NFKBIE mutations are selected by the tumor microenvironment and contribute to immune escape in chronic lymphocytic leukemia.

Loss-of-function mutations in NFKBIE, which encodes for the NF-κB inhibitor IκBε, are frequent in chronic lymphocytic leukemia (CLL) and certain other B-cell malignancies and have been associated with accelerated disease progression and inferior responses to chemotherapy. Using in vitro and in vivo murine models and primary patient samples, we now show that NFKBIE-mutated CLL cells are selected by microenvironmental signals that activate the NF-κB pathway and induce alterations within the tumor microenvironment that can allow for immune escape, including expansion of CD8+ T-cells with an exhausted phenotype and increased PD-L1 expression on the malignant B-cells. Consistent with the latter observations, we find increased expression of exhaustion markers on T-cells from patients with NFKBIE-mutated CLL. In addition, we show that NFKBIE-mutated murine CLL cells display selective resistance to ibrutinib and report inferior outcomes to ibrutinib treatment in NFKBIE-mutated CLL patients. These findings suggest that NFKBIE mutations can contribute to CLL progression through multiple mechanisms, including a bidirectional crosstalk with the microenvironment and reduced sensitivity to BTK inhibitor treatment.

Proteomics Profiling of Bladder Cancer Tissues from Early to Advanced Stages Reveals NNMT and GALK1 as Biomarkers for Early Detection and Prognosis of BCa.

The high recurrence rate and invasive diagnostic and monitoring methods in bladder cancer (BCa) clinical management require the development of new non-invasive molecular tools for early detection, particularly for low-grade and low-stage BCa as well as for risk stratification. By using an in-solution digestion method and label-free data-independent LC-MS/MS coupled with ion mobility, we profiled the BCa tissues from initiation to advanced stages and confidently identified and quantified 1619 proteins (≥2 peptides). A statistically significant difference in abundance (Anova ≤ 0.05) showed 494 proteins. Significant correlation with stage with steady up or down with BCa stages showed 15 proteins. Testing of NNMT, GALK1, and HTRA1 in urine samples showed excellent diagnostic potential for NNMT and GALK1 with AUC of 1.000 (95% CI: 1.000-1.000; p < 0.0001) and 0.801 (95% CI: 0.655-0.947; p < 0.0001), respectively. NNMT and GALK1 also showed very good potential in discriminating non-invasive low-grade from invasive high-grade BCa with AUC of 0.763 (95% CI: 0.606-0.921; p = 0.001) and 0.801 (95% CI: 0.653-0.950; p < 0.0001), respectively. The combination of NNMT and GALK1 increased prognostic accuracy (AUC = 0.813). Our results broaden the range of potential novel candidates for non-invasive BCa diagnosis and prognosis.

Rosuvastatin effects on the HDL proteome in hyperlipidemic patients.

The advancements in proteomics have provided a better understanding of the functionality of apolipoproteins and lipoprotein-associated proteins, with the HDL lipoprotein fraction being the most studied. The focus of this study was to evaluate the HDL proteome in dyslipidemic subjects without an established cardiovascular disease, as well as to test whether rosuvastatin treatment alters the HDL proteome. Patients with primary hypercholesterolemia or mixed dyslipidemia were assigned to 20 mg/day rosuvastatin and blood samples were drawn at study entry and after 12 weeks of treatment. A label-free LC-MS/MS protein profiling was conducted, coupled with bioinformatics analysis. Sixty-nine HDL proteins were identified, belonging to four main biological function clusters: lipid transport and metabolism; platelet activation, degranulation, and aggregation, wound response and wound healing; immune response; inflammatory and acute phase response. Five HDL proteins showed statistically significant differences in the abundance (Anova ≤ 0.05), before and after rosuvastatin treatment. Platelet factor 4 variant (PF4V1), Pregnancy-specific beta-1-glycoprotein 2 (PSG2), Profilin-1 (PFN1) and Keratin type II cytoskeletal 2 epidermal (KRT2) showed decreased expressions, while Integrin alpha-IIb (ITGA2B) showed an increased expression after treatment with rosuvastatin. The ELISA validation of PFN1 segregated the subjects into responders and non-responders, as PFN1 levels after rosuvastatin were shown to mostly depend on the subjects' inflammatory phenotype. Findings from this study introduce novel insights into the HDL proteome and statin pleiotropism.

