Mindaugas Stoskus

Single Nucleotide Polymorphism-Based System Improves the Applicability of Quantitative PCR for Chimerism Monitoring

Recently, several studies demonstrated the feasibility of a real-time quantitative PCR (qPCR) approach for chimerism monitoring. qPCR offers a fast, sensitive, and elegant quantification of genotypes. However, before it becomes an established method for routine chimerism monitoring, a qPCR marker set for every transplant pair should be available. This requirement poses a major challenge since the genetic markers for qPCR--short insertions/deletions (Indels) and single nucleotide polymorphisms (SNPs)--published to-date do not guarantee applicability for every transplant pair. The aim of our study was to design and validate a new SNP allele-specific system to supplement an already existing Indel primer panel and improve applicability of the qPCR approach for chimerism status monitoring. Here, we present an approach for an economical in-house design of SNP allele-specific qPCR primers/probe sets with a locus-individualized reference system that allows for the accurate quantification of the respective informative locus using a simple DeltaDeltaCt method. We designed primers/probe sets specific for seven biallelic SNP loci and validated them in a population of 30 transplant pairs. Repeatability varied depending on the amount of quantifiable genotype. The combination of our SNP-qPCR system and Indel primers increased recipient genotype identification from 86.6% to 96.6% when tested in a population of our transplant pairs. These results demonstrate the feasibility of our SNP-based qPCR approach to improve the applicability of a qPCR for chimerism monitoring.

Recent advances in quantitative chimerism analysis

Quantitative chimerism analysis is a diagnostic tool used to monitor engraftment kinetics after allogeneic stem cell transplantation. It reflects the proportion of recipient and donor genotypes and is based on the identification of genetic markers characteristic to a given transplant pair. Currently, PCR amplification of short tandem repeats and single-nucleotide polymorphism-specific quantitative real-time PCR are the most widely used techniques for this purpose. In this review, we will address advances as well as technology-specific imperfections, of both techniques that have emerged over the recent years. We will discuss new principles that may simplify assay design, and improve its robustness and reliability. A better chimerism assay could then guide clinical interventions and may, eventually, improve the outcome of allogeneic stem cell transplantation.

Identification of characteristic IGF2BP expression patterns in distinct B-ALL entities.

Insulin-like growth factor 2 mRNA-binding proteins IGF2BP1, IGF2BP2, and IGF2BP3 have been shown to have diagnostic and prognostic utility in a number of epithelial and soft tissue tumors. Still, little is known about the expression of these molecules in different types of leukemia and our study aims to fill this gap. By using an RT-qPCR approach, we have systemically analyzed the expression of three IGF2BP coding genes in normal hematopoietic tissues and distinct acute lymphoblastic leukemia (ALL) entities. We show that low/negative IGF2BP1 and IGF2BP3 and high IGF2BP2 levels are characteristic to healthy donor bone marrow and peripheral blood whereas different B-ALL entities displayed characteristic perturbations of IGF2BP expression patterns. Namely, we have identified significant associations of overexpressed IGF2BP1 with ETV6/RUNX1-positive (r(2)=0.7891, y=0.8105x-0.4471, p<0.0001), underexpressed IGF2BP2 with E2A/PBX1-positive (p<0.01), and overexpressed IGF2BP2 and IGF2BP3 with MLL/AF4-positive (r(2)=0.6571, y=0.1507x-0.2722, p<0.0001, and r(2)=0.7022, y=0.6482x-0.7660, p<0.0001, respectively) leukemia. Secondly, based on transcript expression dynamics during follow-up, we conclude that overexpression of only IGF2BP1 is inherent characteristic of ETV6/RUNX1-positive leukemic blasts in contrast to IGF2BP3 which remained stably expressed throughout the monitoring period and upon the achievement of molecular remission. Finally, our data suggest that IGF2BP3 might be a marker of disease aggressiveness in BCR/ABL1-positive ALL as consistently increasing levels of this transcript during follow-up predicted eventual leukemia relapse by three months. Altogether, our results highlight the potential utility of IGF2BP profiling in precursor B lymphoid neoplasms as the functions of IGF2BPs in normal and malignant hematopoiesis are further delineated.

