Cell membrane-camouflaged bufalin targets NOD2 and overcomes multidrug resistance in pancreatic cancer.

AIMS: Multidrug resistance in pancreatic cancer poses a significant challenge in clinical treatment. Bufalin (BA), a compound found in secretions from the glands of toads, may help overcome this problem. However, severe cardiotoxicity thus far has hindered its clinical application. Hence, the present study aimed to develop a cell membrane-camouflaged and BA-loaded polylactic-co-glycolic acid nanoparticle (CBAP) and assess its potential to counter chemoresistance in pancreatic cancer. METHODS: The toxicity of CBAP was evaluated by electrocardiogram, body weight, distress score, and nesting behavior of mice. In addition, the anticarcinoma activity and underlying mechanism were investigated both in vitro and in vivo. RESULTS: CBAP significantly mitigated BA-mediated acute cardiotoxicity and enhanced the sensitivity of pancreatic cancer to several clinical drugs, such as gemcitabine, 5-fluorouracil, and FOLFIRINOX. Mechanistically, CBAP directly bound to nucleotide-binding and oligomerization domain containing protein 2 (NOD2) and inhibited the expression of nuclear factor kappa-light-chain-enhancer of activated B cells. This inhibits the expression of ATP-binding cassette transporters, which are responsible for chemoresistance in cancer cells. CONCLUSIONS: Our findings indicate that CBAP directly inhibits NOD2. Combining CBAP with standard-of-care chemotherapeutics represents a safe and efficient strategy for the treatment of pancreatic cancer.

The epigenetic modifier HDAC2 and the checkpoint kinase ATM determine the responses of microsatellite instable colorectal cancer cells to 5-fluorouracil.

The epigenetic modifier histone deacetylase-2 (HDAC2) is frequently dysregulated in colon cancer cells. Microsatellite instability (MSI), an unfaithful replication of DNA at nucleotide repeats, occurs in about 15% of human colon tumors. MSI promotes a genetic frameshift and consequently a loss of HDAC2 in up to 43% of these tumors. We show that long-term and short-term cultures of colorectal cancers with MSI contain subpopulations of cells lacking HDAC2. These can be isolated as single cell-derived, proliferating populations. Xenografted patient-derived colon cancer tissues with MSI also show variable patterns of HDAC2 expression in mice. HDAC2-positive and HDAC2-negative RKO cells respond similarly to pharmacological inhibitors of the class I HDACs HDAC1/HDAC2/HDAC3. In contrast to this similarity, HDAC2-negative and HDAC2-positive RKO cells undergo differential cell cycle arrest and apoptosis induction in response to the frequently used chemotherapeutic 5-fluorouracil, which becomes incorporated into and damages RNA and DNA. 5-fluorouracil causes an enrichment of HDAC2-negative RKO cells in vitro and in a subset of primary colorectal tumors in mice. 5-fluorouracil induces the phosphorylation of KAP1, a target of the checkpoint kinase ataxia-telangiectasia mutated (ATM), stronger in HDAC2-negative cells than in their HDAC2-positive counterparts. Pharmacological inhibition of ATM sensitizes RKO cells to cytotoxic effects of 5-fluorouracil. These findings demonstrate that HDAC2 and ATM modulate the responses of colorectal cancer cells towards 5-FU.

CIITA-Transduced Glioblastoma Cells Uncover a Rich Repertoire of Clinically Relevant Tumor-Associated HLA-II Antigens.

