may change relative to the effective reproduction number. Our findings indicate that the disparities between patient populations across time are an important consideration when evaluating or deploying diagnostic tests. This is especially relevant to the emergency situation of an evolving pandemic.
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Prueba de Ácido Nucleico para COVID-19/normas , COVID-19 , Ácidos Nucleicos , Bélgica , COVID-19/diagnóstico , Humanos , Ácidos Nucleicos/análisis , ARN Viral/análisis , Reproducibilidad de los Resultados , República de Corea , SARS-CoV-2 , Sensibilidad y Especificidad , Reino UnidoRESUMEN
BACKGROUND: Cutaneous metastasis (CM) is the spread of cancer cells from a primary site to the skin and is rarely the first sign of silent cancer. We investigated the clinicopathological characteristics of CM from internal malignancies in Korean patients treated at our institution over 20 years. METHODS: The clinicopathological findings of 112 patients (62 females, 50 males) with CM diagnosed at Yeungnam University Hospital between 2000 and 2020 were retrospectively reviewed. RESULTS: Mean patient age was 58.6 years (range, 26 to 87 years), and the most common primary cancer site was breast (74.2%) in women and lung (36.0%) in men. Ninety-six patients (85.7%) presented with CM after primary tumor diagnosis. CM from the lung or biliary tract usually occurred within 2 years of primary tumor diagnosis, whereas metastases from the breast and kidney occurred several years later. The chest, abdomen, and scalp were common sites of CM. Breast cancer usually metastasized to chest skin, while gastrointestinal tract cancers commonly metastasized to the abdomen. The scalp was a common location for CM from various tumors. The most common dermatologic presentations were nodules and masses. Immunohistochemical studies helped identify underlying malignancies when primary tumors were unknown. CONCLUSIONS: The relative frequency of CM parallels the overall incidence of primary malignant tumors, and CMs usually occur at anatomic sites close to the primary tumor. CM can be diagnosed based on clinical, radiological, and histological features; however, immunohistochemical study is required in some cases.
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This study was undertaken to investigate immunohistochemical expression of the senescence-associated secretory phenotype (SASP) in invasive breast cancer (IBC) tissues and to determine relationships between SASP positivity and tumor microenvironments and the clinicopathological characteristics of IBC. Immunohistochemistry for senescence markers, that is, high mobility group box-1 (HMGB1), p16, p15, and decoy receptor 2 (DCR2), was performed in tissue microarrays of 1140 IBC samples. Cases positive for at least one of these four markers were considered SASP-positive. Relations between SASP and tumor characteristics, including immune microenvironments (stromal tumor-infiltrating lymphocytes [sTILs] density and numbers of intraepithelial CD103-positive [iCD103 + ] lymphocytes) and clinical outcomes were retrospectively evaluated. HMGB1, p16, p15, or DCR2 was positive in 6.7%, 26.6%, 21.1%, and 26.5%, respectively, of the 1,140 cases. Six hundred and five (53.1%) cases were SASP positive, and SASP positivity was significantly associated with histologic grade 3, high-sTIL and iCD103 + lymphocyte counts, absence of ER or PR, and a high Ki-67 index. Although SASP did not predict breast cancer-specific survival (BCSS) or disease-free survival (DFS) in the entire cohort, SASP positivity in luminal A IBC was associated with poor BCSS and DFS. However, patients with SASP-positive TNBC showed better survival than those with SASP-negative TNBC. In multivariate analysis, SASP positivity was an independent prognostic factor in both luminal A IBC and TNBC, although the effect on prognosis was the opposite. In conclusion, SASP would be involved in the modulation of immune microenvironments and tumor progression in IBC, and its prognostic significance depends on molecular subtype.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Linfocitos Infiltrantes de Tumor/inmunología , Fenotipo Secretor Asociado a la Senescencia , Microambiente Tumoral , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/cirugía , Carcinoma Ductal de Mama/inmunología , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/cirugía , Femenino , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Pronóstico , Receptor ErbB-2/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
BACKGROUND: The COVID-19 pandemic caused by the severe acute SARS-CoV-2 virus has undeniably highlighted the importance of reliable nucleic acid quantification. Digital PCR (dPCR) is capable of the absolute quantification of nucleic acids. METHOD: By using the droplet dPCR (QX200) and the digital real-time PCR (LOAA), the copy numbers were compared via multiple assays for three distinct targerts; EGFR DNA, SARS-CoV-2 and HIV-1 RNA. RESULTS: The droplet dPCR and digital real-time PCR showed similar copy numbers for both DNA and RNA quantification. When the limit of detection (LOD) and limit of quantitation (LOQ) of each method were estimated for DNA and RNA targets, the digital real-time PCR showed a higher sensitivity and precision especially with low copy number targets. CONCLUSION: The breath of nucleic acid testing in diagnostic applications continues to expand. In this study we applied common diagnostic targets to a novel digital real-time PCR methodology. It performed comparably to the established dPCR method with distinctive advantages and disadvantages for implementing in laboratories. These rapidly developing dPCR systems can be applied to benefit the accurate and sensitive nucleic acid testing for various clinical areas.
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COVID-19 , SARS-CoV-2 , ADN , Receptores ErbB , Humanos , Pandemias , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
PURPOSE: The programmed death ligand 1 (PD-L1) SP142 assay with a 1% immune cell (IC) cutoff is approved for the selection of advanced triple-negative breast cancer (TNBC) patients for atezolizumab treatment. We aimed to evaluate the interobserver concordance of PD-L1 scoring and inter-assay variability of various PD-L1 assays in TNBC. METHODS: Thirty patients with primary TNBC were selected, and SP142, SP263, 22C3, and E1L3N assays were performed. PD-L1 staining in ICs and tumor cells (TCs) was scored by 10 pathologists who were blinded to the assay. The interobserver concordance among pathologists and the inter-assay variability of the four PD-L1 assays were analyzed. For SP142, the intraobserver concordance among the six pathologists was analyzed after training. RESULTS: The adjusted means of PD-L1 IC scoring ranged from 6.2% to 12.9% for the four assays; the intraclass correlations showed moderate (0.584-0.649) reader concordance. The PD-L1 IC scoring with a 1% cutoff resulted in identical scoring in 40.0%-66.7% of cases and a poor to moderate agreement (Fleiss κ statistic [FKS] = 0.345-0.534) for the four assays. The SP142 assay had the widest range of positive rate (56.5%-100.0%), lowest number of cases with identical scoring, and lowest FKS at 1% cutoff. Pairwise comparison of adjusted means showed significantly decreased PD-L1 staining in SP142 compared with the other assays in both ICs and TCs. As for the intraobserver concordance in the SP142 assay, the overall percent agreement was 87.8% with a 1% IC cutoff. After training, the proportion of cases with identical scoring at a 1% IC cutoff increased to 70.0%; the FKS also increased to 0.610. CONCLUSION: The concordance of PD-L1 IC scoring among pathologists was low, at the 1% cutoff for the SP142 assay without training. SP142 showed the lowest PD-L1 expression in both IC and TC.