Application of immune checkpoint inhibitors and microsatellite instability in gastric cancer

In this editorial we comment on the article by Li published in the recent issue of the World Journal of Gastroenterology.We focus specifically on the application of immune checkpoint inhibitors(ICIs)and microsatellite instability(MSI)in gastric cancer(GC).The four pillars of GC management have long been considered,including surgery,chemotherapy,radiotherapy and targeted therapy.However,immunotherapy has recently emerged as a“fifth pillar”,and its use is rapidly expanding.There are four principal strategies for tumor immunotherapy:ICIs,tumor vaccines,adoptive immunotherapy and nonspecific immunomodulators.Of them,ICIs are the most advanced and widespread type of cancer immunotherapy for GC.Recent breakthrough results for ICIs have paved the way to a new era of cancer immunotherapy.In particular,inhibition of the PD-1/PD-L1 axis with ICIs,including nivolumab and pembrolizumab,has emerged as a novel treatment strategy for advanced GC.Unfortunately,these therapies are sometimes associated with often subtle,potentially fatal immune-related adverse events(irAEs),including dermatitis,diarrhea,colitis,endocrinopathy,hepatotoxicity,neuropathy and pneumonitis.We must be aware of these irAEs and improve the detection of these processes to prevent inappropriate discharges,emergency department revisits,and downstream complications.Recent studies have revealed that MSI-high or mismatch-repair-deficient tumors,regardless of their primary site,have a promising response to ICIs.So,it is important to detect MSI before applying ICIs for treatment of GC.

Tislelizumab in previously treated,locally advanced unresectable/metastatic microsatellite instability-high/mismatch repair-deficient solid tumors

Objective:The open-label,phase II RATIONALE-209 study evaluated tislelizumab(anti-programmed cell death protein 1 antibody)as a tissue-agnostic monotherapy for microsatellite instability-high(MSI-H)/mismatch repair-deficient(dMMR)tumors.Methods:Adults with previously treated,locally advanced unresectable or metastatic MSI-H/dMMR solid tumors were enrolled.Patients received tislelizumab 200 mg intravenously every 3 weeks.Objective response rate(ORR;primary endpoint),duration of response(DoR),and progression-free survival(PFS)were assessed by independent review committee(Response Evaluation Criteria in Solid Tumors v1.1).Results:Eighty patients were enrolled and treated;75(93.8%)patients had measurable disease at baseline.Most had metastatic disease and received at least one prior therapy for advanced/metastatic disease(n=79;98.8%).At primary analysis(data cutoff July 8,2021;median follow-up 15.2 months),overall ORR[46.7%;95%confidence interval(95%CI),35.1−58.6;one-sided P<0.0001]and ORR across tumor-specific subgroups[colorectal(n=46):39.1%(95%CI,25.1–54.6);gastric/gastroesophageal junction(n=9):55.6%(95%CI,21.2−86.3);others(n=20):60.0%(95%CI,36.1−80.9)]were significantly greater with tislelizumab vs.a prespecified historical control ORR of 10%;five(6.7%)patients had complete responses.Median DoR,PFS,and overall survival were not reached with long-term follow-up(data cutoff December 5,2022;median follow-up 28.9 months).Tislelizumab was well tolerated with no unexpected safety signals.Treatment-related adverse events(TRAEs)of grade≥3 occurred in 53.8%of patients;7.5%of patients discontinued treatment due to TRAEs.Conclusions:Tislelizumab demonstrated a significant ORR improvement in patients with previously treated,locally advanced unresectable or metastatic MSI-H/dMMR tumors and was generally well tolerated.

