[Clinicopathological analysis of gastric adenocarcinoma with elevated serum alpha-fetoprotein and enteroblastic differentiation].

Objective: To investigate the immunophenotypic and molecular biological characteristics of patients with elevated serum alpha-fetoprotein (AFP) and enteroblastic differentiated gastric adenocarcinoma (GAED). Methods: The clinicopathological data of 13 patients with elevated serum AFP and GAED admitted to Shanxi Cancer Hospital from 2018 to 2020 were collected. Immunohistochemistry (IHC) and next-generation sequencing (NGS) were used to analyze the immune markers and molecular biological characteristics of the pathological tissues of the patients. Kaplan-Meier method and log rank test were used for survival analysis. Results: Among the 13 patients with GAED, 12 were male and 1 was female, aged 41-70 years, with a median age of 64 years. The lesions were mainly located in the gastric antrum (5 cases) and gastric body (4 cases). IHC results showed that the tumor embryonic protein (AFP, SALL4, GPC3), intestinal epithelial differentiation protein (CDX-2, CD10), and some original intestinal epithelial phenotype markers (OCT3/4, Claudin6) were expressed in the tumor tissues. Combined application of multiple markers can reduce the rate of missed diagnosis. Among the 13 patients, 12 had at least one mutation (1 mutation: 1 case, 2-5 mutations: 3 cases, 6-15 mutations: 8 cases), and 1 case was not detected. The gene with the highest mutation frequency was TP53 (10 cases), and other mutant genes included EPHB1 (3 cases), ATRX (2 cases), EPHA5 (2 cases), GATA3 (2 cases), LRP1B (2 cases) and MAP2K4 (2 cases) were also detected. Three of the 13 patients had structural variations, which were C14orf177-GNAS, AIM1-FGFR3, and EPHA6-ROS1 gene rearrangements. All 13 patients had copy number variation, and 11 patients had copy number variation of more than 2 genes. The common amplification genes were IRS2 (5 cases), PTEN (5 cases), GNAS (4 cases), CCNE1 (3 cases), CEBPA (3 cases), PCK1 (3 cases) and ERBB2 (2 cases). The common deletion genes were SOX2 (5 cases) and MYC (5 cases). Among the 13 patients, 4 died, and 2 of the dead patients had liver metastasis. There were 4 patients with disease-free survival and 5 patients with disease progression, including 3 cases of abdominal metastasis and 2 cases of liver metastasis. The 3-year survival rate of patients was 65.9 %, and the 3-year progression-free survival rate was 30.7 %. Gene LRP1B point mutation was associated with poor prognosis (P＜0.001). There was no significant improvement in the prognosis of patients treated with immunotherapy compared with those treated with chemotherapy alone (P=0.595), but the prognosis of patients treated with postoperative chemotherapy or postoperative chemotherapy plus immunotherapy was better than that of patients treated with surgery alone (P＜0.05). Conclusions: Elevated serum AFP with GAED is a highly invasive tumor with unique molecular characteristics, often accompanied by multiple molecular events. TP53 mutation is the most common type of gene mutation. In addition, some cases are accompanied by HER2 amplification and gene rearrangement.

Association between remote diffusion-weighted imaging lesions and cerebral small vessel disease in primary intracerebral hemorrhage.

