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.

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.

Prevalence of alcohol use in late pregnancy.

BACKGROUND: Prenatal alcohol exposure (PAE) can result in detrimental developmental complications. The objective of this study was to estimate the most recent PAE prevalence data for the state of West Virginia (WV) and associated factors. METHOD: In all, 1830 newborn residual dried blood spots (DBS) in the WV Newborn Screening Repository were analyzed for phosphatidylethanol (PETH). Data were matched with Project WATCH data (94% match, N = 1729). RESULTS: The prevalence of late pregnancy PAE was 8.10% (95%CI: 6.81, 9.38) for all births, 7.61% (95%CI: 6.26, 8.97) for WV residents only, and ranged from 2.27 to 17.11% by region. The significant factors associated with PAE included smoking (OR: 2.03, 95% CI: 1.40, 2.94), preterm births (OR: 1.88; 95% CI: 1.23, 2.89), birth weight of ≤2000 g vs. >3000 g (OR: 2.62, 95%CI: 1.19, 5.79), no exclusive breastfeeding intention (OR: 1.45, 95% CI: 1.02, 2.04), and not exclusively breastfeeding before discharge (OR: 1.61; 95% CI: 1.09, 2.38). CONCLUSION: The prevalence of PAE is higher than previously shown for the state. Accurate and timely estimates are vital to inform public health workers, policymakers, researchers, and clinicians to develop and promote effective prevention strategies to lower PAE prevalence and provide targeted interventions and treatment services for infants affected by PAE.

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.

Capturing the statewide incidence of neonatal abstinence syndrome in real time: the West Virginia experience.

BACKGROUND: Neonatal abstinence syndrome (NAS) is one of the consequences at birth affecting the newborn after discontinuation of prenatal drug exposure to mainly opioids. The objective of this study was to determine the extent of the problem in the state of West Virginia (WV) using a real-time statewide surveillance system. METHODS: Project WATCH is a surveillance tool that since 1998 collects data on all infants born in the state of WV. NAS surveillance item was added to the tool in October 2016. This study examined all births (N = 23,667) in WV from October to December 2017. The data from six WV birthing facilities were audited for 1 month to evaluate how well this tool was capturing NAS data using κ-statistics. RESULTS: The 2017 annual incidence rate of NAS was 51.3 per 1000 live births per year for all births and 50.6 per 1000 live births per year for WV residents only. The κ-coefficient between the hospital medical records and Project WATCH data was 0.74 (95% confidence interval: 0.66-0.82) for NAS. CONCLUSION: The study provides justification to develop effective systems of care for the mother-infant dyad affected by substance use, especially targeting pregnant women in rural communities.

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.

Patient-Derived Organoids for In Vivo Validation of In Vitro Data.

Patient-derived organoids are promising tumor models for functional validation of next-generation sequencing-based therapy recommendations. In times of rapidly advancing precision oncology approaches in everyday clinical processes, reliable and valid tumor models are required. Tumor organoids consist of tumor "stem" cells, differentiated (epithelial) tumor, and stroma cells. The cellular architecture and interactions closely mimic the original patient tumor. These organoids can be implanted into immunodeficient mice, generating patient-derived organoid-derived xenografts, thus enabling in vitro to in vivo transfer. Most importantly, the high clinical relevance of PDO models is maintained in this conversion. This protocol describes in detail the methods and techniques as well as the materials necessary to generate in vitro PDO and in vivo PDO-derived xenograft models. The elaborate process description starts with the processing of freshly obtained tumor tissue. The proceedings include tissue processing, organoid culturing, PDO implantation into immunodeficient mice, tumor explantation, and finally tumor preservation. All these proceedings are described in this timely chronological order. This protocol will enable researchers to generate PDO models from freshly received tumor tissue and generate PDO-derived xenografts. Models generated according to these methods are suitable for a very broad research spectrum.

Endogenous retrovirus sequences as a novel class of tumor-specific antigens: an example of HERV-H env encoding strong CTL epitopes.