Comparative effectiveness of immunotherapy and chemotherapy in patients with metastatic colorectal cancer stratified by microsatellite instability status

BACKGROUND Immunotherapy have demonstrated promising outcomes in patients with high microsatellite instability(MSI)(MSI-H)metastatic colorectal cancer.However,the comparative effectiveness of Immunotherapy and chemotherapy for patients with low MSI(MSI-L),and microsatellite stable(MSS)metastatic colorectal cancer remains unclear.AIM To investigate immunotherapy vs chemotherapy for treatment of MSI-L/MSS metastatic colorectal cancer,and to evaluate the success of immunotherapy against chemotherapy in managing MSI-H metastatic colorectal cancer during a follow-up of 50 months.METHODS We conducted a retrospective cohort study using the National Cancer Database(NCDB)to evaluate the overall survival(OS)of patients with metastatic colorectal cancer treated with immunotherapy or chemotherapy.The study population was stratified by MSI status(MSI-H,MSI-L,and MSS).Multivariable Cox proportional hazard models were used to assess the association between treatment modality and OS,adjusting for potential confounders.RESULTS A total of 21951 patients with metastatic colorectal cancer were included in the analysis,of which 2358 were MSI-H,and 19593 were MSI-L/MSS.In the MSI-H cohort,immunotherapy treatment(n=142)was associated with a significantly improved median OS compared to chemotherapy(n=860).After adjusting for potential confounders,immunotherapy treatment remained significantly associated with better OS in the MSI-H cohort[adjusted hazard ratio(aHR):0.57,95%confidence interval(95%CI):0.43-0.77,P<0.001].In the MSS cohort,no significant difference in median OS was observed between immunotherapy treatment and chemotherapy(aHR:0.94,95%CI:0.69-1.29,P=0.715).CONCLUSION In this population-based study using the NCDB,immunotherapy treatment was associated with significantly improved OS compared to chemotherapy in patients with MSI-H metastatic colorectal cancer,but not in those with MSI-L/MSS metastatic colorectal cancer.Further studies are warranted to determine the optimal therapeutic approach for patients with MSI-L/MSS metastatic colorectal cancer.

Microsatellite Variations Among Four Populations of Eriocheir sinensis

Using microsatellite markers, we analysed genetic polymorphism in four populations of Eriocheir sinensis, sampled from Jiangsu, Anhui, Liaoning and Tianjin. Twenty-four pairs of primers were used to amplify the target fragments ranging from 80 bp to 445 bp, which included 16 pairs designed in our laboratory and eight pairs published internationally. Two to 10 alleles per locus in four populations were amplified, and there were 155 alleles in all populations. The average number of alleles per locus was 6.458. The average number of effective alleles per locus （Ne） was 4.349l to 4.7234; the average observed heterozygosity （Ho） was 0.5690 to 0.6722; and the average expected heterozygosity （He） was 0.7238 to 0.7546. Hardy-Weinberg equilibrium analysis （Z2 test, P 〈 0.05） revealed that seven loci in the four populations were in equilibrium. The genetic distances between the four populations were calculated and revealed that the Anhui, Jiangsu and Tianjin crabs belong to a Yangtse River population, while the Liaohe crabs form another branch.

Comparative Microsatellite Analysis of Grass Carp Genomes of Two Gynogenetic Groups and the Xiangjiang River Group

The genomes of three groups of grass carp, namely the Xiangjiang River grass carp group （Xiangjiang group）, a one-generation artificially induced meio-gynogenetic grass carp group （meio-gynogenetic-1 group）, and a two-generation artificially induced meio-gynogenetic grass carp group （meio-gynogenetic-2 group）, were comparatively analyzed with microsatellite markers. Genetic polymorphism had been observed in the Xiangjiang group and most of the examined loci had more than two alleles. But the degree of genetic diversity was not very high. Although all the examined genetic loci in the analyzed individuals were in homozygous state, the genotypes of different individuals of the group were not identical in the meio-gynogenetic-1 group. In the meio-gynogenetic-2 group, not only the examined genetic loci of each individual were homozygous but also the genotypes of all the analyzed individuals of the group were the same. These results suggested that the examined meio-gynogenetic-2 group is a homozygous group and homozygous clone could be produced by continuous artificial induction of gynogenesis for two generations. It was found that the polymorphism existed not only at the allele level but also at the locus level; many alleles of the microsatellite loci and some of the microsatellite loci had been lost during the process of artificial gynogenesis. Therefore, both protection of the diversity of natural grass carp resource and selection of homozygous traits with desired economic genotypes are very important aspects for grass carp breeding.