Circulating Variants of SARS-CoV-2 Among Macedonian COVID - 19 Patients in the First Year of Pandemic.

Genomic epidemiology has proven to be a useful tool for investigating pandemic outbreaks and tracking pathogen spread and evolution. This study describes the circulation of SARS-CoV-2 strains in N. Macedonia during a period of one year, encompassing three waves of the COVID-19 pandemic. A certain percentage (2-3%) of positive cases were continuously selected and analyzed by whole genome sequencing (WGS) technology. Using this approach, a total of 337 SARS-CoV-2 genomes were sequenced and 26 different lineages belonging to 7 clades were detected. During the first wave of the pandemic, the most dominant lineage was B.1.1, followed by B.1.1.70, which became the most dominant in the second wave. The B.1.1.7 lineage completely overpassed all other variants in the third wave. Our study strengthens the notion that the progression of COVID-19 pandemic is associated with emergence of new SARS-CoV-2 variants with increased virulence. The measure of the impact of this viral dynamic on the spread of the pandemic should be evaluated in association with other factors that might influence the transmission.

Rhabdomyolysis Associated with Recent SARS-COV-2 Infection in a Patient with Carnitine Palmitoyltransferase II Deficiency.

Carnitine palmitoyltransferase II deficiency (CPT II) is an autosomal recessive inherited disorder of long-chain fatty acid oxidation in the mitochondrial matrix, resulting in an inability to utilize fat for energy in cells. The most frequent myopathic form occurs in young adults and is associated with recurrent episodes of exercise-induced rhabdomyolysis. The myopathic form is caused by the Ser113Leu mutation of the CPT II gene. Rarely, massive rhabdomyolysis could be complicated by acute kidney injury (AKI), cardiomyopathy, and respiratory insufficiency. We present a case of an 18-year old male with myalgia, muscular weakness, and dark-colored urine after prolonged exercise and a recent mildSARS-CoV-2infection. Massive rhabdomyolysis was diagnosed with markedly increased serum concentrations of myoglobin and creatine kinase, with normal kidney function. The patient experienced two similar episodes in the years 2017 and 2018, with rhabdomyolysis and AKI treated with hemodialysis. After excluding autoimmune and infectious diseases as causes of recurrent rhabdomyolysis, the patient was genetically tested and Ser113Leu mutation of the CPT II gene was confirmed. When a patient presents with myalgia and dark-colored urine triggered by minor physical activities, genetic testing for possible CPT II deficiency should be initiated. TheSARS-CoV-2infection could be a factor that triggers the occurrence of rhabdomyolysis and aggravates the severity of the attack in patients with CPT II deficiency.

Potential Role of Seven Proteomics Tissue Biomarkers for Diagnosis and Prognosis of Prostate Cancer in Urine.

As the currently available tests for the clinical management of prostate cancer (PCa) are still far from providing precise diagnosis and risk stratification, the identification of new molecular marker(s) remains a pertinent clinical need. Candidate PCa biomarkers from the published proteomic comparative studies of prostate tissue (2002-2020) were collected and systematically evaluated. AZGP1, MDH2, FABP5, ENO1, GSTP1, GSTM2, and EZR were chosen for further evaluation in the urine of 85 PCa patients and controls using ELISA. Statistically significant differences in protein levels between PCa and BPH showed FABP5 (p = 0.019) and ENO1 (p = 0.015). A biomarker panel based on the combination of FABP5, ENO1, and PSA provided the highest accuracy (AUC = 0.795) for PCa detection. The combination of FABP5, EZR, AZGP1, and MDH2 showed AUC = 0.889 in PCa prognosis, with 85.29% of the samples correctly classified into low and high Gleason score (GS) groups. The addition of PSA to the panel slightly increased the AUC to 0.914. AZGP1, FABP5, and EZR showed significant correlation with GS, stage, and percentage of positive biopsy cores. Although validation using larger patient cohorts will be necessary to establish the credibility of the proposed biomarker panels in a clinical context, this study opens a way for the further testing of more high-quality proteomics biomarkers, which could ultimately add value to the clinical management of PCa.