BACKGROUND AND PURPOSE: The aim of this study was to examine the association amongst remote diffusion-weighted imaging lesions (R-DWILs), imaging markers of cerebral small vessel disease (cSVD) and total cSVD burden in patients with primary intracerebral haemorrhage (ICH). METHODS: In total, 344 consecutive primary ICH patients were enrolled prospectively. R-DWILs on magnetic resonance imaging as well as four imaging markers of cSVD, including cerebral microbleeds (CMBs), white matter hyperintensities (WMHs), lacunes and enlarged perivascular spaces, were rated with validated scales. The total cSVD score was calculated by adding up these four markers. Univariate and multivariate analyses were performed. RESULTS: Remote DWI lesions were detected in 57 (16.6%) primary ICH patients. On multivariate logistic regression analysis, the presence of CMBs [odds ratio (OR) 5.26, 95% confidence interval (CI) 1.72-16.12], of high-grade WMHs (OR 4.68, 95% CI 2.01-10.90), the presence of lacunes (OR 2.69, 95% CI 1.20-6.06), mixed CMBs (OR 2.93, 95% CI 1.35-6.36), mixed lacunes (OR 3.60, 95% CI 1.25-10.37), periventricular WMHs (OR 2.19, 95% CI 1.40-3.44), deep WMHs (OR 1.92, 95% CI 1.24-2.97) and total WMHs (OR 1.52, 95% CI 1.20-1.94) were associated with the presence of R-DWILs. A significant association was also found between high-grade total cSVD score and R-DWILs (OR 1.97, 95% CI 1.36-2.84). This association remained significant in patients stratified by an age of 60 years or more than 60 years. CONCLUSIONS: Remote DWI lesions are correlated with the severity of each imaging marker of cSVD and with the total burden of cSVD.

First report of Giardia duodenalis genotypes in Zangxiang pigs from China.

Giardia duodenalis is an important zoonotic intestinal protozoan of animals and humans. We collected 450 faecal specimens from four age groups (pre-weaned piglets, weaned piglets, juveniles, adults) of Zangxiang pigs from Shaanxi and Qinghai provinces, to investigate the prevalence and genetic diversity of G. duodenalis at the ß-giardin (bg), triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh) loci using nested PCRs in the present study. A total of 28 faecal samples were positive for presence of G. duodenalis, with an overall prevalence of 6.2%. Giardia duodenalis was detected in pigs from all age groups and in both investigated provinces. Significant differences (P < 0.0001) in prevalence were observed among the four age groups with prevalence decreasing with age. Sequence analysis indicated existence of genetic diversity of G. duodenalis isolates from Zangxiang pigs, with 4, 2 and 4 haplotypes at the bg, tpi and gdh loci, respectively. Two assemblages were identified, including the zoonotic assemblage B and assemblage E, with the latter as the predominant assemblage found in both locations and all age groups except adults. The present study expanded the host range of G. duodenalis and provided fundamental data for controlling G. duodenalis infection in Zangxiang pigs.

Morphological and molecular confirmation of the validity of Trichuris rhinopiptheroxella in the endangered golden snub-nosed monkey (Rhinopithecus roxellana).

The golden snub-nosed monkey (Rhinopithecus roxellana) is an endangered species endemic to China. Relatively little is known about the taxonomic status of soil-transmitted helminths (STH) in these monkeys. Trichuris spp. (syn. Trichocephalus) are among the most important STHs, causing significant socio-economic losses and public health concerns. To date, five Trichuris species have been reported in golden monkeys, including a novel species, T. rhinopiptheroxella, based on morphology. In the present study, molecular and morphological analysis was conducted on adult Trichuris worms obtained from a dead golden snub-nosed monkey, to better understand their taxonomic status. Morphology indicated that the adult Trichuris worms were similar to T. rhinopiptheroxella. To further ascertain their phylogenetic position, the complete mitochondrial (mt) genome of these worms was sequenced and characterized. The mt genome of T. rhinopiptheroxella is 14,186 bp, encoding 37 genes. Phylogenetic analysis based on the concatenated amino acids of 12 protein-coding genes (with the exception of atp8) indicated that T. rhinopiptheroxella was genetically distinct and exhibited 27.5-27.8% genetic distance between T. rhinopiptheroxella and other Trichuris spp. Our results support T. rhinopiptheroxella as a valid Trichuris species and suggest that mt DNA could serve as a marker for future studies on the classification, evolution and molecular epidemiology of Trichuris spp. from golden snub-nosed monkeys.

Correction to: Molecular characterization of Blastocystis sp. in captive wild animals in Qinling Mountains.

There were errors in Fig. 1 of the originally published article. Correct fig. 1 is presented here.

Molecular characterization of Blastocystis sp. in captive wild animals in Qinling Mountains.

