Pembrolizumab in microsatellite instability high or mismatch repair deficient cancers: updated analysis from the phase II KEYNOTE-158 study.

BACKGROUND: Pembrolizumab demonstrated durable antitumor activity in 233 patients with previously treated advanced microsatellite instability high (MSI-H) or mismatch repair deficient (dMMR) advanced solid tumors in the phase II multicohort KEYNOTE-158 (NCT02628067) study. Herein, we report safety and efficacy outcomes with longer follow-up for more patients with previously treated advanced MSI-H/dMMR noncolorectal cancers who were included in cohort K of the KEYNOTE-158 (NCT02628067) study. PATIENTS AND METHODS: Eligible patients with previously treated advanced noncolorectal MSI-H/dMMR solid tumors, measurable disease as per RECIST v1.1, and Eastern Cooperative Oncology Group performance status of 0 or 1 received pembrolizumab 200 mg Q3W for 35 cycles or until disease progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR) as per RECIST v1.1 by independent central radiologic review. RESULTS: Three hundred and fifty-one patients with various tumor types were enrolled in KEYNOTE-158 cohort K. The most common tumor types were endometrial (22.5%), gastric (14.5%), and small intestine (7.4%). Median time from first dose to database cut-off (5 October 2020) was 37.5 months (range, 0.2-55.6 months). ORR among 321 patients in the efficacy population (patients who received ≥1 dose of pembrolizumab enrolled ≥6 months before the data cut-off date) was 30.8% [95% confidence interval (CI) 25.8% to 36.2%]. Median duration of response was 47.5 months (range, 2.1+ to 51.1+ months; '+' indicates no progressive disease by the time of last disease assessment). Median progression-free survival was 3.5 months (95% CI 2.3-4.2 months) and median overall survival was 20.1 months (95% CI 14.1-27.1 months). Treatment-related adverse events (AEs) occurred in 227 patients (64.7%). Grade 3-4 treatment-related AEs occurred in 39 patients (11.1%); 3 (0.9%) had grade 5 treatment-related AEs (myocarditis, pneumonia, and Guillain-Barre syndrome, n = 1 each). CONCLUSIONS: Pembrolizumab demonstrated clinically meaningful and durable benefit, with a high ORR of 30.8%, long median duration of response of 47.5 months, and manageable safety across a range of heavily pretreated, advanced MSI-H/dMMR noncolorectal cancers, providing support for use of pembrolizumab in this setting.

Development and application of ELISA for the detection of IgG antibodies to lymphocytic choriomeningitis virus.

Lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen, which can cause severe illnesses in humans. The most vulnerable are the human foetus and immunosuppressed individuals. Since there is no commercially available enzyme-linked immunosorbent assay (ELISA) for the diagnosis of anti-LCMV antibodies in human sera, we developed a sandwich ELISA method detecting anti-nucleoprotein IgG antibodies, using a specific monoclonal anti-nucleoprotein antibody and cells persistently infected with LCMV strain MX as antigen. In the present study we show standardization of this ELISA protocol, determination of its clinical specificity and sensitivity and its application on 30 clinical samples from multiorgan donors. Comparison of these results to the indirect immunofluorescence antibody test (IFA) demonstrates that ELISA is more sensitive. The developed ELISA assay provides a fast, simple and efficient tool for the clinical detection of anti-nucleoprotein antibodies in human sera.

Analysis of HLA-G expression in serum and biopsy samples of kidney transplant recipients.