The Identification and Assessment on Genetic Characteristics in Grading Breeding Sheep Populations with Microsatellite Markers

[Objective] The aim of the research was to identify and assess the genetic characteristics of grading breeding sheep populations in Ba Yan Nur City. [Method] Genetic polymorphism and aggregation of seven sheep populations, including three breeding sheep populations (breeding F1, F2 and Bamei mutton sheep), three introduced mutton sheep breeds (Texel, Dorset and German Merino sheep) and one local female parent population (Mongolia sheep), were assessed using 10 microsatellite markers. [Result] By cluster analysis, the seven sheep populations can be divided into two groups. The F1 and German Merino sheep were closely related, which were clustered with F2, Bamei mutton sheep and Mongolia sheep to form one group while Texel and Dorset to form another group. The genetic aggregation of the seven breeds was assessed by Bayesian discrimination. And the results show that the genetic aggregation of F1 and F2 were lower while that of Bamei mutton sheep, Texel, Dorset and German Merino sheep were higher. [Conclusion] Better genetic stability has been formed in Bamei mutton sheep.

Microsatellite DNA Marker Analysis of Genetic Diversity in Wild Common Carp (Cyprinus carpio L.) Populations

Thirty microsatellite loci were used for analyzing six wild populations of common carp （Cyprinus carpio L.）. Observed （Ho） and expected （He） heterozygosity values, polymorphic information content （PIC）, and number of effective alleles （Ae） were all detected. Genetic similarity index and genetic distance were computed based on the allele frequency. The Hardy-Weinberg Equilibrium was checked according to the test of χ^2. Genetic differentiation and hierarchical partition of genetic diversity were evaluated by FST and Nm. A clustering dendrograrn was made based on the results of UPGMA methods using the PHYLIP software package （version 3.63）. There were totally 8,136 fragments ranging from 125 bp to 414 bp in length. Three to thirteen alleles were amplified in 30 loci and 210 alleles in all six populations. The average number of alleles in each locus was seven. The result showed that 1） the level of genetic variability was moderate in the six populations. Polymorphic information contents of the six wild common carp populations were 0.44, 0.52, 0.53, 0.57, 0.63, and 0.64 respectively. Effective alleles were from 1.04 to 4.72, the average numbers in each population were 2.19, 2.60, 2.42, 2.43, 2.45, and 2.33. The average expected heterozygosity values were 0.50, 0.59, 0.56, 0.56, 0.57, and 0.54 respectively; 2） the highest genetic similarity index that came from the populations of BR and ZL was 0.8511 and the lowest index was 0.6688, and it came from the populations of BR and HN. There was a correlation between the clustering result and the geographical distribution.

Genetic Diversity of Microsatellite DNA Loci of Tibetan Antelope(Chiru,Pantholops hodgsonii)in Hoh Xil National Nature Reserve,Qinghai,China

The Tibetan antelope （Pantholops hodgsonii）, indigenous to China, became an endangered species because of considerable reduction both in number and distribution during the 20th century. Presently, it is listed as an Appendix Ⅰ species by CITES and as Category I by the Key Protected Wildlife List of China. Understanding the genetic diversity and population structure of the Tibetan antelope is significant for the development of effective conservation plans that will ensure the recovery and future persistence of this species. Twenty-five microsatellites were selected to obtain loci with sufficient levels of polymorphism that can provide information for the analysis of population structure. Among the 25 loci that were examined, nine of them showed high levels of genetic diversity. The nine variable loci （MCM38, MNS64, IOBT395, MCMAL TGLA68, BM1329, BMSI341, BM3501, and MB066） were used to examine the genetic diversity of the Tibetan antelope （n = 75） in Hoh Xil National Nature Reserve（HXNNR）, Qinghai, China. The results obtained by estimating the number of population suggested that all the 75 Tibetan antelope samples were from the same population. The mean number of alleles per locus was 9.4 ± 0.5300 （range, 7-12） and the mean effective number of alleles was 6.519± 0.5271 （range, 4.676-9.169）. The observed mean and expected heterozygosity were 0.844 ± 0.0133 （range, 0.791-0.897） and 0.838 ± 0.0132 （range, 0.786-0.891）, respectively. Mean Polymorphism Information Content （PIC） was 0.818 ± 0.0158 （range, 0.753-0.881）. The value of Fixation index （Fis） ranged from -0.269 to -0.097 with the mean of -0.163 ± 0.0197. Mean Shannon＇s information index was 1.990 ± 0.0719 among nine loci （range, 1.660-2.315）. These results provide baseline data for the evaluation of the level of genetic variation in Tibetan antelope, which will be important for the development of conservation strategies in future.

