Az aberráns hipermutáció okozta genetikai instabilitás szerepe a B-sejtes non-Hodgkin lymphomák kialakulásában és transzformációjában = The role of genetic instability caused by aberrant hypermutation in the development and transformation of B-cell non-Hodgkin lymphomas

A kutatás eredményeként bizonyítottuk, hogy a krónikus lymphocytás leukémia (CLL) és a follicularis lymphoma (FL) kifejezett malignitású lymphomába történő transzformációjában, valamint a mediastinalis nagy B-sejtes lymphoma (MLBL) kialakulásában szerepet játszik egy napjainkban leírt, aberráns szomatikus hypermutációnak (ASH) nevezett genetikai instabilitás. Ugyancsak bizonyítottuk, hogy azokban a lymphoma transzformációkban és MLBL-ban, ahol ASH van jelen, a centrum germinativum B-sejtek szomatikus hypermutációjáért felelős aktiváció indukálta citidin deamináz (AID) enzim expressziója figyelhető meg. Ezen adataink arra utalnak, hogy a B-sejtes lymphomák lefolyása során az AID enzim aktiválódhat és az egész genomra kiterjedő, köztük a c-MYC, a PAX-5 és a RhoH géneket érintő genetikai instabilitás alakulhat ki. Ugyancsak a pályázat keretében karakterizáltuk a WHO által meghatározott, krónikus lymphocytás leukémia emelkedett számú prolymphocytával (CLL/PL) nevezett entitás biológiai sajátosságait. IgVH gén, FISH és high resolution comparative genomic hybridization (HR-CGH) módszerek segítségével magas arányú genomikus amplifikáció és deléció arányokat valamint intenzív klonális szelekciót és evolúciót igazoltunk. Ezen eredmények arra utalnak, hogy a CLL/PL kialakulásában genomikus instabilitás és klonális evolúció játszhat szerepet. | As a result of grant support we have provided evidences that in high-grade transformation of chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL) and development of mediastinal large B-cell lymphoma (MLBL), a recently described genetic instability called aberrant somatic hypermutation (ASH) has a central role. We have also demonstrated that in lymphoma transformation and in MLBL, where tumor cells display ASH, the enzyme which is involved in the somatic hypermutation and switch recombination of germinal center B-cells called activation-induced cytidine deminase (AID) have high expression. These results suggest that activation of AID enzyme in the natural history of B-cell lymphomas may generate a genome wide genetic instability causing mutations of several genes including c-MYC, PAX-5 and RhoH. We have also characterized the biological features of a WHO lymphoma entity called chronic lymphocytic leukemia with increased prolymphocytes (CLL/PL). Using IgVH gene, FISH and high resolution comparative genomic hybridization (HR-CGH) analysis we have shown high number of genomic imbalances and intensive clonal evolution and selection in the course of the CLL/PL suggesting that a genome wide instability of the neoplastic cells may play a role in the development of the disease

A tumorsejtek szabályozási zavarai - a diagnosis és a terápia alapja = Disturbances in cancer cell regulation - a basis to design diagnosis and therapy

A project a daganatok viselkedését befolyásoló olyan molekuláris történésekkel foglalkozott, amelyek felhasználhatók diagnosztikai markerként vagy terápiás célpontként. Ennek eredményeként mutattuk ki NSCLC-ben az EGFR mutációk jellegét és gyakoriságát, valamint azt, hogy EGFR-gátlókkal szemben rezisztens tüdőráksejtekben a TRAIL apoptózist tud kiváltani, különösen IKK-gátlóval együtt. A TGFb ugyancsak apoptózist indukált limfomákban, amely rapamycinnel (mTOR-gátló) fokozható volt. A daganatnövekedés szempontjából szolid tumorokban elengedhetetlen az angiogenezis. Kiderült, hogy a vérerek pl. májmetasztázisok esetében több módon is biztosíthatják a daganatsejtek táplálását, ami eltérő mechanizmusra utal és eltérő terápiát igényel. Felhívtuk a figyelmet a limfangiogenezis fontosságára is. A daganat matrixában levő szabályozók közül a szindekán-1 különböző konstrukcióit sikerült előállítani és ezekkel vizsgálni az egyes domének hatását. A daganatok kialakulásával vagy transzformációjukkal kapcsolatos vizsgálatok során limfomákban vált bizonyítottá az aberráns szomatikus mutáció és az aktiváció indukált citidin deamináz szerepe, a májrák megjelenésekor pedig az ovális sejtek és a Hering csatornák jellegzetességeinek tisztázása, részben az őssejtekhez való viszonyuk, részben marker jellegük alapján. A vizsgálatokba a 3 év során 15 PhD hallgató kapcsolódott be, és 7-en sikeresen megvédték értekezésüket. | The project intended to study molecular events influencing behavior of cancer cells in order to use them either as diagnostic markers or therapeutic targets. Along this line the frquency and types of EGFR mutations were described in NSCLC (adenocc), and also the successful use of TRAIL to induce apoptosis, especially in combination with IKK-inhibitors, in lung cancer cells resistant against anti-EGFR agents. Another key regulator, TGFb was also able inducing apoptosis in lymphoma cells, given with rapamycin (mTOR-inhibitor). For solid tumors angiogenesis is essential for survival. It was shown that liver metastases can obtain vasculature in different manners, which suggest different mechanisms and the necessity of alternative approaches in anti-angiogenesis. The importance of lymphangiogenesis was also documented. Besides vessels tumor matrix contains several regulatory factors, as syndecan-1. Some of its artificial constructs were applied to understand the functions of the structural domains. Many experiments were concerned with development and transformation of cancer. In lymphomas the role of aberrant somatic mutations and the activation induced cytosine deaminase served special attention. In the liver the relation of oval cells and Hering chanelles to stem cells was described and proposed as diagnostic markers. This project sponsored the scientific activity of 15 PhD students and helped in some cases (7) to defend successfully their thesis

Molecular cytogenetic analysis of Xq critical regions in premature ovarian failure.