Macrophage- and BCR-derived but not TLR-derived signals support the growth of CLL and Richter syndrome murine models in vivo.

A large amount of circumstantial evidence has accumulated suggesting that Toll-like receptor (TLR) signals are involved in driving chronic lymphocytic leukemia (CLL) cell proliferation, but direct in vivo evidence for this is still lacking. We have now further addressed this possibility by pharmacologically inhibiting or genetically inactivating the TLR pathway in murine CLL and human Richter syndrome (RS) patient-derived xenograft (PDX) cells. Surprisingly, we show that pharmacologic inhibition of TLR signaling by treatment with an IRAK1/4 inhibitor delays the growth of the transplanted malignant cells in recipient mice, but genetic inactivation of the same pathway by CRISPR/Cas9-mediated disruption of IRAK4 or its proximal adaptor MyD88 has no effect. We further show that treatment with the IRAK1/4 inhibitor results in depletion of macrophages and demonstrate that these cells can support the survival and enhance the proliferation of both murine Eµ-TCL1 leukemia and human RS cells. We also show that genetic disruption of the B-cell receptor (BCR) by CRISPR/Cas9 editing of the immunoglobulin M constant region gene inhibits the growth of human RS-PDX cells in vivo, consistent with our previous finding with murine Eµ-TCL1 leukemia cells. Finally, we show that genetic disruption of IRAK4 does not result in negative selection of human CLL cell lines xenografted in immunodeficient mice. The obtained data suggest that TLR signals are unlikely to represent a major driver of CLL/RS cell proliferation and provide further evidence that signals from macrophages and the BCR promote the growth and survival of CLL and RS cells in vivo.

Comparative proteomics analysis of human FFPE testicular tissues reveals new candidate biomarkers for distinction among azoospermia types and subtypes.

Understanding molecular mechanisms that underpin azoospermia and discovery of biomarkers that could enable reliable, non-invasive diagnosis are highly needed. Using label-free data-independent LC-MS/MS acquisition coupled with ion mobility, we compared the FFPE testicular proteome of patients with obstructive (OA) and non-obstructive azoospermia (NOA) subtypes hypospermatogenesis (Hyp) and Sertoli cell-only syndrome (SCO). Out of 2044 proteins identified based on ≥2 peptides, 61 proteins had the power to quantitatively discriminate OA from NOA and 30 to quantitatively discriminate SCO from Hyp and OA. Among these, H1-6, RANBP1 and TKTL2 showed superior potential for quantitative discrimination among OA, Hyp and SCO. Integrin signaling pathway, adherens junction, planar cell polarity/convergent extension pathway and Dectin-1 mediated noncanonical NF-kB signaling were significantly associated with the proteins that could discriminate OA from NOA. Comparison with 2 transcriptome datasets revealed 278 and 55 co-differentially expressed proteins/genes with statistically significant positive correlation. Gene expression analysis by qPCR of 6 genes (H1-6, RANBP1, TKTL2, TKTL1, H2BC1, and ACTL7B) with the highest discriminatory power on protein level and the same regulation trend with transcriptomic datasets, confirmed the proteomics results. In summary, our results suggest some underlying pathways in azoospermia and broaden the range of potential novel candidates for diagnosis. SIGNIFICANCE: Using a comparative proteomics approach on testicular tissue we have identified several pathways associated with azoospermia and a number of testis-specific and germ cell-specific proteins that have the potential to pinpoint the type of spermatogenesis failure. Furthermore, comparison with transcriptomics datasets based on genome-wide gene expression analyses of human testis specimens from azoospermia patients identified proteins that could discriminate between obstructive and non-obstructive azoospermia subtypes on both protein and mRNA levels. Up to our knowledge, this is the first integrated comparative analysis of proteomics and transcriptomics data from testicular tissues. We believe that the data from our study contributes significantly to increase the knowledge of molecular mechanisms of azoospermia and pave the way for new investigations in regards to non-invasive diagnosis.

