Sven Peine

Persistent high-risk human papillomavirus (hr-HPV) infection drives cervical carcinogenesis, yet improved molecular biomarkers are needed to define high-risk groups. Circulating microRNAs (miRNAs), stable in blood and involved in carcinogenic pathways, represent promising liquid biopsy biomarkers. This study assessed five miRNAs for distinguishing high-grade squamous cell intraepithelial lesions (HSILs) and cervical cancer from healthy controls and for HPV stratification. Circulating miRNAs were quantified in blood samples from 80 women (38 HSIL, 10 cervical cancer, and 32 controls). Relative expression by disease and HPV status was measured by RT-qPCR and normalized to miRNA-23a. Diagnostic performance of single and combined miRNAs was evaluated by logistic regression and ROC curve analysis. Three circulating miRNAs (miR-21, miR-205, and miR-218) were found to be significantly differentially dysregulated in the patient cohorts. A combination of the three markers showed the best diagnostic value for HSIL (AUC of 0.81, sensitivity of 79%, and specificity of 71%) and cancer (AUC of 0.81, sensitivity of 90%, and specificity of 65%). Whereas miR-205 was significantly associated with HPV16/18 in HSIL patients, the combined model had the highest diagnostic performance for multiple HPV infections. Circulating miRNA signatures show promise as liquid biopsy biomarkers for detecting cervical dysplasia and stratifying for HPV status in HSIL, warranting validation in larger prospective studies.

Infection with Hepatitis E Virus (HEV) is often asymptomatic but can also lead to chronic infection in immunosuppressed individuals. Although fecal–oral transmission of HEV is well established, transmission by blood transfusion has also been reported. Here, we studied HEV seroprevalence in a cohort of 1000 blood donors (50% male, age 18–73 years, mean 35 years) at the University Medical Center Hamburg–Eppendorf in Germany. We found a seroprevalence of anti-HEV IgG of 16.6%. Interestingly, 1.3% of the blood donors had positive IgM serology despite testing negative for HEV by polymerase chain reaction (PCR). Analysis of preceding and follow-up samples showed persistence of IgM antibodies for up to seven months in asymptomatic individuals. In eight individuals, anti-HEV IgM positivity persisted for 0 to 7 months (median 2 months), as confirmed by testing stored samples. This study demonstrates that anti-HEV IgM positivity can persist for more than six months in individuals who had neither clinically overt hepatitis E nor a viremia duration that would allow PCR positivity to be detected.

Lymphomas, particularly aggressive non-Hodgkin lymphomas (NHL), remain challenging due to poor outcomes in a subset of patients who fail initial therapy. Current minimally invasive biomarkers for risk stratification need further improvement. Immune checkpoint inhibitors (ICIs), while with limited efficacy in NHL, highlight the immune system's crucial role in cancer control. This study investigated the predictive and prognostic potential of soluble immune checkpoints (sICs) and their membrane-bound isoforms in the peripheral blood of lymphoma patients. Levels of sCD27, sCD28, and sCD80 were measured using multiplex immunoassay in 100 lymphoma patients and 62 healthy donors (HD). Expression of membrane-bound isoforms on PBMCs was analyzed via flow cytometry in 40 patients and 10 HD. All three sICs were significantly higher in lymphoma patients compared to HD. High levels were associated with significantly impaired disease control (DC) and survival at 12 and 24 months, progression free-survival (PFS) and overall survival (OS). The highest prediction capacity was achieved when assessing all three molecules in a combined score (p_PFS < 0.001, p_OS = 0.013, for score 0 vs 3: DC_12mo:85% vs 18.5%, OS_24mo:87% vs 27%). Integrating expression of related membrane-bound markers on PBMCs led to a highly robust peripheral blood-based biomarker score comprising the soluble and membrane-bound compartments (2COMPIC-Score:3 sICs + 7 PBMC subsets) (p_PFS < 0.001, HR_PFS = 18.1;p_OS = 0.001,HR_OS = 19.2), corroborated by multivariate analysis, outperforming the clinically validated NCCN-IPI prognostic score. We unveil the potential of combining different minimally invasive liquid biopsy-based immune checkpoint assessments into a robust clinical score for prognostic prediction in lymphoma, mainly B(cell)-NHL. Pending prospective validation, our findings could aid clinicians in guiding therapeutic decisions.

Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality. HCC is characterized by high heterogeneity and, subsequently, adaptation by developing resistance to current treatments. Applying individualized models is crucial to understanding the potential of approved therapies. Therefore, we classify a primary-like cell line derived from the core region of an HCC with underlying HIV-HCV co-infection employing deep analysis on the pathway regulation level. Methods: We employed DEG analysis, followed by pathway analysis, to characterize the preservation level of the LC4 cells and the level of adoption. Next, we classify the new model for HCC research by employing healthy donor samples, commonly used HCC cell lines, and global RNAseq datasets. Results: LC4 cells reflect the characteristics of the parental cancer region, including immunosuppression and metabolic reprogramming, characterized by the downregulation of drug-metabolizing enzymes compared to healthy individuals, indicating a transition to alternate metabolic pathways. Moreover, we identified specific biomarkers equally regulated in the parental tissue, in global datasets of the same entities as well as in LC4 cells. Conclusions: We classified LC4 cells as an individual immunosuppressive and highly progressive primary-like HCC cell line. LC4 cells are applicable as a model for preclinical drug testing, minimizing the lack of preclinical models in HCV-HIV-induced HCC research.

Background Hepatocellular carcinoma represents a significant global health challenge, affecting over a million patients annually, arising mainly from chronic liver diseases, with a majority being related to viral infections. However, despite the groundbreaking clinical results of immune checkpoint blockades and adoptive cell therapies, we still face non-responders accompanied by high rebound rates after resection. Considering that the main concern is to overcome a highly specialized immunosuppressive tumor microenvironment of the individual patient, the characterization of the particular tumor microenvironment and the source of the immune cells used in ACT is of immense importance. Approved ACT therapies mainly use modified peripheral blood cells from individuals. At the same time, tumor-infiltrating lymphocytes are underrepresented even if they have garnered interest due to their potential to target tumor-specific antigens more effectively. Methods In this study, we employed allogenic and autologous immune cell sources for expansion and stimulation, resulting in adoptive T-cell transfer experiments determining the effector cell differentiation and the related anti-tumor effects by the possible implementation of re-stimulation. Results We determined a high success rate in expanding and stimulating tumor-infiltrating lymphocytes with consistent CD8 T-cell fractions from HCC patients. To showcase the effectiveness of stimulated T-cells from different sources, we generated cell lines derived from the margin and center of an HBV-induced HCC with a highly immune-suppressive TME. We found effective immune responses supported by cell death induction, ferroptosis, proptosis and apoptosis triggered by all sources of T-cells depending on the area of derived tumor cells. Conclusion Effector T-cell fractions derived from tumor-infiltrating lymphocytes present a viable source for cell-based immune therapy combined with immune checkpoint inhibitors in HCC patients, especially after resection, to suppress rebound strategies of the parental tumor on an individual level.

Background The hepatocellular carcinoma is one of the leading causes of cancer-related mortality and is characterized by high heterogeneity and subsequently adaptation by developing resistance to current treatments. In this scenario the application of individualized models is crucial to understand the potential of approved therapies. Recently, we established a series of individual cell lines derived from patients who developed HCC on different entities, serving as a platform for individual approaches. In this study, we classified the LC4 cells derived from the center region of a HCC with underlying HIV-HCV co-infection, by using deep analysis on the pathway regulation level. Methods We employed DEG analysis, followed by pathway analysis to characterize the preservation level of the LC4 cells and the level of adoption. Next, we classify the model, by employing healthy donor samples, commonly used HCC cell lines and global RNAseq data sets. Results We showed that the LC4 cells reflect significant characteristics of the parental region, including the replication of the immuno-suppressive and the proliferative milieu. The LC4 cells exhibit a metabolic reprogramming characterized by the downregulation of drug-metabolizing CYP enzymes compared to healthy individuals, indicating a transition to alternate metabolic pathways. Moreover, we identified common Biomarkers in the parental tissue, global datasets and the LC4 cells. Conclusion We showed that the LC4 cell line is applicable as an individual model for pre-clinical testing of treatment regimens in HCC driven research.

