Higher risk of mismatch repair-deficient colorectal cancer in alpha(1)-antitrypsin deficiency carriers and cigarette smokers

Microsatellite instability (MSI) is a genomic alteration observed in 15-30% of colorectal cancer (CRC). Two MSI phenotypes have been defined for CRC: MSI-H is characterized by MSI at > or =30% of the examined loci and MSI-L by MSI at 1-30% of the loci. An absence of MSI at any examined loci has been defined as a microsatellite stable (MSS) phenotype. Current data suggest the majority of MSI tumors are the result of defective DNA mismatch repair (MMR). In this study, we have determined the alpha(1)-antitrypsin deficiency carrier (alpha(1)ATD-ht) status of 161 CRC patients whose MSI phenotype and protein expression states had previously been determined. Cases were selected to enrich a larger number of MSI-H cases. Among 51 CRC patients with MSI-H tumors, the alpha(1)ATD-ht rate was 21.6%; among 110 patients with MSI-L/MSS tumors, the rate was 9.1% (MSI-H vs MSI-L/MSS, P = 0.02); and among the 191 population-based controls the alpha(1)ATD-ht rate was 9.4% (MSI-H vs controls, P = 0.02). The estimated relative risk of having MSI-H CRC among alpha(1)ATD-ht was 3.1 after adjusting for age, gender, and smoking history. The risk of having MSI-H CRC among current and past smokers was 6.6 and 2.7, respectively. Patients who were alpha(1)ATD-ht and smoked had a 20-fold increased risk of developing an MSI-H CRC compared to nonsmokers who were homozygous normal at the alpha(1)ATD locus. Our findings suggest an etiologic link between alpha(1)ATD alleles and development of CRC with defective MMR, and a synergistic effect between smoking and alpha(1)ATD allele in the development of MSI-H CRC.     

Distinct clinicopathologic and genetic profiles in sporadic gastric cancer with different mutator phenotypes

A subset of sporadic gastric cancers (GC) exhibits microsatellite instability (MSI). To define the precise role of MSI in GC, a total of 100 patients with sporadic GC were classified into three groups, i.e., high-frequency MSI (MSI-H), low-frequency MSI (MSI-L), and microsatellite stable (MSS), based on 10 microsatellite markers. Mutational analyses of TGFbetaRII, IGFIIR, BAX, MSH3, MSH6, E2F4, MSH2, MLH1, and TP53 genes, and methylation and protein expression of MLH1 and MSH2 were performed and correlated. Twenty-seven percent of GC showed MSI at least in one locus and could be further graded as MSI-H (14%) and MSI-L (13%). No clinicopathologic difference was noted between GC with MSI-L and MSS. Compared with GC with MSI-L or MSS, GC with MSI-H had a significantly higher frequency of antral location, intestinal subtype, H. pylori seropositivity, but a lower incidence of lymph node metastasis, and displayed a higher frequency of frameshift mutations of TGFbetaRII, IGFIIR, BAX, MSH3, and E2F4 genes but a lower incidence of TP53 mutations. Furthermore, hypermethylation of the MLH1 promoter was responsible for the loss of protein function in 13 of 14 MSI-H tumors. It was concluded that a specific phenotype and a distinct profile of genetic alterations exist in MSI-H GC. We speculate that epigenetic inactivation of MLH1 by methylation plays a crucial role in initiating such a pathway of carcinogenesis. In contrast, GCs with MSS and MSI-L exhibit clinicopathologic features that are distinct from MSI-H tumors and have a higher frequency of TP53 mutations, suggesting that they may evolve through an entirely different pathway.     

Higher Risk of Mismatch Repair-Deficient Colorectal Cancer in α1-Antitrypsin Deficiency Carriers and Cigarette Smokers

