Bevezetés: A T-sejt-receptor-klonalitás vizsgálata régóta alkalmazott rutin diagnosztikai módszer T-sejtes lymphoproliferatiókban, amely a T-sejt-receptor génátrendeződésének vizsgálatán alapul. Előnye a klonalitás molekuláris igazolása, hátránya azonban – az áramlási citometriához képest – a kisebb érzékenység, a viszonylag hosszú vizsgálati időtartam, valamint az, hogy nem ad fenotípusos információt. A T-sejt-receptor-klonalitás vizsgálata egy új, nemzetközi ajánlás szerinti áramlási citometriai módszerrel is végezhető. Ilyen egyszerűsített és hatékony megközelítés korábban nem állt rendelkezésre a rutin klinikai diagnosztikában. Célkitűzés: Célunk az irodalomban leírt legújabb ajánlás alapján a T-sejt-receptor konstans β-láncához tervezett antitestek együttes használata során szerzett tapasztalatok gyűjtése és azok összevetése a polimeráz-láncreakciót alkalmazó génátrendeződés-vizsgálatok eredményeivel. Módszer: A vizsgálat alapja a T-sejt-receptor konstans β-láncához tervezett, fluoreszcens festékkel konjugált kétféle monoklonális antitest alkalmazása, amelyek – a B-sejtes neoplasiák diagnosztizálásakor használatos kappa-lambda immunoglobulin-könnyűláncok vizsgálatához hasonlatosan – a normál expressziós arányoktól való eltérésen alapul. Eredmények: Az 54 mintából 20 esetben lymphoproliferativ betegség nem volt kimutatható, 11 esetben találtunk klonális populációt, amelyek jó összhangban voltak a konvencionális vizsgálati módszer eredményeivel. Egyéb 23 esetben találtunk kisebb fenotípusos eltéréseket, sejtarány-eltolódásokat, minor monoklonális populáció jelenlétét. Megbeszélés és következtetés: Az 54 minta eredményei alapján elmondható, hogy a vizsgálat nemcsak egyértelmű T- és NKT-sejtes klónoknak, hanem minor klonális populációknak az azonosítására is alkalmas, és számos esetben lehetővé teszi a T-sejtes kórképek gyorsabb diagnózisát. Orv Hetil. 2026; 167(15): 576–584.

INTRODUCTION:T-cell receptor clonality testing is a routine diagnostic method based on the detection of T-cell receptor gene rearrangements. Its advantage is the molecular confirmation of clonality, but its disadvantage - compared to flow cytometry - is the lower sensitivity, the actual long testing duration, and the lack of phenotypic information. More recently, T-cell receptor clonality testing can also be performed using a new, internationally recommended flow cytometry method. A simplified and efficient approach was previously not available in routine clinical diagnostics. OBJECTIVE:Our aim was to collect the experiences gained from the combined use of antibodies designed for the constant beta-chain of the T-cell receptor based on the latest recommendations described in the literature and to compare them with the results of conventional T-cell receptor clonality testing method. METHOD:The test is based on the use of two types of monoclonal antibodies conjugated with a fluorescent dye designed for the constant beta-chain of the T-cell receptor and investigates the deviations from normal expression ratios, similar to the testing of kappa-lambda immunoglobulin light chains used in the diagnosis of B-cell neoplasias. RESULTS:Among the 54 samples analyzed, no lymphoproliferative disorder was detected in 20 cases. In 11 cases, a clonal population was identified, showing good concordance with the results of conventional diagnostic methods. In the remaining 23 cases, minor phenotypic abnormalities, shifts in cell population ratios, and the presence of minor monoclonal populations were observed. DISCUSSION AND CONCLUSION:Based on the results of the 54 samples, the test proved to be suitable not only for the identification of clear T and NKT cell clones, but also for the identification of minor clonal populations, furthermore, it may offer faster diagnosis of T-cell diseases. Orv Hetil. 2026; 167(15): 576-584.