In this study, we analyzed HLA-G expression in serum and graft biopsies of renal transplant patients to find out whether there is any relationship between HLA-G and renal graft acceptance. The transplant patients were divided into two groups: those without any rejection episode (n=32) and those with acute rejection (n=33). Patient sera were collected 1 day before and at various intervals after transplantation. Soluble HLA-G (sHLA-G) in serum was determined using ELISA. In time-course experiment we found that in all patients (with and without rejection) the pre-transplantation level of sHLA-G declined in the early post-transplant period (1-2 weeks). In sera collected over 1-12 months after transplantation, a substantial increase of sHLA-G was detected in patients without rejection while no change or additional decline was observed in recipients with graft rejection. In sera collected after more than 1 year post-transplantation, sHLA-G levels increased in both groups of patients (with or without graft rejection). The time-course of serum sHLA-G antigens in patients with graft rejection was in good correlation with the course of total HLA-G mRNA determined in graft biopsy samples isolated from patients with acute rejection. We further demonstrated that serum sHLA-G values were significantly higher in patients without graft rejection than with rejection (P=0.0058). This observation supports the assumption that the increase of serum sHLA-G may contribute to allograft acceptance.

Aerobic training lasting for 10 weeks elevates the adipose tissue FABP4, Giα, and adiponectin expression associated by a reduced protein oxidation.

OBJECTIVE: The chronic moderate exercise positively alters the systemic glucose homeostasis, enhances the insulin action, and ameliorates the oxidative damage in the skeletal muscle and liver. The aim of this study was to investigate the effect of an intermittent aerobic training on the metabolic parameters of the white adipose tissue in the obese Zucker rats. METHODS: Obese Zucker rats, 8 week old, were subjected to running on a 4-channel treadmill (1 h/day 5 times/week 20 m/min at maximum) for 10 weeks, except the weekends, (Trained Obese Zucker, TOZ) or were placed to the turned-off treadmill (Sedentary Obese Zucker, SOZ) for the same period. The serum insulin, glucose, and triglyceride were determined. The gene expression of the renin-angiotensin system (RAS) components and selected metabolic parameters were quantified by real-time qPCR in the liver and epididymal and retroperitoneal adipose tissues. The content of the protein carbonyl groups was assayed in the liver and epididymal fat depot. RESULTS: The gene expression of the adipocyte fatty acid binding protein 4 (FABP4) was significantly elevated in the epididymal and retroperitoneal adipose tissues of the TOZ rats. The level of the adiponectin mRNA was increased in the retroperitoneal adipose tissue while leptin and inhibitory G-protein α mRNA were elevated in the epididymal adipose tissue after exercise. The aerobic training led to a decrease in the amount of protein carbonyl groups in the epididymal adipose depot. Transcription of the angiotensinogen, angiotensin-converting enzyme (ACE), and AT1 receptor genes in the epididymal adipose tissue was not influenced by the exercise. In the liver, only the AT1 receptor gene expression increased significantly. The serum glucose, insulin, and triglycerides concentrations were not changed in the TOZ rats when compared to SOZ animals. CONCLUSIONS: Data of the present study indicate that an intermittent moderate exercise in the hyperphagic obese Zucker rats lasting for 10 weeks improves some of the morphometric and metabolic parameters of the white adipose tissue and decreases the protein oxidation implying a general beneficial effect of the long-lasting exercising.

First-line chemotherapy with liposomal doxorubicin plus cisplatin for patients with advanced malignant pleural mesothelioma: phase II trial.

BACKGROUND: Chemotherapy based on platinum is the standard treatment for unresectable malignant pleural mesothelioma (MPM). Liposomal doxorubicin (LD) consists of pegylated phospholipid vesicles that encapsulate doxorubicin-enhancing liposome deposition in the tumour. We evaluated the toxicity profile and anti-tumour activity of cisplatin plus LD in untreated patients with MPM, as well as (99m)Tc-LD distribution in MPM lesions after chemotherapy administration. METHODS: A total of 38 patients with non-resectable MPM received LD 40 mg m(-2) and cisplatin 60 mg m(-2) every 21 days. Gamma camera images of (99m)Tc-LD were acquired to evaluate LD accumulation in measurable tumour tissue. The study was registered in Clinical Trials (NCT00886028). RESULTS: In all, 72% of patients were stage III and 28% were stage IV. Eighty four percent and 16% have high and low risk acording EORTC respectively. The median time to progression was 4.6 months (95% confidence interval (95% CI: 3.4-5.9 months), and median overall survival (OS) was 19.6 months (15.2-37.2 months). Patients that responded to chemotherapy treatment had better survival than patients who did not. Functional physical scales, dysnea, cough, and chest/arm pain demonstrated improvement. The accumulation ratio of LD in tumour and soft tissues vs liver was 0.78±0.16 and 0.29±0.09, respectively. After 1 h of administration, LD uptake in tumour tissue was higher than in soft tissue (P< 0.001). CONCLUSION: The combination of LD and cisplatin results in an active therapeutic regimen for unresectable MPM, with an acceptable toxicity profile and improvement in quality of life. (99m)Tc-LD showed higher levels of tumour uptake as compared with surrounding tissues.

