Objective To perform single-molecule, real-time sequencing of ceftazidime-avibactam (CAZ-AVI)-resistant Pseudomonas aeruginosa and to investigate the mechanism underlying ceftazidime-avibactam resistance in P. aeruginosa. Methods The susceptibility of 89 P. aeruginosa isolates randomly sampled from clinical specimens in Sanming First Hospital Affiliated to Fujian Medical University from November 2021 through July 2023 to common antimicrobial agents was tested, and the minimum inhibitory concentration (MIC) of CAZ-AVI was determined against P. aeruginosa with a broth microdilution assay, with CAZ-AVI MICs of 8 mg/L and lower defined as susceptible and 16 mg/L and higher as resistant. The expression of drug-resistant genes ampC, oxa-488, oprD, mexA, oxa-10, oxa-14, vim and tem was quantified in P. aeruginosa using a real-time quantitative reverse transcription PCR (qPCR) assay. CAZ-AVI-susceptible and -resistant P. aeruginosa isolates from the same case were selected for PacBio single-molecule, real-time sequencing, and sequencing results were subjected to genome structure and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotations. Results The 89 P. aeruginosa isolates showed a relatively high level of resistance to meropenem (75.28%) and imipenem (74.16%) and the highest susceptibility to amikacin (91.01%). There were 49 CAZ-AVI-resistant P. aeruginosa isolates and 40 susceptible isolates. qPCR assay detected lower oprD gene expression in CAZ-AVI-resistant P. aeruginosa isolates [0.104 (2.385)] than in susceptible isolates [0.551 (17.885)] (Z = -2.958, P < 0.01), and there were no significant differences between CAZ-AVI-susceptible and -resistant P. aeruginosa isolates in terms of ampC, oxa-488, mexA or tem gene expression (all P values > 0.05), while oxa-10, oxa-14 and vim gene was expressed in few P. aeruginosa isolates. There were 1 729, 3 936, 3 737 and 3 955 genes in CAZ-AVI-resistant P. aeruginosa isolates PA-762 and PA-M174 and susceptible isolates PA-885 and PA-808 that were annotated to GO terms, with the highest numbers of genes enriched in the molecular function of catalytic activity, high numbers of genes enriched in biological processes of metabolic process, single-organism process and cellular process, and high numbers of genes enriched in cellular components of cell and cell membranes. There were 1 803, 4 084, 3 915 and 4 066 genes in the PA-762, PA-M174, PA-885 and PA-808 isolates enriched in the KEGG signaling pathway, and the majority of genes were enriched in four primary signaling pathways of metabolism, genetic information processing, environmental information processing and cellular process, with the highest number of genes associated with metabolic pathways. Both CAZ-AVI-resistant P. aeruginosa isolates PA-762 and PA-M174 carried multiple efflux pumps systems, including MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM. Single nucleotide substitution was found at position 169 in the DNA sequence of the PA-762 isolate, leading to substitution of serine for glycine at position 57 in the protein sequence, and there are deletions of two bases at positions 307 and 308 in the DNA sequence of the PA-M174 isolate, leading to substitution of threonine for arginine at position 103 in the protein sequence. Conclusion Mutation or downregulation of oprD gene may lead to CAZ-AVI resistance in P. aeruginosa.