During pregnancy, immune tolerance toward the semi-allogeneic fetus must be established. γδT cells constitute a distinct T-cell lineage characterized by expression of the γδT-cell receptor (γδTCR). They combine rapid innate-like responses with adaptive immune functions and contribute to feto-maternal immune regulation; however, their peripheral phenotypic characteristics, including chemokine receptor (ChR) profiles potentially relevant to decidual recruitment, remain incompletely characterized. Peripheral blood from healthy non-pregnant and first-trimester pregnant women was analyzed by flow cytometry to assess γδT-cell phenotypes and CCR5/CCR6 ChR expression. Analysis of γδT-cell subsets defined by lower (γδTCRdim) or higher (γδTCRbright) surface γδTCR expression showed that CCR5-/CCR6- cells were more frequent in γδTCRbright cells, whereas CCR5+ phenotypes were more prominent in γδTCRdim cells; CD4/CD8 and CD56/CD8 distributions were comparable. Resting γδT cells were mainly CD4-/CD8-, whereas recently activated CD69+ γδT cells had increased CD4+ or CD8+ single-positive frequencies. Compared with non-γδT cells, γδT cells showed distinct CD4/CD8-linked CCR5/CCR6 organization, including a CCR5+/CCR6- preference. Unlike non-γδT cells, conventional CD56- and NK-like CD56+ γδT cells retained similar CCR5/CCR6 profiles across CD4/CD8-defined subsets. PMA/ionomycin-stimulated samples generally exhibited lower CCR5+ and higher CCR5- frequencies across CD4- conventional and NK-like γδT cell subsets. Pregnancy-related circulating γδT-cell phenotype changes were negligible, possibly reflecting decidual immune adaptation. Overall, the study highlights γδTCR intensity-linked ChR heterogeneity and, in contrast to non-γδT cells, conserved CCR5/CCR6 organization across CD56-defined γδT-cell subsets. Broader ChR phenotyping and functional migration analyses could clarify the biological relevance of these subset-specific differences.

IntroductionCD20+ T-cells were described firstly in peripheral blood and later in bone marrow in patients with hematological tumors, and certain immune-mediated diseases. During our hematological diagnostic work, this peculiar subgroup of lymphocytes has been consistently observed associated with untreated monoclonal gammopathy of undetermined significance (MGUS) and myeloma (MM). Despite the expanding literature data, the exact function of CD20+ T cells remains unclear.MethodsWe investigated the incidence of CD20+ T-cells in MGUS (n=27), and MM using a larger cohort (n=125) and compared it with control bone marrow samples (n=39). We examined their presence before and after treatment in 32 cases with flow cytometry. Comprehensive flow cytometric analysis included the examination of functional (T-cell activation, cytotoxic molecules and T-cell exhaustion) and maturation markers in a large number of cases. In addition RNA sequencing and subsequent bioinformatics analyses were carried out to detect differentially expressed (DE) genes of FACS sorted CD20+ T-cells versus CD20- T-cells.Results and discussionWe found that CD20+ T-cells are phenotypically and transcriptionally different from CD20- T-cells. Elevated incidence of CD20+ T-cells in MGUS and MM and the expression of CD8, NKG2D, and CD28 suggests anti-tumor functionality. Increased PD-1 expression indicates T-cell exhaustion which was mostly detected in the samples of patients with a higher tumor percentage. The majority of CD20+ T-cells are effector or effector memory T-cells. Some of the differentially expressed genes suggest antitumor function via regulating T-cell activation pathways, while other genes involved in tumor escape from immune surveillance by suppressing T-cells or by reprogramming T-cells toward T-cell exhaustion. Our findings suggest that CD20+ T-cells may play a vital role both in immune surveillance and immune escape contributing to progression of multiple myeloma.