Antioxidative Effects of Rosuvastatin in Low-to-Moderate Cardiovascular Risk Subjects.

Background: Although vast clinical evidence supports the oxidative CVD hypothesis, little is known on the effects of statins on LDL/HDL oxidative functionality. Therefore, the aim of this study was to evaluate the antioxidative effects of rosuvastatin by monitoring the susceptibility of LDL to oxidation and the antioxidative HDL potential in low-to-moderate CV risk subjects. Methods: 40 adult ambulatory patients (aged 53.8±10.9 years, 27 women and 13 men) were included in the study. Data was collected from patients' records, physical examination, and blood sampling. Subjects were prescribed rosuvastatin at 20mg/day. Traditional risk-factors/indicators, lipid parameters, inflammatory/immune markers, LDL susceptibility to oxidation and HDL antioxidative potential were monitored and statistically analyzed with t-test, Chi-square test, one-way ANOVA, Mann-Whitney, and Kruskal-Wallis tests. Multivariate logistic regression analyses were made. Results were considered significant when p≤0.05. Results: 67% of the patients showed lower susceptibility of LDL to oxidation after rosuvastatin treatment (p=0.03), with no significant effect on baseline LDL oxidation and lag time. All three LDL oxidative indices were seen to be dependent on the subjects' lipid profile, hemoglobin levels and the IL-1α and IL-8 pro-inflammatory marker levels. 53% of the patients showed higher HDL antioxidative capacity after treatment, but without statistical significance (p=0.07). Increased antioxidative potential of HDL with rosuvastatin treatment was more likely in males (OR=9.350; p=0.010), and subjects achieving lower post-treatment CV relative risk levels (higher CV risk reduction) (OR=0.338; p=0.027). Conclusions: This study suggests the need of a comprehensive approach when investigating oxidative stress and LDL/HDL functions, especially in low-to-moderate CVD risk subjects.

Germline MBD4 deficiency causes a multi-tumor predisposition syndrome.

We report an autosomal recessive, multi-organ tumor predisposition syndrome, caused by bi-allelic loss-of-function germline variants in the base excision repair (BER) gene MBD4. We identified five individuals with bi-allelic MBD4 variants within four families and these individuals had a personal and/or family history of adenomatous colorectal polyposis, acute myeloid leukemia, and uveal melanoma. MBD4 encodes a glycosylase involved in repair of G:T mismatches resulting from deamination of 5'-methylcytosine. The colorectal adenomas from MBD4-deficient individuals showed a mutator phenotype attributable to mutational signature SBS1, consistent with the function of MBD4. MBD4-deficient polyps harbored somatic mutations in similar driver genes to sporadic colorectal tumors, although AMER1 mutations were more common and KRAS mutations less frequent. Our findings expand the role of BER deficiencies in tumor predisposition. Inclusion of MBD4 in genetic testing for polyposis and multi-tumor phenotypes is warranted to improve disease management.

Anti-inflammatory and immunomodulatory effects of rosuvastatin in patients with low-to-moderate cardiovascular risk.

Statins have shown anti-inflammatory pleiotropic effects in subjects with/at risk of cardiovascular disease. The aim of this study was to evaluate the inflammatory/immunomodulatory properties of rosuvastatin in subjects at low-to-moderate cardiovascular risk. Data was collected from patients' records, physical examination and blood sampling. Subjects were assigned to rosuvastatin 20 mg per day. Rosuvastatin significantly decreased C-reactive protein (p = 0.045), and increased vascular endothelial growth factor (p = 0.004) and epidermal growth factor (p = 0.009). A multivariate analysis identified total cholesterol (p = 0.027) and vascular endothelial growth factor (p = 0.011) to be independently associated with rosuvastatin treatment. Given beneficial/harmful role of growth factors, vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), in cardiovascular disease, one would suggest the need for routine monitoring of growth factor levels, especially in patients on long-term statin therapy.

