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.

Association between Helicobacter pylori infection,mismatch repair,HER2 and tumor-infiltrating lymphocytes in gastric cancer

BACKGROUND The influence of Helicobacter-pylori(H.pylori)infection and the characteristics of gastric cancer(GC)on tumor-infiltrating lymphocyte(TIL)levels has not been extensively studied.Analysis of infiltrating-immune-cell subtypes as well as survival is necessary to obtain comprehensive information.AIM To determine the rates of deficient mismatch-repair(dMMR),HER2-status and H.pylori infection and their association with TIL levels in GC.METHODS Samples from 503 resected GC tumors were included and TIL levels were evaluated following the international-TILs-working-group recommendations with assessment of the intratumoral(IT),stromal(ST)and invasive-border(IB)compartments.The density of CD3,CD8 and CD163 immune cells,and dMMR and HER2-status were determined by immunohistochemistry(IHC).H.pylori infection was evaluated by routine histology and quantitative PCR(qPCR)in a subset of samples.RESULTS dMMR was found in 34.4%,HER2+in 5%and H.pylori-positive in 55.7%of samples.High IT-TIL was associated with grade-3(P=0.038),while ST-TIL with grade-1(P<0.001),intestinal-histology(P<0.001)and no-recurrence(P=0.003).dMMR was associated with high TIL levels in the ST(P=0.019)and IB(P=0.01)compartments,and STCD3(P=0.049)and ST-CD8(P=0.05)densities.HER2-was associated with high IT-CD8(P=0.009).H.pylorinegative was associated with high IT-TIL levels(P=0.009)when assessed by routine-histology,and with high TIL levels in the 3 compartments(P=0.002-0.047)and CD8 density in the IT and ST compartments(P=0.001)when assessed by qPCR.A longer overall survival was associated with low IT-CD163(P=0.003)and CD8/CD3(P=0.001 in IT and P=0.002 in ST)and high IT-CD3(P=0.021),ST-CD3(P=0.003)and CD3/CD163(P=0.002).CONCLUSION TIL levels were related to dMMR and H.pylori-negativity.Low CD8/CD3 and high CD163/CD3 were associated with lower recurrence and longer survival.

Comprehensive analysis of gene mutations and mismatch repair in Chinese colorectal cancer patients

BACKGROUND RAS,BRAF,and mismatch repair(MMR)/microsatellite instability(MSI)are crucial biomarkers recommended by clinical practice guidelines for colorectal cancer(CRC).However,their characteristics and influencing factors in Chinese patients have not been thoroughly described.AIM To analyze the clinicopathological features of KRAS,NRAS,BRAF,and PIK3CA mutations and the DNA MMR status in CRC.METHODS We enrolled 2271 Chinese CRC patients at the China-Japan Friendship Hospital.MMR proteins were tested using immunohistochemical analysis,and the KRAS/NRAS/BRAF/PIK3CA mutations were determined using quantitative polymerase chain reaction.Microsatellite status was determined using an MSI detection kit.Statistical analyses were conducted using SPSS software and logistic regression.RESULTS The KRAS,NRAS,BRAF,and PIK3CA mutations were detected in 44.6%,3.4%,3.7%,and 3.9% of CRC patients,respectively.KRAS mutations were more likely to occur in patients with moderate-to-high differentiation.BRAF mutations were more likely to occur in patients with right-sided CRC,poorly differentiated,or no perineural invasion.Deficient MMR(dMMR)was detected in 7.9% of all patients and 16.8% of those with mucinous adenocarcinomas.KRAS,NRAS,BRAF,and PIK3CA mutations were detected in 29.6%,1.1%,8.1%,and 22.3% of patients with dMMR,respectively.The dMMR was more likely to occur in patients with a family history of CRC,aged<50 years,right-sided CRC,poorly differentiated histology,no perineural invasion,and with carcinoma in situ,stage I,or stage II tumors.CONCLUSION This study analyzed the molecular profiles of KRAS,NRAS,BRAF,PIK3CA,and MMR/MSI in CRC,identifying key influencing factors,with implications for clinical management of CRC.

