[Call attention to the overall benefit of digital pathology and promote its development].

The successful operation of the complete digital pathology(CDP) in foreign countries indicates that the full digital pathology process has entered the full implementation stage. Digital pathology in China started late and progressed slowly, so far there has not been a truly meaningful CDP. The pathologist's understanding of digital pathology is not comprehensive enough, and there are still doubts about the time efficiency and cost effectiveness of digital pathology processes. Therefore,a comprehensive analysis of the process, overall advantages and cost-effectiveness of CDP was made by drawing on international successful experience and hands-on practice experience, in order to promote the construction and development of the CDP in our country.

Bioinformatics screening of gene expression profile and diagnostic application of meningeal carcinoma.

OBJECTIVE: The aim of this study was to research gene expression profiles and diagnostic applications of meningeal carcinoma based on bioinformatics. MATERIALS AND METHODS: We used the Gene Expression Omnibus (GEO) database to obtain the GSE43290 dataset based on the expression data of normal meninges and meningiomas consisting of 51 samples divided into two groups (47 samples of meningioma tumors and four samples of normal meninges). We used the GEO2R tool to identify differentially expressed genes (DEGs) by setting the log2 fold change as greater than two and an adjusted p-value lower than 0.05. We used the database for annotation, visualization and integrated discovery (DAVID) to perform gene ontology, biological pathways and functional annotation of the DEGs. A search Tool for the Retrieval of Interacting Gene database (STRING) was used to obtain Protein-Protein Interaction (PPI) and modular networks based on the Markov clustering algorithm. RESULTS: Our study identified 358 significant DEGs, of which 343 were downregulated genes while 15 were upregulated. Five significant hub genes (CXCL8, AGT, CXCR4, CXCL12 and CXCL2) were associated with various biological pathways, molecular functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The DEGs were enriched in biological pathways of chemokine-mediated signaling, positive regulation of leukocyte chemotaxis, second messenger-mediated signaling, induction of positive chemotaxis, CXCR chemokine receptor binding and activities of cytokines. CONCLUSIONS: These hub genes and pathways could be targeted in clinical research to discover new treatments for meningeal carcinoma.

[The trend of accurate pathology diagnosis of breast cancer].

Accurate pathology diagnosis of breast cancer is the premise of personalized treatment. In recent years, the pathology diagnosis of breast cancer have been updated and optimized to provide better guidance and basis for clinical treatment. In this paper, we provide an overview on the advances in histological classification of breast cancer, the progress of biomarker detection related to novel antibody-drug conjugates and immunotherapy in breast cancer, the pathology evaluation of breast cancer specimen after neoadjuvant therapy and sentinel lymph nodes, the progress of genetic testing in breast cancer, and the application of artificial intelligence in breast pathology.

[Correlation between balloon volume and Meckel's cave size and its influence of percutaneous microballoon compression for trigeminal neuralgia].