Microsatellite instability (MSI) is a genomic alteration observed in 15–30% of colorectal cancer (CRC). Two MSI phenotypes have been defined for CRC: MSI-H is characterized by MSI at ≥30% of the examined loci and MSI-L by MSI at 1–30% of the loci. An absence of MSI at any examined loci has been defined as a microsatellite stable (MSS) phenotype. Current data suggest the majority of MSI tumors are the result of defective DNA mismatch repair (MMR). In this study, we have determined the α₁-antitrypsin deficiency carrier (α₁ATD-ht) status of 161 CRC patients whose MSI phenotype and protein expression states had previously been determined. Cases were selected to enrich a larger number of MSI-H cases. Among 51 CRC patients with MSI-H tumors, the α₁ATD-ht rate was 21.6%; among 110 patients with MSI-L/MSS tumors, the rate was 9.1% (MSI-H vs MSI-L/MSS, P = 0.02); and among the 191 population-based controls the α₁ATD-ht rate was 9.4% (MSI-H vs controls, P = 0.02). The estimated relative risk of having MSI-H CRC among α₁ATD-ht was 3.1 after adjusting for age, gender, and smoking history. The risk of having MSI-H CRC among current and past smokers was 6.6 and 2.7, respectively. Patients who were α₁ATD-ht and smoked had a 20-fold increased risk of developing an MSI-H CRC compared to nonsmokers who were homozygous normal at the α₁ATD locus. Our findings suggest an etiologic link between α₁ATD alleles and development of CRC with defective MMR, and a synergistic effect between smoking and α₁ATD allele in the development of MSI-H CRC.     

Colorectal carcinogenesis: MSI-H versus MSI-L

Microsatellite instability (MSI) is a well-recognized phenomenon that is classically a feature of tumors in the hereditary non-polyposis colorectal syndrome. Ten to 15% of sporadic colorectal cancers, however, will have MSI. Microsatellite unstable tumors can be divided into two distinct MSI phenotypes: MSI-high (MSI-H) and MSI-low (MSI-L). MSI sporadic colorectal cancers with a high level of MSI (MSI-H) form a well defined group with distinct clinicopathologic features characterized by an overall better long-term prognosis. These sporadic MSI-H colorectal tumors most often arise from the epigenetic silencing of the mismatch repair gene MLH1. In contrast, MSI-L colorectal tumors have not been shown to differ in their clinicopathologic features or in most molecular features from microsatellite stable (MSS) tumors. Unlike MSI-H tumors, MSI-L tumors appear to arise through the chromosomal instability carcinogenesis pathway, similar to MSS tumors. Some groups have reported more frequent mutations in K-ras and in the methylation of methylguanine transferase in MSI-L tumors, but others have questioned these findings. Therefore, although the use of the MSI-L category is widespread, there continues to be some debate as to whether a discrete MSI-L group truly exists. Rather, it has been suggested that MSI-L tumors differ quantitatively from MSS tumors but do not differ qualitatively. Future studies will need to evaluate the specific mutations in non-MSI-H tumors in an attempt to sub-classify MSI-L tumors with regard to MSS tumors so that subtle differences between these two sub-groups can be identified.     

Concerted promoter hypermethylation of hMLH1, p16INK4A,and E-cadherin in gastric carcinomas with microsatellite instability

In most sporadic gastric carcinomas, microsatellite instability (MSI) originates from inactivation of the hMLH1 gene by promoter hypermethylation. However, the methylation patterns of other genes and their consequences in high MSI (MSI-H) gastric carcinomas are not well characterized. To address the aberrant promoter methylation profiles of MSI-H gastric carcinomas, promoter methylation of six genes (hMLH1, p16(INK4A), E-cadherin, Rb, RASSF1A, and VHL) and CpG island methylator phenotype (CIMP) were explored in 36 MSI-H gastric carcinomas and the results were compared with those of 43 microsatellite-stable (MSS) gastric carcinomas. Frequent promoter hypermethylation was found in hMLH1, p16(INK4A), and E-cadherin and the frequency was significantly higher in MSI-H gastric carcinomas. Promoter hypermethylation of hMLH1, E-cadherin, and p16(INK4A) was found in 89%, 78%, and 33% of MSI-H gastric carcinomas and in 16%, 32%, and 11% of MSS carcinomas, respectively (p = 0.01). Selective absent or decreased expression of the gene product related to the hypermethylated promoter was found for hMLH1 and p16(INK4A) in MSI-H carcinoma, whereas the expression of E-cadherin was generally decreased both in the MSI-H and in the MSS carcinomas. MSI-H gastric carcinomas were also related to the high CIMP (CIMP-H, three or more of the five loci examined showing methylation). Twenty-two (61%) MSI-H gastric carcinomas were CIMP-H, compared with only seven (16%) MSS carcinomas (p = 0.001). These findings indicate that hMLH1 is one of the frequent methylation targets in CIMP-H gastric carcinomas and that inactivation of hMLH1 through promoter hypermethylation results in tumours following the MSI pathway.     