Blastocystis is one common protist inhabiting in gastrointestinal tracts of animals and humans. Examining the subtypes has important implications for assessing the zoonotic potential of Blastocystis and intestinal health of hosts. In the present study, a total of 497 fecal samples collected from 37 wild animal species in Qinling Mountains were investigated for the presence and subtypes of Blastocystis. Of them, 200 (40.2%) were positive for Blastocystis and 13 subtypes were found, including eight known subtypes (STs1-3, 5, 10, 12-14) and five possible novel subtypes (temporarily named as STs18-22), with ST10 as the predominate subtype and the subtype ST5 was detected in an ostrich for the first time. These findings indicated the wide distribution and specific subtype characteristics of Blastocystis in wild animals of Qinling Mountains.

[Effects of perioperative transfusion of blood components on the long-term prognosis of hepatocellular carcinoma].

Objective: To investigate the effects of perioperative transfusion of blood components on the long-term prognosis of hepatocellular carcinoma (HCC) patients. Methods: A total of 339 patients with primary HCC who underwent curative hepatectomy between January 2003 and December 2010 at the Cancer Hospital, Chinese Academy of Medical Sciences were enrolled. The clinical data of the patients were retrospectively analyzed. These patients were divided into non-transfusion, fresh frozen plasma (FFP) transfusion only and concentrated red cells (CRC) transfusion groups. Disease-free survival and overall survival were estimated using the Kaplan-Meier method, and Cox regression was performed to identify clinicopathological factors related with survival. Results: Among the 339 patients, the 1-, 3- and 5-year disease-free survival rates were 63.1%, 35.4% and 22.4%, respectively, and the median disease-free survival was 22 months. While the 1-, 3- and 5-year overall survival rates were 90.5%, 69.5% and 56.4%, respectively, and the median overall survival was 72 months. The median disease-free survivals of the non-transfusion (n=181), FFP transfusion only (n=48) and CRC transfusion (n=110) groups were 28, 22 and 12 months, respectively, while the median overall survivals of the three groups were 99, 63 and 40 months respectively. Significant differences in the disease-free and overall survivals were observed among the three groups (both P<0.01). Multivariate Cox regression analyses showed that FFP transfusion only (HR=1.658, P=0.026), CRC transfusion (HR=1.470, P=0.030), serum alpha-fetoprotein>400 µg/L (HR=1.686, P=0.002), albumin<35 g/L (HR=1.782, P=0.047), tumor capsule (HR=0.597, P=0.012), tumor necrosis (HR=1.820, P=0.001) and the TNM stage Ⅲ or above (HR=2.537, P=0.000) were independent predictors of overall survival after hepatectomy. Conclusion: Both perioperative FFP only transfusion and CRC transfusion may have detrimental effect on the long-term prognosis of HCC.

Multilocus genotyping of Giardia duodenalis and Enterocytozoon bieneusi in dairy and native beef (Qinchuan) calves in Shaanxi province, northwestern China.

Giardia duodenalis and Enterocytozoon bieneusi are two common protozoa that parasitize the intestinal epithelium of animals and humans. Calves have been identified as important reservoirs of these two pathogens, but limited data is available for these two pathogens in calves in China. In the present study, the prevalence and assemblages/genotypes of both parasites in calves of dairy and native beef (Qinchuan) cattle in Shaanxi province, northwestern China, were analyzed using multilocus genotyping (MLST). Of 371 fecal samples collected from calves (including 198 dairy calves and 173 Qinchuan calves), the respective overall prevalence of G. duodenalis and E. bieneusi was 18.87 (70 of 371) and 19.68 % (73 of 371). Both the zoonotic G. duodenalis assemblage A and animal adapted assemblage E were found in dairy and Qinchuan calves. Seventeen, eight, five, and two G. duodenalis subtypes were detected at the triose phosphate isomerase (tpi), ß-giardin (bg), glutamate dehydrogenase (gdh), and small subunit ribosomal RNA (SSU-rRNA) loci, with five and two novel subtypes detected at the tpi and bg loci, forming 25 multiple genotypes (MLGs) (15 and 11 in dairy and Qinchuan calves, respectively). Of 73 samples that were positive for E. bieneusi at the ribosomal RNA internal transcribed spacer (ITS) locus, five ITS genotypes were found, including three known zoonotic genotypes (I, J, CHN1) and two novel genotypes (CSX1 and CSX2). MLST analysis of three microsatellite loci (MS1, MS3, MS7) and one minisatellite locus (MS4) detected six, two, two, and two genotypes at the MS1, MS3, MS4, and MS7 loci, respectively, forming ten MLGs (seven and four in dairy and Qinchuan calves, respectively). These results indicate complex population structures of G. duodenalis and E. bieneusi in calves in Shaanxi province and the zoonotic potential of these two pathogens in calves in this province.

