Accurate diagnosis of colorectal cancer based on histopathology images using artificial intelligence.

BACKGROUND: Accurate and robust pathological image analysis for colorectal cancer (CRC) diagnosis is time-consuming and knowledge-intensive, but is essential for CRC patients' treatment. The current heavy workload of pathologists in clinics/hospitals may easily lead to unconscious misdiagnosis of CRC based on daily image analyses. METHODS: Based on a state-of-the-art transfer-learned deep convolutional neural network in artificial intelligence (AI), we proposed a novel patch aggregation strategy for clinic CRC diagnosis using weakly labeled pathological whole-slide image (WSI) patches. This approach was trained and validated using an unprecedented and enormously large number of 170,099 patches, > 14,680 WSIs, from > 9631 subjects that covered diverse and representative clinical cases from multi-independent-sources across China, the USA, and Germany. RESULTS: Our innovative AI tool consistently and nearly perfectly agreed with (average Kappa statistic 0.896) and even often better than most of the experienced expert pathologists when tested in diagnosing CRC WSIs from multicenters. The average area under the receiver operating characteristics curve (AUC) of AI was greater than that of the pathologists (0.988 vs 0.970) and achieved the best performance among the application of other AI methods to CRC diagnosis. Our AI-generated heatmap highlights the image regions of cancer tissue/cells. CONCLUSIONS: This first-ever generalizable AI system can handle large amounts of WSIs consistently and robustly without potential bias due to fatigue commonly experienced by clinical pathologists. It will drastically alleviate the heavy clinical burden of daily pathology diagnosis and improve the treatment for CRC patients. This tool is generalizable to other cancer diagnosis based on image recognition.

[Liquid chromatography-tandem mass spectrometry method for the determination of paraquat and diquat in plasma and urine].

Objective: To establish a LC-MS/MS method for determination of paraquat and diquat in plasma and urine samples. Methods: Plasma is precipitated by acetonitrile then diluent with phosphate buffer (pH=7) , urine is diluent with phosphate buffer (pH=7) , then diluent samples extracted with Oasis WCX solid-phase extraction column. Samples were analyzed using LC-MS/MS in multiple reaction monitoring (MRM) mode. The analytical column was XBridge®BEH-HILIC (100 mm×2.1 mm×2.5 µm) and the mobile phase were 100 mmol ammonium formate add 0.5% formic acid and acetonitrile. Paraquat was quantified by internal standard method and diquat by external standard method. Results: The calibration curves of paraquat and diquat were linear in the concentration range of 10.0~120.0 µg/L, the correlation coefficient (r) were 0.9985~0.9994. The limit of detection of paraquat in plasma and urine were 1.98 µg/L and 1.00 µg/L, respectively, the recovery rate were 100.2%~107.3%, the RSD were 1.6%~3.3%. The limit of detection of diquat in plasma and urine were 1.80 µg/L and 2.77 µg/L, respectively, the recovery rate were 85.3%~93.1%, the RSD were 1.8%~5.5%. Conclusion: This method is sensitive and accurate, and can simultaneously determine paraquat and diquat in plasma and urine.

[The study on the detection method for mercury in blood with direct mercury analyzer].

Objective: To establish a method for determining mercury in blood with direct mercury analyzer. Methods: After the whole blood sample was extracted by solvent and removed by nitric acid, it was then measured by direct mercury analyzer. Results: After optimizing the conditions of the instrument, the linear range was 0.3-60.0 µg/L and the curve correlation coefficient was higher than 0.999. The lower limit of quantitations was 0.3 µg/L and the minimum quantitative concentration was 3.0 µg/L. The recovery and relative standard deviations (RSD) was 95.2%-97.6% and 1.4%-3.3%. Conclusion: The method is stable, reliable, easy to operate and has high sensitive. It can be used to determine mercury in blood.

[Elemental and isotopic determination of lead in whole blood by ISIS-ICP-MS].

Objective: To establish a method for determination of lead and istope ratios in the blood by ISIS-ICP-MS. Methods: After wet digestion, the blood sample was on-line addition of thallium as internal standard and analyzed by ISIS-ICP-MS. Results: The limit of detection was 0.03 µg/L and the lower limit of quantification was 0.08 µg/L. The detection concentration was 0.45 µg/L and the minimum quantitative concentration was 1.49 µg/L. The relative standard deviations (RSD) were 0.3%~1.7%. The recovery was between 91.0% and 103.4%. The precision of the major lead isotope ratios was better than 0.3%. The calibrated isotope ratios of the standard liquid are close to the certificate. Conclusion: The method has a low detection limit, good precision and high accuracy, it is feasible for determination of lead concentration and isotope ratios in the bloune.