Robust microsatellite instability (MSI) analysis by denaturing high-performance liquid chromatography (DHPLC)

Microsatellite instability (MSI) plays an important biological role in various types of cancers, and especially in colorectal cancers. This study aimed to develop a simple, efficient, new method for robust MSI analysis. DNA was extracted from 175 (105 proximal colon and 70 distal colorectal) cancer samples and matched normal tissues, and five Bethesda microsatellite markers (BAT-25, BAT-26, D5S346, D2S123, and D17S250) were examined for MSI by denaturing high-performance liquid chromatography (DHPLC) analysis at a temperature of 50°C and a flow rate of 0.9 ml/min. It took just 9 min per PCR product to determine MSI or microsatellite stability (MSS) using the new protocol. The DHPLC results were confirmed with conventional gel-based electrophoresis and capillary-based sequencing method. Of 175 samples, 45 (26%) showed high microsatellite instability (MSI-H), 12 (7%) showed low microsatellite instability (MSI-L), and 118 (67%) showed MSS. All MSI samples were deletion mutants and all 12 MSI-L cases had MSI in dinucleotide markers (D5S346, D2S123, and D17S250). MSI was significantly associated with proximal colon cancers (p<0.0001), as previously reported. The MSI-H tumors were also associated with tumor node metastasis (TNM) I/II stages (p=0.05) and high-grade tumors (p<0.01). Here, we propose a DHPLC-based method as an alternative for MSI analysis.This work was presented in part at the meeting of AACR Oncogenomics 2003, Phoenix, AZ, USA, January 2003.     

Fatty acid synthase overexpression in colorectal cancer is associated with microsatellite instability, independent of CpG island methylator phenotype

Overexpression of fatty acid synthase (FASN), a key enzyme for de novo lipogenesis, is observed in many cancers including colorectal cancer and is associated with poor clinical outcomes. Cellular FASN expression is physiologically upregulated in a state of energy excess. Obesity and excess energy balance have been known to be risk factors for colorectal cancer. High degree of microsatellite instability (MSI-H) is a distinct phenotype in colorectal cancer, associated with CpG island methylator phenotype (CIMP). Previous data suggest that obesity or altered energy balance may potentially modify risks for MSI-H cancers and microsatellite stable (MSS) cancers differently. However, the relationship between MSI and FASN overexpression has not been investigated. Using 976 cases of population-based colorectal cancer samples from 2 large prospective cohort studies, we correlated FASN expression (by immunohistochemistry) with MSI, KRAS and BRAF mutations, p53 expression (by immunohistochemistry), and CIMP status [determined by MethyLight for 8 CIMP-specific gene promoters including CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1]. Marked (2+) FASN overexpression was observed in 110 (11%) of the 976 tumors and was significantly more common in MSI-H tumors (21% [28/135]) than MSI-low (5.6% [4/72], P = .004) and MSS tumors (11% [72/678], P = .001). The association between FASN overexpression and MSI-H persisted even after stratification by CIMP status. In contrast, FASN overexpression was not correlated with CIMP after stratification by MSI status. Fatty acid synthase overexpression was not significantly correlated with sex, tumor location, p53, or KRAS/BRAF status. In conclusion, FASN overexpression in colorectal cancer is associated with MSI-H, independent of CIMP status.     

MET is overexpressed in microsatellite instability-high gastric carcinoma

Background

MET is a tyrosine kinase receptor for the hepatocyte growth factor, and its overexpression is a poor prognostic factor in gastric carcinomas. Programmed death-ligand 1 (PD-L1) is an important biomarker of immunotherapy and is frequently positive in microsatellite instability-high (MSI-H) gastric carcinomas. In lung cancers, MET activation up-regulates PD-L1 expression. In this study, we investigated expression of MET and PD-L1 in MSI-H gastric carcinoma and the effects on prognosis.

Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis.