CD4+ T cell responses are crucial for inducing and maintaining effective anticancer immunity, and the identification of human leukocyte antigen class II (HLA-II) cancer-specific epitopes is key to the development of potent cancer immunotherapies. In many tumor types, and especially in glioblastoma (GBM), HLA-II complexes are hardly ever naturally expressed. Hence, little is known about immunogenic HLA-II epitopes in GBM. With stable expression of the class II major histocompatibility complex transactivator (CIITA) coupled to a detailed and sensitive mass spectrometry-based immunopeptidomics analysis, we here uncovered a remarkable breadth of the HLA-ligandome in HROG02, HROG17, and RA GBM cell lines. The effect of CIITA expression on the induction of the HLA-II presentation machinery was striking in each of the three cell lines, and it was significantly higher compared with interferon gamma (IFNÉ£) treatment. In total, we identified 16,123 unique HLA-I peptides and 32,690 unique HLA-II peptides. In order to genuinely define the identified peptides as true HLA ligands, we carefully characterized their association with the different HLA allotypes. In addition, we identified 138 and 279 HLA-I and HLA-II ligands, respectively, most of which are novel in GBM, derived from known GBM-associated tumor antigens that have been used as source proteins for a variety of GBM vaccines. Our data further indicate that CIITA-expressing GBM cells acquired an antigen presenting cell-like phenotype as we found that they directly present external proteins as HLA-II ligands. Not only that CIITA-expressing GBM cells are attractive models for antigen discovery endeavors, but also such engineered cells have great therapeutic potential through massive presentation of a diverse antigenic repertoire.

Glucose Influences the Response of Glioblastoma Cells to Temozolomide and Dexamethasone.

OBJECTIVE: Current research indicates that weakness of glucose metabolism plays an important role in silencing of invasiveness and growth of hypoxic tumors such as GBM. Moreover, there are indications that DXM, frequently used in treatment, may support GBM energy metabolism and provoke its recurrence. METHODS: We carried out in vitro experiments on the commercial T98G cell line and two primary GBM lines (HROG02, HROG17) treated with TMZ and/or DXM in physiological oxygen conditions for GBM (2.5% oxygen) and for comparison, in standard laboratory conditions (20% oxygen). The influence of different glucose levels on selected malignancy features of GBM cells-cellular viability and division, dynamic of cell culture changes, colony formation and concentration of InsR have been elevated. RESULTS: Under 2.5% oxygen and high glucose concentration, an attenuated cytotoxic effect of TMZ and intensification of malignancy features in all glioblastoma cell lines exposed to DXM was seen. Furthermore, preliminary retrospective analysis to assess the correlation between serum glucose levels and Ki-67 expression in surgical specimens derived from patients with GBM (IV) treated with radio-chemotherapy and prophylactic DXM therapy was performed. CONCLUSION: The data suggest a link between the in vitro study results and clinical data. High glucose can influence on GBM progression through the promotion of the following parameters: cell viability, dispersal, InsR expression and cell proliferation (Ki-67). However, this problem needs more studies and explain the mechanism of action studied drugs.

Establishment and Characterization of Novel Human Intestinal In Vitro Models for Absorption and First-Pass Metabolism Studies.

Commonly used intestinal in vitro models are limited in their potential to predict oral drug absorption. They either lack the capability to form a tight cellular monolayer mimicking the intestinal epithelial barrier or the expression of cytochrome P450 3A4 (CYP3A4). The aim of this study was to establish a platform of colorectal cancer patient-derived cell lines for evaluation of human intestinal drug absorption and metabolism. We characterized ten 2D cell lines out of our collection with confluent outgrowth and long-lasting barrier forming potential as well as suitability for high throughput applications with special emphasis on expression and inducibility of CYP3A4. By assessment of the transepithelial electrical resistance (TEER) the cells barrier function capacity can be quantified. Very high TEER levels were detected for HROC60. A high basal CYP3A4 expression and function was found for HROC32. Eight cell lines showed higher CYP3A4 induction by stimulation via the vitamin D receptor compared to Caco-2 cells (5.1- to 16.8-fold change). Stimulation of the pregnane X receptor led to higher CYP3A4 induction in two cell lines. In sum, we identified the two cell lines HROC183 T0 M2 and HROC217 T1 M2 as useful tools for in vitro drug absorption studies. Due to their high TEER values and inducibility by drug receptor ligands, they may be superior to Caco-2 cells to analyze oral drug absorption and intestinal drug-drug interactions. Significance statement: Selecting appropriate candidates is important in preclinical drug development. Therefore, cell models to predict absorption from the human intestine are of the utmost importance. This study revealed that the human cell lines HROC183 T0 M2 and HROC217 T1 M2 may be better suited models and possess higher predictive power of pregnane X receptor- and vitamin D-mediated drug metabolism than Caco-2 cells. Consequently, they represent useful tools for predicting intestinal absorption and simultaneously enable assessment of membrane permeability and first-pass metabolism.

