Identification of a DNA-methylome-based signature for prognosis prediction in driver gene-negative lung adenocarcinoma.

"Driver gene-negative" lung adenocarcinoma (LUAD) was of rare treatment options and a poor prognosis. Presently, for them, few biomarkers are available for stratification analysis to make appropriate treatment strategy. This study aimed to develop a DNA-methylome-based signature to realize the precise risk-stratifying. Here, an Illumina MethylationEPIC Beadchip was applied to obtain differentially methylated CpG sites (DMCs). A four-CpG-based signature, named as TLA, was successfully established, whose prognosis-predicting power was well verified in one internal (n = 78) and other external (n = 110) validation cohorts. Patients with high-risk scores had shorter overall survival (OS) in all cohorts [hazard ratio (HR): 11.79, 5.16 and 2.99, respectively]. Additionally, it can effectively divide patients into low-risk and high-risk groups, with significantly different OS in the diverse subgroups stratified by the standard clinical parameters. As an independent prognostic factor, TLA may assist in improving the nomogram's 5-year OS-predicting ability (AUC 0.756, 95% CI:0.695-0.816), superior to TNM alone (AUC 0.644, 95% CI: 0.590-0.698). Additionally, the relationship of TLA-related genes, TAC1, LHX9, and ALX1, with prognosis and tumour invasion made them serve as potential therapy targets for driver gene-negative LUAD.

Mechanisms of vemurafenib-induced anti-tumor effects in ATC FRO cells.

Background: Anaplastic Thyroid Carcinoma (ATC) is a rare and deadly malignant tumor in humans. It is prone to developing resistance to radiotherapy and chemotherapy. Molecular targeted therapy offers a novel way to treat ATC. The BRAF mutation is closely associated with many cancers, including thyroid carcinoma. Vemurafenib, a small-molecule inhibitor, is specifically designed to target the mutant serine/threonine kinase BRAF. The objective of this study is to elucidate the regulatory mechanisms underlying the effects of vemurafenib on human anaplastic thyroid carcinoma cell line FRO and to assess its potential therapeutic role. Methods: The effects of vemurafenib on the proliferation of FRO cells were assessed by the CCK-8 method and Colony-forming assay. Transwell chambers and scratch tests were employed to examine the impact of vemurafenib on the invasion and migration of FRO cells. Apoptosis and cycle distribution of FRO cells were analyzed by tunel assay and flow cytometry. The effects of vemurafenib on the expression of BRAF-activated non-protein coding RNA (BANCR), Bax, Bcl2, and E-cadherin were evaluated by qRT-PCR. Furthermore, the effects of vemurafenib on the expression of phosphoinositol-3-kinase (PI3K)/phosphoinositol-3-kinase (AKT) pathway-related proteins, BRAF, CyclinD1, Bcl-2, Bax, and E-cadherin proteins in FRO cells were investigated through the western-blot method. All experiments were conducted in three replicates. Results: Vemurafenib was observed to inhibit proliferation and induce apoptosis in a dose- and time-dependent manner (P < 0.05). The formation of FRO cell colonies, as well as migration and invasion, all showed a dose-dependent reduction (P < 0.05). Flow cytometric analysis indicated G0/G1 cell cycle arrest (P < 0.05). QRT-PCR revealed that vemurafenib could suppress the expression of BANCR and Bcl2 while increasing the expression of Bax and E-cadherin in a dose-dependent manner (P < 0.05). The protein expression levels of Bax and E-cadherin were up-regulated significantly, and the expression levels of BRAF, CyclinD1, Bcl-2, p-PI3K, p-AKT, and p-mTOR were markedly down-regulated with increasing concentrations of vemurafenib (P < 0.05). Conclusions: The proliferation and metastasis of FRO cells can be suppressed by vemurafenib through the silencing of BRAF and BANCR expression, inhibition of PI3K/AKT signaling pathway activation, induction of apoptosis, and cell cycle arrest.

Co-Delivery of Gemcitabine and Honokiol by Lipid Bilayer-Coated Mesoporous Silica Nanoparticles Enhances Pancreatic Cancer Therapy via Targeting Depletion of Tumor Stroma.