[Determination of Sevoflurane, Isoflurane and Enflurane in the air of workplace by gas chromatography].

Objective: To establish a solvent desorption gas chromatographic method for determination of Sevoflurane, Isoflurane and Enflurane in the air of the Workplace. Methods: Sevoflurane, Isoflurane and Enflurane were collected with activated carbon tube and desorbed with dichloromethane, separated with DB-1 capillary columns, and then detected with flame ionization detector. Results: The linearity ranges were 1.9-304.8 µg/ml for Sevoflurane, 2.1-300.4 µg/ml for Isoflurane and 1.7-305.2 µg/ml for Enflurane, The correlation coefficient was both >0.999. Their limits of detection were 0.6 µg/ml, 0.6 µg/ml and 0.5 µg/ml, and Their limits of quatification were 1.9 µg/ml, 2.1 µg/ml and 1.7 µg/ml, and their minimum detectable concentrations were 0.1、0.2 and 0.1 mg/m(3) per 4.5 L of air. Their relative standard deviations (RSD) were 2.5%-3.0%, 2.3%-3.1% and 2.2%-3.0%. The average desorption efficiencies were 101.1%-103.3%, 100.7%-102.7% and 101.0%-102.9%. The sampling efficiency was both 100%. The breakthrough volume of 100 mg actived carbon was 3.7 mg, 3.4 mg and 3.4 mg. Sevoflurane, Isoflurane and Enflurane in activated carbon tube could be kept at least 10 days at room temperature without significant losses. Conclusion: The method shows lower detection limit, high accuracy and precision. It is feasible for determination of Sevoflurane, Isoflurane and Enflurane in the air of workplace.

[Direct determination of manganese in urine by Graphite Furnace Atomic Absorption Spectrometry].

Objective: To establish a method for the determination of manganese in urine by graphite furnace atomic absorption spectrometry (AAS) without the use of matrix modifier. Methods: The urine samples were 5 times diluted with 1% nitric acid then directly determined by AAS. Zeeman was used for background correction. Results: The linear range for determination of manganese in urine was 5~60 µg/L (urine) . The correlation coefficient was greater than 0.995 with the detection limit of 1.5 µg/L and with the lower limit of quantification of 5.0 µg/L. The relative standard deviations (RSDs) of within-run precision was between 1.1%~4.3%, the RSDs of between-run precision was between 3.3%~7.0%. The average recovery was 102.6%. The samples can be stored for 14 days at room temperature, 4℃, -8 ℃ and -35 ℃. Conclusion: The method is feasible for determination of manganese in urine.

[Simultaneous determination of various volatile organic compounds in urine by headspace GC-MS].

Objective: To establish a method for determination of acetone, dichloromethane, hexane, 1, 1, 1-trichloroethane, 1, 2-dichloroethane, benzene, toluene, ethylbenzene etc organic compounds in urine by headspace gas chromatography-mass spectrometry (GC-MS) . Methods: Headspace gases of urine samples were injected into GC and determined by mass. Results: Determination of urine components were in a good linear range in their concentration range of this method. The correlation coefficients were between 0.996 and 1.000 with the detection limits between 0.1 µg/L and 4.5 µg/L, the precisions were between 1.3% and 4.6%, the recovery rates were between 86.2% and 97.4%. Conclusion: This method has the advantages of low detection limits, high accuracy, high precision and simple pretreatment, which is suitable for the determination of the content of various volatile organic compounds in urine.

[Determination of tin dioxide in workplace air by atomic fluorescence spectrometry after alkali fusion].

OBJECTIVE: To establish the method of atomic fluorescence spectrometry (AFS) after alkali fusion for determination of tin dioxide in workplace air. METHODS: Tin dioxide in workplace air was collected with microporous membrane, directly digested by alkali fusion with solid sodium hydroxide heated by electric furnace, and determined by AFS. RESULTS: The linear range of tin dioxide (as Sn) determined by AFS was 1.5~100 µg/L (excluding zero) , and the correlation coefficient was 0.9993. The detection limit of this method was 0.5 µg/L, the lower limit of quantification was 1.5 µg/L, and the minimum detectable concentration was 0.05 mg/m(3) (the volume of the air sample was 75 L) . The relative standard deviation was 1.94%~3.55%, and the average recovery of standard addition was 95.0%~96.0%. CONCLUSION: The method of AFS after alkali fusion for determination of tin dioxide in workplace air is proved to be simple, rapid, sensitive, and accurate, with complete digestion.