B-cell receptor signaling and genetic lesions in TP53 and CDKN2A/CDKN2B cooperate in Richter transformation.

B-cell receptor (BCR) signals play a critical role in the pathogenesis of chronic lymphocytic leukemia (CLL), but their role in regulating CLL cell proliferation has still not been firmly established. Unlike normal B cells, CLL cells do not proliferate in vitro upon engagement of the BCR, suggesting that CLL cell proliferation is regulated by other signals from the microenvironment, such as those provided by Toll-like receptors or T cells. Here, we report that BCR engagement of human and murine CLL cells induces several positive regulators of the cell cycle, but simultaneously induces the negative regulators CDKN1A, CDKN2A, and CDKN2B, which block cell-cycle progression. We further show that introduction of genetic lesions that downregulate these cell-cycle inhibitors, such as inactivating lesions in CDKN2A, CDKN2B, and the CDKN1A regulator TP53, leads to more aggressive disease in a murine in vivo CLL model and spontaneous proliferation in vitro that is BCR dependent but independent of costimulatory signals. Importantly, inactivating lesions in CDKN2A, CDKN2B, and TP53 frequently co-occur in Richter syndrome (RS), and BCR stimulation of human RS cells with such lesions is sufficient to induce proliferation. We also show that tumor cells with combined TP53 and CDKN2A/2B abnormalities remain sensitive to BCR-inhibitor treatment and are synergistically sensitive to the combination of a BCR and cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor both in vitro and in vivo. These data provide evidence that BCR signals are directly involved in driving CLL cell proliferation and reveal a novel mechanism of Richter transformation.

Insight in the Current Progress in the Largest Clinical Trials for Covid-19 Drug Management (As of January 2021).

The outbreak of the COVID-19 pandemic has generated the largest global health crisis of the 21st century, evolving into accelerating socioeconomic disruption. In spite of all rapidly and widely emerging scientific data on epidemiology, diagnosis, prevention and treatment of the COVID-19 disease, severe acute respiratory coronavirus 2 (SARS-CoV-2) is continuing to propagate in lack of definitive and specific therapeutic agents. Current therapeutic strategies are mainly focused on viral inhibition by antiviral drugs and hampering the exuberant immune response of the host by immunomodulatory drugs. In this review, we have studied the reports of the largest clinical trials intended to COVID-19 treatment published during the first year of the pandemics. In general, these results concentrate on seven therapeutic options: remdesivir, chloroguine/hydroxychloroquine, lopinavir-ritonavir combination, corticosteroids, tocilizumab, convalescent plasma and monoclonal antibodies. In line with the reviewed data, as of January 2021, most of the evidence support the use of remdesivir in hospitalized patients with moderate and severe forms of the disease and provide reliable data on the substantial beneficial effect of corticosteroids in patients requiring supplemental oxygen. Moreover, preliminary RECOVERY trial results have demonstrated the efficacy of tociluzumab in the treatment of critically ill patients. The reports presenting the outcomes of the other immune-based therapies under investigation are enthusiastically awaited.

Comparative evaluation of two methods for LC-MS/MS proteomic analysis of formalin fixed and paraffin embedded tissues.