Mismatch repair genes (hMLH1, hPMS1, hPMS2, GTBP/hMSH6,HMSH2) in the pathogenesis of hepatocellular carcinoma

AIM: DMA mismatch repair (MMR) is an important mechanism for maintaining fidelity of genomic DNA. Abnormalities in one or more MMR genes are implicated in the development of many cancers. We investigated the role of expression of MMR genes (hMLH1, hPMS1, hPMS2, GTBP/hMSH6, hMSH2) in hepatocellular carcinogenesis. METHODS: We evaluated the expression level of MMR genes in 33 hepatocellular carcinoma (HCC) cases using the multiplex reverse transcription (RT) PCR assays, as well as in 16 cases of normal adjacent hepatic tissues. β-actin gene was used as an internal control and calibrator for quantification of gene expression. RESULTS: Out of the 33 studied cases, 25 were HCV positive and 30 (90.9%) showed reduced expression in one or more of the studied MMR genes. Reduced expression was found in hMSH2(71.9%), hMLH1 (53.3%), GTBP(51.1%), hPMS2 (33.3%) and hPMS1 (6%). A significant correlation was found between reduced expression of hPMS2(P= 0.0069) and GTBP(P= 0.0034), hPMS2 and non-cirrhosis (P= 0.0197), hMLH1 and high grade. On the other hand, 57.1%, 50%, 20%, 18.8%, and 6% of the normal tissues distant to tumors showed reduced expression of hMSH2, hMLH1, GTBP, hPMS2, and hPMS1 respectively. Multivariate analysis revealed a significant correlation between the expression level of hMSH2(P= 0.008), hMLH1 (P= 0.001) and GTBP (P= 0.032) and HCC, between hPMS2, GTBP and HCV-associated HCC (P<0.001, 0.002). CONCLUSION: Reduced expression of MMR genes seems to play an important role in HCV-associated HCC. hPMS2 is likely involved at an early stage of hepatocarcinogenesis since it was detected in normal adjacent tissues. Reduced expression of hPMS2 provides a growth advantage and stimulates proliferation which encourages malignant transformation in non-cirrhotic HCV-infected patients via acquisition of more genetic damages.

Helicobacter pylori infection and expression of DNA mismatch repair proteins

AIM： TO determine the expression of DNA （MMR） proteins, including hMLH1 and hMSH2, in gastric epithelial cells in the patients with or without Helicobacter pylori （H pylori）-infected gastritis. METHODS： Fifty Hpylori-positive patients and 50 H pylori-negative patients were enrolled in the study. During endoscopy of patients with non-ulcer dyspepsia, two antral and two corpus biopsies were taken for histological examination （Giemsa stain） and for immunohistochemical staining of hMLH1 and hMSH2. RESULTS： The percentage of epithelial cell nuclei that demonstrated positivity for hMLH1 staining was 84.14 ± 7.32% in Hpylori-negative patients, while it was 73.34 ±10.10% in Hpylori-positive patients （P 〈 0.0001）. No significant difference was seen between the two groups regarding the percentage of epithelial cell nuclei that demonstrated positivity for hMSH2 staining （81.16±8.32% in H pylori-negative versus 78.24 ± 8.71% in Hpylori-positive patients; P = 0.09）. CONCLUSION： This study indicates that Hpylori might promote development of gastric carcinoma at least in part through its ability to affect the DNA MMR system

Relationship between mismatch repair protein, RAS, BRAF, PIK3CA gene expression and clinicopathological characteristics in elderly colorectal cancer patients