Hepatitis B virus x protein induces autophagy via activating death-associated protein kinase.

Hepatitis B virus x protein (HBX), a product of hepatitis B virus (HBV), is a multifunctional protein that regulates viral replication and various cellular functions. Recently, HBX has been shown to induce autophagy; however, the responsible mechanism is not fully known. In this study, we established stable HBX-expressing epithelial Chang cells as the platform to study how HBX induced autophagy. The results showed that the overexpression of HBX resulted in starvation-induced autophagy. HBX-induced autophagy was related to its ability to dephosphorylate/activate death-associated protein kinase (DAPK). The block of DAPK by its siRNA significantly counteracted HBX-mediated autophagy, confirming the positive role of DAPK in this process. HBX also induced Beclin 1, which functions at the downstream of the DAPK-mediated autophagy pathway. Although HBX could activate JNK, a kinase known to participate in autophagy in certain conditions, the change in JNK failed to influence HBX-induced autophagy. In conclusion, HBX induces autophagy via activating DAPK in a pathway related to Beclin 1, but not JNK. This new finding should help us to understand the role of autophagy in HBX-mediated pathogenesis and thus may provide targets for intervening HBX-related disorders.

Elevated expressions of MMP7, TROP2, and survivin are associated with survival, disease recurrence, and liver metastasis of colon cancer.

PURPOSE: Colorectal cancer is one of the most common cancers worldwide. We tested the hypothesis that differences in the expression of certain molecular markers of colon cancer may account for different clinical outcomes. METHODS: Tissue microarray technology was used to assay the expression of 17 biological markers [beta-catenin, CD44v7, c-myc, cyclin D1, estrogen receptor beta, mitogen-activated protein kinase/extracellular signal-regulated kinase, maspin, matrix metalloproteinase-7 (MMP7), p53, Pin1, peroxisome proliferators-activated receptor-gamma, survivin, T cell transcription factor 4 (TCF4), transforming growth factor beta receptor II (TGFbetaR II), TGFbeta, TROP2, and Wnt] by immunohistochemistry in 620 colon cancer patients. The Cox proportional hazards regression model was applied to analyze the lifetime data, including time to death, time to recurrence, and time to liver metastasis. RESULTS: All the markers were present at significantly higher expression levels in tumor specimens than in normal colonic specimens. Kaplan-Meier analysis showed that high expression of TROP2, MMP7, and survivin were related to decreased survival; TCF4 and TROP2 were related to disease recurrence; and CD44v7, cyclin D1, MMP7, p53, survivin, and TCF4 were related to liver metastasis. However, the results of the multivariate analysis only showed that expression of MMP7, survivin, and TROP2 were significant predictors of lower patient survival, while TROP2 and MMP7 were significantly related to disease recurrence and liver metastasis, respectively. CONCLUSIONS: We conclude that elevated survivin, MMP7, and TROP2 expression levels are related to decreased survival. In addition, elevated MMP7 and TROP2 expression levels are predictors of disease recurrence and liver metastasis, respectively.

[Interpretation of the fourth edition of WHO pathological classification of the thyroid tumors in 2017].

In recent years, the incidence of thyroid carcinoma gradually increased in China. The pathology diagnosis and classification was based on WHO classification of Tumors of Endocrine Organs, the third edition which published in 2004. The fourth edition, WHO classification of Tumors of Endocrine Organs was published in July 2017. Compared with the third, some important aspects (or points) were revised: the ICD-O code of hyalinizing trabecular tumor was changed from 0 to 1; three other encapsulated follicular-patterned thyroid tumors were added; the variants of well differentiation thyroid carcinoma (including papillary carcinoma and follicular carcinoma ) which was originated from thyroid epithelial cells were updated; oncocytic cell tumors were separated from follicular tumors; the ICD-O code of ectopic thymoma was changed from 1 to 3. Refinement and standardization part of the concepts and diagnostic criterias were done which can solve practical problems in pathology diagnosis.