Our genome consists to about 8% of human endogenous retroviral (HERV) sequences. These HERVs have been discussed to be linked to human diseases for decades. Recently, a detailed analysis of a HERV-H sequence located on chromosome Xp22.3 revealed a strong expression in a subset of gastrointestinal cancers whereas expression in normal tissues and in other cancer entities was low. In the present study, we used the reverse immunology approach to test the immunological potential of this HERV-H ORF on Xp22.3. A total of ten peptides displaying HLA-A2.1-binding motifs were selected from the predicted env protein sequence. Stimulation of peripheral T cells with retroviral peptides (RVPs) presented by autologous antigen-presenting cells clearly resulted in sustained proliferation of predominantly CD8(+) T cells. High numbers of IFN-γ-secreting T cells were detectable after several weekly stimulations with RVP mixes. Reactivity observed in RVP-Mix-stimulated cultures was attributable to RVP03, RVP09 and to a lower extend to RVP08, suggesting those to be highly immunogenic epitopes. Besides killing of RVP-loaded target cells, up to 40% specific lysis of colorectal carcinoma cell lines endogenously expressing this HERV-H Xp22.3 ORF was achieved. These data demonstrate that human T cells can be sensitized toward HERV peptides and moreover posses a high lytic potential toward HERV-H expressing CRC cells. Additionally, these data hint toward endogenous ENV protein expression followed by proteasomal degradation and presentation in the context of HLA molecules. Finally, our data strengthen the view that HERV-encoded sequences should be considered as a new class of tumor-specific antigens.

Global Association of Cause-specific Mortality between the Major Gastrointestinal Cancers and Parkinson's Disease for the First Two Decades of the New Millennium.

Parkinson's disease (PD) and gastrointestinal (GI) cancers are both "age-related diseases" sharing several environmental risk factors, but possess opposite underlying biological mechanisms. Aim of this study was to evaluate the correlations between GI cancers and PD using national cause-specific mortality data of 183 countries extracted from the Global Health Observatory database. The association between PD- and GI cancers- (i.e. esophagus cancer, EC; stomach cancer, SC; colorectal cancer, CRC; liver cancer, LC and pancreatic cancer, PC) specific mortality on the country level was evaluated using Spearman correlation and logistic regression analysis. A global increase in mortality from 2000 to 2019 was observed in PD, CRC and PC, whereas in EC, SC and LC it decreased. We see the consistent diminishment of correlation intensities between PD and GI cancer mortalities from 2000 to 2019 as a positive development. In 2019, PD inversely correlated with CRC (rs = -0.39) and PC (rs = -0.40, all P < 0.001) but not with EC and SC. Of note, an exceptionally positive correlation of PD with LC (rs = 0.26, P < 0.001) and its two hepatitis B and C virus-associated subtypes was revealed. Logistic regression analysis further determined that PD associated negatively with CRC (OR = 0.25) and PC (OR = 0.21, both P < 0.001), but positively with LC (OR = 2.27, P = 0.007). Consequently, future research aiming to unravel the functional biological link between neurodegeneration, hepatitis and tumor development holds great potential for developing novel therapeutics.

Recent advances in diagnosis and treatment of gastroenteropancreatic neuroendocrine neoplasms.

Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) are a rare group of tumors originating from neuroendocrine cells of the digestive system. Their incidence has increased over the last decades. The specific pathogenetic mechanisms underlying GEP-NEN development have not been completely revealed. Unfunctional GEP-NENs are usually asymptomatic; some grow slowly and thus impede early diagnosis, which ultimately results in a high rate of misdiagnosis. Therefore, many GEP-NEN patients present with later staged tumors. Motivated hereby, research attention for diagnosis and treatment for GEP-NENs increased in recent years. The result of which is great progress in clinical diagnosis and treatment. According to the most recent clinical guidelines, improved grading standards can accurately define poorly differentiated grade 3 neuroendocrine tumors and neuroendocrine carcinomas (NECs), which are subclassified into large and small cell NECs. Combining different functional imaging methods facilitates precise diagnosis. The expression of somatostatin receptors helps to predict prognosis. Genetic analyses of mutations affecting death domain associated protein (DAXX), multiple endocrine neoplasia type 1 (MEN 1), alpha thalassemia/intellectual disability syndrome X-linked (ATRX), retinoblastoma transcriptional corepressor 1 (RB 1), and mothers against decapentaplegic homolog 4 (SMAD 4) help distinguishing grade 3 NENs from poorly differentiated NECs. The aim of this review is to summarize the latest research progress on diagnosis and treatment of GEP-NENs.