Characterization of MICA gene polymorphism of HLA complex in the Slovak population.

BACKGROUND: The function of the MHC class I polypeptide-related sequence A (MICA) gene, which belongs to the MHC class I chain-related genes, is to trigger cytolysis of target cells mediated by NKG2D receptor recognition in NK (Natural Killer) cells and CD8 T-lymphocytes. The MICA gene has a high degree of polymorphism, especially observed in exons 2-5. MICA allelic diversity has been reported in association with some autoimmune diseases such Behcet's disease, psoriasis and diabetes, as well as with organ rejections. AIM: The aim of this study was to analyse MICA gene polymorphism in the Slovak population, to establish frequencies of MICA alleles and to compare the results with those found in other Western Eurasian populations. No such study has been performed previously in the Slovak population. SUBJECTS AND METHODS: This study examined DNA samples from 124 unrelated Slovak individuals (51 women and 73 men with an average age of 40.3 years) using direct sequencing of MICA exons 2-5. Allele and genotype frequencies were calculated by direct counting and statistical analysis was carried out using Arlequin software. RESULTS: This study identified 15 out of 71 MICA alleles. The most frequent allele was MICA(*)008 (37.1%) followed by alleles MICA(*)002 (16.5%) and MICA(*)009 (11.3%). The rarest alleles were MICA(*)027, MICA(*)006 (both 0.8%) and MICA(*)057 (0.4%), respectively. The most frequent genotypes were 008/008 and 008/002, both with a frequency of 13.7%. Exon 5 microsatellite polymorphism screening revealed five MICA alleles, namely A4, A5, A5.1, A6 and A9. The most frequent was allele A5.1 (37.1%) and the rarest A5 (8.1%). Finally it was found that haplotype MICA*008 A5.1 was the most frequent (37.1%). CONCLUSION: A comparison of these results with those reported in the literature revealed similarity in MICA polymorphism to that found in other Western Eurasian populations. The data will be useful for further association studies on MICA polymorphism and its function.

Demethylating agent 5-aza-2'-deoxycytidine activates HLA-G expression in human leukemia cell lines.

HLA-G is a non-classical HLA class I antigen primarily expressed in the extravillous cytotrophoblast. HLA-G can also be expressed at some pathological circumstances and may thus contribute to inhibition of efficient immune responses. Complex regulation of HLA-G expression also involves epigenetic mechanisms as DNA methylation. Here we demonstrate that treatment with demethylating agent 5-aza-2'-deoxycytidine (AdC) resulted in HLA-G transcription in 18 out of 20 examined leukemia cell lines. HLA-G protein synthesis was detected in 10 cell lines expressing significant level of HLA-G transcripts following AdC treatment.

Modulation of HLA-G expression.