Syndecan-1 (SDC1) modified lipid bilayer (LB)-coated mesoporous silica nanoparticles (MSN) to co-deliver gemcitabine (GEM) and honokiol (HNK) were prepared for the targeting treatment of pancreatic cancer. The encapsulation efficiencies of GEM and HNK in SDC1-LB-MSN-GEM/HNK were determined to be 60.3 ± 3.2% and 73.0 ± 1.1%. The targeting efficiency of SDC1-LB-MSN-GEM/HNK was investigated in BxPC-3 cells in vitro. The fluorescence intensity in the cells treated with SDC1-LB-MSN-Cou6 was 2-fold of LB-MSN-Cou6-treated cells, which was caused by SDC1/IGF1R-mediated endocytosis. As anticipated, its cytotoxicity was significantly increased. Furthermore, the mechanism was verified that SDC1-LB-MSN-HNK induced tumor cell apoptosis through the mitochondrial apoptosis pathway. Finally, the biodistribution, tumor growth inhibition, and preliminary safety studies were performed on BALB/c nude mice bearing BxPC-3 tumor models. The tumor growth inhibition index of SDC1-LB-MSN-GEM/HNK was 56.19%, which was 1.45-fold and 1.33-fold higher than that of the free GEM/HNK and LB-MSN-GEM/HNK treatment groups, respectively. As a result, SDC1-LB-MSN-GEM/HNK combined advantages of both GEM and HNK and simultaneously targeted and eliminated pancreatic cancerous and cancer-associated stromal cells. In summary, the present study demonstrated a new strategy of synergistic GEM and HNK to enhance the therapeutic effect of pancreatic cancer via the targeting depletion of tumor stroma.

Preoperative Prediction of Perineural Invasion in Oesophageal Squamous Cell Carcinoma Based on CT Radiomics Nomogram: A Multicenter Study.

RATIONALE AND OBJECTIVES: To investigate the value of computed tomography (CT) radiomics nomogram in the preoperative prediction of perineural invasion (PNI) in oesophageal squamous cell carcinoma (ESCC) through a multicenter study. MATERIALS AND METHODS: We retrospectively collected postoperative pathological data of 360 ESCC patients with definite PNI status (131 PNI-positive and 229 PNI-negative) from two centres. Radiomic features were extracted from the arterial-phase CT images, and the least absolute shrinkage and selection operator and logistic regression algorithm were used to screen valuable features for identifying the PNI status and calculating the radiomics score (Rad-score). A radiomics nomogram was established by integrating the Rad-score and clinical risk factors. A receiver operating characteristic curve was used to evaluate model performance, and decision curve analysis was used to evaluate the predictive performance of the radiomics nomogram in the training, internal validation, and external validation sets. RESULTS: Twenty radiomics features were extracted from a full-volume tumour region of interest to construct the model, and the radiomics nomogram combined with radiomics features and clinical risk factors was superior to the clinical and radiomics models in predicting the PNI status of ESCC patients. The area under the curve values of the radiomics nomogram in the training, internal validation, and external validation sets were 0.856 (0.794-0.918), 0.832 (0.742-0.922), and 0.803 (0.709-0.898), respectively. CONCLUSION: The radiomics nomogram based on CT has excellent predictive ability; it can non-invasively predict the preoperative PNI status of ESCC patients and provide a basis for preoperative decision-making.

[Expression and Prognostic Impact of HK2 in Bone Marrow of Patients with Newly Diagnosed Acute Myeloid Leukemia].