During pregnancy, the maternal immune system must allow and support the growth of the developing placenta while maintaining the integrity of the mother’s body. The trophoblast’s unique HLA signature is a key factor in this physiological process. This study focuses on decidual γδT cell populations and examines their expression of receptors that bind to non-classical HLA molecules, HLA-E and HLA-G. We demonstrate that decidual γδT cell subsets, including Vδ1, Vδ2, and double-negative (DN) Vδ1-/Vδ2- cells express HLA-specific regulatory receptors, such as NKG2C, NKG2A, ILT2, and KIR2DL4, each with varying dominance. Furthermore, decidual γδT cells produce cytokines (G-CSF, FGF2) and cytotoxic mediators (Granulysin, IFN-γ), suggesting functions in placental growth and pathogen defense. However, these processes seem to be controlled by factors other than trophoblast-derived non-classical HLA molecules. These findings indicate that decidual γδT cells have the potential to actively contribute to the maintenance of healthy human pregnancy.

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

[This corrects the article DOI: 10.3389/fimmu.2020.596489.].

Background: Based on previous retrospective results, we investigated the association of coagulation FXIII subunit A (FXIII-A) expression pattern on survival and correlations with known prognostic factors of B-cell progenitor (BCP) childhood acute lymphoblastic leukemia (ALL) as a pilot study of the prospective multi-center BFM ALL-IC 2009 clinical trial. Methods: The study included four national centers (n = 408). Immunophenotyping by flow cytometry and cytogenetic analysis were performed by standard methods. Copy number alteration was studied in a subset of patients (n = 59). Survival rates were estimated by Kaplan-Meier analysis. Correlations between FXIII-A expression patterns and risk factors were investigated with Cox and logistic regression models. Results: Three different patterns of FXIII-A expression were observed: negative (<20%), dim (20–79%), and bright (≥80%). The FXIII-A dim expression group had significantly higher 5-year event-free survival (EFS) (93%) than the FXIII-A negative (70%) and FXIII-A bright (61%) groups. Distribution of intermediate genetic risk categories and the “B-other” genetic subgroup differed significantly between the FXIII-A positive and negative groups. Multivariate logistic regression confirmed independent association between the FXIII-A negative expression characteristics and the prevalence of intermediate genetic risk group. Conclusions: FXIII-A negativity is associated with dismal survival in children with BCP-ALL and is an indicator for the presence of unfavorable genetic alterations.

Abstract: Introduction: Plasma cell myeloma is a hematological malignancy with heterogeneous genomic landscape and diverse clinical course. Recurrent chromosomal and subchromosomal aberrations commonly occur in this entity and are associated with the pathogenesis and progression of the disease. The identification of these alterations aids genetic characterization, classification and prognostication of patients. Aim: Molecular cytogenetic investigations of plasma cell myeloma patients treated at the University of Pécs Clinical Center and János Balassa County Hospital of Tolna County, Szekszárd, between 2005 and 2018 were evaluated in our study. Method: 231 patients were screened for genetic aberrations using fluorescence in situ hybridization. Translocations involving the immunoglobulin heavy chain gene, losses of 1p and 17p chromosome arms, gains of 1q chromosome arm and unbalanced aberrations of chromosome 13 were investigated. Losses and gains of 1p, 1q, 5q, 12p, 13q, 16q and 17p chromosome arms were analyzed using multiplex ligation-dependent probe amplification in 42 patients. During the investigated period, 116 bone marrow karyotyping was also performed. Results: In total, 233 genetic aberrations were identified using our targeted approaches; the frequency of specific aberrations correlated with data of the recent literature. Concordance of results gained by fluorescence in situ hybridization and multiplex ligation-dependent probe amplification was 96.2% by analyzing the same chromosome arms. The latter technique revealed 21 additional genetic aberrations in 16/42 patient samples (38%) as compared to fluorescence in situ hybridization. Conclusions: Our results suggest that the combined application of the two molecular cytogenetic methods may facilitate a more detailed characterization of genetic aberrations of plasma cell myeloma patients in Hungary. Orv Hetil. 2019; 160(24): 944–951.