Isolation of CA/GT Microsatellites from the Paralichthys olivaceus Genome

A library rich in CA/GT microsatellites was constructed from the Paralichthys olivaceus genome by combining biotin capture method and radioactive labeling hybridization. Five hundred and twenty six positive clones were obtained through twice screens. Sequencing confirmed 133 microsatellite loci （number of repeats t〉 5） in 119 positive clones. Of these microsatellites, two （1.5%） had compound repeat motifs, 63 （47.37%） had perfect motifs and 68 （51.13%） had imperfect motifs. Primer pairs were designed in the flanking regions of 22 microsatelites and subjected to PCR amplification. In 8 artificial gynogenesis families, four pairs failed to amplification, one pair was monomorphic, and the rest were polymorphic with an average of 5.2 alleles per locus. Heterozygosities ranged between 0. 375 and 0. 846, PIC ranged between 0. 305 and 0. 823. The results suggested that most of the microsatellites we isolated were qualified to be applied to the population genetic studies of P. olivaceus.

Searching for Protein-coding Genes Using Microsatellites in Common Carp by Comparing to Zebrafish EST Database

In this study, an in silico approach was utilized to identify homologies existing between common carp microsatellite sequences and GenBank database using Blastn and Blastx searches. About 875 microsatellite sites with flanking sequences over 50bp of common carp were first compared to the zebrafish EST database. The results showed that 121 homologies were found using Blastn. Subsequent Blastx searches confirmed 94 sites recorded in the protein database. Except for 33 hypothetical proteins and three unknown proteins, seven out of 58 characterized proteins have been mapped to two linkage maps. In addition, two polymorphic STS markers were developed using matched zebrafish EST sequences by PCR-SSCP method, of which one marker HLJZe33 was mapped successfully. This study was a pilot for comparative studies between common carp and zebrafish, and the results demonstrated that more genetic and genomic resources of zebrafish can be used for the genome research of common carp.

Study on Population Genetic Characteristics of Qinchuan Cows Using Microsatellite Markers

To evaluate the genetic polymorphisms and to search for available molecular markers for Qinchuan cattle, 90 Qinchuan cows were genotyped with 12 microsatellite markers. A total of 247 alleles were detected, with the number of alleles ranging from 13 （INRA005） to 33 （HEL13）, giving a mean number of 21 alleles per locus. The total and mean effective allele number were 142.6229 and 11.8852, respectively. Mean sampling variance of the allele frequency was 2.6036 × 10^-4. Allele size ranges of the 12 microsatellite loci were different. The observed heterozygosity and expected heterozygosity were from 0.7842 （INRA005） to 0.9775 （BM315） and 0.7952 （BM315） to 0.9446 （HEL13）, respectively. Mean observed heterozygosity and mean expected heterozygosity were 0.9117 and 0.9047, respectively. Polymorphism information content values were from 0.7653 （INRA005） to 0.9420 （HEL13）, and mean polymorphism information content of the 12 microsatellite loci was 0.8965. All the 12 microsatellite loci were highly polymorphic, which showed that there were rich genetic polymorphisms at these detected microsatellite loci in Qinchuan cows. At the 12 microsatellite loci, the mean fixation index was -0.0076, reflecting that the degree of heterozygote defect at these loci was not high and deviations from Hardy-Weinberg equilibrium were not significant.

Genetic Analysis of Six Endangered Local Duck Populations in China Based on Microsatellite Markers

Seventeen microsatellite loci were used to assess the genetic variation and population structure of six endangered duck populations in China. PIC, heterozygosity with two estimators of genetic differentiation （FST and GST）, and Nei＇s standard genetic distance were evaluated. The results showed that these six endangered duck populations showed high polymorphism. The propor- tion of inter-population subdivision among the six duck populations ranged between 17.0 and 14.7%. The average heterozygosity was 0.706, 0.702, 0.691, 0.676, 0.604, and 0.660 in the Gaoyou, Liancheng, Jinding, Beijing, Shaoxing, and Jianchang ducks, respectively. The average PIC ranged from 0.561 to 0.663. Finally, the results showed higher genetic diversity and difference above the levels of genetic variation among all the populations.