miR-625 suppresses tumour migration and invasion by targeting IGF2BP1 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies and the third leading cause of cancer-related deaths worldwide. Tumour metastasis is one of the major causes of high mortality. microRNAshave been implicated in HCC metastasis. In this study, we found that miR-625 was frequently downregulated in HCC samples. A decrease in miR-625 was significantly correlated with lymph node anddistance metastasis (P=0.013), the presence of portal venous invasion (P=0.036), tumor-node-metastasis (TNM) stage (P=0.027) and unfavourable overall survival (P=0.003). Compared with primary tumours, miR-625 expression was markedly reduced in portal venous metastatic tumours. Re-expression of miR-625 in HCC cells was remarkably effective in suppressing cell migration andinvasiveness in vitro and in vivo. Mechanistically, miR-625 was confirmed to downregulate IGF2 mRNA-binding protein 1(IGF2BP1) directly, the expression of which was inversely correlated with the level of miR-625 in HCC cell lines and tissues. High expression of IGF2BP1 was frequently found in HCC samples, and associated with poor prognosis. Knockdown of endogenous IGF2BP1 by siRNA exhibited similar effects as the overexpression of miR-625, whereas overexpression of IGF2BP1 (without the 3'-UTR) abrogated miR-625-mediated metastasis inhibition. Interference of the PTEN/HSP27 pathway contributed to miR-625-mediated metastasis inhibition. Taken together, our data suggest that miR-625 might function as an antimetastatic miRNA to have an important role in HCC progression by modulating the IGF2BP1/PTEN pathway. The newly identified miR-625/IGF2BP1 axis represents a new potential therapeutic target for HCC treatment.

Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells.

The events of cell death and the expression of nuclear matrix protein (NMP) have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide. By means of TUNEL assay, the nuclei displayed a characteristic morphology change, and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h. Nucleosomal DNA fragmentation was observed after treatment for 4 h. The morphological change of HL-60 cells, thus, occurred earlier than the appearance of DNA ladder. Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis. Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells. Western blotting was then performed on three nuclear matrix acssociated proteins, PML, HSC70 and NuMA. The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment, while NuMA, a nuclear mitotic apparatus protein, was down regulated. These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process.

Bcl-2 and Bax proteins are nuclear matrix associated proteins.

Bcl-2 and Bax proteins are implicated in the regulation of apoptosis. Nuclear matrix has been demonstrated to be associated with a vast array of functional and regulatory properties of cells. NuMA is one member of a class of nuclear matrix proteins that resides in both the nucleus and mitotic apparatus. The nuclear lamins appear to form a thin fibrous structure immediately underlying the inner nuclear membrane of eukaryotic cell nuclei. The association of bcl-2 and Bax protein with nuclear matrix in glioblastoma cell line U343 was studied by confocal microscopy and Western blotting. Confocal microscopic images display that bcl-2 was localized at the peripheral of the nuclear matrix and Bax protein was located in the nuclear matrix. Western blotting detected a 26 kDa bcl-2 band and a specific band of Bax at around 66 kDa in nuclear matrix proteins. Our results suggest that bcl-2 and Bax proteins are nuclear matrix associated proteins.

The measurement of AgNORs in human glioma cells.

Sixty gliomas obtained by neurosurgical resections were examined. Paraffin blocks were retrieved from pathological files of the Second Affiliated Hospital in Guangzhou Medical College. The methods of argyrophilic technique for AgNORs staining, and Image Analysis System for measurement of AgNORs were used. Six parameters, which included hcount, count, narea, agnrea, agpern and agperc were used to correlated well with histopathological grades (compared grade 2 & 3, grade 3 & 4, and grade 2 & 4, respectively). We concluded that AgNORs is useful in evaluating proliferative activity and assessing the malignancy of human gliomas. It may also be used as a target for anti-neoplastic drugs in the treatment of gliomas.

Association of EGFR gene fragments with nuclear matrix proteins in high and low metastatic melanoma cell lines.

Two melanoma cell lines with different metastatic potential were used to study the association of EGFR gene fragments with the nuclear matrix and its role in cancer metastasis by polymerase chain reaction. A 940 bp positive amplification by PCR using primers I-II was demonstrated in a high metastatic cell line, WM451. A 110 bp positive amplification was shown using primers III-IV in both high and low metastatic cell lines. This finding demonstrates that EGFR gene fragments are tightly bound to the nuclear matrix and suggests that binding ability of this EGFR gene fragment to nuclear matrix seems to be closely related to metastatic potential in melanoma cell lines WM45 1 and WM35.

SPAG5 upregulation predicts poor prognosis in cervical cancer patients and alters sensitivity to taxol treatment via the mTOR signaling pathway.