The proteomics of formalin-fixed, paraffin-embedded (FFPE) samples has advanced significantly during the last two decades, but there are many protocols and few studies comparing them directly. There is no consensus on the most effective protocol for shotgun proteomic analysis. We compared the in-solution digestion with RapiGest and Filter Aided Sample Preparation (FASP) of FFPE prostate tissues stored 7 years and mirroring fresh frozen samples, using two label-free data-independent LC-MS/MS acquisitions. RapiGest identified more proteins than FASP, with almost identical numbers of proteins from fresh and FFPE tissues and 69% overlap, good preservation of high-MW proteins, no bias regarding isoelectric point, and greater technical reproducibility. On the other hand, FASP yielded 20% fewer protein identifications in FFPE than in fresh tissue, with 64-69% overlap, depletion of proteins >70 kDa, lower efficiency in acidic and neutral range, and lower technical reproducibility. Both protocols showed highly similar subcellular compartments distribution, highly similar percentages of extracted unique peptides from FFPE and fresh tissues and high positive correlation between the absolute quantitation values of fresh and FFPE proteins. In conclusion, RapiGest extraction of FFPE tissues delivers a proteome that closely resembles the fresh frozen proteome and should be preferred over FASP in biomarker and quantification studies. SIGNIFICANCE: Here we analyzed the performance of two sample preparation methods for shotgun proteomic analysis of FFPE tissues to give a comprehensive overview of the obtained proteomes and the resemblance to its matching fresh frozen counterparts. These findings give us better understanding towards competent proteomics analysis of FFPE tissues. It is hoped that it will encourage further assessments of available protocols before establishing the most effective protocol for shotgun proteomic FFPE tissue analysis.

Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed "satellites," were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

Clinical Characteristics and Treatment Results of Childhood Acute Lymphoblastic Leukemia in North Macedonia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. This study was designed to determine the clinical, biological features and outcomes among children with ALL treated at the only pediatric hematology-oncology center in North Macedonia. PATIENTS AND METHODS: Seventy four consecutive children age 1 to 14 years, diagnosed with ALL between January 1, 2010 and October 31, 2017 and treated according to ALL IC BFM 2002 protocol were retrospectively evaluated. RESULTS: The median age at diagnosis was 5 years and males were predominant (60.8%). Precursor B-cell ALL was diagnosed in 81.1% of patients, while 18.9% had T cell ALL. CNS involvement at the time of diagnoses was present in 6.8% of patients. Complete remission was achieved in 93.2% of patients. The induction death rate was 5.4%. The rate of death during first complete remission was 4.1%. Relapse occurred in 13.5% of patients. After a median observation time of 44 months, the 5-year overall survival (OS) and event-free survival (EFS) rates (± standard error) were 79.4% ± 5.2% and 74% ± 5.7%, respectively. The 5-year EFS rate for patients categorized as standard risk by NCI criteria was significantly higher than for high risk patients (83.3% versus 46.7%; P<0.001). Patients with precursor B-cell ALL and negative minimal residual disease (MRD) status at the end of induction had the best prognoses. CONCLUSION: Our study demonstrated that the treatment results of childhood ALL in North Macedonia are comparable to those obtained in the ALL IC BFM 2002 trial.

The use of remdesivir outside of clinical trials during the COVID-19 pandemic.

With a scientific background from filoviruses, paramyxoviruses, SARS-CoV, and MERS-CoV, remdesivir entered into the COVID-19 battle to become one of the favorable therapeutic candidates with potential antiviral activity in the treatment of this disease. Globally, remdesivir was accessed and investigated through clinical research (clinical trials) and clinical practice (compassionate use, expanded access, early access scheme, and emergency use). Currently, remdesivir approval status differs between states. This paper aims to review and analyze regulatory approaches for accessing and investigating remdesivir, by communicating regulatory variability between countries in terms of terminology, modalities, and protocols.

Detection of TMPRSS2-ERG Fusion Transcript in Biopsy Specimen of Prostate Cancer Patients: A Single Centre Experience.