Host iron deficiency is protective against severe malaria as the human malaria parasite Plasmodium falciparum depends on bioavailable iron from its host to proliferate. The essential pathways of iron acquisition, storage, export, and detoxification in the parasite differ from those in humans, as orthologs of the mammalian transferrin receptor, ferritin, or ferroportin, and a functional heme oxygenase are absent in P. falciparum . Thus, the proteins involved in these processes may be excellent targets for therapeutic development, yet remain largely unknown. Here, we show that parasites cultured in erythrocytes from an iron-deficient donor displayed significantly reduced growth rates compared to those grown in red blood cells from healthy controls. Sequencing of parasite RNA revealed diminished expression of genes involved in overall metabolism, hemoglobin digestion, and metabolite transport under low-iron versus control conditions. Supplementation with hepcidin, a specific ferroportin inhibitor, resulted in increased labile iron levels in erythrocytes, enhanced parasite replication, and transcriptional upregulation of genes responsible for merozoite motility and host cell invasion. Through endogenous GFP tagging of differentially expressed putative transporter genes followed by confocal live-cell imaging, proliferation assays with knockout and knockdown lines, and protein structure predictions, we identified six proteins that are likely required for ferrous iron transport in P. falciparum . Of these, we localized Pf VIT and Pf ZIPCO to cytoplasmic vesicles, Pf MRS3 to the mitochondrion, and the novel putative iron transporter Pf E140 to the plasma membrane for the first time in P. falciparum . Pf NRAMP/ Pf DMT1 and Pf CRT were previously reported to efflux Fe ²⁺ from the digestive vacuole. Our data support a new model for parasite iron homeostasis, in which Pf E140 is involved in iron uptake across the plasma membrane, Pf MRS3 ensures non-redundant Fe ²⁺ supply to the mitochondrion as the main site of iron utilization, Pf VIT transports excess iron into cytoplasmic vesicles, and Pf ZIPCO exports Fe ²⁺ from these organelles in case of iron scarcity. These results provide new insights into the parasite’s response to differential iron availability in its environment and into the mechanisms of iron transport in P. falciparum as promising candidate targets for future antimalarial drugs.

Background: Lung cancer is the leading cause of cancer-related deaths worldwide, with brain metastasis (BM) occurring in 40% of advanced non-small cell lung cancer (NSCLC) patients. In 15% of these patients, the brain is the only affected organ (oligo-metastasis), corresponding to improved prognosis compared to widespread disease. Thus far, it is unknown if the metastatic dissemination to the brain without systemic metastases is a consequence of the immune system’s ability to control systemic tumor outgrowth. Methods: Here, we investigated the local and peripheral immune cell composition in NSCLC BM patients, and identified new immune patterns related to the occurrence of brain metastases either as oligo- or poly-metastatic disease. Results: The multi-parametric immune phenotyping of peripheral blood revealed a downregulation of KLRG1 in CD8 ⁺ T-cells and an increase in CD4 ⁺ T _H 17 cells and elevated IL-17 levels in the blood of all NSCLC BM patients compared to healthy individuals. In addition, BM patients CD4 ⁺ T cells showed less CD73 expression with reduced effector memory differentiation. Furthermore, we observed less intra-tumoral infiltration in tumor tissues and a distinctive CD4+ T-cell profile in oligo-synchronous BM, both in the tumor microenvironment and peripheral blood compared to poly-metastatic BM patients. Moreover, 5′-ectonucleotidase CD73 was significantly upregulated in CD4 and T-regulatory cells of oligo-synchronous BM. Conclusions: These results indicate that oligo-synchronous BM exhibits a more pronounced alteration in the CD4 T-cell immune profile both locally at the tumor site and systemically. Key Points: BM patients exhibit a skewed systemic immune profile, characterized by downregulation of KLRG1 in CD8 ⁺ and induction of T _H 17/IL-17 axis and CD73 in CD4 ⁺ T-cells. Oligo-synchronous BM displayed a distinct CD4 ⁺ T-cell profile in both TME and peripheral blood. Importance of the Study: This study presents a novel insight into immune profiles of brain metastasis types in NSCLC patients. Examining tissues and PBMCs sheds light on the disease and uncovers unique immune responses within distinct brain metastasis patterns. This research offers valuable knowledge for improved understanding and identifying potential prognosis markers.

SARS-CoV-2 serosurveillance is important to adapt infection control measures and estimate the degree of underreporting. Blood donor samples can be used as a proxy for the healthy adult population. In a repeated cross-sectional study from April 2020 to April 2021, September 2021, and April/May 2022, 13 blood establishments collected 134,510 anonymised specimens from blood donors in 28 study regions across Germany. These were tested for antibodies against the SARS-CoV-2 spike protein and nucleocapsid, including neutralising capacity. Seroprevalence was adjusted for test performance and sampling and weighted for demographic differences between the sample and the general population. Seroprevalence estimates were compared to notified COVID-19 cases. The overall adjusted SARS-CoV-2 seroprevalence remained below 2% until December 2020 and increased to 18.1% in April 2021, 89.4% in September 2021, and to 100% in April/May 2022. Neutralising capacity was found in 74% of all positive specimens until April 2021 and in 98% in April/May 2022. Our serosurveillance allowed for repeated estimations of underreporting from the early stage of the pandemic onwards. Underreporting ranged between factors 5.1 and 1.1 in the first two waves of the pandemic and remained well below 2 afterwards, indicating an adequate test strategy and notification system in Germany.