HROP68: A rare case of medullary pancreatic cancer-characterization and chemosensitivity of the first patient-derived cell line.

Introduction: Medullary pancreatic carcinoma (MPC) is a rare subtype of pancreatic ductal adenocarcinoma. MPCs represent less than 1% of all pancreatic cancers, and, with only 26 cases in the literature, knowledge regarding drug response and treatment outcome is very limited. Material and methods: We present the case of a 64-year-old male patient with MPC who was treated by left pancreatic resection and adjuvant chemotherapy. Due to local recurrence, the patient underwent intended curative reoperation. From both surgical specimens, patient-derived xenografts (PDXs) and, from the recurrence, a patient-derived cell line (PDCL) were established. We subsequently performed an in-depth characterization of this cell line including phenotypic characterization, surface protein expression, growth, and migratory performance as well as mutational analysis using whole-exome sequencing (WES). Additionally, in vitro drug sensitivity toward the standard-of-care chemotherapeutic regimen and selected targeted therapies was evaluated. Results: The pathological and molecular properties of this rare MPC case observed in the patient's tumors are preserved in the corresponding PDX and the PDCL of HROP68Tu2. Despite displaying an "immunogenic phenotype" with marked T-cell infiltration and a high-level expression of HLA II and Programmed death-ligand 1 (PD-L1), molecular analysis revealed microsatellite stability but a multitude of mutations affecting KRAS, TP53, KAT6B, FOXG1, RUNX1, and GRIK2 among others. Furthermore, HROP68Tu2 cells were susceptible toward 5-FU, irinotecan, oxaliplatin, gemcitabine, paclitaxel, and erlotinib as single agents, but only a moderate synergistic response was seen to the drugs of the FOLFIRINOX regimen. Even worse, the drugs of the two combinations gemcitabine plus paclitaxel and gemcitabine plus erlotinib showed antagonistic effects. Moreover, lapatinib, PRIMA-Met1, and olaparib selected as targeted therapeutics according to the mutational profiles and protein expression inhibited HROP68Tu2 cells' growth. Conclusion: This study illustrates the establishment of the first preclinical MPC models as well as the first in-depth characterization of an MPC PDCL. Since the scientific and clinical knowledge of this rare pancreatic cancer type is very limited, the presented models contribute to a better understanding of MPC and might be a valuable tool for the development of future treatment options.

Microsatellite Status and IκBα Expression Levels Predict Sensitivity to Pharmaceutical Curcumin in Colorectal Cancer Cells.

Clinical utilization of curcumin in colorectal cancer (CRC) was revived as a result of the development of novel curcumin formulations with improved bioavailability. Additionally, identification of biomarkers for curcumin sensitivity would also promote successful clinical applications. Here, we wanted to identify such biomarkers in order to establish a predictive model for curcumin sensitivity. Thirty-two low-passage CRC cell lines with specified tumor characteristics were included. Curcumin suppressed cell proliferation, yet sensitivity levels were distinct. Most curcumin-sensitive CRC cell lines were microsatellite stable and expressed high levels of IκBα. The predictive capacity of this biomarker combination possessed a statistical significance of 72% probability to distinguish correctly between curcumin-sensitive and -resistant CRC cell lines. Detailed functional analyses were performed with three sensitive and three resistant CRC cell lines. As curcumin's mode of action, inhibition of NF-κB p65 activation via IκBα was identified. In consequence, we hypothesize that novel curcumin formulations-either alone or, more likely, in combination with standard therapeutics-can be expected to prove clinically beneficial for CRC patients with high IκBα expression levels.