BACKGROUND Colorectal cancer(CRC)is common in elderly patients.Mismatch repair(MMR)protein deletion is one of the causes of CRC.The RAS(KRAS/NRAS),BRAF,and PIK3CA genes are important gene targets in CRC treatment and are closely related to the prognosis and survival of patients.However,little is known regarding the relationship between the expression of MMR,RAS,BRAF,PIK3CA and the clinicopathological features in CRC patients.AIM To analyze the relationship between the expression of MMR,RAS,BRAF,PIK3CA and the clinicopathological features in CRC.METHODS A total of 327 elderly patients with CRC were enrolled,and immunohistochemistry was used to detect the MMR protein.Real-time quantitative polymerase chain reaction was used to detect the RAS(KRAS/NRAS),BRAF,and PIK3CA genes.The clinicopathological data of the patients were recorded and analyzed by SPSS 19.0 statistical software.RESULTS In 327 elderly patients with CRC,the rate of MMR protein loss was 9.79%(32/327),and the deletion rate of four MMR proteins(MSH2,MSH6,MLH1,PMS2)was 1.83%(6/327),3.06%(10/327),7.65%(25/327),and 7.65%(25/327),respectively.There were no significant differences between MMR protein deletion and sex,pathological type,tumor morphology,differentiation degree or lymph node metastasis(P>0.05),but there was a significant difference between MMR protein deletion and tumor diameter and tumor location(P=0.048/P=0.000).The mutation rates of the KRAS,NRAS,BRAF and PIK3CA genes in elderly CRC patients were 44.95%(147/327),2.45%(8/327),3.36%(11/327)and 2.75%(9/327),respectively;the KRAS gene mutation was closely related to tumor morphology(P=0.002)but not to other clinicopathological features(P>0.05),and there were no significant differences between NRAS gene mutation and clinicopathological features(P>0.05).The BRAF gene mutation showed a significant difference in pathological type,tumor location,differentiation degree and lymph node metastasis(P<0.05),but was not correlated with sex,tumor size and tumor morphology(P>0.05).The PIK3CA gene mutation showed no significant differences in the above clinicopathological characteristics(P>0.05).Significant differences were observed between MMR protein deletion and KRAS,BRAF,and PIK3CA gene mutations in elderly CRC patients(P=0.044,P=0.000,P=0.003,respectively),but there was no significant difference between MMR protein deletion and NRAS mutation(P>0.05).CONCLUSION In elderly CRC patients,the tumor is mainly located in the right colon,and the deletion rate of MMR protein is higher when the tumor diameter is greater than or equal to 5 cm;the deletion rate of MLH1 and PMS2 is more common;the mutation rate of KRAS gene is higher than that of the NRAS,BRAF and PIK3CA genes,the BRAF gene mutation has different degrees of correlation with clinicopathological characteristics;when the MMR protein is deleted,the BRAF and PIK3CA gene mutations are often present,and the KRAS gene mutation rate is low.

Complementary analysis of microsatellite tumor profile and mismatch repair defects in colorectal carcinomas

Microsatellite instability (MSI) is a prognostic factor and a marker of defi cient mismatch repair (MMR) in colorectal adenocarcinomas (CRC). However, a proper application of this marker requires understanding the following: (1) The MSI concept: The PCR approach must amplify the correct locus and accurately identify the microsatellite pattern in the patient’s normal tissue. MSI is demonstrat- ed when the length of DNA sequences in a tumor differs from that of nontumor tissue. Any anomalous expansion or reduction of tandem repeats results in extra-bands normally located in the expected size range (100 bp, above or below the expected product), differ from the germline pattern by some multiple of the repeating unit, and must show appropriate stutter. (2) MSI mechanisms: MMR gene inactivation (by either mutation or protein down-regulation as frequently present in deep CRC com- partments) leads to mutation accumulation in a cell with every cellular division, resulting in malignant transforma- tion. These mechanisms can express tumor progression and result in a decreased prevalence of aneuploid cells and loss of the physiologic cell kinetic correlations in the deep CRC compartments. MSI molecular mechanisms are not necessarily independent from chromosomal in- stability and may coexist in a given CRC. (3) Because of intratumoural heterogeneity, at least two samples from each CRC should be screened, preferably from the su- perfi cial (tumor cells above the muscularis propria) and deep (tumor cells infi ltrating the muscularis propria) CRC compartments to cover the topographic tumor hetero- geneity. (4) Pathologists play a critical role in identify- ing microsatellite-unstable CRC, such as occur in young patients with synchronous or metachronous tumors or with tumors showing classic histologic features. In these cases, MSI testing and/or MMR immunohistochemistry are advisable, along with gene sequencing and genetic counseling if appropriate. MSI is an excellent functional and prognostically useful marker, whereas MMR immuno- histochemistry can guide gene sequencing.