Pregnancy is an immunological enigma where paternal antigens are present at the fetomaternal interface. What regulates the local immunotolerance, which is necessary to prevent rejection of the conceptus, is still under strong investigation. Gamma/delta T cells are believed to play a role in the local regulation of this immunotolerance towards the semiallogenic fetus. Gamma/delta T cells from the uterus and spleen of pregnant and nonpregnant mice were analyzed by flow cytometry. We confirmed that the rate of γδT cells in the decidua increases during murine pregnancy and half of decidual γδT cells are CD4+. Furthermore, we found a unique association of CD4 or CD8 coreceptor expression with their γδTCR intensity, where in all investigated groups CD4- or CD8-positive γδT cells seemed principally to be γδTCRdim. In addition, compared to peripheral γδT lymphocytes, a greater proportion of decidual γδT cells expressed the cytotoxic marker CD107a and markers of Th1 or Th2 polarization (TIM-3, TIM-1), where decidual γδTCRbright cells were characterized by high TIM-3 and TIM-1 receptor expression. On the other hand, no difference in the expression of CD160, a receptor with dual function affecting cytotoxicity and T cell inhibition, was detected. Within lymphocytes expressing CD107a, TIM-1, or CD160, the rate of γδT cells was significantly higher in the decidua. According to our results, cytotoxic potential of decidual γδTCRbright cells could be regulated by TIM-3 ligation, while the TIM-1 receptor seems to be able to influence the Th1-Th2 balance at the fetomaternal interface. These mechanisms could play a part in the active maternal immunotolerance towards the fetus, allowing an efficient protection against pathogens during healthy murine pregnancy.

Introduction: Plasma cell myeloma is a hematological malignancy with heterogeneous genomic landscape and diverse clinical course. Recurrent chromosomal and subchromosomal aberrations commonly occur in this entity and are associated with the pathogenesis and progression of the disease. The identification of these alterations aids genetic characterization, classification and prognostication of patients. Aim: Molecular cytogenetic investigations of plasma cell myeloma patients treated at the University of Pecs Clinical Center and Janos Balassa County Hospital of Tolna County, Szekszard, between 2005 and 2018 were evaluated in our study. Method: 231 patients were screened for genetic aberrations using fluorescence in situ hybridization. Translocations involving the immunoglobulin heavy chain gene, losses of 1p and 17p chromosome arms, gains of 1q chromosome arm and unbalanced aberrations of chromosome 13 were investigated. Losses and gains of 1p, 1q, 5q, 12p, 13q, 16q and 17p chromosome arms were analyzed using multiplex ligation-dependent probe amplification in 42 patients. During the investigated period, 116 bone marrow karyotyping was also performed. Results: In total, 233 genetic aberrations were identified using our targeted approaches; the frequency of specific aberrations correlated with data of the recent literature. Concordance of results gained by fluorescence in situ hybridization and multiplex ligation-dependent probe amplification was 96.2% by analyzing the same chromosome arms. The latter technique revealed 21 additional genetic aberrations in 16/42 patient samples (38%) as compared to fluorescence in situ hybridization. Conclusions: Our results suggest that the combined application of the two molecular cytogenetic methods may facilitate a more detailed characterization of genetic aberrations of plasma cell myeloma patients in Hungary.