Recent studies demonstrated that HLA-G transcription is in some cells silenced by epigenetic mechanisms as DNA methylation and histone modification. Accordingly HLA-G gene transcriptions can be activated in such cells by demethylating agent or by inhibitors of histone deacetylation. In addition to epigenetic alterations HLA-G gene transcription can be activated by stress. In the present study these aspects of HLA-G expression are re-examined and a new inhibitor of histone deacetylation (valproic acid) and hypoxia mimetic chemical (CoCl2) are included. The highest activation of HLA-G transcription was achieved by treatment of choriocarcinoma JAR and lymphoblastoid RAJI cell lines with demethylating agent 5-aza-2 - deoxycytidine. Treatment of JAR and RAJI cells with histone deacetylase inhibitors (sodium butyrate and valproic acid) also enhanced HLA-G transcription. Nevertheless this increase in HLA-G expression was low as compared with activation by 5-aza-2 - deoxycytidine. The hypoxia mimetic agents (desferrioxamine or CoCl2) had no detectable effect on HLA-G gene transcription in examined cells. Relatively high increase of HLA-G transcription was detected in JAR and RAJI cells exposed to heat shock treatment. Interestingly heat shock induced high expression of HLA-G6 transcript in JAR cells. Heat shock treatment had no effect on alternative splicing of constitutively expressed HLA-G mRNA in choriocarcinoma cell line JEG-3. HLA-G1 protein expression was induced in JAR and RAJI cell lines by 5-aza-2 - deoxycytidine. In agreement with the differences in the levels of HLA-G transcripts JAR cells express more of HLA-G1 protein than RAJI cells.

HLA class II allele frequencies in type 1A diabetes mellitus Slovak patients.

BACKGROUND: Diabetes mellitus type 1A (DM-1A) is an autoimmune disease in which the immune response is directed to pancreatic islet cells. DM-1A occurs in genetically predisposed individuals. Among type 1A diabetes associated genes, those of the HLA region have the greatest effect. OBJECTIVES: The aim of our study was to obtain a comprehensive survey of the HLA-DRB1 and HLA-DQB1 allele frequencies in Slovak patients suffering from DM-1A. METHODS: HLA class II genotyping was performed on genomic DNA by the PCR-SSP method according to the 12th Workshop protocol. RESULTS: Our report gives the first presentation of the distribution of HLA-DRB1 alleles (including complete DRB1*04 subtypes) and that of HLA-DQB1 alleles in the Slovak diabetic patients diagnosed at 0-18 years of age. Susceptibility is significantly associated with the alleles DQB1*0302 (OR = 7.8), DRB1*04 (OR = 4.9), DRB1*0301 (OR = 4.2) and DQB1*02 (OR = 2.2), whereas the alleles DQB*0602 (OR = 0.05), DRB1*11 (OR = 0.2), DRB1*15 (OR = 0.2) and DQB1*0301 (OR = 0.3) were found to be protective. CONCLUSIONS: Our results, consistent with other studies, show increased frequencies of known positively associated HLA class II alleles in our type 1A diabetes mellitus patients compared to the general (nondiabetic) population. The protective effect of previously reported alleles was confirmed as well. Results of our population-based study serve in clinical practice for the identification of subjects at risk of developing DM-1A among the first-degree relatives (Tab. 2, Ref. 12).

Effects of sizofiran on endotoxin-enhanced cold ischemia-reperfusion injury of the rat liver.

Kupffer cells (KC), resident macrophages of the liver, have been strongly implicated in lipopolysaccharide (LPS)-induced liver graft injury. However, our recent study showed that sizofiran (schizophyllan glucan) (SPG), which activates KC, did not influence cold ischemia-reperfusion liver injury of LPS-exposed rats. Here we investigated some mechanisms by which SPG does not aggravate LPS-enhanced cold ischemia-reperfusion rat liver injury. Control and SPG-treated rats were exposed to LPS for 2 h prior to hepatectomy. The livers were cold-preserved in University of Wisconsin solution followed by reperfusion with Krebs-Henseleit buffer. We found that SPG dramatically inhibited LPS-induced increases of tumor necrosis factor-alpha (TNF-alpha) in the plasma and bile in vivo. Moreover, LPS-induced TNF- release into the washout solution after cold ischemia was also abrogated by SPG pretreatment. However, SPG increased TNF- release into the perfusate after reperfusion. On the other hand, SPG completely abolished expression of c-myc protooncogene, which is known to sensitize cells to TNF-alpha cytotoxicity. In conclusion, inhibition of both TNF- release after LPS challenge and c-myc expression may explain why activation of KC with SPG does not aggravate endotoxin-enhanced cold ischemia-reperfusion liver injury.