Pharmaceutical immunoglobulin G impairs anti-carcinoma activity of oxaliplatin in colon cancer cells.

BACKGROUND: Recent evidence proves that intravenous human immunoglobulin G (IgG) can impair cancer cell viability. However, no study evaluated whether IgG application benefits cancer patients receiving chemotherapeutics. METHODS: Influence of pharmaceutical-grade human IgG on the viability of a series of patient-derived colon cancer cell lines with and without chemotherapeutic intervention was determined. Cell death was analysed flow cytometrically. In addition, the influence of oxaliplatin and IgG on the ERK1/2-signalling pathway was evaluated by western blots. RESULTS: We evaluated the effects of pharmaceutical IgG, such as PRIVIGEN® IgG and Tonglu® IgG, in combination with chemotherapeutics. We did not observe any significant effects of IgG on tumour cell viability directly; however, human IgG significantly impaired the anti-tumoral effects of oxaliplatin. Primary cancer cell lines express IgG receptors and accumulate human IgG intracellularly. Moreover, while oxaliplatin induced the activation of ERK1/2, the pharmaceutical IgG inhibited ERK1/2 activity. CONCLUSIONS: The present study demonstrates that pharmaceutical IgG, such as PRIVIGEN® IgG and Tonglu® IgG, can impair the anti-carcinoma activity of oxaliplatin. These data strongly suggest that therapeutic IgG as co-medication might have harmful side effects in cancer patients. The clinical significance of these preclinical observations absolutely advises further preclinical, as well as epidemiological and clinical research.

Epidemiologic trends and prognostic risk factors of patients with pancreatic neuroendocrine neoplasms in the US: an updated population-based study.

Background: We aimed to evaluate the incidence, mortality and survival outcome for patients with pancreatic neuroendocrine neoplasms (pNEN). Methods: Patients with pNEN were collected from the Surveillance, Epidemiology, and End Results (SEER) database. Incidence, mortality and average annual percentage change (AAPC) were calculated using SEER stat 8.3.6 and Joinpoint software. Survival outcome was estimated using Kaplan-Meier and Cox proportional hazard model. Results: During 2000-2016, the incidence of pNEN significantly rose from 0.2647 to 1.0618 per 100,000 persons with an AAPC of 9.4; AAPC of mortality was 6.7. Prognostic improvement was revealed in 2010-2016, but not for late-stage pNEN, which had the highest risk of death. Conclusion: Efforts to improve prognosis of pNEN patients must focus on not only early detection, but also on improving therapy for late-stage disease.

Cancer-Cell-Derived IgG and Its Potential Role in Tumor Development.

Human immunoglobulin G (IgG) is the primary component of the human serum antibody fraction, representing about 75% of the immunoglobulins and 10-20% of the total circulating plasma proteins. Generally, IgG sequences are highly conserved, yet the four subclasses, IgG1, IgG2, IgG3, and IgG4, differ in their physiological effector functions by binding to different IgG-Fc receptors (FcγR). Thus, despite a similarity of about 90% on the amino acid level, each subclass possesses a unique manner of antigen binding and immune complex formation. Triggering FcγR-expressing cells results in a wide range of responses, including phagocytosis, antibody-dependent cell-mediated cytotoxicity, and complement activation. Textbook knowledge implies that only B lymphocytes are capable of producing antibodies, which recognize specific antigenic structures derived from pathogens and infected endogenous or tumorigenic cells. Here, we review recent discoveries, including our own observations, about misplaced IgG expression in tumor cells. Various studies described the presence of IgG in tumor cells using immunohistology and established correlations between high antibody levels and promotion of cancer cell proliferation, invasion, and poor clinical prognosis for the respective tumor patients. Furthermore, blocking tumor-cell-derived IgG inhibited tumor cells. Tumor-cell-derived IgG might impede antigen-dependent cellular cytotoxicity by binding antigens while, at the same time, lacking the capacity for complement activation. These findings recommend tumor-cell-derived IgG as a potential therapeutic target. The observed uniqueness of Ig heavy chains expressed by tumor cells, using PCR with V(D)J rearrangement specific primers, suggests that this specific part of IgG may additionally play a role as a potential tumor marker and, thus, also qualify for the neoantigen category.