Dynamics of Th17 associating cytokines in Cryptosporidium parvum-infected mice.

Cryptosporidium parvum commonly inhabits the intestinal tract of animals and humans and can cause acute watery diarrhea and weight loss. However, host immune responses to Cryptosporidium infections are not fully understood. IL-17 (also called IL-17A) is a pro-inflammatory cytokine of Th17 cells that plays a role in the host response to Cryptosporidium baileyi infection. The present study examined levels of IL-17-specific messenger RNA (mRNA) and Th17 associating cytokines in C. parvum-infected immune-suppressed BALB/c mice using real-time quantitative PCR (qPCR). Levels of IL-17 protein were determined by ELISA. The results showed that levels of IL-17 mRNA and Th17 cell-related cytokines, namely TGF-ß, IL-6, STAT-3, RORγt, IL-22, TNF-α, and IL-23, were significantly increased (P < 0.05) in gut-associated lymphoid tissue (GALT) and spleen. IL-17 protein levels in GALT were also significantly increased (P < 0.05) after infection. The present study suggested that Th17 cells play a role in host-C. parvum interaction. These results could inform future studies of the immune response against C. parvum infection in transient immunosuppressed populations.

The complete mitochondrial genome of Pseudanoplocephala crawfordi and a comparison with closely related cestode species.

Pseudanoplocephala crawfordi is an important zoonotic cestode of economic significance and public health concern. In spite of its significance as a pathogen, the systematics, genetics, epidemiology and biology of this parasite remain poorly understood. In the present study, we sequenced and characterized the complete mitochondrial (mt) genome of P. crawfordi, which is 14,192 bp long and encodes 36 genes, including 12 protein-coding genes, 22 transfer RNA genes and two ribosomal RNA genes. Phylogenetic analysis of the concatenated amino acid sequences using the Bayesian inference (BI) method showed that P. crawfordi was closely related to the family Hymenolepididae. Considering that the taxonomic status of P. crawfordi has been controversial when based only on morphological features, the mt genome obtained here will provide novel molecular markers to ascertain the phylogenetic position of this parasite accurately.

Molecular approaches to differentiate three species of Nematodirus in sheep and goats from China based on internal transcribed spacer rDNA sequences.

Internal transcribed spacer (ITS) rDNA sequences of three Nematodirus species from naturally infected goats or sheep in two endemic provinces of China were analysed to establish an effective molecular approach to differentiate Nematodirus species in small ruminants. The respective intra-specific genetic variations in ITS1 and ITS2 rDNA regions were 0.3-1.8% and 0-0.4% in N. spathiger, 0-6.5% and 0-5.4% in N. helvetianus, and 0-4.4% and 0-6.1% in N. oiratianus from China. The respective intra-specific variations of ITS1 and ITS2 were 1.8-4.4% and 1.6-6.1% between N. oiratianus isolates from China and Iran, 5.7-7.1% and 6.3-8.3% between N. helvetianus samples from China and America. For N. spathiger, compared with samples from China, sequence differences in ITS1 rDNA were 0.3-2.4% in isolates from America, 0.3-2.9% in New Zealand and 2.1-2.4% in Australia. Genetic variations in ITS2 rDNA of N. spathiger were 0-0.4% between samples from China and America, and 0-0.8% between samples from China and New Zealand. Using mutation sites, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and specific PCR techniques were developed to differentiate these three Nematodirus species. The specific PCR assay allowed the accurate identification of N. oiratianus from other common nematodes with a sensitivity of 0.69 pg and further examination of Nematodirus samples demonstrated the reliability of these two molecular methods.

Characterization of Dicrocoelium dendriticum isolates from small ruminants in Shaanxi Province, north-western China, using internal transcribed spacers of nuclear ribosomal DNA.