High-level microsatellite instability (MSI-H) is demonstrated in 10 to 15% of sporadic colorectal cancers and in most cancers presenting in the inherited condition hereditary nonpolyposis colorectal cancer (HNPCC). Distinction between these categories of MSI-H cancer is of clinical importance and the aim of this study was to assess clinical, pathological, and molecular features that might be discriminatory. One hundred and twelve MSI-H colorectal cancers from families fulfilling the Bethesda criteria were compared with 57 sporadic MSI-H colorectal cancers. HNPCC cancers presented at a lower age (P < 0.001) with no sporadic MSI-H cancer being diagnosed before the age of 57 years. MSI was less extensive in HNPCC cancers with 72% microsatellite markers showing band shifts compared with 87% in sporadic tumors (P < 0.001). Absent immunostaining for hMSH2 was only found in HNPCC tumors. Methylation of hMLH1 was observed in 87% of sporadic cancers but also in 55% of HNPCC tumors that showed loss of expression of hMLH1 (P = 0.02). HNPCC cancers were more frequently characterized by aberrant beta-catenin immunostaining as evidenced by nuclear positivity (P < 0.001). Aberrant p53 immunostaining was infrequent in both groups. There were no differences with respect to 5q loss of heterozygosity or codon 12 K-ras mutation, which were infrequent in both groups. Sporadic MSI-H cancers were more frequently heterogeneous (P < 0.001), poorly differentiated (P = 0.02), mucinous (P = 0.02), and proximally located (P = 0.04) than HNPCC tumors. In sporadic MSI-H cancers, contiguous adenomas were likely to be serrated whereas traditional adenomas were dominant in HNPCC. Lymphocytic infiltration was more pronounced in HNPCC but the results did not reach statistical significance. Overall, HNPCC cancers were more like common colorectal cancer in terms of morphology and expression of beta-catenin whereas sporadic MSI-H cancers displayed features consistent with a different morphogenesis. No individual feature was discriminatory for all HNPCC cancers. However, a model based on four features was able to classify 94.5% of tumors as sporadic or HNPCC. The finding of multiple differences between sporadic and familial MSI-H colorectal cancer with respect to both genotype and phenotype is consistent with tumorigenesis through parallel evolutionary pathways and emphasizes the importance of studying the two groups separately.     

Distinct patterns of KRAS mutations in colorectal carcinomas according to germline mismatch repair defects and hMLH1 methylation status

In sporadic colorectal tumours the BRAFV600E is associated with microsatellite instability (MSI-H) and inversely associated to KRAS mutations. Tumours from hereditary non-polyposis colorectal cancer (HNPCC) patients carrying germline mutations in hMSH2 or hMLH1 do not show BRAFV600E, however no consistent data exist regarding KRAS mutation frequency and spectrum in HNPCC tumours. We investigated KRAS in 158 HNPCC tumours from patients with germline hMLH1, hMSH2 or hMSH6 mutations, 166 MSI-H and 688 microsatellite stable (MSS) sporadic carcinomas. All tumours were characterized for MSI and 81 of 166 sporadic MSI-H colorectal cancer (CRCs) were analysed for hMLH1 promoter hypermethylation. KRAS mutations were observed in 40% of HNPCC tumours, and the mutation frequency varied upon the mismatch repair gene affected: 48% (29/61) in hMSH2, 32% (29/91) in hMLH1 and 83% (5/6) in hMSH6 (P = 0.01). KRAS mutation frequency was different between HNPCC, MSS and MSI-H CRCs (P = 0.002), and MSI-H with hMLH1 hypermethylation (P = 0.005). Furthermore, HNPCC CRCs had more G13D mutations than MSS (P < 0.0001), MSI-H (P = 0.02) or MSI-H tumours with hMLH1 hypermethylation (P = 0.03). HNPCC colorectal and sporadic MSI-H tumours without hMLH1 hypermethylation shared similar KRAS mutation frequency, in particular G13D. In conclusion, we show that depending on the genetic/epigenetic mechanism leading to MSI-H, the outcome in terms of oncogenic activation may be different, reinforcing the idea that HNPCC, sporadic MSI-H (depending on the hMLH1 status) and MSS CRCs, may target distinct kinases within the RAS/RAF/MAPK pathway.     

Postoperative radiotherapy for laryngeal cancer. The prognostic role of programmed death-ligand 1: An immune microenvironment-based cluster analysis

BackgroundThe prognostic role of programmed death-ligand 1 (PD-L1) expression and the tumor’s immune microenvironment has yet to be investigated in the specific setting of adjuvant postoperative radiotherapy (PORT) for laryngeal carcinoma (LSCC). The main aim of this exploratory study was to investigate, also by cluster analysis, whether PD-L1 expression (in terms of combined positive score [CPS]), tumor-infiltrating lymphocytes (TIL), and tertiary lymphoid structures (TLS) correlated prognostically with response to PORT in a cohort of consecutive LSCC patients.MethodsPD-L1, TIL and TLS were assessed in 24 consecutive patients with LSCC who underwent PORT. Cluster analysis was used to classify cases on the strength of these parameters.ResultsA CPS ≥ 1 was associated with a significantly lower recurrence rate (p = 0.033), and longer disease-free survival (DFS) (p = 0.012) than a CPS < 1. Two clusters of prognostic relevance emerged from our analysis. Cluster 1 was characterized by a mean CPS of 23.0 ± 37.9, a mean TIL count of 68.0 ± 16.4, and the presence of TLS in all cases. Cluster 2 featured a mean CPS of 3.1 ± 7.3, a mean TIL count of 23.9 ± 16.5, and no cases with TLS. Cluster 1 showed a trend towards a lower recurrence rate (p = 0.071) and longer DFS (p = 0.054) than cluster 2.ConclusionsJudging from this preliminary investigation, assessing PD-L1 and immune microenvironment markers seems a promising approach for identifying patients at higher risk of LSCC recurrence after PORT, who might reasonably benefit from adjuvant postoperative chemo-RT, or immunotherapy.     