Objective: To investigate the correlation between balloon volume and Meckel's cave size during percutaneous puncture microballoon compression (PMC) for trigeminal neuralgia and the influence of the compression coefficient (the ratio of balloon volume/Meckel's cave size) on the prognosis. Methods: Seventy-two patients (28 males and 44 females) aged (62±11) years who underwent PMC under general anesthesia for trigeminal neuralgia in the First Affiliated Hospital of Zhengzhou University from February 2018 to October 2020 were retrospectively collected. All patients underwent preoperative cranial magnetic resonance imaging (MRI) to measure Meckel's cave size, intraoperative balloon volume was recorded, and the compression coefficient was calculated. Follow-up visits were performed preoperatively (T0) and 1 d (T1), 1 month (T2), 3 months (T3), and 6 months (T4) postoperatively, either in the outpatient clinic or by telephone, and the Barrow Neurological Institute pain scale (BNI-P) score, the Barrow Neurological Institute facial numbness (BNI-N) score and the occurrence of complications were recorded and compared at each time point. Patients were divided into 3 groups according to different prognoses: patients in group A (n=48) were with no recurrence of pain and mild facial numbness, patients in group B (n=19) were with no recurrence of pain but severe facial numbness, while those in group C (n=5) had recurrence of pain. The differences in balloon volume, Meckel's cave size, and compression coefficient were compared among the three groups, and the correlation between balloon volume and Meckel's cave size in each group was analyzed by Pearson correlation. Results: The effective rate of PMC for trigeminal neuralgia was 93.1% (67/72). At time points from T0 to T4, patients had BNI-P scores [M (Q1, Q3)] of 4.5 (4.0, 5.0), 1.0 (1.0, 1.0), 1.0 (1.0, 1.0), 1.0 (1.0, 1.0) and 1.0 (1.0, 1.0), and BNI-N scores [M (Q1, Q3)] of 1.0 (1.0, 1.0), 4.0 (3.0, 4.0), 3.0 (3.0, 4.0), 3.0 (2.0, 4.0) and 2.0 (2.0, 3.0), respectively. Compared with those at T0, patients had lower BNI-P scores and higher BNI-N scores from T1 to T4 (all P<0.05). In all patients, group A, group B, and group C, the balloon volume was (0.65±0.15), (0.67±0.15), (0.59±0.15) and (0.67±0.17) cm3, respectively, with no statistically significant difference (P>0.05), while the Meckel's cave size was (0.42±0.12), (0.44±0.11), (0.32±0.07), and (0.57±0.11) cm3, with a statistically significant difference (P<0.001). The balloon volumes and Meckel's cave sizes were all linearly and positively correlated (r=0.852, 0.924, 0.937 and 0.969, all P<0.05). The compression coefficient in group A, B and C was (1.54±0.14), (1.84±0.18) and (1.18±0.10), respectively, with a statistically significant difference (P<0.001). There were no serious intraoperative complications such as death, diplopia, arteriovenous fistula, cerebrospinal fluid leak, and subarachnoid hemorrhage. Conclusions: Intraoperative balloon volume during PMC for trigeminal neuralgia is linearly and positively correlated with the volume of the patient's Meckel's cave. The compression coefficient varies among patients with different prognoses and the compression coefficient may be a factor affecting the patient's prognosis.

[Clinical observation of 8 cases with central nervous system anaerobic infection diagnosed by metagenomic next-generation sequencing of cerebrospinal fluid].

The current study summarized the clinical characteristics of 8 patients with central nervous system anaerobic infections who visited the Second Hospital of Hebei Medical University from June 2019 to June 2021. Among the 8 patients, 7 cases had headache and fever as the first symptoms, and 1 case had left-sided limb weakness as the first symptom. Four patients had a previous history of diabetes (1 patient had a history of tooth extraction before the onset of the disease), 1 patient had a previous history of sinusitis, 1 patient had a history of tooth extraction before the onset of the disease, and 2 patients had chronic extensive periodontitis or periapical periodontitis. Five cases showed abnormal intracranial and/or meningeal enhancement on cranial magnetic resonance imaging (MRI), and 1 case showed swelling of the whole brain tissue on cranial computed tomography (CT). After treatment, symptoms of 4 cases were improved, symptoms of 1 case were aggravated, and 3 cases died. Anaerobic infections can occur in various parts of the body:the central nervous system, oral cavity, head and neck, chest, abdomen, pelvis, skin and soft tissues. It is generally believed that anaerobic bacteria rarely cause central nervous system infection, therefore, anaerobic culture of cerebrospinal fluid is usually not performed. Moreover, anaerobic culture is affected by many factors, and thus it is difficult to obtain positive results. Metagenomic next-generation sequencing can identify multiple pathogens (viral, bacterial, fungal, or parasitic) from samples based on DNA and/or RNA sequences and has advantages in diagnosing anaerobic infections of the central nervous system. Prompt empirical antimicrobial therapy is essential for the prognosis of the patients.

[Diagnostic value of MDM2 RNA in situ hybridization in atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma].