Background: Leukemic B-cell precursor (BCP) lymphoblasts were identified as a novel expression site for coagulation factor XIII subunit A (FXIII-A). Flow cytometry (FC) revealed three distinct expression patterns, i.e., FXIII-A negative, FXIII-A dim, and FXIII-A bright subgroups. The FXIII-A negative subgroup was significantly associated with the “B-other” genetic category and had an unfavorable disease outcome. Methods: RNA was extracted from bone marrow lymphoblasts of 42 pediatric patients with BCP-acute lymphoblastic leukemia (ALL). FXIII-A expression was determined by multiparameter FC. Genetic diagnosis was based on conventional cytogenetic method and fluorescence in situ hybridization. Affymetrix GeneChip Human Primeview array was used to analyze global expression pattern of 28,869 well-annotated genes. Microarray data were analyzed by Genespring GX14.9.1 software. Gene Ontology analysis was performed using Cytoscape 3.4.0 software with ClueGO application. Selected differentially expressed genes were validated by RT-Q-PCR. Results: We demonstrated, for the first time, the general expression of F13A1 gene in pediatric BCP-ALL samples. The intensity of F13A1 expression corresponded to the FXIII-A protein expression subgroups which defined three characteristic and distinct gene expression signatures detected by Affymetrix oligonucleotide microarrays. Relative gene expression intensity of ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, NUP43, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2 followed the pattern of change in the intensity of the expression of the F13A1 gene. Common enhancer elements of these genes revealed by in silico analysis suggest that common transcription factors may regulate the expression of these genes in a similar fashion. PLAC8 was downregulated in the FXIII-A bright subgroup. Gene expression signature of the FXIII-A negative subgroup showed an overlap with the signature of “B-other” samples. DFFA, GIGYF1, GIGYF2, and INTS3 were upregulated and CD3G was downregulated in the “B-other” subgroup. Validated genes proved biologically and clinically relevant. We described differential expression of genes not shown previously to be associated with pediatric BCP-ALL. Conclusions: Gene expression signature according to FXIII-A protein expression status defined three novel subgroups of pediatric BCP-ALL. Multiparameter FC appears to be an easy-to-use and affordable method to help in selecting FXIII-A negative patients who require a more elaborate and expensive molecular genetic investigation to design precision treatment.

Aim: Molecular cytogenetic investigations of plasma cell myeloma patients treated at the University of Pécs Clinical Center and János Balassa County Hospital of Tolna County, Szekszárd, between 2005 and 2018 were evaluated in our study. Method: 231 patients were screened for genetic aberrations using fluorescence in situ hybridization. Translocations involving the immunoglobulin heavy chain gene, losses of 1p and 17p chromosome arms, gains of 1q chromosome arm and unbalanced aberrations of chromosome 13 were investigated. Losses and gains of 1p, 1q, 5q, 12p, 13q, 16q and 17p chromosome arms were analyzed using multiplex ligation-dependent probe amplification in 42 patients. During the investigated period, 116 bone marrow karyotyping was also performed. Result s : In total, 233 genetic aberrations were identified using our targeted approaches; the frequency of specific aberrations correlated with data of the recent literature. Concordance of results gained by fluorescence in situ hybridization and multiplex ligation-dependent probe amplification was 96.2% by analyzing the same chromosome arms. The latter technique revealed 21 additional genetic aberrations in 16/42 patient samples (38%) as compared to fluorescence in situ hybridization. Conclusions: Our results suggest that the combined application of the two molecular cytogenetic methods may facilitate a more detailed characterization of genetic aberrations of plasma cell myeloma patients in Hungary.