Microsatellite alterations in differentiated-type adenocarcinomas and precancerous lesions of the stomach with special reference to cellular phenotype

To elucidate the relationship between genetic alterations and cellular phenotype of differentiated-type adenocarcinomas and precancerous lesions of the stomach, we phenotyped 61 gastric tumors consisting of 33 noninvasive lesions and 28 submucosal invasive carcinomas by histochemical and immunohistochemical techniques, including analysis of mucin expression. We then analyzed loss of heterozygosity (LOH) at tumor suppressor loci, examined microsatellite instability (MSI), and compared the results according to cellular phenotype. Of the 61 gastric tumors studied, 7% (4 of 61) were classified as tumors with a gastric foveolar epithelial phenotype (foveolar-type), 8% (5 of 61) as tumors with a complete-type intestinal metaplastic phenotype (CIM-type), and the remaining 85% (52 of 61) as tumors with an ordinary phenotype (ordinary-type). Forty-two percent (26 of 61) of the tumors showed LOH on at least 1 chromosomal arm. Although LOH was rare in foveolar-type tumors, it was present at variable frequencies at each tumor suppressor loci in tumors with other cellular phenotypes. p53 overexpression was observed in 0% (0 of 4) of foveolar-type, 48% (25 of 52) of ordinary-type, and 80% (4 of 5) of CIM-type tumors. With regard to MSI, all (4 of 4) of the foveolar-type tumors were classified as having high-rate MSI (MSI-H), whereas all (5 of 5) of the CIM-type tumors were microsatellite stable (MSS). Of 52 ordinary-type tumors, 19% (10 of 52) were classified as MSI-H, 12% (6 of 52) as low-rate MSI (MSI-L), and 69% (36 of 52) as MSS. The incidence of MSI-H was found to be significantly higher in foveolar-type tumors (100%; 4 of 4) than in ordinary-type (19%; 10 of 52) or CIM-type tumors (0%; 0 of 5) (P < .01). An inverse correlation between MSI-H and p53 overexpression was also noticed (P < .01). Results suggested that each cellular phenotype followed a different genetic pathway; foveolar-type tumors followed the "mutator" pathway, characterized by MSI, CIM-type tumors followed the "suppressor" pathway, characterized by LOH of tumor suppressor loci and p53 overexpression, and ordinary-type tumors appeared to show mixed genetic alterations of both types.     

Expression of programmed cell death-ligand 1 in oral squamous cell carcinoma and oral leukoplakia is associated with disease progress and CD8+ tumor-infiltrating lymphocytes

ObjectiveIn recent years, monoclonal antibodies targeting programmed cell death-ligand 1 (PD-L1) have become a promising cancer immunotherapy. However, the role of PD-L1 in oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMDs), including oral leukoplakia (OLK), remains controversial. The aim of the present study was to investigate the expression level of PD-L1 in OSCC and OPMDs, and examine its relationship with CD8 expression and different clinicopathological features.MethodExpression of PD-L1 and CD8 were conducted in 41 OSCC, 21 OLK, and 25 normal mucosa samples by immunohistochemistry. Then, the density of PD-L1 expression was measured, and its correlation with CD8 expression and different clinicopathological features was analyzed.ResultsPD-L1 protein was detected in 97.6% of OSCC, 61.9% of OLK, and 0% of normal tissues. PD-L1 was highly expressed in human OSCC tissue (P < 0.0001), when compared to both OLK and control tissues. PD-L1 positivity was significantly associated with CD8 density (P < 0.0001, r = 0.8491). The PD-L1 high expression OSCC group displayed a trend for improved overall survival (OS) and disease-free survival (DFS) compared to the low expression group, although the differences were not significant. Moreover, the expression level of PD-L1 in OSCC was positively correlated with the pathological grade (P < 0.0001), but it was independent of age, gender, smoking, drinking, tumor size, lymph node status, or recurrence (P > 0.05). Also, there was a significant upregulation of PD-L1 expression observed in the OLK group compared to the control group (P < 0.0001). PD-L1 positivity in OLK patients was associated with gender and smoking habits (P < 0.05), but it did not correlate with age, drinking, or dysplasia (P > 0.05).ConclusionThe upregulation of PD-L1 may be associated with disease progress and CD8+ tumor-infiltrating lymphocytes in oral premalignant and malignant lesions.     