Objective:To investigate the value of CT-radiomics based machine learning model in predicting the abundance of tumor infiltrating CD8 +T cells and the prognosis of pancreatic cancer patients. Methods:A total of 150 pancreatic cancer patients who underwent surgical excision and confirmed by pathology from Fudan University Shanghai Cancer Center between December 2011 and January 2017 were retrospectively enrolled. The patients were randomly divided into the training set ( n=105) and the validation set ( n=45) in a 7∶3 ratio with simple random sampling. The immunohistochemical method was used to assess the abundance of tumor infiltrating CD8 +T cells, and the patients were then divided into high infiltrating group ( n=75) and low infiltrating group ( n=75) according to the median. The prognosis between the 2 groups was evaluated using Kaplan-Meier method and log-rank test. Radiomic features were extracted from preoperative venous-phase enhanced CT images in the training set. The Wilcoxon test, the max-relevance and min-redundancy algorithm were used to select the optimal feature set. Three supervised machine learning models (decision tree, random forest and extra tree) were established based on the optimal feature set to predict the abundance of tumor infiltrating CD8 +T cells. Performance of above-mentioned models to predict the abundance of tumor infiltrating CD8 +T cells in pancreatic cancer was tested in the validation set. The evaluation parameters included area under the receiver operating characteristic curve (AUC), F1-score, accuracy, precision and recall. Results:The median overall survival time of patients in high infiltrating group and low infiltrating group were 875 days and 529 days, respectively (χ2=11.53, P<0.001). The optimal feature set consisted of 10 radiomic features in training set. In the validation set, the decision tree, random forest and extra tree model showed the AUC of 0.620, 0.704 and 0.745, respectively; corresponding to a F1-score of 0.457, 0.667 and 0.744, the accuracy of 57.8%, 68.9% and 75.6%, the precision of 66.7%, 73.7% and 80.0%, the recall of 34.8%, 60.9% and 69.6%. Conclusions:Pancreatic cancer patients with high tumor infiltrating CD8 +T cells have better prognosis than those with low tumor infiltrating CD8 +T cells. The radiomics-based extra tree model is valuable in predicting the CD8 +T cells infiltrating level in pancreatic cancer.

OBJECTIVE: To explore the genetic basis for a child with Rothmund-Thomson syndrome (RTS). METHODS: The child has featured poikeloderma, short stature, cataract, sparse hair and skeletal malformation. Peripheral blood samples of the child and her family members were collected and subjected to whole exome sequencing. Candidate variants were verified by Sanger sequencing. RESULTS: The child was found to harbor compound heterozygous variants of the RECQL4 gene, namely c.1048_1049delAG and c.2886-1G>A, among which c.2886-1G>A was unreported previously. According to the ACMG guidelines, the c.1048_1049delAG was predicted to be pathogenic (PVS1+PM3_Strong+PM2), while the c.2886-1G>A was predicted to be likely pathogenic (PVS1+PM2). CONCLUSION The compound heterozygous variants of the RECQL4 gene probably underlay the pathogenesis of RTS in this patient. Above finding has enriched the mutational spectrum of the RECQL4 gene.
Child ; Family ; Female ; Humans ; Mutation ; RecQ Helicases/genetics* ; Rothmund-Thomson Syndrome/genetics* ; Whole Exome Sequencing

Objective: To investigate the clinicopathological features and outcome of gastroenteropancreatic high-grade neuroendocrine tumors. Methods: A total of 60 gastroenteropancreatic high-grade neuroendocrine tumors were collected from January 1st, 2013 to December 31th, 2018 at Fudan University Shanghai Cancer Center, with available pathology databases and clinic follow-up information. At the same time, 157 cases of gastrointestinal pancreatic neuroendocrine neoplasm (NEN) diagnosed at the hospital in 2018 were collected and the incidence of NEN at all grades was compared. Results: There were 32 males and 28 females, aged 13-80 years (mean 54 years). Pancreas primary was the most common (48%, 29/60). Nodal metastatic rate was 9/16 and distant metastatic rate was 41%(18/44). Liver was the most common site of metastasis. Among all the gastroenteropancreatic neuroendocrine neoplasms diagnosed in the hospital in 2018, the incidence of high-grade neuroendocrine tumors was the lowest (7%, 11/157). High-grade neuroendocrine tumors had typical pathologic features of well-differentiated/moderate neuroendocrine tumors, but with significant differences in mitotic rates. By immunohistochemical staining, most of the tumors expressed neuroendocrine markers and somatostatin receptor 2 was positive in 60% (12/20) of the cases. The average Ki-67 index was 30%-40%, and there was significant difference between cases (18%-80%). The overall survival of high-grade neuroendocrine tumors was 43 months, and the disease-free survival was 12 months. Conclusions High-grade neuroendocrine tumor is a rare group of neuroendocrine tumors, with unique clinicopathological features and good prognosis. Pathological classification and grading of gastroenteropancreatic neuroendocrine neoplasms can help clinicians to select appropriate treatment and accurately evaluate prognosis.