OBJECTIVE: To detect the expression level of HK2 gene in the bone marrow of newly diagnosed patients with acute myeloid leukemia (AML) and investigate its influence on the clinical characteristics and prognosis. METHODS: The expression level of HK2 gene in the bone marrow of 90 newly diagnosed patients with AML that accompanying clinical characteristics and survival status were detected by RT-qPCR, and compared with 18 allogeneic hematopoietic stem cell transplantation (allo-HSCT) donors. The Chi-square test, Kaplan-Meier survival analysis, and Cox proportional hazards regression model were used to analyze the correlation of HK2 expression level with clinical characteristics and prognosis. RESULTS: Compared with allo-HSCT donors, the HK2 expression was significantly increased in newly diagnosed AML patients (P <0.01). Compared with patients with total response (OR, complete response + complete response with incomplete hematologic recovery) after 2 courses of induction chemotherapy, the expression of HK2 in patients without OR was significantly increased (P <0.05). There was a significant difference in the relative expression of HK2 between patients with and without OR after 2 courses of induction therapy (P <0.001). The median survival time of patients with high expression of HK2 was significantly shorter than that of patients with low expression of HK2 (P <0.05). The multivariate Cox proportional hazards regression analysis showed that prognostic stratification, the expression level of HK2, and whether two courses of induction therapy achieved OR were independent factors affecting the prognosis of AML patients (P <0.05). CONCLUSIONS: Compared with allo-HSCT donors, the expression level of HK2 gene is increased in the bone marrow of newly diagnosed AML patients. The prognosis of patients with high expression of HK2 is poor. The expression level of HK2 is an independent factor affecting the prognosis of AML patients.

Multiple brain regions are involved in reaction to acute restraint stress in CYLD-knockout mice.

The lysine 63 deubiquitinase cylindromatosis (CYLD) is expressed at high levels in the brain and is considered to be involved in anxious and depressive behavior, cognitive inflexibility, and autism disorders. Previous research was limited in some brain regions, including the hippocampus, striatum, and amygdala. To better understand whether CYLD plays a role in adaptation to stress and which brain regions are involved, we analyzed the behavior of CYLD-knockout mice in the elevated plus maze (EPM) and light-dark box test (LDT) after acute restraint stress (ARS) and mapped their c-Fos immunoreactivity in brain sections. Here we report that CYLD deficiency leads to an unexpected reaction to ARS in mice, and is accompanied by significant neuronal activation of brain regions including the medial prefrontal cortex (mPFC), dorsal striatum (DS), nucleus accumbens (NAc), and basal lateral amygdala (BLA), but not ventral hippocampus (vHPC). Our findings show that CYLD participates in ARS-induced anxious behavior and that this involves multiple brain regions.

The pyruvate dehydrogenase complex regulates mitophagic trafficking and protein phosphorylation.

The mitophagic degradation of mitochondrial matrix proteins in Saccharomyces cerevisiae was previously shown to be selective, reflecting a pre-engulfment sorting step within the mitochondrial network. This selectivity is regulated through phosphorylation of mitochondrial matrix proteins by the matrix kinases Pkp1 and Pkp2, which in turn appear to be regulated by the phosphatase Aup1/Ptc6. However, these same proteins also regulate the phosphorylation status and catalytic activity of the yeast pyruvate dehydrogenase complex, which is critical for mitochondrial metabolism. To understand the relationship between these two functions, we evaluated the role of the pyruvate dehydrogenase complex in mitophagic selectivity. Surprisingly, we identified a novel function of the complex in regulating mitophagic selectivity, which is independent of its enzymatic activity. Our data support a model in which the pyruvate dehydrogenase complex directly regulates the activity of its associated kinases and phosphatases. This regulatory interaction then determines the phosphorylation state of mitochondrial matrix proteins and their mitophagic fates.

Altered regional brain activity and functional connectivity in resting-state brain networks associated with psychological erectile dysfunction.