PD-L1 immuno-expression assay in thymomas: Study of 84 cases and review of literature

Background and aimsProgrammed death ligand 1 (PD-L1), an immune check point inhibitor, is known to be expressed in several malignancies and is being considered as a prognostic factor and a potential immunotherapeutic target. The aim of this study was to characterize PD-L1 expression in thymomas and to determine correlation with clinicopathological features and previously published studies in the literature.MethodsTissue microarrays were prepared from selected blocks of thymomas and immunohistochemistry (IHC) for PD-L1 was performed. Cases were considered as PD-L1 positive or negative depending on whether the percentage of stained thymic epithelial cells were <25 or >25%. Results were compared clinically and with previously published studies using Google and Pubmed search engines.ResultsOf 84 cases of thymoma, 69 (82.1%) revealed PD-L1 positivity in >25% cells. 94.23% of type B thymoma subtypes (B1/B2/B3) were PD-L1 positive (P < 0.001). There was no correlation of PD-L1 with age, gender, myasthenia gravis, the tumor size or stage of disease. Nine studies were available in the literature; most of which showed PD-L1 expression in higher stage and B subtype however percentage positivity varied from 53.7% to over 90%.ConclusionsPD-L1 expression is frequent in type B (B1/B2/B3) thymomas. It can be easily evaluated by IHC even on small biopsies in unresectable cases, thereby enabling improved clinical evaluation as well as prognostic stratification of patients. It will serve as a potential indicator for benefit from anti-PD-L1 antibody immunotherapy in thymomas.     

PD-L1 expression in carcinoma of the esophagogastric junction is positively correlated with T-cell infiltration and overall survival

IntroductionIn recent years antitumor immunity and inhibition of checkpoint molecules, such as PD-1 and PD-L1, have emerged as potential therapeutic strategies in advanced stages of various malignancies. We investigated PD-L1 expression in adenocarcinomas of the esophagogastric junction and correlated the results with densitiy of intratumoral T-lymphocytes.MethodsImmunohistochemical staining for PD-L1 was carried out on 135 samples using a tissue microarray. Scoring was done according to the combined positivity score.Results48.1% of tumors (65 cases) showed PD-L1 positivity with a score ≥ 1 while 51.9% were PD-L1 negative (70 cases). A positive correlation between PD-L1 negativity and mucinous and poorly cohesive carcinoma could be shown (p = 0.043), while no association existed for either gender, T-stage, N-stage, grading, surgical resection status, neoadjuvant therapy, distant metastases, lymphovascular or perineural invasion. No correlation of PD-L1 expression and overall survival could be detected (p = 0.497). Again, when stratified according to presence or absence of neoadjuvant therapy, no survival differences could be shown for either group (p = 0.540 and p = 0.736). When PD-L1 expression was correlated with density of tumor-infiltrating T-lymphocytes a positive correlation between PD-L1 positivity and denser T-cell infiltration could be shown (p = 0.001). Concerning overall survival, in PD-L1 negative cases, denser CD8-positive T-cell infiltrates were associated with prolonged survival times (p = 0.045). No differences could be shown for PD-L1 positive cases or CD103-positive T-cells.ConclusionPD-L1 expression is frequent in esophagogastric adenocarcinoma and - when combined with dense CD8 infiltration – PD-L1 negativity correlates with prolonged overall survival.     