Expression of Cyclooxygenase-2 and Its Relationship with Mismatch Repair and Microsatellite Instability in Hereditary Nonpolyposis Colorectal Cancer

Objective To investigate cyclooxygenase-2 (COX-2) expression and its relationship with mismatch repair (MMR) protein expression and microsatellite instability (MSI) in hereditary nonpolyposis colorectal cancer (HNPCC). Methods A total of 28 cases of colorectal adenoma and 14 cases of colorectal carcinoma were collected between July 2003 and July 2007 from 33 HNPCC families. Sporadic colorectal adenoma (n=32) and carcinoma patients (n=24) served as controls. With samples of tumor tissues and normal colonic mucosa collected from the patients, the protein expressions of COX-2 and MMR (hMLH1, hMSH2, and hMSH6) were examined with immunohistochemical assay. Frequency of MSI in five standard MSI loci BAT25, BAT26, D2S123, D5S346, and D17S250 were analyzed by means of polymerase chain reaction. Results The rate of COX-2 high-expression was 53.6% (15/28) and 42.9% (6/14) in HNPCC adenoma and carcinoma; 62.5% (20/32) and 91.7% (22/24) in sporadic adenoma and carcinoma, respectively. That rate was lower in HNPCC carcinoma than in sporadic carcinoma (P<0.05). MMR-deletion rate and percentage of high-frequency MSI (MSI-H) in HNPCC carcinoma were higher than those in sporadic colorectal carcinoma [both 71.4% (10/14) vs. 12.5% (3/24), both P<0.01]. Among the 10 MMR-deficient HNPCC carcinoma patients, COX-2 low-expression was observed in 8 cases (80.0%), while COX-2 high-expression was observed in all of the 4 MMR-positive HNPCC carcinoma cases (P<0.05). In comparison to MMR positive HNPCC carcinoma, HNPCC adenoma, and sporadic carcinoma, COX-2 expression was significantly lower in corresponding MMR-deficient cases (all P<0.05). The rates of COX-2 low-expression in HNPCC adenoma, HNPCC carcinoma, and sporadic carcinoma with MSI-H were significantly higher than those in the cases with microsatellite stability (all P<0.05). Conclusion COX-2 is expressed at a low level in HNPCC carcinoma, different from the high COX-2 expression in sporadic carcinoma.

Mismatch repair enzyme expression in primary and castrate resistant prostate cancer

Objective:Although the utility of immunohistochemistry(IHC)for assessing mismatch repair(MMR)protein expression has been demonstrated in solid tumors including primary prostate cancer(PCa),its utility has not been assessed in castration-resistant PCa(CRPC).Methods:Tissue microarrays were constructed from 127 radical prostatectomies and 155 CRPC metastases from 50 patients.MMR(MLH1,MSH2,MSH6,and PMS2)expression was assessed by IHC and gene expression arrays.Associations between MMR protein expression in PCa and CRPC and biochemical recurrence(BCR)or time from diagnosis to death respectively were determined.Results:There was no correlation between levels of MMR protein and BCR.Absence of MSH2 and MSH6 was the most pronounced at 15%and 22%in PCa and 17.8%and 16%in CRPC patients,respectively.MSH2 and MSH6 protein were absent in 9.4%and 8%of PCa and CRPC respectively.Absence of individual MMR proteins did not correlate with BCR or time from diagnosis to death.However absent MSH2/MSH6 in CRPC was associated with shorter time to death(pZ0.0006).Loss of MSH2 was verified at the gene expression level.This finding correlated with microsatellite instability previously reported in this CRPC cohort.

Evaluation of 30 DNA damage response and 6 mismatch repair gene mutations as biomarkers for immunotherapy outcomes across multiple solid tumor types