The genetic variations in internal transcribed spacers (ITS) spanning ITS-1, 5.8S and ITS-2 rDNA of Dicrocoelium dendriticum, isolated from sheep and goats in four geographical regions in Shaanxi province, were examined. The lengths of ITS-1, 5.8S and ITS-2 rDNA sequences for D. dendriticum were 749 bp, 161 bp and 234 bp, respectively. Intra-specific sequence variations of D. dendriticum were 0-0.5% for ITS-1 and 0-1.3% for ITS-2 rDNA, while the inter-specific variations among species in genus Dicrocoelium in ITS-2 rDNA were 3.4-12.3%. Phylogenetic analysis based on sequences of ITS-2 rDNA showed that all D. dendriticum isolates in the present study were grouped with reference D. dendriticum isolates from sheep and goats, and D. dendriticum isolates from cattle and Japanese serow were clustered in a sister clade. However, the phylogenetic tree could not reveal geographically genetic relationships of D. dendriticum isolates in different origins and hosts. These findings provided basic information for further study of molecular epidemiology and control of D. dendriticum infection in Shaanxi province as well as in the world.

Characterization of Trichuris trichiura from humans and T. suis from pigs in China using internal transcribed spacers of nuclear ribosomal DNA.

Trichuris trichiura and Trichuris suis parasitize (at the adult stage) the caeca of humans and pigs, respectively, causing trichuriasis. Despite these parasites being of human and animal health significance, causing considerable socio-economic losses globally, little is known of the molecular characteristics of T. trichiura and T. suis from China. In the present study, the entire first and second internal transcribed spacer (ITS-1 and ITS-2) regions of nuclear ribosomal DNA (rDNA) of T. trichiura and T. suis from China were amplified by polymerase chain reaction (PCR), the representative amplicons were cloned and sequenced, and sequence variation in the ITS rDNA was examined. The ITS rDNA sequences for the T. trichiura and T. suis samples were 1222-1267 bp and 1339-1353 bp in length, respectively. Sequence analysis revealed that the ITS-1, 5.8S and ITS-2 rDNAs of both whipworms were 600-627 bp and 655-661 bp, 154 bp, and 468-486 bp and 530-538 bp in size, respectively. Sequence variation in ITS rDNA within and among T. trichiura and T. suis was examined. Excluding nucleotide variations in the simple sequence repeats, the intra-species sequence variation in the ITS-1 was 0.2-1.7% within T. trichiura, and 0-1.5% within T. suis. For ITS-2 rDNA, the intra-species sequence variation was 0-1.3% within T. trichiura and 0.2-1.7% within T. suis. The inter-species sequence differences between the two whipworms were 60.7-65.3% for ITS-1 and 59.3-61.5% for ITS-2. These results demonstrated that the ITS rDNA sequences provide additional genetic markers for the characterization and differentiation of the two whipworms. These data should be useful for studying the epidemiology and population genetics of T. trichiura and T. suis, as well as for the diagnosis of trichuriasis in humans and pigs.

Characterization of Oesophagostomum asperum and O. columbianum by internal transcribed spacers of nuclear ribosomal DNA.

In the present study, the internal transcribed spacers (ITS) of ribosomal DNA (rDNA) of Oesophagostomum asperum and O. columbianum were amplified and sequenced. The ITS-1, 5.8S and ITS-2 rDNA sequences of O. asperum were 374 bp, 153 bp and 259 bp in length, respectively, and the corresponding sequences of O. columbianum were 259, 153 and 218 bp in length, respectively. Sequence differences in the ITS-1 and ITS-2 rDNA between the two Oesophagostomum species were 9.5-10.2% and 12.7-13.9%, respectively. Sequence differences in the ITS-1 and ITS-2 rDNA among members of the genus Oesophagostomum were 2.5-11.6% and 6.8-22.3%, respectively. Based on genetic markers in the ITS rDNA, an effective polymerase chain reaction (PCR) approach was developed to differentiate O. columbianum from O. asperum with a sensitivity of 0.2 ng/µl DNA. Since accurate characterization of parasites at different taxonomic levels is essential for population genetic studies and control of parasitosis, the present findings have important implications for studying epidemiology, taxonomy and population biology, as well as for the control of oesophagostomiasis.