PD-L1 expression in vulvar cancer: a systematic review and meta-analysis

Programmed cell death ligand-1 (PD-L1) expression in cancer may predict clinical response to immunotherapeutic treatment with PD-1/PD-L1 inhibitors. Within the vulvar cancer field, PD-L1 expression has only been assessed by a few studies. We conducted a meta-analysis to examine the prevalence of PD-L1 positivity in vulvar cancer. PubMed, Embase, and Cochrane were searched for articles reporting on PD-L1 expression in vulvar cancer. Study selection and data extraction were performed independently by two authors. We extracted data on PD-L1 prevalence in vulvar cancer according to combined positive score (CPS) and tumour proportion score (TPS). Cutoff values for positivity were ≥1 or ≥10 for CPS and ≥1% and ≥5% for TPS. Random-effects models were used to estimate pooled PD-L1 prevalence, with 95% confidence intervals (CIs). Tests of between-study heterogeneity were evaluated by the I² statistics. Sources of heterogeneity were explored by subgroup analyses and meta-regression. In total, 19 studies were included. Pooled PD-L1 prevalence in vulvar cancer was 83.4% (95% CI: 70.8–91.3; I² = 80.0) and 53.9% (95% CI: 37.4–69.6; I² = 93.0) according to CPS and TPS, respectively. Based on TPS, human papillomavirus (HPV)-associated vulvar squamous cell carcinomas (SCC) showed a lower PD-L1 prevalence (39.9%; 95% CI: 13.3–74.2) compared with HPV-independent SCC (62.6%; 95% CI: 33.7–84.6), but meta-regression showed no significant variation in PD-L1 prevalence by HPV status. PD-L1 prevalence was similar in advanced (44.9%; 95% CI: 29.8–61.1) and localized vulvar cancer (56.7%; 95% CI: 18.9–76.7). In conclusion, PD-L1 expression in vulvar cancer is frequent but between-study heterogeneity was high. Based on a subgroup of heterogenous studies, we found no strong variation in PD-L1 prevalence according to HPV status and stage.     

The expression of programmed death-ligand 1 and programmed death-ligand 2 in endometrial carcinosarcoma: Correlation with mismatch repair protein expression status,tumor-infiltrating lymphocyte infiltration,and clinical outcomes

BackgroundEndometrial carcinosarcomas have high malignant potential with a high recurrence rate and poor prognosis. Immunotherapy may be a promising treatment option. The aim of this study is to evaluate the expression of PD-L1/PD-L2 and its relationship to mismatch repair (MMR) protein status and tumor-infiltrating lymphocyte (TIL) density.MethodsWe performed immunohistochemical analyses of PD-L1 (clone 22C3), PD-L2 (clone TY25), MSH-2, MSH-6, PMS-2, and MLH-1 in 77 tumors. We count TILs using CD8 antibody. Clinicopathologic features were recorded and statistically correlated with immunohistochemical results. Kaplan-Meier analyses were used to analyze the prognosis.ResultsWhile PD-L1 positivity was seen more commonly in MMR protein deficient tumors (p = 0.010), PD-L2 positivity was seen more commonly in MMR protein proficient tumors (p = 0.003). PD-L1 positivity was also found to be more common in carcinosarcoma with high TIL infiltration. PD-L2 positivity was associated with decreased overall survival (OS) rates (p = 0.043, p = 0.043, respectively), whereas the PD-L1 positivity and TIL density were not significantly associated with OS rate. The OS rate of patients with MMR protein proficient tumors was significantly lower compared with those with MMR protein deficient tumors (p = 0.042). The lower TILs infiltration was associated with a shorter disease-free survival (DFS) rate. PD-L1 and PD-L2 positivity did not affect the DFS rate.ConclusionsPD-L1/PD-L2 might be a better target for immunotherapy in endometrial carcinosarcoma. PD-L2 positivity was also associated with a worse clinical outcome in patients with endometrial carcinosarcoma, suggesting that PD-L2 status can be used to predict clinical behavior. Further studies are needed to elucidate the relationship between PD-L1/PD-L2 expression and therapeutic response.     

Increased PD-L1 expression in high-grade bladder cancer with squamous cell differentiation in Bulgarian and French patients' samples

