Novel PD-L1- and collagen-expressing patient-derived cell line of undifferentiated pleomorphic sarcoma (JBT19) as a model for cancer immunotherapy.

Soft tissue sarcomas are aggressive mesenchymal-origin malignancies. Undifferentiated pleomorphic sarcoma (UPS) belongs to the aggressive, high-grade, and least characterized sarcoma subtype, affecting multiple tissues and metastasizing to many organs. The treatment of localized UPS includes surgery in combination with radiation therapy. Metastatic forms are treated with chemotherapy. Immunotherapy is a promising treatment modality for many cancers. However, the development of immunotherapy for UPS is limited due to its heterogeneity, antigenic landscape variation, lower infiltration with immune cells, and a limited number of established patient-derived UPS cell lines for preclinical research. In this study, we established and characterized a novel patient-derived UPS cell line, JBT19. The JBT19 cells express PD-L1 and collagen, a ligand of the immune checkpoint molecule LAIR-1. JBT19 cells can form spheroids in vitro and solid tumors in immunodeficient nude mice. We found JBT19 cells induce expansion of JBT19-reactive autologous and allogeneic NK, T, and NKT-like cells, and the reactivity of the expanded cells was associated with cytotoxic impact on JBT19 cells. The PD-1 and LAIR-1 ligand-expressing JBT19 cells show ex vivo immunogenicity and effective in vivo xenoengraftment properties that can offer a unique resource in the preclinical research developing novel immunotherapeutic interventions in the treatment of UPS.

In silico prediction of maximum perineal muscle strain during vaginal delivery by design of experiment.

BACKGROUND AND OBJECTIVE: The prevalence of pelvic floor muscle injuries induced by childbirth is higher than 23 % in the general women population. Such injuries can lead to prolapses and other pathologies in future female life. Leveraging computational biomechanics, the study implements an advanced female pelvic floor model for computing the maximum pelvic muscle strain, which serves as an injury risk indicator. The design of experiment method, abbreviated as DoE, is used to compute the maximum strain for boundary values of bony pelvis dimensions, namely the anterior-posterior diameter (abbreviated as APD) and the transverse diameter (abbreviated as TD). This is done in combination with small, medium and large percentiles of fetal head circumference (abbreviated as HC). METHODS: We utilized a previously developed finite element model of a female pelvic floor, as a reference, and enhanced it with new features, including a more detailed tissue geometry and advanced constitutive material models. The APD and TD dimensions were sourced from the set of MRI of 64 nulliparous women. This data was used to estimate the boundary dimensions of the female bony pelvis, combining both small and large values of APD and TD. Together with the 10th and the 95th percentiles for HC, a three-dimensional domain was constructed to assess the maximum pelvic muscle strain. In boundary cases, the maximum pelvic muscle strain was computed across 8 full-factorial design models (each situated at one corner of the domain, thereby combining the minimum and the maximum values of APD, TD and HC). This was done to define a response surface that predicts the maximum pelvic muscle strain within the domain. The accuracy of this response surface prediction was validated using 15 additional intermediate design models. These models were placed at the center of the domain (1 point), the centres of the domain boundary surfaces (6 points), and midway along each domain boundary edge (8 points). RESULTS: The maximum strain results for 8 combinations of APD, TD, and HC were employed to construct a linear response surface as a function of APD, TD, and HC. Tests at an additional 19 domain points served to evaluate the efficiency of the response surface prediction. The response surface demonstrated strong predictability, with an absolute average error of 1.52 %, an absolute median error of 1.52 %, and an absolute maximum error of 11.11 %. HC emerged as the most influencing dimension, accounting for 16 % of influence. CONCLUSIONS: The reference finite element pelvic floor model was scaled to 8 full-factorial female-specific pelvic floor models, which represent the combination of boundary values for APD, TD, and HC. The maximum pelvic floor muscle strain from these 8 models was used to design a response surface. When implementing the DoE approach to construct the response, there was consistent predictability for the maximum perineal muscle strain, as validated by the additional 19 intermediate design models. As a result, the response surface methodology can serve as an initial predictor for potential childbirth-induced pelvic floor muscle injury.

Finite element modeling of maximum stress in pelvic floor structures during the head expulsion (FINESSE) study.