Objective:DNA damage response(DDR)genes have low mutation rates,which may restrict their clinical applications in predicting the outcomes of immune checkpoint inhibitor(ICI)treatment.Thus,a systemic analysis of multiple DDR genes is needed to identify potential biomarkers of ICI efficacy.Methods:A total of 39,631 patients with mutation data were selected from the cBioPortal database.A total of 155 patients with mutation data were obtained from the Fudan University Shanghai Cancer Center(FUSCC).A total of 1,660 patients from the MSK-IMPACT cohort who underwent ICI treatment were selected for survival analysis.A total of 249 patients who underwent ICI treatment from the Dana-Farber Cancer Institute(DFCI)cohort were obtained from a published dataset.The Cancer Genome Atlas(TCGA)level 3 RNA-Seq version 2 RSEM data for gastric cancer were downloaded from cBioPortal.Results:Six MMR and 30 DDR genes were included in this study.Six MMR and 20 DDR gene mutations were found to predict the therapeutic efficacy of ICI,and most of them predicted the therapeutic efficacy of ICI,in a manner dependent on TMB,except for 4 combined DDR gene mutations,which were associated with the therapeutic efficacy of ICI independently of the TMB.Single MMR/DDR genes showed low mutation rates;however,the mutation rate of all the MMR/DDR genes associated with the therapeutic efficacy of ICI was relatively high,reaching 10%–30%in several cancer types.Conclusions:Coanalysis of multiple MMR/DDR mutations aids in selecting patients who are potential candidates for immunotherapy.

Microsatellite instability and expression of DNA mismatch repair genes in malignant astrocytic tumors from adult and pediatric patients

Microsatellite instability (MSI) is used as a molecular marker for defective DNA mismatch repair (MMR) genes.We report here alterations of MSI in 15 malignant astrocytomas (WHO grade Ⅲ) and glioblastomas (GBM; WHO grade Ⅳ) of pediatric patients (2-21 years) and 12 GBM from adults (44-68 years) by comparative analysis of BAT25/BAT26 loci and 10 other microsatellite markers. High-level microsatellite instability (MSI-H) occurred in 4 of the 15 pediatric cases (26.7%) and in 1 of the 12 adult GBM cases (8.3%). Low-level mi-

The Effect of Curcumin on Mismatch Repair(MMR) Proteins hMSH2 and hMLH1 after Ultraviolet(UV) Irradiation on HL-60 Cells

To understand the expression and effect of mismatch repair genes, hMSH2 and hMLH1, and to investigate anti-leukemic cell proliferation mechanism of curcumin, the levels of both genes were detected by multiple comparative RT-PCR. The protein of hMSH2 was determined by flow cy-tometry (FCM) and the gene mutation of hMSH2 and hMLH1 were detected by PCR-SSCP and mi-crosatellite instability assay. After UV irradiation, the gene expression of hMSH2 and hMLHl was not increased and showed no response. This phenomenon was not ascribed to gene mutation, because PCP-SSCP and microsatellite instability assay revealed no abnormal gel-shift band in both genes. After irradiation and addition of curcumin, the expression of hMSH2 mRNA increased and the cellular apoptotic rate also increased at the same time. The difference was statistically significant as compared with groups without addition of curcumin and control groups (P<0. 05). Our results suggested that when MMR system was inhibited by the same agents, curcumin can remove this suppression and switch to cellular apoptosis.

Immunohistochemical expression of mismatch repair genes:A screening tool for predicting mutator phenotype in liver fluke infection-associated intrahepatic cholangiocarcinoma

AIM： To clarify possible contributions of DNA mismatch repair （MMR） system in carcinogenesis of liver fluke infection-associated intrahepatic cholangiocarcinoma （ICC） by using immunohistochemical assay. METHODS： A total of 29 ICC samples, which had been assessed for genomic instability by a PCR-based method, were used for study. They were examined immunohistochemically to demonstrate protein expression of two MMR genes, hMSH2 and hMLH1. Results obtained were compared with their mutator phenotype assessed previously. RESULTS： Either hMSH2 or hMLH1 protein was obviously expressed in 28 of 29 （96.6%） ICC samples. Positive nuclear localization of hMSH2 or hMLH1 protein was observed in 86.2% （25/29） or 93.1% （27/29） ICC cases, respectively, while their negative nuclear reactivity was only detected in 13.8% （4/29） or 6.9% （2/29） ICC cases analyzed, respectively. CONCLUSION： Our study, probably for the first time, showed through immunohistochemical detection of hMSH2 and hMLH1 gene that DNA MMR system does not play a prominent role in liver fluke infection-associatedcholangiocarcinogenesis. These results confirm previous findings on mutational status of these genes assessed through a PCR-based method. The immunohistochemical analysis has proven to be an effective and sensitive approach for screening MMR deficiency regardless of somatic inactivation or promoter hypermethylation of hMSH2 and/or hMLH1 gene. Furthermore, immunohistochemistry is more advantageous compared to mutator phenotyping assay in terms of simplicity, less time consuming and cost effectiveness for screening possible involvements of target MMR genes in tumorigenesis.