Cure rates of childhood acute lymphoblastic leukemia in Lithuania and the benefit of joining international treatment protocol

BACKGROUND Childhood acute lymphoblastic leukemia (ALL) represents the largest group of pediatric malignancies with long-term survival rates of more than 80% achieved in developed countries. Epidemiological data and survival rates of childhood ALL in Lithuania were lacking. Therefore, the aim of this study was to analyze the population-based long-term treatment results of childhood ALL in Lithuania during 1992-2012. MATERIALS AND METHODS Data of all 459 children with T-lineage and B-cell precursor ALL treated in Lithuania from 1992 to 2012 were collected and analyzed. Results were compared among four time-periods: 1992-1996 (N=132), 1997-2002 (N=136), 2003-2008 (N=109) and 2009-2012 (N=82). RESULTS The incidence of childhood ALL in Lithuania was 3.2-3.6 cases per 100000 children per year during the study period. Five-year probability of event-free survival increased from 50%± 4% in 1992-1996 to 71%± 4% in 2003-2008 (P<0.001). Five-year cumulative incidence of relapses reduced from 27%± 4.5% in 1992-1996 to 14%± 3.6% in 2003-2008 (P=0.042). After introduction of high-dose methotrexate of 5 g/m(2), cumulative incidence of CNS-involving relapses reduced from 17%± 3.9% in 1992-1996 to 1%± 1.0% in 2003-2008 (P<0.001). Trend for further improvement in survival was seen in 2009-2012 when Lithuania joined international the Nordic Society of Pediatric Hematology and Oncology (NOPHO) ALL-2008 treatment protocol. CONCLUSIONS Cure rates of childhood ALL in Lithuania are improving steadily and are now approaching those reported by the largest international study groups. The reasons for such a positive effect are both better financial support for treatment of children with cancer in Lithuania and international collaboration with joining international treatment protocol for childhood ALL.

ETV6/RUNX1 transcript is a target of RNA-binding protein IGF2BP1 in t(12;21)(p13;q22)-positive acute lymphoblastic leukemia.

The oncofetal RNA-binding protein IGF2BP1 (IGF2 mRNA binding protein 1) is overexpressed in a subset of cancers and promotes cell cycle, migration and aggressive phenotype by regulating post-transcriptionally a number of key mRNAs (e. g, ACTB, CD44, CTNNB1, KRAS, MAPK4, MYC, PTEN and others). IGF2BP1 is also overexpressed in t(12;21)(p13;q22)-positive acute lymphoblastic leukemia (ALL), but the biological significance of this phenomenon has not been addressed so far. We have identified leukemia fusion gene ETV6/RUNX1 mRNA to be highly enriched in immunoprecipitated fraction of endogenous IGF2BP1 from a model cell line REH and t(12;21)(p13;q22)-positive ALL samples. Furthermore, downregulation of IGF2BP1 by two-fold has resulted in a corresponding decrease of ETV6/RUNX1 mRNA validating this transcript as a target of IGF2BP1 protein in t(12;21)(p13;q22)-positive ALL. These data infer that IGF2BP1 is a potent regulator of ETV6/RUNX1 mRNA stability and potentially link this evolutionary-highly conserved protein to cell transformation events in ETV6/RUNX1-mediated leukemogenesis of t(12;21)(p13;q22)-positive ALL.

Defining the significance of IGF2BP1 overexpression in t(12;21)(p13;q22)-positive leukemia REH cells