Introduction: Erectile dysfunction (ED), especially psychological ED (pED), is usually accompanied with psychological factors, which are related to abnormal activity in brain regions involved in sexual behavior. However, the mechanisms underlying functional changes in the brain of pED are still unclear. The present study aimed to explore the abnormalities of brain function, as well as their relationships with sexual behavior and emotion in pED patients. Materials and methods: Resting state functional magnetic resonance imaging (rs-fMRI) data were collected from 31 pED patients to 31 healthy controls (HCs). The values of amplitude of fractional amplitude of low-frequency fluctuation (fALFF) and functional connectivity (FC) were calculated and compared between groups. In addition, the associations between abnormal brain regions and clinical features were evaluated by Pearson correlation analyses. Results: Compared to HCs, pED patients demonstrated decreased fALFF values in the left medial superior frontal gyrus (had decreased FC values with the left dorsolateral superior frontal gyrus), the left lingual gyrus (had decreased FC values with the left parahippocamal gyrus and insula), the left putamen (had decreased FC values with the right caudate) and the right putamen (had decreased FC values with the left putamen and the right caudate). The fALFF values of the left medial superior frontal gyrus were negatively correlated with the fifth item scores of International Index of Erectile Function (IIEF-5). Negative relationships were found between fALFF values of the left putamen and the second item scores of Arizona Sexual Scale (ASEX). FC values between the right putamen and caudate were negatively related to the state scores of State-Trait Anxiety Inventory (STAI-S). Conclusion: Altered brain function were found in the medial superior frontal gyrus and caudate-putamen of pED patients, which were associated with sexual function and psychological condition. These findings provided new insights into the central pathological mechanisms of pED.

Combination immunotherapy of PEG-modified preladenant thermosensitive liposomes and PD-1 inhibitor effectively enhances the anti-tumor immune response and therapeutic effects.

Immunotherapy is a promising cancer treatment strategy. In contrast, programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) inhibitors are associated with low response rates and are only useful in a small group of cancer patients. A combination of treatments may be effective for overcoming this clinical issue. Preladenant is an adenosine (ADO) receptor inhibitor that can block the ADO pathway and improve the tumor microenvironment (TME), thereby enhancing the immunotherapeutic effect of PD-1 inhibitors. However, its poor water solubility and low targeting limit its clinical applications. We designed a PEG-modified thermosensitive-liposome (pTSL) loaded with ADO small molecule inhibitor preladenant (P-pTSL) to overcome these problems and enhance the effect of PD-1 inhibitor on breast cancer immunotherapy. The prepared P-pTSL was round and uniformly distributed with a particle size of (138.9 ± 1.22) nm, PDI: 0.134 ± 0.031, and zeta potential (-10.1 ± 1.63) mV; preladenant was released slowly at 37 °C but released fast at 42 °C from P-pTSL, which was 76.52 ± 0.44%. P-pTSL has good long-term and serum stability and excellent tumor-targeting ability in mice. Moreover, the combination with PD-1 inhibitor significantly enhanced the anti-tumor effect, and the improvement of related factors in serum and lymph was more obvious under the condition of 42 °C thermotherapy in vitro.

Deubiquitinase CYLD regulates excitatory synaptic transmission and short-term plasticity in the hippocampus.

The fate of proteins is determined by the addition of various forms of polyubiquitin during ubiquitin-mediated proteasomal degradation. Cylindromatosis (CYLD), a K63-specific deubiquitinase, is enriched in postsynaptic density fractions of the rodent central nervous system (CNS), but the synaptic role of CYLD in the CNS is poorly understand. Here we show that CYLD deficiency (Cyld-/-) results in reduced intrinsic hippocampal neuronal firing, a decrease in the frequency of spontaneous excitatory postsynaptic currents and a decrease in the amplitude of field excitatory postsynaptic potentials. Moreover, Cyld-/- hippocampus shows downregulated levels of presynaptic vesicular glutamate transporter 1 (vGlut1) and upregulated levels of postsynaptic GluA1, a subunit of the AMPA receptor, together with an altered paired-pulse ratio (PPR). We also found increased activation of astrocytes and microglia in the hippocampus of Cyld-/- mice. The present study suggests a critical role for CYLD in mediating hippocampal neuronal and synaptic activity.

The inflammation repressor TNIP1/ABIN-1 is degraded by autophagy following TBK1 phosphorylation of its LIR.

