The aim of the present study was to evaluate the clinical relevance of mutations in tumor suppressor genes using whole-exome sequencing data from centenarians and young healthy individuals. Two pools, one of centenarians and one of young individuals, were constructed and whole-exome sequencing was performed. We examined the whole-exome sequencing data of Bulgarian individuals for carriership of tumor suppressor gene variants. Of all variants annotated in both pools, 5080 (0.06%) are variants in tumor suppressor genes but only 46 show significant difference in allele frequencies between the two studied groups. Four variants (0.004%) are pathogenic/risk factors according to single nucleotide polymorphism database: rs1566734 in PTPRJ, rs861539 in XRCC3, rs203462 in AKAP10, and rs486907 in RNASEL. Based on their high minor allele frequencies and presence in the centenarian group, we could reclassify them from pathogenic/risk factors to benign. Our study shows that centenarian exomes can be used for re-evaluating the clinically uncertain variants.

Background/Objectives: Immunosenescence implies innate and adaptive immunity dysfunction, which naturally occurs with aging. It is a complex multifactorial process which can be triggered by either genetic changes, immune changes or both. Numerous research studies have shown that the process of senescence goes alongside chronic immune activation. The purpose of this study is to analyze the changes in the expression of genes associated with adaptive and innate immune responses in order to identify reliable biomarkers for immune aging. Methods: For that aim, 55 clinically healthy individuals of active age (21–65 years) were distributed based on immunophenotyping in two groups, with and without signs of premature senescence. A gene expression analysis was subsequently made on those two groups, and the differentially expressed genes were presented and interpreted. Results: Altogether, forty-eight (48) genes exhibited differential expression between the two groups, most of which showed up-regulation (45) (fold change more than 2), and only three were down-regulated (fold change less than −2). The highest positive fold change showed IL-1β (10.76), BCL6 (13.25) and CCL4 (15.91), while the highest negative fold changes were documented for IL23R (−3.10), IL5 (−2.66) and PTGS2 (COX-2) (−2.15). Conclusions: Our results reveal that immunosenescence is positively associated with chronic inflammation, which is typical for the aging process. On the other hand, we identified markers of possible protective effects against oxidative stress and tumorigenesis. These findings can aid the early diagnosis of chronic degenerative diseases in subclinical phase, as well as the development of strategies to prevent the processes of premature immune aging.

Lymphoplasmacytic lymphoma (LPL) is a malignant proliferation of small lymphocytes, lymphoplasmocytoid cells and plasmocytes affecting the bone marrow, lymph nodes and spleen. Its incidence is 1/100,000 and represents 8% of all lymphomas. A total of ~5% of patients with LPL may secrete non‑IgM of IgG, IgA, kappa or lambda type or be non‑secretory. In the present study, a case of a 62‑year‑old female patient who was diagnosed with non‑IgM LPL with kappa light chain monoclonal paraprotein production and normal serum immunoglobulin levels was reported. The MYD88 L265P mutation was detected by molecular genetic analysis using a sample of the bone marrow. The patient underwent initial treatment with a combination of Bendamustine‑Rituximab, and later on, Ibrutinib (a Bruton kinase inhibitor) was added to the treatment protocol. The authors' aim was to describe a case of a rare type of LPL studied and cured at the University Hospital ‘St. Ivan Rilski’, as well as to show the methods used for its diagnosis and their applicability. The difficulty in diagnosing such rare cases of LPL which are associated with marked plasmacytic differentiation and IgA paraprotein secretion resembling plasma cell neoplasia was addressed. From the other side, the characteristic features in favor of LPL diagnosis are the immunophenotype profile of plasmocytes, as well as the presence of MYD88 L265P mutation. Finally, the methods of management and treatment of this type of lymphoma were reported, highlighting the favorable effect of the treatment with Bruton TK inhibitor (Ibrutinib).