The IGF2 mRNA binding protein 1 (IGF2BP1) belongs to a family of regulatory RNA-binding proteins and controls stability, transport or translation of its target transcripts. Re-expression of IGF2BP1 is frequently found in different tumors and has been associated with aggressive disease phenotypes. IGF2BP1 has also been identified to be exclusively specific for t(12;21)(p13;q22)-positive acute lymphoblastic leukemia (ALL) but biological significance of IGF2BP1 overexpression has not been investigated to date. We have recently reported that ETV6/RUNX1 transcript is a target of RNA-binding protein IGF2BP1 in t(12;21)(p13;q22)-positive ALL suggesting a direct role of IGF2BP1 in ETV6/RUNX1-mediated leukemogenesis. To address this question we have employed stable clones of REH cells - a model cell line of t(12;21)(p13;q22)-positive ALL - with downregulated IGF2BP1 expression. Here we show that downregulation of IGF2BP1 impairs proliferation by attenuating cell cycle progression and increasing the rate of spontaneous cell death. We also provide evidence that downregulation of IGF2BP1 induce reduction of STAT3 mRNA levels and augments sensitivity to STAT3 selective inhibitor S3I-201. These data imply that IGF2BP1 indirectly potentiates ETV6/RUNX1-RAC1-STAT3 signaling axis by sustaining appropriate ETV6/RUNX1 and STAT3 transcript levels in REH cells. Further studies are warranted to specify the role of IGF2BP1 in t(12;21)(p13;q22)-positive ALL.

The value of the repeated examination of BRAF V600E mutation status in diagnostics of papillary thyroid cancer.

INTRODUCTION Nodular thyroid disease is one of the most frequently diagnosed pathologies of the adult population in iodine-deficient regions. Approximately 30% of thyroid aspirates are classified as nondiagnostic/unsatisfactory or indeterminate. However, patients with indeterminate cytology still undergo surgery. The object of this study was to determine the diagnostic value of re-examining the BRAF V600E mutation in papillary thyroid carcinoma patients. MATERIAL AND METHODS All patients underwent ultrasound guided fine-needle aspiration of a thyroid nodule. They were assigned to one of the four groups (indeterminate or positive for malignant cells) of the Bethesda System for Reporting Thyroid Cytopathology. Genetic investigation of the BRAF V600E mutation was performed for all of the fine-needle aspiration cytology specimens. All of the patients underwent surgery. Subsequently, histological investigation of the removed tissues was performed. Additional analysis of the BRAF V600E mutation from the histology specimen was then performed for the initially BRAF-negative cases. RESULTS Two hundred and fourteen patients were involved in the study. One hundred and six (49.53%) patients were diagnosed with thyroid cancer. Of these 106 patients, 95 (89.62%) patients were diagnosed with papillary thyroid cancer. The BRAF V600E mutation was positive in 62 (65.26%) and negative in 33 (34.74%) histologically confirmed papillary thyroid cancer cases. After the genetic investigation, a total of 74 (77.89%) papillary thyroid cancer cases were positive for the BRAF V600E mutation and 21 (22.11%) were negative. CONCLUSIONS Repeated examination of the BRAF V600E mutation status in the fine-needle aspiration may potentially increase the sensitivity of papillary thyroid cancer diagnostics.

The utility of the Bethesda category and its association with BRAF mutation in the prediction of papillary thyroid cancer stage

PurposeThis study aims to determine the utility of the Bethesda category and its association with BRAF mutation in prediction of the papillary thyroid cancer (PTC) stage.MethodsA prospective study analyzed patients who had ultrasound-suspicious thyroid nodules, underwent FNA and cytological examination, and were classified according to the Bethesda system. Patients from Undetermined Significance Or Follicular Lesion Of Undetermined Significance (AUS/FLUS), Follicular Neoplasm or Suspicious for a Follicular Neoplasm (FN/SFN), Suspicious for Malignant Cells (SMC), and Positive for Malignant Cells (PMC) groups were examined for the BRAF mutation and had a thyroid surgery. Demographical and histological features and stage of the disease were evaluated for PTC patients in accordance with the Bethesda category and its association with BRAF mutation.ResultsThree hundred eight of all patients underwent operation. One hundred forty-three (46.4%) of them were diagnosed with PTC. In 14 (9.8%) PTC cases, FNA biopsies were classified as AUS/FLUS, 23 (16.1%) as FN/SFN, 41 (28.7%) as SMC, and 65 (45.5%) as PMC. I–II stages of PTC were diagnosed for 88 (61.5%) patients and III–IVA for 55 (38.5%). Patients from the SMC and PMC groups had larger tumors, higher incidence of lymph node metastases, classical PTC type, B-type Raf (BRAF) positive, and III–IVA stage cancer, than patients from the AUS/FLUS and FN/SFN groups. When comparing 27 (18.9%) BRAF-negative patients from the AUS/FLUS and FN/SFN groups with 116 (81.1%) BRAF-negative patients from the SMC and PMC groups and all BRAF-positive patients, the prediction of more aggressive histological features and stage was slightly improved.ConclusionsHigher Bethesda categories are associated with higher stages of PTC. Association of the Bethesda category with BRAF mutation can slightly improve the value of stage prediction.