NSG mice as hosts for oncological precision medicine.

Patient-derived xenograft (PDX) models have been rediscovered as meaningful research tool. By using severely immunodeficient mice, high-engraftment rates can be theoretically achieved, permitting clinical stratification strategies. Apart from engraftment efficacy, tolerability towards certain cytostatic drugs varies among individual mouse strains thus impeding large-scale screenings. Here, we aimed at optimizing an in vivo treatment schedule using the widely applied cytostatic drug 5-fluoruracil (5-FU) for exemplary response prediction in colorectal cancer (CRC) PDX models. Four different individual CRC PDX models were engrafted into NOD.Cg-PrkdcscidIl2rgtm1Wjl (NSG) mice. Mice with established PDX were allocated to different treatment groups, receiving 5-FU, the oral prodrug Capecitabine, or 5-FU/leucovorin (LV) at different doses. Body weight, tumor size, and general behavior were assessed during therapy. Ex vivo analyses were done from blood samples, liver, as well as tumor resection specimen. Engraftment efficacy was high as expected in NSG mice, yielding stable PDX growth for therapy stratification. However, overall tolerability towards 5-FU was unexpectedly low, whereas the prodrug Capecitabine as well as the combination of 5-FU/LV at low doses were well tolerated. Accompanying plasma level determination of DYPD, the rate-limiting enzyme for 5-FU-mediated toxicity, revealed reduced activity in NSG mice compared with other common laboratory mouse strains, offering a likely explanation for the drug incompatibility. Also, the De Ritis quotient was highly elevated in treated mice, reflecting overall organ injury even at low doses. Summarizing these findings, NSG mice are ideal hosts for in vivo engraftment studies. However, the complex immunodeficiency reduces tolerance to certain drugs, thus making those mice especially sensitive. Consequently, such dose finding and tolerance tests constitute a necessity for similar cancer precision medicine approaches.

Patient-individual cancer cell lines and tissue analysis delivers no evidence of sequences from DNA viruses in colorectal cancer cells.

BACKGROUND: Several DNA viruses are highly suspicious to have oncogenic effects in humans. This study investigates the presence of potentially oncogenic viruses such as SV40, JCV, BKV and EBV in patient-derived colorectal carcinoma (CRC) cells typifying all molecular subtypes of CRC. METHODS: Sample material (gDNA and cDNA) of a total of 49 patient-individual CRC cell lines and corresponding primary material from 11 patients, including normal, tumor-derived and metastasis-derived tissue were analyzed for sequences of SV40, JVC, BKV and EBV using endpoint PCR. In addition, the susceptibility of CRC cells to JCV and BKV was examined using a long-term cultivation approach of patient-individual cells in the presence of viruses. RESULTS: No virus-specific sequences could be detected in all specimens. Likewise, no morphological changes were observed and no evidence for viral infection or integration could be provided after long term CRC cell cultivation in presence of viral particles. CONCLUSIONS: In summary, the presented data suggest that there is no direct correlation between tumorigenesis and viral load and consequently no evidence for a functional role of the DNA viruses included into this analysis in CRC development.

Frameshift mutational target gene analysis identifies similarities and differences in constitutional mismatch repair-deficiency and Lynch syndrome.