The inflammatory repressor TNIP1/ABIN-1 is important for keeping in check inflammatory and cell-death pathways to avoid potentially dangerous sustained activation of these pathways. We have now found that TNIP1 is rapidly degraded by selective macroautophagy/autophagy early (0-4 h) after activation of TLR3 by poly(I:C)-treatment to allow expression of pro-inflammatory genes and proteins. A few hours later (6 h), TNIP1 levels rise again to counteract sustained inflammatory signaling. TBK1-mediated phosphorylation of a TNIP1 LIR motif regulates selective autophagy of TNIP1 by stimulating interaction with Atg8-family proteins. This is a novel level of regulation of TNIP1, whose protein level is crucial for controlling inflammatory signaling.

Prevalence of Body Somatic Deformities in Plastic Surgery Patients: A Systematic Review with Meta-analysis.

BACKGROUND: Plastic surgery is a surgical specialty that focuses on restoring, reconstructing, or changing the human body. Somatic deformities (SD) are defined by a distorted impression of one's own body image and are rather frequent. The majority of people with SD have some level of social and vocational impairment, with obsessive concerns about appearance leading to compulsive behaviors and, in more severe situations, suicidal thoughts. OBJECTIVE: The current study aims to confirm the prevalence of SD in plastic surgery patients using a systematic review of the literature and a meta-analysis. METHODOLOGY: We have searched for electronic databases with MeSH terms, and the studies for analysis were selected based on inclusion and exclusion criteria and quality assessment. The study was conducted as per the PRISMA guidelines. The pooled prevalence was calculated using fixed and random effect model. The publication bias was assessed qualitatively (funnel plot) as well as quantitatively (Begg, Egger and Harbord tests). All analysis was done using Stats Direct (version 3). RESULTS: The pooled prevalence of somatic deformities in plastic surgery with 95% confidence interval using random effect model was found to be 0.19 [0.12, 0.27] which indicates a significant association of somatic deformities in plastic surgery. The heterogeneity among studies was found to be high as indicated by Cochran Q (P < 0.0001) and I2 tests (98.6%). The qualitative and quantitative analysis has also shown significant involvement of publication bias. CONCLUSION: Based on available evidence, there is a significant association of somatic deformities in plastic surgery. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Pan-Cancer Analysis of Histone Methyltransferase KMT2D with Potential Implications for Prognosis and Immunotherapy in Human Cancer.

BACKGROUND: Pan-cancer analysis is an efficient tool to obtain a panoramic view of cancer- related genes and identify their oncogenic processes, facilitating the development of new therapeutic targets. Lysine methyltransferase 2D (KMT2D), acting as a major enhancer coactivator for mammalian cells, is one of the most frequently mutated genes across various cancer types and is considered an oncogene and a rationale for epigenetic therapeutic targets. OBJECTIVE: This study was designed to explore the potential role of KMT2D in human cancer through a pan-cancer analysis. METHODS: The expression of KMT2D was assessed in normal tissues and cell lines, and pancancers from The Cancer Genome Atlas (TCGA), Cancer Cell Line Encyclopedia (CCLE), and Genotype-Tissue Expression (GTE) datasets were used to explore its correlation with prognosis, immune cell infiltration, tumor mutation burden, microsatellite instability, and mismatch repair. RESULTS: KMT2D expression was heterogeneous across different cancer types. Increased KMT2D indicated a worse prognosis in adrenocortical carcinoma (ACC), brain lower-grade glioma (LGG), and mesothelioma (MESO), while patients with high KMT2D expression showed better outcomes in renal clear cell carcinoma (KIRC). Moreover, KMT2D expression was positively correlated with immune cell infiltration and negative tumor mutation burden in multiple cancers. In addition, a significant correlation between KMT2D and immune checkpoint-related genes or mismatch repair genes was identified. CONCLUSIONS: These findings support the hypothesis that KMT2D is not only a potential biomarker for prognosis and immunotherapy response prediction but also an essential immune regulator in human cancer.

Clinical Significance of a Circulating Tumor Cell-based Classifier in Stage IB Lung Adenocarcinoma: A Multicenter, Cohort Study.