Objective: To investigate the value of MDM2 RNA in situ hybridization (RNA-ISH) in diagnosing atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) and dedifferentiated liposarcoma (DDL). Methods: A total of 26 ALT/WDL/DDLs diagnosed from March 2017 to May 2019 in West China Hospital, Sichuan University, Chengdu, China and 18 control cases were included. MDM2 RNA-ISH was performed on all samples and compared with the fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) regarding their performance in detecting MDM2. Results: All samples were detected successfully using the three methods. Among 26 ALT/WDL/DDLs, all cases showed MDM2 amplification and positivity for MDM2 RNA-ISH (26/26, 100%). Twenty-four (24/26, 92.3%) of the 26 tested cases were positive for MDM2 IHC while two of them were negative. Eighteen control cases were all negative for MDM2 FISH and RNA-ISH, and 15 (15/18) cases were negative for MDM2 IHC. The sensitivity and specificity of RNA-ISH were both 100%, and those of MDM2 IHC were 92.3% and 83.3%, respectively. Diffuse staining was identified in all MDM2 RNA-ISH positive ALT/WDL/DDLs, but identified in only 8/24 (33.3%) of the MDM2 IHC positive cases. Among the 11 ALT/WDL/DDL samples evaluated on tissue microarray, the positive rate of MDM2 RNA-ISH was 100% with diffuse staining in all cases. The positive rate of MDM2 IHC was 9/11 while only 1 of the 9 cases showed diffuse staining. The result of MDM2 RNA-ISH was identical to that of MDM2 FISH and was overall consistent with that of MDM2 IHC (Kappa=0.763, P<0.001). Conclusions: In ALT/WDL/DDLs, results of MDM2 RNA-ISH are highly consistent with those of FISH. MDM2 RNA-ISH is more sensitive and more specific and has more diffuse positive signals than the IHC. The findings indicate that MDM2 RNA-ISH is highly valuable for the diagnosis and differential diagnosis of ALT/WDL/DDLs.

[Prognostic values of spindle checkpoint protein BUB1B in triple negative breast cancer].

Objective: To identify important prognostic molecular markers of triple negative breast cancer (TNBC) using high throughput sequencing technology and to explore the correlation of spindle checkpoint protein BUB1B and clinicopathological features with patients' prognosis. Methods: The clinicopathological data and prognostic information of TNBC diagnosed at the West China Hospital of Sichuan University from 2009 to 2017 were collected. Forty-seven fresh tumor samples and 139 formalin fixed paraffin-embedded samples were selected. The fresh tumor samples were subject to RNA sequencing (RNA-seq). The enrichment analysis and protein-protein interaction (PPI) analysis were performed after intersection of difference analysis between RNAseq and GEO (Gene Expression Omnibus) datasets GSE38959 and GSE65194. Kaplan-Meier plotter database was used to analyze the relationship between expression of BUB1B and prognosis. Immunohistochemical staining was used to verify its expression in TNBC and correlation with clinicopathological features and prognosis. Results: Using edgeR to perform differential expression analysis between 47 TNBC tumor tissues and 12 normal tissues, 1 559 up-regulated genes and 1 376 down-regulated genes were identified, while only 131 differentially expressed genes were overlapping with those in GSE38959 and GSE65194. Enrichment analysis was mainly enriched in cell cycle, JAK-STAT signaling pathway and p53 signaling pathway. The top 10 genes ranked by degree of association were TOP2A, BUB1B, MKI67, PLK1, RRM2, PCNA, KPNA2, SMC4, PBK and IGF1. Kaplan-Meier plotter database analysis showed that the expression of BUB1B was significantly correlated with the prognosis of TNBC [overall survival, hazard ratio (HR)=0.52, 95%CI (0.35-0.77), P=0.001; distant metastasis-free, HR=0.72, 95%CI (0.52-0.98), P=0.038]. The immunohistochemical analyses of 139 formalin fixed paraffin-embedded samples showed that the low expression of BUB1B was correlated with poor prognosis in TNBC [HR=0.41, 95%CI (0.18-0.95), P=0.024]. Conclusions: The low expression of BUB1B protein is associated with poor prognosis in TNBC patients, and the molecular mechanism related with prognosis and potential therapeutic targets need to be further studied.