Mismatch-repair deficient (MMR-D) malignancies include Lynch Syndrome (LS), which is secondary to germline mutations in one of the MMR genes, and the rare childhood-form of constitutional mismatch repair-deficiency (CMMR-D); caused by bi-allelic MMR gene mutations. A hallmark of LS-associated cancers is microsatellite instability (MSI), characterized by coding frameshift mutations (cFSM) in target genes. By contrast, tumors arising in CMMR-D patients are thought to display a somatic mutation pattern differing from LS. This study has the main goal to identify cFSM in MSI target genes relevant in CMMR-D and to compare the spectrum of common somatic mutations, including alterations in DNA polymerases POLE and D1 between LS and CMMR-D. CMMR-D-associated tumors harbored more somatic mutations compared to LS cases, especially in the TP53 gene and in POLE and POLD1, where novel mutations were additionally identified. Strikingly, MSI in classical mononucleotide markers BAT40 and CAT25 was frequent in CMMR-D cases. MSI-target gene analysis revealed mutations in CMMR-D-associated tumors, some of them known to be frequently hit in LS, such as RNaseT2, HT001, and TGFßR2. Our results imply a general role for these cFSM as potential new drivers of MMR-D tumorigenesis.

Optimized creation of glioblastoma patient derived xenografts for use in preclinical studies.

BACKGROUND: Glioblastoma multiforme (GBM) is the most common and lethal brain tumor in adults, highlighting the need for novel treatment strategies. Patient derived xenografts (PDX) represent a valuable tool to accomplish this task. METHODS: PDX were established by implanting GBM tissue subcutaneously. Engraftment success was compared between NMRI Foxn1nu and NOD/SCID as well as between fresh and cryopreserved tissue. Established PDX were analyzed histologically and molecularly. Five PDX were experimentally treated with different drugs to assess their potential for preclinical drug testing. RESULTS: Establishment of PDX was attempted for 36 consecutive GBM cases with an overall success rate of 22.2% in NMRI Foxn1nu mice. No difference was observed between fresh or cryopreserved (20-1057 days) tissue in direct comparison (n = 10 cases). Additionally, engraftment was better in NOD/SCID mice (38.8%) directly compared to NMRI Foxn1nu mice (27.7%) (n = 18 cases). Molecular data and histology of the PDX compare well to the primary GBM. The experimental treatment revealed individual differences in the sensitivity towards several clinically relevant drugs. CONCLUSIONS: The use of vitally frozen GBM tissue allows a more convenient workflow without efficiency loss. NOD/SCID mice appear to be better suited for initial engraftment of tumor tissue compared to NMRI Foxn1nu mice.

MicroRNA-mRNA interactions in colorectal cancer and their role in tumor progression.

MicroRNAs (miRNA/miR) play an important role in gene regulatory networks through targeting mRNAs. They are involved in diverse biological processes such as cell proliferation, differentiation, angiogenesis, and apoptosis. Due to their pivotal effects on multiple genes and pathways, dysregulated miRNAs have been reported to be associated with different diseases, including colorectal cancer (CRC). Recent evidence indicates that aberrant miRNA expression is tightly linked with the initiation and progression of CRC. To elucidate the influence of miRNA regulation in CRC, it is critical to identify dysregulated miRNAs, their target mRNA genes and their involvement in gene regulatory and signaling networks. Various experimental and computational studies have been conducted to decipher the function of miRNAs involved in CRC. Experimental studies that are used for this purpose can be classified into two categories: direct/individual and indirect/high-throughput gene expression studies. Here we review miRNA target identification studies related to CRC with an emphasis on experimental data based on Luciferase reporter assays. Recent advances in determining the function of miRNAs and the signaling pathways they are involved in have also been summarized. The review helps bioinformaticians and biologists to find extensive information about downstream targets of dysregulated miRNAs, and their pro-/anti-CRC effects.

Assessing quality and functionality of DNA isolated from FFPE tissues through external quality assessment in tissue banks.