Mutation screening of mismatch repair gene Mlh3 in familial esophageal cancer

AIM： To shed light on the possible role of mismatch repair gene MIh3 in familial esophageal cancer （FEC）. METHODS： A total of 66 members from 10 families suggestive of a genetic predisposition to hereditary esophageal cancer were screened for germline mutations in MIh3 with denaturing high performance liquid chromatography （DHPLC）, a newly developed method of comparative sequencing based on heteroduplex detection. For all samples exhibiting abnormal DHPLC profiles, sequence changes were evaluated by cycle sequencing. For any mutation in family members, we conducted a segregation study to compare its prevalence in sporadic esophageal cancer patients and normal controls. RESULTS： Exons of MIh3 in all samples were successfully examined. Overall, 4 missense mutations and 3 polymorphisms were identified in 4 families. MIh3 missense mutations in families 9 and 10 might be pathogenic, but had a reduced penetrance. While in families 1 and 7, there was no sufficient evidence supporting the monogenic explanations of esophageal cancers in families. The mutations were found in 33% of high-risk families and 50% of low-risk families.CONCLUSION： MIh3 is a high risk gene with a reduced penetrance in some families. However, it acts as a low risk gene for esophageal cancer in most families. Mutations of MIh3 may work together with other genes in an accumulated manner and result in an increased risk of esophageal tumor. DHPLC is a robust and sensitive technique for screening gene mutations.

Cost-effective screening using a two-antibody panel for detecting mismatch repair deficiency in sporadic colorectal cancer

BACKGROUND The microsatellite instability(MSI)test and immunohistochemistry(IHC)are widely used to screen DNA mismatch repair(MMR)deficiency in sporadic colorectal cancer(CRC).For IHC,a two-antibody panel of MLH1 and MSH2 or four-antibody panel of MLH1,MSH2,PMS2,and MSH6 are used.In general,MSI is known as a more accurate screening test than IHC.AIM To compare two-and four-antibody panels of IHC in terms of accuracy and cost benefit on the basis of MSI testing for detecting MMR deficiency.METHODS We retrospectively analyzed patients with CRC who underwent curative surgery between 2015 and 2017 at a tertiary referral center.Both IHC with four antibodies and MSI tests were routinely performed.The sensitivity and specificity of a fourand two types of two-antibody panels(PMS2/MSH6 and MLH1/MSH2)were compared on the basis of MSI testing for detecting MMR deficiency.RESULTS High-frequency MSI was found in 5.5%(n=193)of the patients(n=3486).The sensitivities of the four-and two types of two-antibody panels were 97.4%,92.2%,and 87.6%,respectively.The specificities of the three types of panels did not differ significantly(99.6%for the four-antibody and PMS2/MSH6 panels,99.7%for the MLH1/MSH2 panel).Based on Cohen's kappa statistic(κ),four-and twoantibody panels were in almost perfect agreement with the MSI test(κ>0.9).The costs of the MSI test and the four-and two-antibody panels of IHC were approximately$200,$160,and$80,respectively.CONCLUSION Considering the cost of the four-antibody panel IHC compared to that of the twoantibody panel IHC,a two-antibody panel of PMS2/MSH6 might be the best choice in terms of balancing cost-effectiveness and accuracy.