[Cerebrospinal fluid TP53 gene mutation in patients with lung cancer associated meningitis and its clinical implications].

Objective: To observe the role of cerebrospinal fluid (CSF) TP53 gene mutation in lung cancer associated meningitis. Methods: A retrospective analysis was performed on 35 patients diagnosed with lung cancer associated meningitis at the Second Hospital of Hebei Medical University from December 2015 to December 2018.All patients underwent the next-generation sequencing of CSF, and TP53 gene was found to be mutant or wild type, including 23 patients with TP53 mutant type and 12 patients with TP53 wild type. The clinical characteristics, CSF leukocyte, protein, glucose, chloride, Karnofsky performance (KPS) and overall survival were observed. Results: Headache, nausea and vomiting were the main clinical manifestations in both groups.There were no significant differences in CSF pressure, leukocyte, biochemical indicators and KPS between the two groups. The average time from diagnosis of lung cancer to diagnosis of lung cancer associated meningitis in the TP53 mutant group was significantly shorter than that in the TP53 wild type group (5.79 months vs 25.5 months).The median survival time of patients in the TP53 mutant group from lung cancer diagnosis to the observation endpoint was 19.77 months, while it was 88.73 months in the TP53 wild type group, and the difference was statistically significant (P=0.043). Conclusions: Mutation in the tumor suppressor gene TP53 can be detected in the CSF of patients with lung cancer associated meningitis. Patients with such mutation have earlier meningeal metastasis and shorter median survival time.

[Imaging characteristics of patients with large vestibular aqueduct syndrome and its relationship with the acoustically evoked short latency negative response].

Objective: To explore the imaging characteristics of large vestibular aqueduct syndrome (LVAS) patients and their relationship with the acoustically evoked short latency negative response (ANSR), so as to provide reference for the diagnosis of LVAS. Methods: Clinical data of 174 patients(334 ears) with LVAS diagnosed and treated by the Department of Otorhinolaryngology Head and Neck Surgery of the First Affiliated Hospital of Guangxi Medical University, from October 2009 to December 2017 were retrospectively analyzed, including 117 males and 57 females, aged from 5 months to 47 years old, with the median age of 4 years and 4 months. ABR and imaging data of patients were collected. Midpoint diameter and the outlet diameter of the vestibular aqueduct were measured on CT images, the midpoint diameter of the intraosseous parts and the extraosseous parts of enlarged endolymphatic sac(EES) were measured on MRI images. The correlation between the above measurements was analyzed by Pearson test using SPSS 17.0. According to whether ASNR was detected in ABR, the above data were divided into two groups, and the differences of the above imaging measurements were compared by the Independent-Sample Test. Results: The average midpoint diameter of the vestibular aqueduct was (1.87±0.58) mm (x±s, the following was the same), and the outlet diameter was (3.07±0.99) mm on CT; the average midpoint diameter of the intraosseous parts in enlarged endolymphatic sac(EES) was (2.39±1.37) mm, and the extraosseous parts was (2.50±2.18) mm on MRI. There was a correlation between the four measurements (P<0.05), among which the midpoint diameter of vestibular aqueduct was strongly positively correlated with the outlet diameter (r=0.760), and the remaining pairs were weakly correlated. ASNR was detected in 241 ears (72.16%,241/334) and undetected in 93 ears (27.84%, 93/334) of the 334 ears with LVAS. Midpoint diameter and the outlet diameter of the vestibular aqueduct in no ASNR group were smaller than the ASNR group, and the difference was statistically significant (t value was 2.814 and 2.754, P<0.05). There was no significant difference in the midpoint diameter of the intraosseous parts and the extraosseous parts of enlarged endolymphatic sac between the two groups, and the difference was no statistically significant(t value was 0.101 and 0.683, P>0.05). Conclusions: There is a strong positive correlation between the midpoint diameter of vestibular aqueduct and the outlet diameter in LVAS patients. There is a certain correlation between the size of vestibular aqueduct and the size of endolymphatic sac. The smaller the diameter of vestibular aqueduct, the lower the occurrence rate of ASNR.