BACKGROUND: Biobanks are becoming increasingly important for assessment of disease risk as well as identification and validation of new diagnostic biomarkers and druggable targets. The validity of data obtained from biobanks is critically limited by the biomaterial quality of the biological samples. External quality assessment (EQA) programs suitable to comprehensively measure the biomaterial quality in archived materials are currently lacking. We report on quantitative assay designs for the analysis of both structural and functional integrity of DNAs that were applied in a first pilot EQA within the priority program on tumor tissue biobanking funded by the German Cancer Aid. METHODS: Participating biobanks isolated DNAs from a standardized set of 10 samples comprising sections of four different formalin-fixed paraffin-embedded tissues using their standard operating procedures. Isolated DNAs and analytical results were returned and analyzed centrally for nucleic acids yield, purity, fragmentation and amplificability at a quantitative level using dedicated assay designs. RESULTS: The amount of extracted DNA varied in isolates ranging between 1.5 µg and 25.8 µg. Quantification of DNA fragmentation and amplificability allowed to highlight considerable discrepancies in DNA quality. Amplicons yielded from the isolates of these identical EQA samples ranged from 105 to 411 bp suggesting differences between residual inhibitors of downstream enzymatic reactions. CONCLUSIONS: The quality of extraction of bioanalytes from biomaterial archives is heterogeneous even for stable biomolecules like DNA isolated with highly standardized methods. EQAs are appropriate tools to uncover strengths and weaknesses in biobanks in a systematic fashion. Biomaterial integrity is insufficiently reflected by standard methods, but needs to be assessed to improve biobank interoperability. Finally, our results also point towards the problem of measuring the quality of more delicate biomolecules like proteins or metabolites.

Stage-specific frequency and prognostic significance of aneuploidy in patients with sporadic colorectal cancer--a meta-analysis and current overview.

PURPOSE: Aneuploidy has long been suggested as an independent prognostic marker for colorectal cancer (CRC) patients and could thus aid for individualized medicine. However, due to a large spectrum of deviating studies, expert panels do not recommend ploidy assessment. In order to clarify a potential bias of stage-specific frequency of aneuploidy, we now conducted a meta-analysis combined with a systematic review regarding aneuploidy and prognosis. METHODS: A systematic, web-based search process retrieved 1935 studies published in English between 1990 and 2011. The defined endpoint for the meta-analysis was an increase in aneuploidy frequency between early- (Dukes A, B and UICC I, II; n = 3632 samples) and late-stage (Dukes C, D and UICC III, IV; n = 3440 samples) colorectal carcinomas. RESULTS: Of 1935 studies initially identified, 17 image (2130 patients) and 20 (7023 patients) flow cytometric studies were analyzed in detail. The meta-analysis (7072 patients) revealed late-stage CRC to be more frequently aneuploid than early-stage CRC (odds ratio 1.51, 95 % CI 1.37-1.67; p = 0.0007). Independent of tumor stage, the overall range of aneuploidy was 39 to 81 % (median 58 %), and altogether, 21 (54.1 %) studies described a significant prognostic impact of aneuploidy for overall, disease-specific, and recurrence-free survival, respectively. CONCLUSIONS: A substantial number of studies showed a prognostic importance of aneuploidy in CRC. Furthermore, the higher frequency of aneuploidy in late-stage CRC implies an increase in genomic instability with CRC progression, indicating aneuploidy to be also a stage-specific prognostic marker.

The impact of pyrvinium pamoate on colon cancer cell viability.