Unusual immunohistochemical“null”pattern of four mismatch repair proteins in gastric cancer:A case report

BACKGROUND Immunohistochemical(IHC)staining for mismatch repair(MMR)proteins is useful for gastric cancer treatment and prognosis.Different IHC staining patterns reflect the complex biological phenomena underlying MMR deficiency.We herein report a rare IHC staining pattern of four MMR-related proteins in gastric cancer.CASE SUMMARY A“null”IHC staining pattern of four MMR-related proteins,including MLH1,PMS2,MSH2,and MSH6,in a 67-year-old male patient with gastric cancer pT3N3aM0 revealed promoter hypermethylation of MLH1.Next-generation sequencing showed that these four genes exhibited changes.One of these was the somatic mutation of the missing copy number in exon 14 of MSH2.Mutation analysis using peripheral blood showed no germline mutations in these four genes.The patient had no history of personal or family tumor history.We classified this case as sporadic.The patient returned to normal after operation,and there were no signs of tumor metastasis and recurrence.After six cycles of adjuvant chemotherapy,the patient was discharged in a stable condition.The patient had a mild reaction to chemotherapy and a good prognosis.At present,16 mo after the operation,the patient's condition is stable.CONCLUSION Abnormal MMR protein expression,helpful for individualized follow-up care,helped identify a sporadic case lacking familial clinical management implications.

Programmed cell death-1 inhibitor combination treatment for recurrent proficient mismatch repair/miscrosatellite-stable type endometrial cancer:A case report

BACKGROUND Endometrial cancer(EC)is one of the most common cancers of the female reproductive tract,and the incidence is increasing rapidly.Immunotherapy using programmed cell death-1(PD-1)inhibitors is an emerging research topic and treatment strategy for refractory gynecological malignancies.However,clinical management of EC with checkpoint inhibitors requires improvement.Herein,we discuss a case of refractory proficient mismatch repair(pMMR)/miscrosatellitestable(MSS)EC treated with a combination of PD-1 and angiogenesis inhibitors and offer a review of the pathophysiology and clinical outcomes based on previous studies.CASE SUMMARY A 62-year-old woman diagnosed with invasive or metastatic EC in 2015 was treated with six courses of chemotherapy and refused further radiotherapy.Four years later,she developed chest pain,and lung biopsy indicated thyroid transcription factor-1(-),Napsin A(-),estrogen receptor(+),progesterone receptor(+),anaplastic lymphoma kinase(D5F3)(-),and receptor tyrosine kinase(D4D6)(-)metastatic EC.Genetic testing results showed low tumor mutation burden,pMMR,PD ligand 1(-),MSS,and HLA-class 1 heterogeneous disease.The patient was started on toripalimab combined with nab-paclitaxel for seven cycles(every 3 wk),but this regimen was terminated because of an intolerable chemotherapy adverse event.The disease progressed in 2020,and the patient’s treatment was switched from nab-paclitaxel to anlotinib,while immunotherapy using toripalimab was continued.The patient achieved a major partial response with well-tolerated toxicities,and treatment is ongoing.CONCLUSION Molecular testing is advised for clinical classifications of EC owing to its high heterogeneity.In this case,the patient had pMMR/MSS EC and achieved a positive outcome with combination PD-1 inhibitor treatment.These results warrant further clinical exploration.

Mismatch repair gene hMSH2 protein as predictive maker for gastric carcinoma

Objective: To study the possibility of mismatch repair gene hMSH2 as a marker to predict the occurrence of gastric carcinoma. Methods: Immunohistochemical method was used to detect hMSH2 protein expressions of gastric carci- nomas (carcinoma group) and their surrounding epithelia (surrounding epithelium group) in patients and the epithelia (normal epithelium group) in persons without carcinoma. Results: The positive rates of hMSH2 protein expression in nucleus of carcinoma, surrounding epithelium and normal epithelium groups were 44.94% (71/158), 28.48% (45/158) and 11.76% (4/34), respectively (P=0.000); the positive rates of hMSH2 protein expression in plasma of the 3 groups were 37.97% (60/158), 27.85% (44/158) and 23.53% (8/34), respectively (P=0.084); the positive rates of hMSH2 protein expression in both nucleus and plasma of the 3 groups were 20.89% (33/158), 17.72% (28/158) and 2.94% (1/34), respectively (P=0.045). Although the difference of positive rates between carcinoma and surrounding epithelium groups was not significant (P=0.476), both of them were higher than that of normal epithelium group (P=0.018). Conclusion: Our findings show that the detection of hMSH2 protein expression in gastric epithelia may help to predict and diagnose gastric carcinoma.