Introduction: Subunit A of blood coagulation factor XIII (FXIIIA) may be expressed in leukemic B-cell precursor (BCP) lymphoblasts in addition to platelets, megakaryocytes, monocytes and macrophages (Kiss F. et al Thromb Haemost 2006;96:172-82.). In a retrospective single-center cohort of children with acute lymphoblastic leukemia (ALL) treated with the BFM ALL-IC 2002 protocol, expression of FXIIIA was correlated with statistically significant survival advantage (Kárai B et al. Pathol Oncol Res 2018; 24:345-52.). Our aim was to investigate the impact of FXIIIA expression pattern on EFS and its correlation with known clinical and genetic prognostic factors in a multi-center pilot study within the frames of the BFM ALL-IC 2009 clinical trial (EuDract: 2010-019722-13). Patients and Methods: We examined 317 children with BCP-ALL between 2011 and 2018 at institutions in Poland (n=188), Hungary (n=116), and Slovakia (n=13). Patients with Down-syndrome, t(9;22), and infants (<1 year) were excluded. Immunophenotype and minimal residual disease (MRD) were determined by flow cytometry (FC). Cytogenetic analysis and fluorescence in situ hybridization were performed according to standard methods. DNA extracted from bone marrow samples was processed for SALSA multiplex ligation-dependent probe amplification P335-B2 ALL-IKZF1 probe mix (MRC-Holland, Amsterdam, The Netherlands) analysis according to manufacturer's instructions. Statistical analysis: Survival analysis was carried out by the Kaplan-Meier estimator. More than 2 groups were analyzed by the Kruskal-Wallis test. Dunn's multiple comparison was applied as post host test. Dichotomous categorical variables were compared by Chi square test and logistic regression to analyze multiple variables. P<0.05 was considered significant. Results: We observed 3 different patterns of FXIIIA expression in leukemic lymphoblasts: negative pattern (<20%), moderate positive expression (20-79%), and strong positive pattern (>80%). Evaluation of FC histograms of the moderate positive pattern showed that FXIIIA expression increased continuously which excluded the existence of distinct FXIIIA negative and FXIIIA positive subpopulations within the moderate positive expression group (Fig.1). In contrast to the previous single-center retrospective cohort, EFS of patients with FXIIIA positive BCP-ALL was not significantly different from that of patients of FXIIIA negative BCP-ALL (Fig.2A). However, a significant EFS advantage of patients with moderate FXIIIA positivity was demonstrated when compared with patients with FXIIIA negative BCP-ALL (p=0.019), and with patients with FXIIIA strong positive BCP-ALL (p=0.001) (Fig.2B). The 3 different FXIIIA expression patterns did not correlate significantly with either risk stratification according to BFM ALL-IC 2009 or FC MRD categories. Intermediate genetic risk categories (low hyperdiploidy, t(1;19), and "B-other") were significantly more prevalent (p=0.042) among patients with FXIIIA negative BCP-ALL than among patients with strong FXIIIA positive BCP-ALL. The "B-other" subgroup was also significantly more prevalent (p=0.022) among patients of the above 2 groups (Table 1). Distribution of the "B-other" genetic group and the prednisone response differed significantly between the FXIIIA positive and negative groups. Intermediate genetic risk group is mostly made up of patients assigned to the "B-other" group explaining the significant difference between the FXIIIA positive and negative groups, in terms that FXIIIA negative patients had 50% lower chance of facing low instead of intermediate genetic risk compared to FXIIIA positive patients. The multivariate logistic regression analysis confirmed the association between the FXIIIA characteristics and the intermediate genetic risk group. This association persisted after adjusting for categorical variables, i.e. gender, age, WBC, and BFM ALL-IC 2009 stratification (Table 2). High risk copy number alterations were detected in 4/7 samples of the FXIIIA negative group and in 1/17 of the FXIIIA positive group. Conclusions: FXIIIA expression status of leukemic lymphoblasts can easily be determined by FC. Patients with FXIIIA negative lymphoblasts should be further investigated with sophisticated and more expensive genetic and molecular methods. Grant sponsor: OTKA K108885. No relevant conflicts of interest to declare.

Multiple myeloma (MM) is a genetically heterogeneous disease with a diverse clinical outcome. Copy number alterations (CNAs), including whole chromosome and subchromosomal gains and losses, are common contributors of the pathogenesis and have demonstrated prognostic impact in MM. We tested the performance of digital multiplex ligation-dependent probe amplification (digitalMLPA), a novel technique combining MLPA and next-generation sequencing, to detect disease-related CNAs. Copy number status at 371 genomic loci was simultaneously analyzed in 56 diagnostic bone marrow samples, which were also examined by conventional MLPA and interphase fluorescence in situ hybridization (iFISH). On average, digitalMLPA identified 4.4 subchromosomal CNAs per patient. The increased number of probes compared with conventional MLPA allowed a detailed mapping of CNAs, especially on chromosome 1, where 24 different patterns were observed in 38 patients harboring loss(1p) and/or gain(1q). iFISH, MLPA, and digitalMLPA results at loci investigated by multiple methods showed a congruency of 95%. Besides precise characterization of hyperdiploid karyotypes not efficiently achievable by iFISH or MLPA, digitalMLPA unraveled 156 CNAs not detected by the other two methods in 45 patients (80%). In addition, we provide proof of principle that digitalMLPA can detect known point mutations, in this case the BRAFV600E. Our study demonstrates the robustness of digitalMLPA to profile CNAs and to screen point mutations in MM, which could efficiently be used in myeloma diagnostics.