Negative Magnetic Sorting Preserves the Functionality of Ex Vivo Cultivated Non-Adherent Human Monocytes.

BACKGROUND: Monocyte-derived macrophages or dendritic cells are of increasing interest for cellular therapeutic products to treat inflammation-related diseases and cancer. However, the isolation method and the culture conditions applied influence the functionality of cells. For some approaches, the adhesion-induced differentiation into macrophages must be prevented to maintain functions attributed to circulating monocytes. The effects of the isolation method on the functionality of non-adherent peripheral monocytes have not yet been investigated. METHODS: The present study examines the impact of the isolation method on cell viability, growth, metabolism, inflammation-induced cytokine response, migratory capacity, and adherence of non-adherent human peripheral monocytes. The monocytes were isolated by magnetic sorting using either positive or negative selection and cultured in cell-repellent plates. RESULTS: The purity and yield of monocytes were higher after positive selection. However, the adherence and migratory capacity, cytokine response, and metabolic activity were decreased compared to negatively selected monocytes. The impaired functionality presented in combination with cell shrinking, thus, indicates the start of cell viability loss. Negatively selected non-adherent monocytes showed no impairment in functionality, and the viability remained high. In conclusion, this approach is better suited for conducting ex vivo modifications of monocytes prior to the intended experimental setup or therapeutic application.

T cells of colorectal cancer patients' stimulated by neoantigenic and cryptic peptides better recognize autologous tumor cells.

BACKGROUND: Patients with cancers that exhibit extraordinarily high somatic mutation numbers are ideal candidates for immunotherapy and enable identifying tumor-specific peptides through stimulation of tumor-reactive T cells (Tc). METHODS: Colorectal cancers (CRC) HROC113 and HROC285 were selected based on high TMB, microsatellite instability and HLA class I expression. Their HLA ligandome was characterized using mass spectrometry, compared with the HLA ligand atlas and HLA class I-binding affinity was predicted. Cryptic peptides were identified using Peptide-PRISM. Patients' Tc were isolated from either peripheral blood (pTc) or tumor material (tumor-infiltrating Tc, TiTc) and expanded. In addition, B-lymphoblastoid cells (B-LCL) were generated and used as antigen-presenting cells. pTc and TiTc were stimulated twice for 7 days using peptide pool-loaded B-LCL. Subsequently, interferon gamma (IFNγ) release was quantified by ELISpot. Finally, cytotoxicity against autologous tumor cells was assessed in a degranulation assay. RESULTS: 100 tumor-specific candidate peptides-97 cryptic peptides and 3 classically mutated neoantigens-were selected. The neoantigens originated from single nucleotide substitutions in the genes IQGAP1, CTNNB1, and TRIT1. Cryptic and neoantigenic peptides inducing IFNγ secretion of Tc were further investigated. Stimulation of pTc and TiTc with neoantigens and selected cryptic peptides resulted in increased release of cytotoxic granules in the presence of autologous tumor cells, substantiating their improved tumor cell recognition. Tetramer staining showed an enhanced number of pTc and TiTc specific for the IQGAP1 neoantigen. Subpopulation analysis prior to peptide stimulation revealed that pTc mainly consisted of memory Tc, whereas TiTc constituted primarily of effector and effector memory Tc. This allows to infer that TiTc reacting to neoantigens and cryptic peptides must be present within the tumor microenvironment. CONCLUSION: These results prove that the analyzed CRC present both mutated neoantigenic and cryptic peptides on their HLA class I molecules. Moreover, stimulation with these peptides significantly strengthened tumor cell recognition by Tc. Since the overall number of neoantigenic peptides identifiable by HLA ligandome analysis hitherto is small, our data emphasize the relevance of increasing the target scope for cancer vaccines by the cryptic peptide category.

Multidrug-resistance proteins are weak tumor associated antigens for colorectal carcinoma.