KRAS and BRAF gene mutations and DNA mismatch repair status in Chinese colorectal carcinoma patients

AIM:To investigate gene mutations and DNA mismatch repair(MMR) protein abnormality in Chinese colorectalcarcinoma(CRC) patients and their correlations with clinicopathologic features.METHODS:Clinical and pathological information for 535 patients including 538 tumors was reviewed and recorded.Mutation analyses for exon 2 of KRAS gene and exon 15 of BRAF gene were performed by Sanger sequencing except that in 9 tumors amplification refractory mutation system PCR was used.Expression of MMR proteins including MHL1,MSH2,MSH6 and PMS2 was evaluated by immunohistochemistry.Correlations of KRAS and BRAF mutation status and the expression status of MMR proteins with age,gender,cancer stage,location,and histology were analyzed.Correlations between KRAS or BRAF mutations and MMR protein expression were also explored.RESULTS:The overall frequencies of KRAS and BRAF mutations were 37.9% and 4.4%,respectively.KRAS mutations were more common in patients ≥ 50 years old(39.8% vs 22% in patients < 50 years old,P < 0.05).The frequencies of BRAF mutants were higher in tumors from females(6.6% vs males 2.8%,P < 0.05),located in the right colon(9.6% vs 2.1% in the left colon,1.8% in the rectum,P < 0.01),with mucinous differentiation(9.8% vs 2.8% without mucinous differentiation,P < 0.01),or being poorly differentiated(9.5% vs 3.4% well/moderately differentiated,P < 0.05).MMR deficiency was strongly associated with proximal location(20.5% in the right colon vs 9.2% in the left colon and 5.1% in the rectum,P < 0.001),early cancer stage(15.0% in stages Ⅰ-Ⅱ vs 7.7% in stages Ⅲ-Ⅳ,P < 0.05),and mucinous differentiation(20.2% vs 9.2% without mucin,P < 0.01).A higher frequency of MLH1/PMS2 loss was found in females(9.2% vs 4.4% in males,P < 0.05),and MSH2/MSH6 loss tended to be seen in younger(<50 years old) patients(12.0% vs 4.0% ≥ 50 years old,P < 0.05).MMR deficient tumors were less likely to have KRAS mutations(18.8% vs 41.7% in MMR proficient tumors,P < 0.05) and tumorswith abnormal MLH1/PMS2 tended to harbor BRAF mutations(15.4% vs 4.2% in MMR proficient tumors,P < 0.05).CONCLUSION:The frequency of sporadic CRCs having BRAF mutation,MLH1 deficiency and MSI in Chinese population may be lower than that in the Western population.

ARID1A expression in gastric adenocarcinoma:Clinicopathological significance and correlation with DNA mismatch repair status

AIM:To analyze the mismatch repair(MMR)status and the ARID1A expression as well as their clinicopathological significance in gastric adenocarcinomas.METHODS:We examined the expressions of MMR proteins and ARID1A by immunohistochemistry in consecutive 489 primary gastric adenocarcinomas.The results were further correlated with clinicopathological variables.RESULTS:The loss of any MMR protein expression,indicative of MMR deficiency,was observed in 38cases(7.8%)and was significantly associated with an older age(68.6±9.2 vs 60.4±11.7,P<0.001),a female sex(55.3%vs 31.3%,P=0.004),an antral location(44.7%vs 25.7%,P=0.021),and a differentiated histology(57.9%vs 39.7%,P=0.023).Abnormal ARID1A expression,including reduced or loss of ARID1A expression,was observed in 109 cases(22.3%)and was significantly correlated with lymphatic invasion(80.7%vs 69.5%,P=0.022)and lymph node metastasis(83.5%vs 73.7%,P=0.042).The tumors with abnormal ARID1A expression more frequently indicated MMR deficiency(47.4%vs 20.2%,P<0.001).A multivariate analysis identified abnormal ARID1A expression as an independent poor prognostic factor(HR=1.36,95%CI:1.01-1.84;P=0.040).CONCLUSION:Our observations suggest that the AIRD1A inactivation is associated with lymphatic invasion,lymph node metastasis,poor prognosis,and MMR deficiency in gastric adenocarcinomas.