Objective:To ascertain the basic situation of radiotherapy in Fujian in 2016.Methods:Based on the unified questionnaire, the types and quantity of radiotherapy and its corollary equipment, the number of radiotherapy staff and the work for quality control were surveyed in the radiotherapy units in the whole province, and the data on patients undergoing radiotherapy and other types of patients were collected from 26 hospitals. The total number of radiotherapy patients in the whole province was estimated by the correlation analysis and multiple linear regression analysis.Results:Radiotherapy was performed in a total of 32 hospitals in Fujian province in 2016. Among them, there were 62 sets of radiotherapy equipment, 33 sets of the simulators, 57 sets of treatment planning systems and 762 workers. The total number of 15 156 radiotherapy patients in 26 hospital were available for the survey. Multiple linear regression models showed that the frequency of application of medical electron accelerator was positively correlated with number of outpatients, emergency patients and inpatients, number of radiotherapy staff, number of electron accelerators ( r=0.311, 0.893, 0.956, P<0.05). Meanwhile, the frequency of aterloading brachytherapy was positively correlated with number of outpatients, emergency patients and inpatients, number of radiotherapy staff, number of aterloading brachytherapy units ( r=0.307, 0.966, 0.988, P<0.05). The frequency of radiotherapy was 0.54 patients per 1 000 population in Fujian in 2016. The delivered dose calibration of accelerators was performed in all hospitals involved in line with the relevant regulations, but the number of quality control equipment for radiotherapy was insufficient, such as the QA beam checker or well-type ionization chamber. Conclusions:In recent years, the rapid development of radiotherapy has been seen in Fujian province. The radiotherapy management should focus on standardenized and improved quality control and regulation in future.

Background and purpose:Tumor budding is a poor prognostic factor in colorectal cancer. In this study, we studied the tumor budding by counting the actual number in 10 high power fields and evaluated itsclinical application in predicting lymph node metastasis of T1 colorectal cancer.Methods:Tissue specimens from 307 patients with histologically conifrmed T1 colorectal cancer were enrolled. The clinicopathological characteristics including tumor budding were evaluated for their predictive value in lymph node metastasis. A formula was created to calculate the risk score for prediction of lymph node metastasis which was validated by 14 new cases.Results:In the multivariate analysis, it showed that tumor grade, lymphovascular invasion and the number of tumor budding were signiifcantly associated with lymph node metastasis. The probability of lymph node metastasis was calculated using the following equations:Z=1.571×(lymphovascular state: invasion, 1; no invasion, 0)+2.661×(tumor grade: high grade, 1; low grade, 0)+0.024×(budding counts)-3.885; Probability=1/1+e-Z. The high scores were correlated with the lymph node metastasis in the validations.Conclusion:We can accurately assess the risk of lymph node metastasis by counting the number of tumor budding in 10 high power fields. Therefore tumor budding could potentially assist treatment decision making in T1 colorectal cancer patients with high-risk lymph node metastasis.

Background and purpose:Metastatic colorectal cancer (mCRC) patients with K-ras mutation won’t benefit in the anti-epidermal growth factor receptor (EGFR) treatments. Thus K-ras mutation analysis is mandatory before this treatment. There is controversy that K-ras mutation analysis should be performed on primaries or related metastases. The aim of our study was to evaluate the concordance of K-ras status between primary and related metastases tumors, thus investigate the validity and rigorousness of clinical K-ras testing. Methods:Seventy-six patients with confirmed mCRC treated in Fudan University Shanghai Cancer Center were enrolled. After DNA extraction and PCR amplification, tumor specimens with paired primary tumors and related metastatic sites were put into sequencing analysis. And the K-ras mutation status in exon 2 was assessed. Results: K-ras mutation was detected in 31 out of 76 primary tumours (40.8%) and also 40.8%of the metastatic sites. But discordance was found between primary tumor and metastasis in 15 cases (19.7%):8 primary tumors had a K-ras mutation with a wild-type metastasis, meanwhile 7 primary tumors were wild type with a K-ras-mutated metastasis. Conclusion:Our study indicated that quite a few mCRC cases have different K-ras status between primary tumors and related metastatic sites, and it’s not very rigorous to choose the anti-EGFR treatments merely according to the primary tumor-K-ras mutation.Further study and consultation are needed on this problem.