OBJECTIVE: To investigate the effectiveness of a CTC-based classifier in stratifying stage IB LUAD. SUMMARY OF BACKGROUND DATA: Stage IB LUADs have an approximately 70% 5-year survival rate. The clinical application of ACT is controversial due to inconsistent results in a series of trials and few useful guide biomarkers. Thus, there is a pressing need for robust biomarkers to stratify stage IB patients to define which group would most likely benefit from ACT. Methods: Two hundred twelve stage IB LUAD patients were enrolled and were divided into 3 independent cohorts. The aptamer-modified NanoVelcro system was used to enrich the CTCs. RESULTS: A cutoff of <4 or >4 CTCs as the optimal prognostic threshold for stage IB LUAD was generated to stratify the patients in a 70-patient cohort into low-risk and high-risk groups. Patients with ≥ 4 CTCs in the training cohort had shorter progression-free survival ( P < 0.0001) and overall survival ( P < 0.0001) than patients with <4 CTCs. CTC number remained the strongest predictor of progression-free survival and overall survival even in a multivariate analysis including other clinicopathological parameters. Furthermore, a nomogram based on the CTC count was developed to predict the 3-year and 5-year survival in the training cohort and performed well in the other 2 validation cohorts (C-index: 0.862, 0.853, and 0.877). CONCLUSION: The presence of >4 CTCs can define a high-risk subgroup, providing a new strategy to make optimal clinical decisions for stage IB LUAD.

TBK1 phosphorylation activates LIR-dependent degradation of the inflammation repressor TNIP1.

Limitation of excessive inflammation due to selective degradation of pro-inflammatory proteins is one of the cytoprotective functions attributed to autophagy. In the current study, we highlight that selective autophagy also plays a vital role in promoting the establishment of a robust inflammatory response. Under inflammatory conditions, here TLR3-activation by poly(I:C) treatment, the inflammation repressor TNIP1 (TNFAIP3 interacting protein 1) is phosphorylated by Tank-binding kinase 1 (TBK1) activating an LIR motif that leads to the selective autophagy-dependent degradation of TNIP1, supporting the expression of pro-inflammatory genes and proteins. This selective autophagy efficiently reduces TNIP1 protein levels early (0-4 h) upon poly(I:C) treatment to allow efficient initiation of the inflammatory response. At 6 h, TNIP1 levels are restored due to increased transcription avoiding sustained inflammation. Thus, similarly as in cancer, autophagy may play a dual role in controlling inflammation depending on the exact state and timing of the inflammatory response.

Can Anti-inflammatory Drugs used in Plastic Surgery Procedures Increase the Risk of Hematoma?

BACKGROUND: Anti-inflammatory drugs are commonly used to lower inflammation which is linked to a variety of disorders. It acts by inhibiting cyclooxygenase-mediated prostaglandin synthesis at the molecular level. Hematoma is related with the use of anti-inflammatory medications. However, the specific link is still unknown. Thus, the main objective of the study is to find out the association of hematoma with ant-inflammatory drugs. MATERIAL AND METHODS: The relevant studies were searched in PubMed and screened based on inclusion and exclusion criteria. The quality of full-text studies was assessed using suitable Newcastle-Ottawa Scale. The overall estimate was calculated in terms of odds ratio with 95% confidence interval. The random effect model was used. The qualitative analysis of publication bias was done through funnel plot. RESULTS: The overall estimate measures [OR 1.01 (0.50, 2.06)] have shown non-significant risk of hematoma with use of anti-inflammatory drugs in plastic surgery as compared to non-anti-inflammatory drugs. The heterogeneity among studies was found to be 34%. The subgroup analysis of individual drugs was not done due to availability of a smaller number of studies. CONCLUSION: Based on available data, there is no significant risk of hematoma with use of anti-inflammatory drugs in plastic surgery. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

A compound cantilever beam piezoelectric harvester based on wind energy excitation.