Previously, we found that sperm-associated antigen 5 (SPAG5) was upregulated in pelvic lymph node metastasis-positive cervical cancer. The aim of this study is to examine the role of SPAG5 in the proliferation and tumorigenicity of cervical cancer and its clinical significance in tumor progression. In our study, SPAG5 expression in cervical cancer patients was detected using quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry; cervical cancer cell function with downregulated SPAG5 in vitro was explored using tetrazolium assay, flow cytometry, and colony formation and Transwell assays. SPAG5 was upregulated in tumor tissue compared with paired adjacent noncancerous tissues; SPAG5 upregulation in tumor tissues indicated poor disease-free survival, which was also an independent prognostic indicator for cervical cancer patients. In vitro study demonstrated that SPAG5 downregulation inhibited cell proliferation and growth significantly by G2/M arrest and induction of apoptosis, and hindered cell migration and invasion. Under SPAG5 downregulation, the sensitivity of cervical cancer cells differed according to taxol dose, which correlated with mammalian target of rapamycin (mTOR) signaling pathway activity. In general, SPAG5 upregulation relates to poor prognosis in cervical cancer patients, and SPAG5 is a regulator of mTOR activity during taxol treatment in cervical cancer.

Prognostic significance of low DICER expression regulated by miR-130a in cervical cancer.

Dicer is crucial for the maturation of microRNAs (miRNAs) and its dysregulation may contribute to tumor initiation and progression. The study explored the clinical implications of Dicer and its post-transcriptional regulation by microRNAs in cervical cancer. qRT-PCR and immunohistochemistry investigated Dicer mRNA and protein levels in cervical cancer tissues. The relationship between Dicer expression and survival was analyzed. MiRNA target prediction identified miRNAs that might target Dicer. Luciferase reporter and gain- or loss-of-function assays were performed. The results showed that 36.7% of cervical cancer cases showed low expression of Dicer mRNA and 63.3% cases showed high expression. At the protein level, 51% cases showed negative expression and 49% cases showed positive expression. Dicer mRNA and protein expressions were significantly associated with distant metastasis and recurrence in cervical cancer (P=0.002 and P=0.012, respectively). Multivariate Cox analysis indicated that low Dicer expression (P=0.016) and tumor stage (P=0.047) were independent predictors. Among the miRNAs predicted to target Dicer, 10 were detected by RT-PCR; their expressions were significantly higher in cervical cancers with lower Dicer expression than in those with higher Dicer expression and were negatively correlated with Dicer expression level (P<0.05). In vitro experiments demonstrated that miR-130a directly targeted Dicer mRNA to enhance migration and invasion in SiHa cells. Finally, survival analysis indicated that higher expression of miR-130a was significantly associated with poor disease-free survival. Taken together, Dicer expression regulated by miR-130a is an important potential prognostic factor in cervical cancer.

KPNA2 promotes cell proliferation and tumorigenicity in epithelial ovarian carcinoma through upregulation of c-Myc and downregulation of FOXO3a.

Karyopherin alpha 2 (KPNA2), a member of the karyopherin family, has a central role in nucleocytoplasmic transport and is overexpressed in many cancers. Our previous study identified KPNA2 as significantly upregulated in epithelial ovarian carcinoma (EOC), correlating with poor survival of patients. However, the precise mechanism of this effect remains unclear. The aim of the present study was to examine the role of KPNA2 in the proliferation and tumorigenicity of EOC cells, and its clinical significance in tumor progression. Real-time quantitative RT-PCR analysis revealed high expression levels of KPNA2 in 162 out of 191 (84.8%) fresh EOC tissues, which was significantly correlated with International Federation of Gynecology and Obstetrics (FIGO) stage, differentiation, histological type, recurrence, and prognosis of EOC patients. Our results showed that upregulation of KPNA2 expression significantly increased the proliferation and tumorigenicity of EOC cells (EFO-21 and SK-OV3) in vitro and in vivo, by promoting cell growth rate, foci formation, soft agar colony formation, and tumor formation in nude mice. By contrast, knockdown of KPNA2 effectively suppressed the proliferation and tumorigenicity of these EOC cells in vitro and in vivo. Our results also indicated that the molecular mechanisms of the effect of KPNA2 in EOC included promotion of G1/S cell cycle transition through upregulation of c-Myc, enhanced transcriptional activity of c-Myc, activation of Akt activity, suppression of FOXO3a activity, downregulation of cyclin-dependent kinase (CDK) inhibitor p21Cip1 and p27Kip1, and upregulation of CDK regulator cyclin D1. Our results show that KPNA2 has an important role in promoting proliferation and tumorigenicity of EOC, and may represent a novel prognostic biomarker and therapeutic target for this disease.