Urothelial carcinomas (UC) of the bladder are biologically and clinically heterogeneous and the most common malignancy of the urinary tract in developed countries worldwide, where several checkpoint targets as programmed death ligand-1 (PD-L1) and programmed cell death protein (PD-1) have received the most attention in the treatment of bladder cancer. However, the clinicopathological impact of this biomarker has not yet been established enough.ObjectiveTo evaluate programmed death ligand-1 (PD-L1) expression in UCs of the bladder in Bulgarian and French patients' samples.Materials and methodsUrothelial bladder carcinomas cases from 2016–2020 were retrospectively were analyzed. The cohort included 105 cases: 42 (40%) low grade and 63 (60%) high grade. Immunohistochemical (IHC) staining for PD-L1 expression was performed using an anti-PD-L1 primary antibody clone 22C3pharmDx only to 73/105 cases.ResultsApproximately 21/73 cases (28.8%) of urothelial bladder carcinomas demonstrated positive PD-L1 expression, and in 52/73 cases (71.2%) were negative. Positive PD-L1 expression was associated with high grade and high pathologic stage (p < 0.001). We found that PD-L1 was expressed in a significant percentage in UC with squamous differentiation (40%), followed by classic UC (30%). An association between histological grading systems of bladder UC (WHO1973 and WHO 2016) and the TNM-staging system, estimated by Pearson correlation coefficients (r = 0.590 and r = 0.583, respectively, p < 0.001) was observed.ConclusionsWe found that PD-L1 expression is increased in patients with muscle-invasive UC, and PD-L1 might be a new biomarker that correlates with the pathological stage of urothelial bladder cancer and might predict recurrence-free survival.     

Proximal colon cancers and the serrated pathway: a systematic analysis of precursor histology and BRAF mutation status

Although the serrated neoplasia pathway is thought to give rise to the majority of sporadic microsatellite instability-high (MSI-H) colon cancer, the exact proportion of these tumors that arise from serrated precursors has not been fully studied. Tubular and tubulovillous adenomas with features of the serrated neoplasia pathway have been described, and unlike sessile serrated adenomas, these lesions lack BRAF mutations. The contribution of these adenomas to sporadic MSI-H colon cancer is unclear. To this end, we conducted an analysis of right-sided sporadic MSI-H and microsatellite stable (MSS) colon cancer, with emphasis on precursor lesions. Overall 25% (19/75) of MSI-H colon cancer had a precursor, of which only 4 were recognized histologically as arising from a sessile serrated adenoma, and the remaining were best classified as adenomas. Of the 31 (of 89) MSS colon cancers with a precursor, only 1 was a sessile serrated adenoma (P=0.06). Histological analysis of the precursor adenomas to sporadic MSI-H colon cancer demonstrated a high frequency of crypt serrations compared with MSS colon cancer (93 vs 36%, P<0.001). BRAF mutations were found in 57/75 (76%) sporadic MSI-H and 10/89 (11%) MSS colon cancers (P<0.001). Molecular analysis demonstrated BRAF mutations in 11/12 adenoma and 3/3 sessile serrated adenoma precursors adjacent to BRAF-mutated MSI-H colon cancer. Similarly, all 4 precursors to BRAF-mutated MSS colon cancer were also BRAF mutated. The presence of BRAF mutations in these adenomatous precursors suggests that they represent sessile serrated adenomas with complete cytologic dysplasia. Finally, patients with sporadic MSI-H colon cancer were more likely to harbour synchronous sessile serrated adenomas (20 vs 8%; P=0.023). This is the largest study to rigorously evaluate the precursor and synchronous lesions in patients with right-sided colon cancer. Detailed molecular and histological analysis of these lesions confirms the importance of serrated precursors in the development of sporadic MSI-H colon cancer.     

Origin of Microsatellite Instability in Gastric Cancer

Microsatellite instability (MSI) is observed in 13-44% of gastric carcinoma. The etiology of MSI in gastric carcinoma has not been clearly defined. To assess the role of mismatch repair in the development of MSI in gastric cancer, expression of hMSH2 and hMLH1 was explored. We examined 117 gastric carcinomas for MSI and observed instability at one or more loci in 19 (16%) of these tumors. Of the 19 tumors with MSI, nine exhibited low-rate MSI (MSI-L) with instability at <17% of loci, whereas the remaining 10 exhibited high-rate MSI (MSI-H) with instability at >33% of loci examined. Immunohistochemical staining for hMLH1 and hMSH2 was performed on eight of the tumors with MSI-H, five with MSI-L, and 15 tumors without MSI. All eight tumors with MSI-H showed loss of staining for either hMLH1 (n = 5) or hMSH2 (n = 3). In contrast, tumors with MSI-L or without MSI all showed normal hMSH2 and hMLH1 protein expression patterns. Moreover, all eight of the tumors with MSI-H also showed instability at BAT-26, whereas none of the MSI-L tumors or tumors without instability showed instability at BAT-26. These findings suggest that the majority of high-level MSI in gastric cancer is associated with defects of the mismatch repair pathway. Although larger studies are needed, BAT-26 appears to be a sensitive and specific marker for the MSI-H phenotype in gastric carcinoma.