BACKGROUND: Multidrug resistance (MDR) is a clinically, highly relevant phenomenon. Under chemotherapy many tumors show an increasing resistance towards the applied substance(s) and to a certain extent also towards other agents. An important molecular cause of this phenomenon is an increased expression of transporter proteins. The functional relationship between high expression levels and chemotherapy resistance makes these MDR and MRP (MDR related protein) proteins to interesting therapeutic targets. We here wanted to systematically analyze, whether these proteins are tumor specific antigens which could be targeted immunologically. RESULTS: Using the reverse immunology approach, 30 HLA-A2.1 restricted MDR and MRP derived peptides (MDP) were selected. Stimulated T cell lines grew well and mainly contained activated CD8+ cells. Peptide specificity and HLA-A2.1 restriction were proven in IFN-γ-ELISpot analyses and in cytotoxicity tests against MDP loaded target cells for a total of twelve peptides derived from MDR-1, MDR-3, MRP-1, MRP-2, MRP-3 and MRP-5. Of note, two of these epitopes are shared between MDR-1 and MDR-3 as well as MRP-2 and MRP-3. However, comparably weak cytotoxic activities were additionally observed against HLA-A2.1+ tumor cells even after upregulation of MDR protein expression by in vitro chemotherapy. CONCLUSIONS: Taken together, these data demonstrate that human T cells can be sensitised towards MDPs and hence, there is no absolute immunological tolerance. However, our data also hint towards rather low endogenous tumor cell processing and presentation of MDPs in the context of HLA-A2.1 molecules. Consequently, we conclude that MDR and MRP proteins must be considered as weak tumor specific antigens-at least for colorectal carcinoma. Their direct contribution to therapy-failure implies however, that it is worth to further pursue this approach.

A global assessment of recent trends in gastrointestinal cancer and lifestyle-associated risk factors.

BACKGROUND: Gastrointestinal (GI) cancers were responsible for 26.3% of cancer cases and 35.4% of deaths worldwide in 2018. This study aimed to analyze the global incidence, mortality, prevalence, and contributing risk factors of the 6 major GI cancer entities [esophageal cancer (EC), gastric cancer (GC), liver cancer (LC), pancreatic cancer (PC), colon cancer, and rectal cancer]. METHODS: Using the Global Cancer Observatory and the Global Health Observatory databases, we reviewed the current GI cancer incidence, prevalence, and mortality, analyzed the association of GI cancer prevalence with national human development indices (HDIs), identified the contributing risk factors, and estimated developing age- and sex-specific trends in incidence and mortality. RESULTS: In 2020, the trend in age-standardized rate of incidence of GI cancers closely mirrored that of mortality, with the highest rates of LC, EC, and GC in Asia and of colorectal cancer (CRC) and PC mainly in Europe. Incidence and mortality were positively, but the mortality-to-incidence ratio (MIR) was inversely correlated with the national HDI levels. High MIRs in developing countries likely reflected the lack of preventive strategies and effective treatments. GI cancer prevalence was highest in Europe and was also positively correlated with HDIs and lifestyle-associated risk factors, such as alcohol consumption, smoking, obesity, insufficient physical activity, and high blood cholesterol level, but negatively correlated with hypertension and diabetes. Incidences of EC were consistently and those of GC mostly decreasing, whereas incidences of CRC were increasing in most countries/regions, especially in the younger populations. Incidences of LC and PC were also increasing in all age-gender populations except for younger males. Mortalities were decreasing for EC, GC, and CRC in most countries/regions, and age-specific trends were observed in PC and LC with a decrease in the younger but an increase in the older population. CONCLUSIONS: On the global scale, higher GI cancer burden was accompanied, for the most part, by factors associated with the so-called Western lifestyle reflected by high and very high national HDI levels. In countries/regions with very high HDI levels, patients survived longer, and increasing GI cancer cases were observed with increasing national HDI levels. Optimizing GI cancer prevention and improving therapies, especially for patients with comorbid metabolic diseases, are thus urgently recommended.