PURPOSE: The in vitro and in vivo effects of pyrvinium pamoate (PP), a newly identified WNT signaling inhibitor, were evaluated against colon cancer cell lines and primary colon cancer samples. EXPERIMENTAL DESIGN: Antiproliferative activity of PP and its effects on protein and RNA levels of WNT targets were evaluated on adenomatous polyposis coli (APC (mut)) and ß-catenin(mut) cell lines, one WNT(wt) colon cancer cell line, as well as six primary colon cancer samples with mutant APC in vitro. In addition, the effect of PP on the growth of liver metastasis was examined. RESULTS: PP blocked colon cancer cell growth in vitro in a dose-dependent manner with great differences in the inhibitory concentration (IC(50)), ranging from 0.6 × 10(-6) to 65 × 10(-6) mol/L for colon cancer cells with mutations in WNT signaling. In addition, PP demonstrated a cytotoxic effect on primary colon cancer samples. A combined cytotoxic effect of PP with 5-fluorouracil (5-FU) was observed for two cell lines. PP decreased messenger RNA (mRNA) and protein levels of known WNT target genes as c-MYC and thereby led to the induction of p21. PP inhibited the migration of HCT116 colon cancer cells in vitro and decreased tumor growth in vivo after intraportal injection of HCT116 cells in nude mice. CONCLUSIONS: PP displays promising anticancer activity against a broad panel of human colon cancer cell lines, as well as primary colon cancer samples. However, our findings do not demonstrate a predominant cytotoxic effect of PP on colon cancer cells with mutations in WNT signaling.

Mutation patterns in colorectal cancer and their relationship with prognosis.

Background: Colorectal cancer (CRC) is a prevalent malignancy and a leading cause of cancer-related mortality. Extensive research into the aetiology of CRC has revealed that somatic mutations in certain genes play a crucial role in CRC development.AIM: In this study, we utilized data from public databases to investigate prevalent mutation patterns in CRC and developed a prognostic predictive model for CRC patients based on mutant genetic characteristics and other relevant clinical features. Methods: We initially gathered mutation information from CRC patients by analysing data from 15 datasets to identify genes with a mutation frequency of ≥10 %. Next, log-rank analyses were used to determine the relationship between prognosis and the mutational status of the most commonly mutated genes; the SIGnaling database was utilized to generate a protein‒protein interaction network. We consolidated and classified the gene mutation patterns of CRC patients in the database based on frequently mutated genes related to prognosis. A predictive nomogram was constructed, including age, sex, TNM stage, and mutation partner, based on available clinical, mutational, and prognostic information for CRC patients at our institution. Finally, the reliability of the model was verified using time-dependent ROC curve analysis. Results: The top 7 genes somatically mutated ≥10 % in 4477 samples from 4255 patients were TP53 (67 %), APC (66 %), KRAS (43 %), PIK3CA (18 %), FBXW7 (14 %), SMAD4 (14 %), and BRAF (10 %). Log-rank analysis demonstrated that the mutation status of 5 genes, namely, TP53, APC, PIK3CA, SMAD4, and BRAF, correlated significantly with prognosis. Protein‒protein interaction analysis confirmed functional interactions between these 5 genes, implicating them in tumorigenesis. We exhaustively enumerated the mutation patterns involving these five genes in 4255 patients, resulting in identification of 32 mutational patterns. After consolidation and classification, these patterns were divided into 3 grades based on patient prognosis. Next, a predictive nomogram based on the clinical, mutational, and prognostic information of 107 CRC patients treated at University Medical Center Rostock was constructed. The area under the curve (AUC) values for the model for predicting 1-, 3-, and 5-year overall survival were 0.779, 0.721, and 0.815, respectively. Conclusion: Common mutational patterns based on frequently mutated genes are associated with prognosis in CRC patients. Our study provides a valuable and concise prognostic predictor for determining outcomes in patients with CRC.

Peptide-stimulated T cells bypass immune checkpoint inhibitor resistance and eliminate autologous microsatellite instable colorectal cancer cells.