Isolation and Characterization of Microsatellite DNA Markers from Forest Musk Deer(Moschus berezovskii)

In this report, we describe the isolation and characterization of microsatellite loci for forest musk deer （Moschus berezovskii） through an improved enrichment protocol. Five new markers were isolated from the genomic DNA of forest musk deer and showed high polymorphism with 4 - 13 alleles in 24 sampled individuals from the population of Jinfeng Mountain, Sichuan Province, China. The observed and expected heterozygesities were from 0.429 - 0.957 and 0.587 - 0.902, respectively. The average polymorphism information content （PIC） value in these five loci was 0. 730. This suggests that the five microsatellite loci are a valuable tool for further studies about forest musk deer.

Molecular Tagging of a New Resistance Gene to Maize Dwarf Mosaic Virus Using Microsatellite Markers

With joint analysis based on the parents, F 1, F 2 and backcrosses, the authors found that the resistance of the maize inbred line Huangzaosi to the maize dwarf mosaic virus strain B was conditioned by a major gene and polygene, and identified a new major gene. Bulked segregate and microsatellite analysis of a F 2 progeny from the combination of Huangzaosi×Mo17 were used to identify the resistance gene, mdm1(t), on the long arm of chromosome 6. This new resistance gene is tightly linked to and located between the microsatellite markers loci, phi077 and bnlg391. The linkage distances between phi077-mdm1(t) and mdm1(t)-bnlg391 are 4.74 centiMorgan (cM) and 6.72 cM respectively.

Genetic Variation of Wild and Hatchery Populations of the Pacific Oyster Crassostrea gigas Assessed by Microsatellite Markers

Microsatellite DNA technique was used to detect the genetic variation between five hatchery populations of the Pacific oyster from China and two wild populations from Japan. Seven microsatellite loci screened in this study showed high polymorphism in both hatchery and wild populations, as observed in an average number of allele per locus （19.1-29.9） and average expected heterozygosity （0.916-0.958）. No significant difference in average allelic richness or expected heterozygosity was observed between Chinese hatchery populations and Japanese wild populations. Pairwise Fsr values and heterogeneity tests of allele frequencies showed significant genetic differentiation between all populations. According to the neighbor-joining tree constructed on the basis of the Dc distance, the seven populations fell into three groups showing a clear division between hatchery and wild populations, and between the northern and southern hatchery populations. Assignment tests correctry assigned high percentages （97%-100%） of individuals to their original populations and demonstrated the feasibility of microsatellite analysis for discrimination between populations. The information obtained in this study is useful for designing suitable management guidelines and selective breeding programs for the Pacific oyster in China.

Genetic Quality Analysis of Xiphophorus helleri RR-B Strain Using 18 Microsatellite Loci

[Objective] The study aimed to further understand the genetic quality of Xiphophorus helleri RR-B strain and estimate the feasibility of microsatellite markers in genetic monitoring of aquatic laboratory animals.[Method] Eighteen microsatellite loci which had polymorphism in wild X. helleri were used for genetic quality analysis of RR-B strain (red eyes and red body) by PCR. [Result] All these 18 microsatellite loci displayed single allelic gene band in 30 individuals of X. helleri RR-B strain and the average homozygote rate reached 100%. [Conclusion] These loci could be used as markers in genetic monitoring of X. helleri RR-B strain, which had obtained fairly high genetic homozygosity by inbreeding for more than 20 generations. This study provided basic data for establishing the molecular genetic monitoring standard of aquatic laboratory animals.