[Protection of hypothermic rat liver against gentle manipulation injury]. / Ochrana hypotermickej pecene potkana pred poskodením jemnou manipuláciou.

Hypothermic preservation can increase hepatocyte sensitivity to various insults. Here we studied the hypothesis that hepatocytes are injured by manipulation with cold-preserved liver. Livers from Wistar rats were divided into two groups. In the 1st group (n = 6) the livers were placed after harvesting into a polyethylene (PE) bag. After the preservation period they were placed into the perfusion chamber--developed in our laboratory. Connection of livers from PE bag to the perfusion chamber required a contact manipulation with the liver. This contact manipulation was eliminated in the second group of livers (n = 6) by using the perfusion chamber for preservation. Lavage and perfusion were done in both groups under the same conditions. We found in the lavage solution of livers of the 1st group 2-times more LDH and 3.7-times higher release of TNF-alpha compared to the 2nd group. Further, bile flow of livers from the 1st group during reperfusion was significantly lower compared to the 2nd group (0.179 +/- 0,12 vs 0.398 +/- 0.15 ml/min/g liver). Manipulation with hypothermic liver leads to hepatocyte injury. Our new model of chamber can protect hypothermic liver against manipulation injury and can allow to perform physiological and pharmacological experiments on liver ex vivo.

Protection of the rat liver against rewarming ischemic injury by University of Wisconsin solution.

BACKGROUND/AIM: University of Wisconsin (UW) solution has been proven able to prevent liver injury during cold ischemia. During rewarming ischemia, however, the efficacy of this solution in preserving hepatocyte function is unclear. The aim of the present study was to investigate to what extent UW solution protects rat liver during rewarming ischemia. METHODS: Livers were washed out with cool physiologic saline or with UW solution and subjected to rewarming ischemia for periods of 20 min or 45 min followed by reperfusion using a blood-free perfusion model. RESULTS: In comparison with controls, ischemia for 20 min in saline-treated livers led to mild depression of hepatocyte function, while UW solution afforded complete protection of the liver. In UW-treated livers, compared with saline-treated livers exposed to ischemia for 45 min, portal flow was slightly but significantly higher, bile production was increased by 62%, and lactate dehydrogenase leakage into the perfusate was reduced by 61%. In an attempt to explain mechanisms of liver protection by UW solution, we found that UW solution inhibited conversion of hypoxanthine into uric acid, but this effect was not associated with decreased degradation of adenine nucleotides in the liver during ischemia. Following 30 min reperfusion, UW solution increased tissue levels of adenosine triphosphate (not significantly) and adenosine diphosphate (significantly). Further, UW solution markedly reduced tumor necrosis factor-alpha release by the liver both after ischemia and after reperfusion. CONCLUSIONS: These results create the hypothesis that UW solution may protect liver tissue during ischemia in liver surgery as well as during the implantation stage of liver transplantation.

Improvement of rat liver function by energy repletion after the preservation period: implications for hepatic graft management.

We very recently showed (using a blood-free perfusion model) that cold preservation sensitized rat hepatocyte functions to rewarming ischemic injury and that the injury can be prevented by repleting high-energy adenylates in the liver by short-term oxygenated warm reperfusion. Here we investigated whether short-term reperfusion after the preservation period can improve hepatic graft function in a blood reperfusion model. Eighteen-hour cold-preserved rat livers either untreated (Group A) or pretreated by 30-min oxygenated warm reperfusion after preservation (Group B) were subjected to 20-min ischemic rewarming and then reperfused with blood. Livers in Group B compared to Group A exhibited approx. three times increased bile production and bromosulfophthalein excretion, nearly 7-fold decreased swelling, and 1.2-fold improved blood flow. These results suggest that repletion of the energy by short-term oxygenated reperfusion after prolonged preservation may improve markedly initial hepatic graft function.