INTRODUCTION: Prostate carcinoma is the most frequent malign neoplasm among men with an ever-growing incidence rate. TMPRSS2-ERG fusion transcript leads to the androgen induction of ERG proto-oncogenes expression, representing a high presence of oncogenes alteration among prostate tumour cells. AIM: The aim of this research was to detect and evaluate theTMPRSS2-ERG fuse transcript in the tissues of patients with prostate cancer, and establish a base of material of these samples for further genetic examination. MATERIALS AND METHODS: The research was a prospective clinical study that involved and focused on random sampling of 101 patients (62 with prostate cancer-study group and 39 with benign changes in the prostate-control group). Real time PCR analysis for detection of the TMPRSS2-ERG fusion transcript in prostate tissue was performed and also data from the histopathology results of tissues were used, as well as data for the level of PSA (prostate-specific antigen) in blood. RESULTS: TMPRSS2-ERG fusion transcript was detected in 20 out of 62 (32.2%) patients with prostate carcinoma and among no patients with benign changes whatsoever. There were no significant differences between patients with/without detected TMPRSS2-ERG fusion related to Gleason score. Among 50%, in the study group this score was greater than 7 per/for Median IQR=7 (6-8). Significant difference was recognized, related to the average value of PSA in favour of significantly higher value of PSA in the study group with prostate cancer, but there was also no significant difference between samples with prostate cancer who were with/without detected TMPRSS2-ERG fusion transcript related to PSA level. DISCUSSION: The results from this research are in accordance with the values and results from analyses done in several research centres and oncological institutes. CONCLUSION: The positive findings in small scale studies encourage the implementation of larger scale studies that will be enriched with results of genetic transcript in blood and urine and will define the positive diagnostic meaning of the TMPRSS-ERG fusion transcript.

Molecular Monitoring in Acute Myeloid Leukemia Patients Undergoing Matched Unrelated Donor - Hematopoietic Stem Cell Transplantation: Single Center Experience.

INTRODUCTION: Minimal residual disease (MRD) assessment in acute myeloid leukemia (AML) cases is a complex, multi-modality process and, though much of its clinical implications at different points are extensively studied, it remains even now a challenging area. It is a disease the biology of which governs the modality of MRD assessment; in patients harboring specific molecular targets, high sensitivity techniques can be applied. On the other hand, relapse is considered as the leading cause of treatment failure in AML patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). MATERIALS AND METHODS: Since November 2018 until June 2020, 10 AML patients underwent matched unrelated donor (MUD) HSCT at the University Clinic of Hematology-Skopje, Republic of North Macedonia. Molecular markers were identified in a total of 4 patients; 3 patients expressed chimeric fusion transcripts; two RUNX-RUNX1T1 and one for CBFB-MYH11. One patient harbored mutation in the transcription factor CCAAT/enhancer binding protein α (CEBPA). Post-transplant MRD kinetics was evaluated by using quantitative polymerase chain reaction (RT-qPCR) or multiplex fluorescent-PCR every three months during the first two years after the transplantation. RESULTS: MRD negativity was achieved in three pre-transplant MRD positive patients by the sixth month of HSCT. They sustained hematological and molecular remission for 19, 9 and 7 months, respectively. The fourth patient died due to transplant-related complications. CONCLUSION: According to our experience, when molecularly-defined AML patients undergo HSCT, regular MRD monitoring helps predict impending relapse and direct future treatment strategies.

Clinical Significance of Minimal Residual Disease at the End of Remission Induction Therapy in Childhood Acute Lymphoblastic Leukemia.

BACKGROUND: Detection of minimal residual disease (MRD) in the early phase of therapy is the most powerful predictor of relapse risk in children with acute lymphoblastic leukaemia (ALL). AIM: We aimed to determine the significance of MRD at the end of remission induction therapy in the prediction of treatment outcome in children with ALL. METHODS: Sixty-four consecutive patients aged 1-14 years with newly diagnosed ALL were enrolled in this study from January 2010 to October 2017. All patients were treated according to the ALL IC BFM 2002 protocol. MRD was detected at the end of remission induction therapy (day 33) by multiparameter 6-colour flow cytometry performed on bone marrow specimens with a sensitivity of 0.01%. RESULTS: Overall, 42.2% of patients had detectable MRD on day 33 of therapy. MRD measurements were not significantly related to presenting characteristics but were associated with a poorer blast clearance on day 8 and 15 of remission induction therapy. Patients with negative MRD status on day 33 had a 5-year event-free survival of 94.6% compared with 76.1% for those with positive MRD status (P = 0.044). CONCLUSION: MRD levels at the end of remission induction therapy measured by multiparameter flow cytometry have clinical significance in childhood ALL. High levels of MRD are strongly related to poor treatment outcome.