Two hypermutated colon cancer cases with patient-derived cell lines, peripheral and tumor-infiltrating T cells available were selected for detailed investigation of immunological response.T cells co-cultured with autologous tumor cells showed only low levels of pro-inflammatory cytokines and failed at tumor recognition. Similarly, treatment of co-cultures with immune checkpoint inhibitors (ICI) did not boost antitumor immune responses. Since proteinase inhibitor 9 (PI-9) was detected in tumor cells, a specific inhibitor (PI-9i) was used in addition to ICI in T cell cytotoxicity testing. However, only pre-stimulation with tumor-specific peptides (cryptic and neoantigenic) significantly increased recognition and elimination of tumor cells by T cells independently of ICI or PI-9i.We showed, that ICI resistant tumor cells can be targeted by tumor-primed T cells and also demonstrated the superiority of tumor-naïve peripheral blood T cells compared to highly exhausted tumor-infiltrating T cells. Future precision immunotherapeutic approaches should include multimodal strategies to successfully induce durable anti-tumor immune responses.

A glutamatergic biomarker panel enables differentiating Grade 4 gliomas/astrocytomas from brain metastases.

Background: The differentiation of high-grade glioma and brain tumors of an extracranial origin is eminent for the decision on subsequent treatment regimens. While in high-grade glioma, a surgical resection of the tumor mass is a fundamental part of current standard regimens, in brain metastasis, the burden of the primary tumor must be considered. However, without a cancer history, the differentiation remains challenging in the imaging. Hence, biopsies are common that may help to identify the tumor origin. An additional tool to support the differentiation may be of great help. For this purpose, we aimed to identify a biomarker panel based on the expression analysis of a small sample of tissue to support the pathological analysis of surgery resection specimens. Given that an aberrant glutamate signaling was identified to drive glioblastoma progression, we focused on glutamate receptors and key players of glutamate homeostasis. Methods: Based on surgically resected samples from 55 brain tumors, the expression of ionotropic and metabotropic glutamate receptors and key players of glutamate homeostasis were analyzed by RT-PCR. Subsequently, a receiver operating characteristic (ROC) analysis was performed to identify genes whose expression levels may be associated with either glioblastoma or brain metastasis. Results: Out of a total of 29 glutamatergic genes analyzed, nine genes presented a significantly different expression level between high-grade gliomas and brain metastases. Of those, seven were identified as potential biomarker candidates including genes encoding for AMPA receptors GRIA1, GRIA2, kainate receptors GRIK1 and GRIK4, metabotropic receptor GRM3, transaminase BCAT1 and the glutamine synthetase (encoded by GLUL). Overall, the biomarker panel achieved an accuracy of 88% (95% CI: 87.1, 90.8) in predicting the tumor entity. Gene expression data, however, could not discriminate between patients with seizures from those without. Conclusion: We have identified a panel of seven genes whose expression may serve as a biomarker panel to discriminate glioblastomas and brain metastases at the molecular level. After further validation, our biomarker signatures could be of great use in the decision making on subsequent treatment regimens after diagnosis.

SKP2 promotes the metastasis of pancreatic ductal adenocarcinoma by suppressing TRIM21-mediated PSPC1 degradation.

Despite significant advances in diagnostic techniques and treatment approaches, the prognosis of pancreatic ductal adenocarcinoma (PDAC) is still poor. Previous studies have reported that S-phase kinase-associated protein 2 (SKP2), a subunit of the SCF E3 ubiquitin ligase complex, is engaged in the malignant biological behavior of some tumor entities. However, SKP2 has not been fully investigated in PDAC. In the present study, it was observed that high expression of SKP2 significantly correlates with decreased survival time. Further experiments suggested that SKP2 promotes metastasis by interacting with the putative transcription factor paraspeckle component 1 (PSPC1). According to the results of coimmunoprecipitation and ubiquitination assays, SKP2 depletion resulted in the polyubiquitination of PSPC1, followed by its degradation. Furthermore, the SKP2-mediated ubiquitination of PSPC1 partially depended on the activity of the E3 ligase TRIM21. In addition, inhibition of the SKP2/PSPC1 axis by SMIP004, a traditional inhibitor of SKP2, impaired the migration of PDAC cells. In summary, this study provides novel insight into the mechanisms involved in PDAC malignant progression. Targeting the SKP2/PSPC1 axis is a promising strategy for the treatment of PDAC.