[Reassessment of practice of Chinese surgeons since introduction of the watch and wait strategy after neoadjuvant therapy for rectal cancer].

Objective: To investigate perspectives and changes in treatment selection by Chinese surgeons since introduction of the watch-and-wait approach after neoadjuvant therapy for rectal cancer. Methods: A cross-sectional survey was conducted using a questionnaire distributed through the "Wenjuanxing" online survey platform. The survey focused on the recognition and practices of Chinese surgeons regarding the strategy of watch-and-wait after neoadjuvant therapy for rectal cancer and was disseminated within the China Watch-and-Wait Database (CWWD) WeChat group. This group targets surgeons of deputy chief physician level and above in surgical, radiotherapy, or internal medicine departments of nationally accredited tumor-specialist or comprehensive hospitals (at provincial or municipal levels) who are involved in colorectal cancer diagnosis and treatment. From 13 to 16 December 2023, 321 questionnaires were sent with questionnaire links in the CWWD WeChat group. The questionnaires comprised 32 questions encompassing: (1) basic physician characteristics (including surgical volume); (2) assessment methods and criteria for clinical complete response (cCR); (3) patients eligible for watch-and-wait; (4) neoadjuvant therapies and other measures for achieving cCR; (5) willingness to implement watch-and-wait and factors influencing that willingness; (6) risks and monitoring of watch-and-wait; (7) subsequent treatment and follow-up post watch-and-wait; (8) suggestions for development of the CWWD. Descriptive statistics were employed for data analysis, with intergroup comparisons conducted using the χ2 or Fisher's exact probability tests. Results: The response rate was 31.5%, comprising 101 responses from the 321 individuals in the WeChat group. Respondents comprised 101 physicians from 70 centers across 23 provinces, municipalities, and autonomous regions nationwide, 85.1% (86/101) of whom represented provincial tertiary hospitals. Among the respondents, 87.1% (88/101) had implemented the watch-and-wait strategy. The approval rate (65.6%, 21/32) and proportion of patients often informed (68.8%, 22/32) were both significantly higher for doctors in oncology hospitals than for those in general hospitals (27.7%, 18/65; 32.4%, 22/68) (χ2=12.83, P<0.001; χ2=11.70, P=0.001, respectively). The most used methods for diagnosing cCR were digital rectal examination (90.1%, 91/101), colonoscopy (91.1%, 92/101), and rectal T2-weighted magnetic resonance imaging (86.1%, 87/101). Criteria used to identify cCR comprised absence of a palpable mass on digital rectal examination (87.1%, 88/101), flat white scars or new capillaries on colonoscopy (77.2%, 78/101), absence of evident tumor signals on rectal T2-weighted sequences or T2WI low signals or signals equivalent to the intestinal wall (83.2%, 84/101), and absence of tumor hyperintensity on diffusion-weighted imaging with no corresponding hypointensity on apparent diffusion coefficient maps (66.3%, 67/101). As for selection of neoadjuvant regimen and assessment of cCR, 57.4% (58/101) of physicians preferred a long course of radiotherapy with or without induction and/or consolidation capecitabine + oxaliplatin, whereas 25.7% (26/101) preferred immunotherapy in combination with chemotherapy and concurrent radiotherapy. Most (96.0%, 97/101) physicians believed that the primary lesion should be assessed ≤12 weeks after completion of radiotherapy. Patients were frequently informed about the possibility of achieving cCR after neoadjuvant therapy and the strategy of watch-and-wait by 43.6% (44/101) of the responding physicians and 38.6% (39/101) preferred watch-and-wait for patients who achieved cCR or near cCR after neoadjuvant therapy for rectal cancer. Capability for multiple follow-up evaluations (70.3%, 71/101) was a crucial factor influencing physicians' choice of watch-and-wait after cCR. The proportion who patients who did not achieve cCR and underwent surgical treatment was lower in provincial tertiary hospitals (74.2%, 23/31) than in provincial general hospitals (94.5%, 52/55) and municipal hospitals (12/15); these differences are statistically significant (χ2=7.43, P=0.020). The difference between local recurrence and local regrowth was understood by 88.1% (89/101) of respondents and 87.2% (88/101) agreed with monitoring every 3 months for 5 years. An increase in local excision or puncture rates to reduce organ resections in patients with pCR was proposed by 64.4% (65/101) of respondents. Conclusion: Compared with the results of a previous survey, Chinese surgeons' awareness of the watch-and-wait concept has improved significantly. Oncologists in oncology hospitals are more aware of the concept of watch-and-wait.