INTRODUCTION AND HYPOTHESIS: Several studies have assessed birth-related deformations of the levator ani muscle (LAM) and perineum on models that depicted these elements in isolation. The main aim of this study was to develop a complex female pelvic floor computational model using the finite element method to evaluate points and timing of maximum stress at the LAM and perineum in relation to the birth process. METHODS: A three-dimensional computational model of the female pelvic floor was created and used to simulate vaginal birth based on data from previously described real-life MRI scans. We developed three models: model A (LAM without perineum); model B (perineum without LAM); model C (a combined model with both structures). RESULTS: The maximum stress in the LAM was achieved when the vertex was 9 cm below the ischial spines and measured 37.3 MPa in model A and 88.7 MPa in model C. The maximum stress in the perineum occurred at the time of distension by the suboocipito-frontal diameter and reached 86.7 MPa and 119.6 MPa in models B and C, respectively, while the stress in the posterior fourchette caused by the suboccipito-bregmatic diameter measured 36.9 MPa for model B and 39.8 MPa for model C. CONCLUSIONS: Including perineal structures in a computational birth model simulation affects the level of stress at the LAM. The maximum stress at the LAM and perineum seems to occur when the head is lower than previously anticipated.

WT1 Gene Expression in Peripheral Blood Before and After Allogeneic Stem Cell Transplantation is a Clinically Relevant Prognostic Marker in AML - A Single-center 14-year Experience.

BACKGROUND: This work summarizes our experience with WT1 monitoring before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT). PATIENTS AND METHODS: The expression of WT1 gene was measured by real-time polymerase chain reaction in peripheral blood according to the European Leukemia Net recommendations. Between May 2005 and August 2019, we analyzed 147 consecutive patients with acute myeloid leukemia with high WT1 expression at diagnosis, transplanted in first (CR1) or second (CR2) complete remission. RESULTS: At the time of allo-HSCT, 107 patients had WT1-normal expression (WT1 ≤ 50 copies), and 40 patients had WT1-high expression. The median follow-up was 21 months. The estimated 5-year overall survival and event-free survival was significantly better in the WT1-normal cohort (65% and 57% vs. 37% and 25%; P = .0003 and P < .0001, respectively) and 5-year cumulative incidence of relapse was significantly lower in the WT1-normal group (19% vs. 53%; P < .0001). Five-year non-relapse mortality was not significantly different (20% and 23%). Multivariate analysis revealed WT1-high expression and acute graft-versus-host disease grade 3/4 as significantly negative prognostic factors for OS. Overall, 49 patients developed WT1 molecular relapse in the post-transplant period; in 14 cases, the therapeutic intervention was done. In all but 1 relapsed patient where WT1 minimal residual disease (MRD) was monitored (38 patients), we detected WT1-high levels (sensitivity of 97%). CONCLUSION: The results of the analysis confirmed our previous experience that WT1 status before allo-HSCT is a strong prognostic factor for both OS and relapse risk. In addition, we confirmed the usefulness of this marker for MRD monitoring after allo-HSCT. The main advantage is the possibility of frequent MRD monitoring in peripheral blood and early bone marrow examination based on WT1-high expression.

Chimerism as an important marker in post-transplant monitoring chimerism monitoring.

Cell chimerism determination is important for the monitoring of engraftment dynamics and for relapse prediction. Our cohort of 474 patients was divided into two groups according to the determination methods used over time, and by their chimerism status. A significant difference in survival was observed between mixed vs complete chimerism (P < 0.0001 vs P < 0.0002) in both patient groups, and also vs microchimerism (P = 0.0201) in the second group. Detection of mixed chimerism is thus a high-risk factor, and microchimerism is potentially a risk factor in the post-transplantation course. Methods with a high sensitivity for monitoring cell chimerism significantly improve the assessment of patients post-transplant, and they enable the identification of patients with high relapse risk. Supported by MH CZ-DRO (00023736, UHKT).

Multiplex real-time quantitative polymerase chain reaction assay for rapid and sensitive detection of hematopoietic chimerism.