PROBLEM:CD160, a cell surface co-receptor, is capable of up- or downregulating cell proliferation, cytotoxicity or cytokine production on lymphocytes. Our aim was to investigate CD160+ lymphocytes in the periphery and at the maternal-foetal interface during murine pregnancy.METHOD OF STUDY:CD4+ , CD8+ and gamma/delta T-cell phenotype, TIM3 co-expression and cytotoxic activity of CD160+ lymphocytes of pregnant BALB/c mice were analysed by flow cytometry.RESULTS:The percentage of CD160+ lymphocytes in the decidua was unchanged compared to non-pregnant endometrium; however, the ratio of CD4+ cells within the CD160 population was significantly increased. The co-expression of TIM3 co-inhibitory molecule and cytotoxicity of CD160+ cells were increased in the decidua.CONCLUSION:The expansion of CD4-expressing CD160+ decidual lymphocytes is a new observation suggesting a potential regulatory role of T-cell function during mouse pregnancy. The altered immunological character of CD160+ lymphocytes could play a role in the maintenance of murine pregnancy.

Abstract Introduction: In the progression of colorectal cancer, metastasis is a critical step in the development of systemic disease. Early diagnosis of metastatic progression, however, is not available. We present a novel approach to detect metastatic predisposition early, from patient blood. Background: Metastasis is disseminated by cancer stem cells. They rapidly adapt to hypoxia, they prefer hypoxic metabolism, which is also a major factor in clinical therapy-resistance and dissemination. By studying cancer stem cell differentiation we found that they readily form spheroids (small multicellular clusters or organoids) with hypoxic centers. Hypothesis: We propose that cancer stem cells develop hypoxic spheroids to provide a protected hypoxic micro-niche for survival. Based on the fact that spheroid formation is universally found in vitro, we asked whether cancer stem cells may also form spheroids in vivo, when they enter circulation. Our hypothesis is that in vivo circulating tumorspheres carry cancer stem cells and contribute to metastatic dissemination. To investigate the possibility of circulating cancer spheroids in colorectal and other cancers, we worked out the methodology to detect and characterize them from patient blood. Experimental Procedures: Blood samples were collected from 20 colorectal and 86 mixed cancer cases. 16 of the 20 colorectal and 62 of the 86 mixed cancers were metastatic, respectively. We also tested 44 controls. The samples were analyzed by flow cytometry using forward scatter and SYTO16 fluorescent labels. For spheroid capture we used simple nylon mesh filters (10 um and 20 um pore size). From the collected spheroids RNA was isolated and gene expression assays were performed for stem cell, hypoxia and epithelial markers. We also studied the role of inflammation and immune check point mechanisms in dissemination. Results: We found that spheroids with epithelial lineage develop hypoxia and carry stem cells. By using Partec and Beckman-Coulter flow cytometers we detected circulating spheroids in patient blood. In the majority of colorectal cancer cases (56%) spheroids in blood indicated metastasis, while the controls were all negative. Furthermore, in half of the mixed cancer cases spheroids in blood also correlated with metastasis. All healthy samples and non-metastatic cases were negative. A subset of spheroids was positive for immune checkpoint and platelet markers suggesting immune/inflammation mechanisms in dissemination. Follow-up clinical studies will investigate the prospective significance of the results. Conclusions: We showed that cancer stem cells form spheroid clusters in vivo and can be detected in circulation. We found significant correlation between spheroids in blood (spheremia) and metastasis in colorectal and other cancers. The most critical new finding of our studies is that systemic dissemination of cancer involves a novel spheroid mechanism. Based on this observation we developed a simple blood test that may detect metastatic predisposition early in colon cancer progression. Citation Format: Viktoria Denes, Laszlo Graf, Gabor Barna, Balint Tegze, Pal Jakso, Peter Geck. Blood test of metastatic predisposition in colon cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B03.