[Long-term outcome of patients with rectal cancer who achieve complete or near complete clinical responses after neoadjuvant therapy: a multicenter registry study of data from the Chinese Watch and Wait Database].

Objective: To report the long-term outcomes of Chinese rectal cancer patients after adopting a Watch and Wait (W&W) strategy following neoadjuvant therapy (NAT). Methods: This multicenter, cross-sectional study was based on real-world data. The study cohort comprised rectal cancer patients who had achieved complete or near complete clinical responses (cCRs, near-cCRs) after NAT and were thereafter managed by a W&W approach, as well as a few patients who had achieved good responses after NAT and had then undergone local excision for confirmation of pathological complete response. All participants had been followed up for ≥2 years. Patients with distant metastases at baseline or who opted for observation while living with the tumor were excluded. Data of eligible patients were retrospectively collected from the Chinese Wait-and-Watch Data Collaboration Group database. These included baseline characteristics, type of NAT, pre-treatment imaging results, evaluation of post-NAT efficacy, salvage measures, and treatment outcomes. We herein report the long-term outcomes of Chinese rectal cancer patients after NAT and W&W and the differences between the cCR and near-cCR groups. Results: Clinical data of 318 rectal cancer patients who had undergone W&W for over 2 years and been followed up were collected from eight medical centers (Peking University Cancer Hospital, Fudan University Shanghai Cancer Center, Sun Yat-sen University Cancer Center, Shanghai Changhai Hospital, Peking Union Medical College Hospital, Liaoning Cancer Hospital, the First Hospital of Jilin University, and Yunnan Cancer Hospital.) The participants comprised 221 men (69.4%) and 107 women (30.6%) of median age 60 (26-86) years. The median distance between tumor and anal verge was 3.4 (0-10.4) cm. Of these patients, 291 and 27 had achieved cCR or near-cCR, respectively, after NAT. The median duration of follow-up was 48.4 (10.2-110.3) months. The 5-year cumulative overall survival rate was 92.4% (95%CI: 86.8%-95.7%), 5-year cumulative disease-specific survival (CSS) rate 96.6% (95%CI: 92.2%-98.5%), 5-year cumulative organ-preserving disease-free survival rate 86.6% (95%CI: 81.0%-90.7%), and 5-year organ preservation rate 85.3% (95%CI: 80.3%-89.1%). The overall 5-year local recurrence and distant metastasis rates were 18.5% (95%CI: 14.9%-20.8%) and 8.2% (95%CI: 5.4%-12.5%), respectively. Most local recurrences (82.1%, 46/56) occurred within 2 years, and 91.0% (51/56) occurred within 3 years, the median time to recurrence being 11.7 (2.5-66.6) months. Most (91.1%, 51/56) local recurrences occurred within the intestinal lumen. Distant metastases developed in 23 patients; 60.9% (14/23) occurred within 2 years and 73.9% (17/23) within 3 years, the median time to distant metastasis being 21.9 (2.6-90.3) months. Common sites included lung (15/23, 65.2%), liver (6/23, 26.1%), and bone (7/23, 30.4%) The metastases involved single organs in 17 patients and multiple organs in six. There were no significant differences in overall, cumulative disease-specific, or organ-preserving disease-free survival or rate of metastases between the two groups (all P>0.05). The 5-year local recurrence rate was higher in the near-cCR than in the cCR group (41.6% vs. 16.4%, P<0.01), with a lower organ preservation rate (69.2% vs. 88.0%, P<0.001). The success rates of salvage after local recurrence and distant metastasis were 82.1% (46/56) and 13.0% (3/23), respectively. Conclusion: Rectal cancer patients who achieve cCR or near-cCR after NAT and undergo W&W have favorable oncological outcomes and a high rate of organ preservation. Local recurrence and distant metastasis during W&W follow certain patterns, with a relatively high salvage rate for local recurrence. Our findings highlight the importance of close follow-up and timely intervention during the W&W process.