The increase of mixed chimerism (MC) after allogeneic hematopoietic stem cell transplantation has been associated with a high risk of relapse. A variety of techniques that use polymorphic markers have been established to survey hematopoietic chimerism status. The highest sensitivity is achieved using real-time quantitative polymerase chain reaction (RQ-PCR) analysis of insertion/deletion polymorphism, which allows the detection of disease recurrence and subsequently the earlier initiation of therapeutic intervention. The purpose of this study is the evaluation of multiplex RQ-PCR for MC assessment (six biallelic genetic systems and Y-specific locus), allowing the amplification and detection of target gene of interest and glyceraldehyde-3-phosphate dehydrogenase reference housekeeping gene in a single microtube. With optimized amounts of primers and probe, the quantification of target DNA was shown to be linear throughout the tested range (100%-0.05%). The efficiencies of multiplex RQ-PCR were in a range of 0.89 to 1.07. The sensitivity of individual systems ranged 0.02% to 0.04% with an average of 0.034%. A high degree of linear correlation between the chimerism results obtained by multiplex RQ-PCR vs singleplex RQ-PCR was observed (P < 0.0001, Spearman's coefficient = 0.9927), while correlation between multiplex RQ-PCR vs short tandem repeat analysis was also statistically significant (P < 0.0001, Spearman's coefficient = 0.9769). This new multiplex RQ-PCR assay is a quick, sensitive, reproducible, and cost-effective method for accurate MC assessment.

Primary and recurrent diffuse astrocytomas: genomic profile comparison reveals acquisition of biologically relevant aberrations.

BACKGROUND: Diffuse astrocytomas are characterized by their highly variable biological behavior. The possibility that tumors develop novel aberrations, with relevant biological properties, is often neglected. In this study, we present two cases of diffuse astrocytoma in which additional cytogenetic and epigenetic markers with potential influence on cell proliferation or differentiation were detected at relapse. FINDINGS: The biopsies taken from the primary and recurrent tumors of two patients were analyzed with molecular methods to detect copy number variations (CNVs), gene mutations and epigenetic changes. Both cases were characterized by the R132H mutation in the isocitrate dehydrogenase 1 (IDH1) gene. Features typical of astrocytomas, such as copy-neutral loss of heterozygosity at 17p and the deletion of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, were also detected in both cases. These markers were present in the primary and recurrent lesions. Other aberrations, predominantly deletions or amplifications of chromosomal segments and the hypermethylation of gene promoters, were detected in the recurrent lesions. CONCLUSIONS: The IDH1 mutation was the primary event, as previously reported. According to our observations, the methylation of promoters constituted later events, which may have further disrupted cell proliferation and/or differentiation, together with additional CNVs.

Genetic and epigenetic characterization of low-grade gliomas reveals frequent methylation of the MLH3 gene.

Diffuse astrocytomas and oligodendrogliomas (WHO grade II) are the most common histological subtypes of low-grade gliomas (LGGs). Several molecular and epigenetic markers have been identified that predict tumor progression. Our aim was in detail to investigate the genetic and epigenetic background of LGGs and to identify new markers that might play a role in tumor behavior. Twenty-three patients with oligodendroglioma or oligoastrocytoma (LGO) and 22 patients with diffuse astrocytoma (LGA) were investigated using several molecular-cytogenetic and molecular methods to assess their copy number variations, mutational status and level of promoter methylation. The most frequent findings were a 1p/19q codeletion in 83% of LGO and copy-neutral loss of heterozygosity (CN-LOH) of 17p in 72% of LGA. Somatic mutations in the isocitrate dehydrogenase 1 or 2 (IDH1/IDH2) genes were detected in 96% of LGO and 91% of LGA. The O-6-methylguanine-DNA-methyltransferase (MGMT) promoter was methylated in 83% of LGO and 59% of LGA. MutL homolog 3 (MLH3) promoter methylation was observed in 61% of LGO and 27% of LGA. Methylation of the MGMT promoter, 1p/19q codeletion, mutated IDH1, and CN-LOH of 17p were the most frequent genetic aberrations in LGGs. The findings were more diverse in LGA than in LGO. To the best of our knowledge, this is the first time description of methylation of the MLH3 gene promoter in LGGs. Further studies are required to determine the role of the methylated MLH3 promoter and the other aberrations detected.