In this paper, a compound cantilever beam based piezoelectric energy harvester (CCBPH) is proposed. This piezoelectric energy harvester uses vibrations caused by vortex excitation behind the winding fluid to harvest wind energy. In particular, this structure uses vortex excitation formed behind a vertically suspended cylindrical winding fluid to cause the vibration of the cylindrical winding fluid, which then indirectly excites the piezoelectric element. The CCBPH consists of a fixed support, a cantilever beam, magnet-1 and magnet-2, a support beam, two piezoelectric units-PVDF (polyvinylidene fluoride), a compound cantilever beam, and a cylindrical winding fluid. We investigated the parameters affecting the structure and verify the effectiveness of the energy harvester through the design of the structure, simulation analysis, and experiments. The experimental results show that the CCBPH can obtain the maximum output voltage from the energy harvester at a wind speed of 18 m/s. The maximum output power was achieved with an external load resistance of 2000 kΩ. By comparison, it is found that the maximum output power is 0.095 mW when the distance between two magnets is 20 mm and the mass ratio is 1:2 for copper.

Comparison of immunohistochemistry and RT-qPCR for assessing ER, PR, HER2, and Ki67 and evaluating subtypes in patients with breast cancer.

PURPOSE: Currently, the most commonly applied method for the determination of breast cancer subtypes is to test estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki67 by immunohistochemistry (IHC). However, the IHC method has substantial intraobserver and interobserver variability. ESR1, PGR, ERBB2, and MKi67 mRNA tests by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay may improve the diagnostic objectivity and efficiency. Here, we compared the concordance between RT-qPCR and IHC for assessment of the same biomarkers and evaluated the subtypes. METHODS: A total of 265 eligible cases were divided into a training cohort and a validation cohort, and the expressions of ER/ESR1, PR/PGR, HER2/ERBB2, and Ki67/MKI67 were tested by IHC and RT-qPCR. Then, the appropriate cutoff of RT-qPCR was calculated in the training cohort. The concordance between RT-qPCR and IHC was calculated for individual marker. In addition, we investigated the subtypes based on the RT-qPCR results. RESULTS: The Spearman correlation coefficients between ER/ESR1, PR/PGR, HER2/ERBB2, and Ki67/MKI67 by IHC and RT-qPCR were 0.768, 0.699, 0.762, and 0.387, respectively. The cutoff values for the RT-qPCR assay of ESR1 (1%), PGR (1%), ERBB2, and MKi67 (14%) were 35.539, 32.139, 36.398, and 29.176, respectively. The overall percent agreement (OPA) between ER/ESR1, PR/PGR, HER2/ERBB2, and Ki67/MKI67 by IHC and RT-qPCR was 92.48%, 73.68%, 92.80%, and 74.44%, respectively. A total of 224 (84.53%) specimens were concordant for the breast cancer subtypes (IHC-based type) by RT-qPCR. CONCLUSION: Evaluation of breast cancer biomarker status by RT-qPCR was highly concordant with IHC. RT-qPCR can be used as a supplementary method to detect molecular markers of breast cancer.

Predictive Value of Circulating Tumor Cells Based on Subtraction Enrichment for Recurrence Risk in Stage II Colorectal Cancer.

Current guidelines recommend adjuvant chemotherapy (ACT) for stage II colorectal cancer (CRC) patients by using clinical high risk factors, with which circulating tumor cells (CTCs) were not considered. Here, an assessment to detect CTCs based on subtraction enrichment mediated by magnetic beads conjugated with CD45, immunofluorescence staining of CK, and fluorescence in situ hybridization of CEP8 is established. Both CEP8- and CK-positive CTCs have the potential to improve the risk stratification of stage II CRC patients. Patients with preoperative CTCs of ≥4 had a significantly higher recurrence risk than those with preoperative CTCs of <4 in two external validation cohorts (P < 0.0001). In the subgroup with clinical high risk, when preoperative CTCs were <4, patients did not benefit from ACT (P = 0.5764); however, when preoperative CTCs were ≥4, patients received benefit from ACT (P = 0.0064). Additionally, regardless of clinical risk status and preoperative CTC levels, if postoperative CTC levels were ≥4 for more than three consecutive time points (monitoring time interval, 2-6 months), the recurrence rate was 100%. Our findings suggested that the subtraction enrichment of CTCs could provide a reliable method to stratify the recurrence risk and make therapeutic decisions after surgery in stage II CRC patients.