Myelofibrosis (MF) is characterized by a presence of an extra fibrous tissue in the bone marrow and additional hematopoiesis. The somatic mutation in the Janus kinase 2 ( JAK2 ) gene (V617F) occurs gradually and is detected in about 50.0% of myelofibrosis or essential thrombo-cytopenia (ET) patients. Our aim was to determine the genotype status according to the carriers of the V617F mutation in patients with MF at the Hematology Ward of the University Hospital "Ivan Rilski" in Sofia, Bulgaria. DNA samples were isolated from venous blood of patients with various hematological disorders. DNA was amplified by polymerase chain reaction (PCR) and subsequent restriction analysis was performed using a Bsa XI restriction enzyme. The genotype status was determined on 2.0% agarose gel. We analyzed 38 patients initially suspected of carrying MF or osteomyelofibrosis (OMF). After trepanobiopsy, 20 out of 38 patients were confirmed as myelofibrotic (52.6%), 5/38 (13.2%) were diagnosed as ET, 1/38 (2.6%) was diagnosed as myeloproliferative neoplasm (MPN), 6/38 (15.8%) had polycythemia vera (PV). In six patients, the presence of disease was rejected. Patients with MF were divided into three groups according to the JAK2 V617F genotype status: homozygous for the mutation (3/20 or 15.0%), heterozygous (9/20 or 45.0%) and homozygous for the wild type allele (8/20 or 40.0%). The triggering factor of MF is still unknown. It was considered that this factor could have a genetic nature. Mutations in three genes were mainly accepted as an actual predisposing events to this disease: point mutations leading to amino acid substitutions in JAK2 (V617F) and in MPL (W515L, W515K), as well as insertion or deletion in CALK We have proven that carriers of the V617F mutation prevailed in the group of patients with MF (altogether 12 patients or 60.0%). Previous studies also showed that JAK2 V617F is present in more than half of MF patients within their blood-forming cells. Therefore, the risk of evolution to MF could be associated with V617F-mutant allele burden in patients with MPN.

Introduction: Azacitidine is one of the hypomethylating agents available for the treatment of elderly patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). It is also used as an appropriate treatment of chronic myelomonocytic leukemia (CMML) in the real life setting. As treatment of AML and CMML is not curative, and allogeneic stem cell transplantation (allo-SCT) remains traditionally the only option, significant clinical benefits by hypomethylating agents have been reported. According to the available data, 16% of subjects with MDS who received azacitidine had a complete or partial normalization of blood cell counts and bone marrow morphology, while two-thirds of patients who required blood transfusions no longer needed them. Nevertheless, it can also be hepatotoxic in patients with severe liver impairment and extensive liver tumors. Aim: to summarize the effect of azacitidine treatment in patients in the light of their general condition, blood count parameters, toxicity (general and hematologic), as well as the presence of cytogenetic aberrations. Materials and Methods: Twenty-seven patients of which 15 patients with MDS, 9 patients with CMML and 3 patients with AML received azacitidine treatment. The blood count levels and toxicity were followed for a period of twelve months. Results: 22.2% of our patients (6 of 27) of different hematologic diagnoses showed genetic aberrations in their DNA. All they showed quick disease progression and fatal outcome, four of them also developed hematologic toxicity. The remaining 77.8% had no cytogenetic findings. Of all the cohort, 19.05% developed toxicity during the course of the treatment, 38% – decreased leucocyte levels, 14.3% – decreased thrombocyte levels and 18.2% – decreased hemoglobin level. The erythrocyte levels were not substantially influenced by the treatment. The majority of the patients sustained stable levels of red blood cells, as well as of platelets and hemoglobin without remarkable changes between month 0 and month 6 of the treatment. Conclusion: Our results showed, that the main disadvantage of azacitidine treatment in our patients were progressive leucopenia (in 10/27 patients or 37% of cases) and toxicity (8/27 or 29.6% of cases).

Targeted therapy with tyrosine kinase inhibitors (TKIs) was introduced for the suppression of BCR-ABL fusion protein in chronic myelogenic leukemia (CML) more than 50 years ago. The introduction of TKI was an elegant way to gain control over the disease and opened a horizon to the development of new and safer drugs to better manage the disease progression in CML patients. Although Imatinib (the first TKI) was highly effective for the treatment of CML, the lack of response in some patients and the development of resistance led to the introduction of a second-generation TKI, like Nilotinib (Tasigna®). This study aimed to show the molecular response to Nilotinib after a long-term monitoring of CML patients. We analyzed the molecular response rate of twenty-seven CML patients admitted to the Hematology Ward. All of them were subjected to multiple control measurements of Philadelphia chromosome once in at least three months’ period between January 2017 and March 2020. All patients showed a remarkable decrease in the level of the fusion gene’s expression. The highest drop in the expression level was detected after the first dose admission. Some of the patients even reached an expression of BCR-ABL below the detection limit and it was maintained stably low during further examinations. In our patients with CML, Nilotinib resulted in prolonged and deep molecular response. The expression level of BCR-ABL fusion gene showed a substantial decrease immediately after Nilotinib treatment, revealed as early as the third month of therapy.