The second transcribed spacer rDNA sequence: an effective genetic marker for inter-species phylogenetic analysis of trematodes in the order Strigeata.

In the present study, the second nuclear internal transcribed spacer (ITS-2) rDNA of Schistosoma japonicum isolates in mainland China was amplified, sequenced, and assessed for inferring the intra- and inter-species phylogenetic relationships of trematodes in the order Strigeata. The fragment containing ITS-2 rDNA was obtained from 24 S. japonicum isolates from eight epidemic provinces in mainland China. The length polymorphisms were observed among these ITS-2 rDNA sequences, ranging from 343 to 346 bp, and the intra- and inter-population variations in ITS-2 sequence were 0.0-2.1% among S. japonicum isolates in China. Phylogenetic analyses using the maximum parsimony and maximum likelihood methods revealed that the ITS-2 rDNA sequence is not a suitable marker for studying inter- and intra-population variation in S. japonicum. However, phylogenetic analysis of trematodes in the order Strigeata indicated that the ITS-2 rDNA sequence provides an effective molecular marker for studying inter-species phylogenetic relationships among trematodes in this order.

Characterization of Baylisascaris schroederi from Qinling subspecies of giant panda in China by the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA.

In the present study, a total of 20 nematode isolates, (including 10 male and 10 female worms) representing Baylisascaris schroederi from 5 Qinling subspecies of giant pandas (Ailuropoda melanoleuca) in Shaanxi Province of China, were characterized and grouped genetically by the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA). The rDNA fragment spanning 3' end of 18S rDNA, complete ITS-1 rDNA, and 5' end of 5.8S rDNA were amplified and sequenced. The sequence variability in ITS-1 rDNA was examined within B. schroederi and among parasites in order Ascaridata available in GenBank™, and their phylogenetic relationships were also reconstructed. The sequences of ITS-1 rDNA for all the B. schroederi isolates were 427 bp in length, with no genetic variation detected among these isolates. Phylogenetic analyses based on the ITS-1 rDNA sequences revealed that all the male and female B. schroederi isolates sequenced in the present study were posited into the clade of genus Baylisascaris, sistered to zoonotic nematodes in genus Ascaris, and the ITS-1 rDNA sequence could distinguish different species in order Ascaridata. These results showed that the ITS-1 rDNA provides a suitable molecular marker for the inter-species phylogenetic analysis and differential identification of nematodes in order Ascaridata.

Combined mitochondrial 16S and 12S rDNA sequences: an effective genetic marker for inter-species phylogenetic analysis of zoonotic trematodes.

The present study studied the genetic variation among Schistosoma japonicum isolates from different endemic regions in mainland China and examined the phylogenetic relationships of zoonotic trematodes using the combined mitochondrial 16S and 12S ribosomal DNA sequences. The fragments of 16S and 12S rDNA were amplified from 22 S. japonicum isolates, and sequenced, and the relevant sequences of other nine trematode species belonging to six genera in four families were downloaded from GenBank, and their phylogenetic relationships were re-constructed by unweighted pair-group method with arithmetic averages analyses using the combined 16S and 12S rDNA sequences, with Trichinella spiralis as outgroup. The results showed that the partial sequences of mitochondrial 16S and 12S rDNA of S. japonicum were 757 and 797 bp, respectively, and they were quite conserved among the S. japonicum isolates. Phylogenetic analysis revealed that the combined 16S and 12S rDNA sequences were not able to distinguish S. japonicum isolates in mountainous areas from those in lake/marshland areas in mainland China. However, the combined sequences could distinguish different species of zoonotic trematodes. Therefore, the combined mitochondrial 16S and 12S rDNA sequences provide an effective molecular marker for the inter-species phylogenetic analysis and differential identification of zoonotic trematodes.