Marked difference in tumor necrosis factor-alpha expression in warm ischemia- and cold ischemia-reperfusion of the rat liver.

Although tumor necrosis factor-alpha has been implicated in liver injury after both warm ischemia- and cold ischemia-reperfusion, it is unclear whether reactivity of the liver to these stimuli is similar with regard to cytokine expression. Here we compare the effects of warm and cold ischemia on tumor necrosis factor-alpha expression and test the hypothesis that cold ischemia preceding warm ischemia causes overexpression of this cytokine. Rat livers were flushed out with University of Wisconsin solution and subjected to varying periods of warm ischemia, cold ischemia, or cold ischemia plus warm ischemia followed by reperfusion using a blood-free perfusion model. Tumor necrosis factor-alpha and interleukin-10 release into the perfusate and bile were measured by ELISA, and expression of these cytokines and that of c-fos, c-jun, and c-myc were studied by reverse-transcriptase polymerase chain reaction. We found high levels of tumor necrosis factor-alpha in the perfusates of livers subjected to warm ischemia-reperfusion, whereas minimal or no tumor necrosis factor-alpha was detected in livers subjected to cold ischemia-reperfusion or to cold ischemia plus warm ischemia-reperfusion. Reverse-transcriptase polymerase chain reaction confirmed the above findings and showed that immediate early genes were expressed in reperfused groups of livers. Measurements of cytokine release into bile showed that neither tumor necrosis factor-alpha nor interleukin-10 were upregulated by cold ischemia-reperfusion. The results suggest that (1) warm ischemia- and cold ischemia-reperfusion of rat liver lead to very different outcomes with regard to tumor necrosis factor-alpha expression and (2) cold ischemia preceding warm ischemia prevents upregulation of tumor necrosis factor-alpha.

Familial testicular cancer and developmental anomalies.

Familial occurrence belongs to factors followed in etiology and pathogenesis of testicular germ-cell tumors. Association with abnormal testicular development, or with other risk factors is relatively frequent. In our material 650 patients had been treated for testicular cancer in the period of 1981-1995. Familial occurrence was observed 7-times (1.08%), most frequently in combination with cryptorchidism. Individual families were analyzed in details, including HLA typing. On basis of the observations the supplementation of initial examination of each patient with suspicious testicular cancer with detailed familial history aimed also at the occurrence of urogenital developmental anomalies and tumors has been recommended. The knowledge about familial tumor occurrence in the first-degree relatives in combination with thorough testicular self-examination is being considered of great importance in the secondary prevention.

[Familial occurrence of tumors and developmental anomalies of the testis]. / Familiárny výskyt nádorov a vývojových anomálií testis.

BACKGROUND: Family occurrence ranks belong the factors followed in etiology and pathogenesis of germ-cell tumours of the testis. Its association with abnormal testicular development, respectively with other risk factors is relatively frequent. OBJECTIVES: The aim of this study was to indicate this coherence by means of case histories of author's patients and to propose further procedures. METHODS AND RESULTS: There were 535 patients treated for testicular cancer in the period of 1982-1994. Family occurrence was observed in 6 cases (1.12%), most frequently incombination with maldescensus testis. Individual families were analysed in detail, including HLA typization. Bilaterality of testicular cancer was observed in two brothers who were HLA identical. Other two brothers had the history of bilateral maldescensus testis, one of whom was subdued to bilateral orchiectomy in childhood, the other at the age of 16, a tumour in one testicle following orchidopexy performed in childhood. The history of maldesensus testis was observed in four members of another family, two of whom developed tumours. CONCLUSIONS AND MEANING FOR PRACTICE: Authors recommend supplementation of the initial examination of each patient with suspective testicular cancer with detailed family history aim at the occurrence of urogenital anomalies and tumours. General knowledge of the first-degree relatives about the possibility of family occurrence of tumours, and instructions for testicular self-examination are considered as the most suitable method from the stand point of secondary prevention. (Ref. 21.)