Therapeutic Plasma Exchange in Multiple Sclerosis Patients with Abolished Interferon-beta Bioavailability

Background Neutralizing antibodies (NAb) to interferon-beta (IFN-β) are associated with reduced bioactivity and efficacy of IFN-β in multiple sclerosis (MS). The myxovirus resistance protein A (MxA) gene expression is one of the most appropriate markers of biological activity of exogenous IFN-β. We hypothesized that therapeutic plasma exchange (TPE) can restore the ability of IFN-β to induce the MxA mRNA expression and that maintenance plasmapheresis can sustain the bioavailability of IFN-β. Material/Methods Eligible patients underwent 4 primary separate plasma exchange sessions. After the induction TPE sessions, they were transferred to maintenance plasmapheresis. Bioactivity of IFN-β was expressed as in vivo MxA mRNA induction in whole blood using RT-qPCR. Results Six patients with low IFN-β bioavailability detected by the MxA mRNA response were included. Four patients became biological responders after induction plasmapheresis. In 2 patients an increase of MxA mRNA expression was found, but the values persisted below the cut-off and the patients remained as “poor biological responders”. The effect of maintenance plasmapheresis was transient: MxA mRNA expression values reverted to the baseline levels after 1–2 months. Conclusions Therapeutic plasma exchange is able to restore the bioavailability of IFN-β in the majority of studied patients, but the effect of TPE on the IFN-β bioavailability was transient.

Significance of BRAF V600E Mutation and Cytomorphological Features for the Optimization of Papillary Thyroid Cancer Diagnostics in Cytologically Indeterminate Thyroid Nodules

BACKGROUND Ultrasound guided fine needle aspiration biopsy with cytologic analysis is an initial step in diagnostic of thyroid nodules. Unfortunately, up to 30% of biopsies are indeterminate and diagnostic surgery is required. The aim of this study was to estimate the diagnostic value of BRAF V600E mutation status combined with cytomorphological features for diagnosis of papillary thyroid cancer (PTC) in cytologically indeterminate thyroid nodules. METHODS A prospective study analyzed patients who had ultrasound suspicious thyroid nodules, underwent fine needle aspiration and cytological examination, and were classified according to the Bethesda system. Patients from indeterminate diagnostic categories were examined for BRAF V600E mutation and 22 cytomorphological features, and underwent thyroid surgery. A binary logistic regression model was used to evaluate the diagnostic utility. RESULTS A total of 219 patients met study criteria. After histological examination, 77 (35.2%) patients were diagnosed with PTC and 142 (64.8%) with benign nodular thyroid disease. According to logistic regression model, significant features for PTC diagnosis were: liquid colloid consistency, papillary structures, eosinophilic colloid bodies, and BRAF V600E mutation. Risk groups classified by this model have sensitivity of 80.5% (95% CI: 69.9 to 88.7), specificity of 99.3% (95% CI: 96.1 to 100), positive predictive value of 98.4% (95% CI: 89.8 to 99.8), negative predictive value of 90.4% (95% CI: 85.7 to 93.7), and accuracy of 92.7% (95% CI: 88.4 to 95.8) for PTC diagnosis. CONCLUSIONS Evaluation of BRAF V600E mutation status combined with cytomorphological features for diagnosis of PTC in cytologically indeterminate thyroid nodules can significantly improve diagnostic accuracy and reduce the number of diagnostic operations (calculator available at www.ptc-calc.we2host.lt).