BACKGROUND: One of the frequent reasons for unsuccessful conception is premature ovarian failure/primary ovarian insufficiency (POF/POI) that is defined as the loss of functional follicles below the age of 40 years. Among the genetic causes the most common one involves the X chromosome, as in Turner syndrome, partial X deletion and X-autosome translocations. Here we report a case of a 27-year-old female patient referred to genetic counselling because of premature ovarian failure. The aim of this case study to perform molecular genetic and cytogenetic analyses in order to identify the exact genetic background of the pathogenic phenotype. RESULTS: For premature ovarian failure disease diagnostics we performed the Fragile mental retardation 1 gene analysis using Southern blot technique and Repeat Primed PCR in order to identify the relationship between the Fragile mental retardation 1 gene premutation status and the premature ovarion failure disease. At this early onset, the premature ovarian failure affected patient we detected one normal allele of Fragile mental retardation 1 gene and we couldn't verify the methylated allele, therefore we performed the cytogenetic analyses using G-banding and fluorescent in situ hybridization methods and a high resolution molecular cytogenetic method, the array comparative genomic hybridization technique. For this patient applying the G-banding, we identified a large deletion on the X chromosome at the critical region (ChrX q21.31-q28) which is associated with the premature ovarian failure phenotype. In order to detect the exact breakpoints, we used a special cytogenetic array ISCA plus CGH array and we verified a 67.355 Mb size loss at the critical region which include total 795 genes. CONCLUSIONS: We conclude for this case study that the karyotyping is definitely helpful in the evaluation of premature ovarian failure patients, to identify the non submicroscopic chromosomal rearrangement, and using the array CGH technique we can contribute to the most efficient detection and mapping of exact deletion breakpoints of the deleted Xq region

Unique patterns of CD8+ T-cell-mediated organ damage in the Act-mOVA/OT-I model of acute graft-versus-host disease.

T-cell receptor (TCR)-transgenic models of acute graft-versus-host disease (aGvHD) offer a straightforward and highly controlled approach to study the mechanisms and consequences of T-cell activation following allogeneic hematopoietic stem cell transplantation (aHSCT). Here, we report that aHSCT involving OT-I mice as donors, carrying an ovalbumin-specific CD8+ TCR, and Act-mOVA mice as recipients, expressing membrane-bound ovalbumin driven by the β-actin promoter, induces lethal aGvHD in a CD8+ T-cell-dependent, highly reproducible manner, within 4-7 days. Tracking of UBC-GFP/OT-I graft CD8+ T cells disclosed heavy infiltration of the gastrointestinal tract, liver, and lungs at the onset of the disease, and histology confirmed hallmark features of gastrointestinal aGVHD, hepatic aGvHD, and aGvHD-associated lymphocytic bronchitis in infiltrated organs. However, T-cell infiltration was virtually absent in the skin, a key target organ of human aGvHD, and histology confirmed the absence of cutaneous aGVHD, as well. We show that the model allows studying CD8+ T-cell responses in situ, as selective recovery of graft CD45.1/OT-I CD8+ T cells from target organs is simple and feasible by automated tissue dissociation and subsequent cell sorting. Assessment of interferon-gamma production by flow cytometry, granzyme-B release by ELISA, TREC assay, and whole-genome gene expression profiling confirmed that isolated graft CD8+ T cells remained intact, underwent clonal expansion, and exerted effector functions in all affected tissues. Taken together, these data demonstrate that the OT-I/Act-mOVA model is suitable to study the CD8+ T-cell-mediated effector mechanisms in a disease closely resembling fatal human gastrointestinal and hepatic aGVHD that may develop after aHSCT using HLA-matched unrelated donors

Connexin 43 Communication Channels in Follicular Dendritic Cell Development and in Follicular Lymphomas

Follicular dendritic cells (FDC) show homo- and heterocellular metabolic coupling through connexin 43 (Cx43) gap junctions and support B cell selection and maturation in germinal centers. In follicular lymphomas B cells escape apoptosis while FDC develop abnormally. Here we tested Cx43 channels in reactive FDC development and follicular lymphomas. In culture, the treatment of FDC-B cell clusters (resembling to “ ex vivo ” germinal centers) with Gap27 peptide, mimicking the 2nd extracellular loop of Cx43 protein, significantly impaired FDC-B cell cluster formation and cell survival. In untreated cultures of intact clusters, cell proliferation showed a moderate reduction. In tissues, Cx43 protein levels run parallel with the density of FDC both in reactive germinalcentersandinmalformedfolliclesoffollicularlymphomasandshowedstrongupregulationinnewlygeneratedand/or degrading bi-/multinuclear FDC of rudimentary processes. However, the inverse correlation between Cx43 expression and B cell proliferation seen in reactive germinal centers was not detected in follicular lymphomas. Furthermore, Cx43 levels were not associated with either lymphoma grade or bone marrow involvement. Our results suggest that Cx43 channels are critical in FDC and “ ex vivo ” germinal center development and in the persistence of FDC in follicular lymphomas but do not affect tumor progression