[Comparison of different massive parallel sequencing platforms for mutation profiling in formalin-fixed and paraffin-embedded samples].

Objective: To compare the performance of Miseq and Ion Torrent PGM platforms and library construction method for next-generation sequencing (NGS) technology for formalin-fixed and paraffin-embedded (FFPE) samples. Methods: A total of 204 FFPE cancer samples including 100 non-small cell lung cancers at the First Affiliated Hospital of Zhejiang University, and 104 colorectal cancers at West China Hospital of Sichuan University were retrospectively selected from January 2013 to December 2016. By using the same samples, DNA was extracted, and the same amount of DNA was used for library construction with the same kit, and sequenced on Miseq and Ion Torrent PGM respectively, after passing the quality control. Any discordant mutations between two platforms were validated by amplified refractory mutation system-polymerase chain reaction (ARMS-PCR) method and Sanger sequencing. Results: A total of 204 FFPE samples were included and 197 samples were successfully analyzed by both platforms. The number of reads generated by the samples on Miseq platform sequencing was higher than PGM platform (median 391 634 vs. 298 030, P<0.01). Alignment with human reference genome showed that the mapping rate of Miseq platform was higher than PGM platform (median 100.0% vs. 99.7%, P<0.01). The median sequence depth of samples on Miseq was higher than PGM platform (median 853× vs. 698×, P<0.01). A total of 236 mutations were detected by two platforms, of which 221 were detected on both platforms, with a 93.6% concordance. Miseq platform detected 11 mutations not detected on PGM platform, while PGM platform detected 4 more mutations not detected on Miseq platform. With validation by ARMS-PCR and Sanger sequencing, Miseq platform was more reliable for low-frequency mutations. The main reasons for the discordant mutations between two platforms were that mutation frequency on undetected platform was lower than mutation reporting range (5%) and FFPE samples were stored for a long time. Conclusions: Compared with PGM, Miseq platform shows higher sequencing quality in terms of the number of reads, alignment results and coverage depth, and the test results are more reliable. In clinical practice, the appropriate platform should be chosen based on sample size and actual throughput requirements to aid in the molecular characterization of tumors.

[Feasibility of amplicon-based targeted next-generation sequencing of colorectal cancer in endoscopic biopsies].

Objective: To investigate whether small endoscopic biopsies of colorectal cancer were sufficient for quality and accurate mutational analysis by amplicon-based next-generation sequencing (NGS). Methods: By using an amplicon-based targeted NGS panel for mutational detection on Illumina Miseq platform, a total of 109 formalin-fixed and paraffin-embedded (FFPE) endoscopic biopsies of colorectal cancer were retrospectively selected, based on specific histopathologic criteria, from January 2012 to June 2016 at West China Hospital of Sichuan University and Peking University Third Hospital. Twelve of these biopsies had corresponding FFPE surgical resection specimens. Quality control parameters of NGS testing were analyzed and NGS results were confirmed by other methods. Mutation calls of the 12 paired endoscopic biopsies and surgical resections were compared. Results: Of the endoscopic biopsy specimens, 97.2% (106/109) had sufficient DNA and qualified sequencing library. NGS generated excellent sequencing data, with a median of 848× for median read depth and 95.7% for uniformity. The success rate of NGS was 95.4% (104/109). Conventional methods confirmed the results of NGS for KRAS and BRAF, and the concordance rate was 100.0%. The clinically actionable mutations detected in the 12 paired endoscopic biopsies and surgical resections were concordant. Conclusion: FFPE endoscopic biopsies of colorectal cancer is suitable for targeted NGS, providing quality sequencing data and accurate mutational information to guide targeted therapy.