Genetic Polymorphism in GDF8 Region of HU Sheep Based on Microsatellite DNA

[ Objective ] This study was to analyze the genetic polymorphism in GDF8 Region of HU sheep. [Method] Four microsatellite loci including BMS1591, TEXAN-2, FCB128 and BM81124 mapped on GDF8 region of chromosome No. 2 of sheep that may be correlated with growth performance were chosen to detect the molecular genetics foundation of growth performance of Hu sheep. [ Result] Four microsatellite loci detected were high in heterozygosity, more in effective alleles number and rich in polymorphic information, all the three indices passed through the high polymorphic level （PIC 〉0.5）. [ Conclusion ] The four microsatellite loci detected could be used to estimate the genetic polymorphism of growth performance of Hu sheep.

Isolation of Microsatellite DNA and Preliminary Genomic Analysis of Mud Carp (Cirrhina molitorella)

In order to determine the applicability of microsatellite primers developed from common carp （ Cyprinuscario ） for genomic analysis in mud carp （ Cirrhina molitorella ）, 24 primer pairs from common carp were designed to amplify microsatellite loci in the mud carp containing CA, GA, AT and GGGA sequences. Thirteen primers （54%） successfully amplified specific products in the mud carp and 11 primers （48%） showed high polymorphism in the mud carp population. The results indicated that the average number of alleles per locus in the mud carp stocks was 5.2. Average heterozygosity （Ho）, unbiased expected heterozygosity （He） and polymorphism information content （PIC） in the wild population were 0.61 ± 0.2, 0.8 ± 0.09 and 0.72 ± 0.1 respectively. Several Hardy-Weinberg departure value were significandy departed from Hardy-Weinberg equilibrium. The study showed that microsatellite primers from a species of Cyprinidae can be used for mud carp genetic analysis without much cost or time input.

Genetic Diversity Analysis of Three Guizhou Local Pig Breeds Using Microsatellite DNA Markers

[Objective] The research aimed to provide the genetic basis for the protection, development and utilization of Guizhou local pig breeds. [Method] From 27 pairs of porcine microsatellite primers recommended by Food and Agriculture Organization of the United Nations （FAO） and international Society for Animal Genetics （ISAG）, six pairs （S0155, SW240, IGF1, SW951, SW857, SW24） were selected for microsatellite DNA detection of three Guizhou local pig breeds, including Nuogu Pig, Kele Pig and Guanling Pig. Subsequently, their genetic diversities were analyzed. [ Result] The three pig breeds were high polymorphic at the six microsatellite loci （PIC 〉0.5）. The Nei＇s standard genetic distance of them was 0.206 3 -0.481 5. The genetic distance between Nuogu Pig and Kele Pig Was the closest, and that between Nuogu Pig and Guanling Pig was the furthest. [ Conclusion] The three Guizhou local pig breeds are in high genetic diversities. Nuogu Pig is a special type of Kele Pig, an excellent Chinese local pig breed.

Assessment of Genetic Variability and Inter-specific Relationships among Four Species of Groupers (Genus Epinephelus) from South China Sea Using Microsatellite Markers

[Objective] The study aimed to evaluate the genetic variability and interspecific relationships among four species of groupers from South China Sea, including E. fario, E. merra, E. malabaricus and E. coioides. [Method] Twenty one mircosatellite loci of groupers were selected from GenBank and eight high polymorphic loci were used to further genetic analysis. [Result] The mean number of alleles per locus （A）, effective number of alleles （Ne）, mean polymorphism information content （PIC）, observed heterozygosity （Ho） and expected heterozygosity （He） were 4.38±1.60, 3.69±0.86, 0.69±0.08, 0.67±0.08, 0.72±0.06 in E. malabaricus; 3.88±1.13, 3.55±1.04, 0.66±0.10, 0.68±0.21, 0.70±0.08 in E.coioides; 6.00±1.07, 4.68±0.65, 0.78±0.03, 0.73±0.25, 0.79±0.03 in E. fario; 5.50±1.07, 4.58±0.80, 0.76±0.05, 0.75±0.18, 0.78±0.04 in E. merra, respectively. [Conclusion] We compared the values above, the order of the genetic variability among these grouper species was E. fario E. merra E. malabaricus E. coioides. We found that the level of genetic variability of these groupers species was relatively higher than that of other marine fish, so their genetic status was good. In addition, the analyisis of genetic relationship showed that E. malabaricus and E. coioides was the closest and it was the farthest between E. merra and E. coioides.