Relationship between amide ratio assessed by Fourier-transform infrared spectroscopy: A biomarker candidate for polycythemia vera disease.

The study utilized Fourier transform infrared (FTIR) spectroscopy coupled with chemometrics to investigate protein composition and structural changes in the blood serum of patients with polycythemia vera (PV). Principal component analysis (PCA) revealed distinct biochemical properties, highlighting elevated absorbance of phospholipids, amides, and lipids in PV patients compared to healthy controls. Ratios of amide I/amide II and amide I/amide III indicated alterations in protein structures. Support vector machine analysis and receiver operating characteristic curves identified amide I as a crucial predictor of PV, achieving 100% accuracy, sensitivity, and specificity, while amide III showed a lower predictive value (70%). PCA analysis demonstrated effective differentiation between PV patients and controls, with key wavenumbers including amide II, amide I, and CH lipid vibrations. These findings underscore the potential of FTIR spectroscopy for diagnosing and monitoring PV.

Raman Spectroscopy of Blood Serum for Essential Thrombocythemia Diagnosis: Correlation with Genetic Mutations and Optimization of Laser Wavelengths.

Essential thrombocythemia (ET) is a type of myeloproliferative neoplasm that increases the risk of thrombosis. To diagnose this disease, the analysis of mutations in the Janus Kinase 2 (JAK2), thrombopoietin receptor (MPL), or calreticulin (CALR) gene is recommended. Disease poses diagnostic challenges due to overlapping mutations with other neoplasms and the presence of triple-negative cases. This study explores the potential of Raman spectroscopy combined with machine learning for ET diagnosis. We assessed two laser wavelengths (785, 1064 nm) to differentiate between ET patients and healthy controls. The PCR results indicate that approximately 50% of patients in our group have a mutation in the JAK2 gene, while only 5% of patients harbor a mutation in the ASXL1 gene. Additionally, only one patient had a mutation in the IDH1 and one had a mutation in IDH2 gene. Consequently, patients having no mutations were also observed in our group, making diagnosis challenging. Raman spectra at 1064 nm showed lower amide, polysaccharide, and lipid vibrations in ET patients, while 785 nm spectra indicated significant decreases in amide II and C-H lipid vibrations. Principal Component Analysis (PCA) confirmed that both wavelengths could distinguish ET from healthy subjects. Support Vector Machine (SVM) analysis revealed that the 800-1800 cm-1 range provided the highest diagnostic accuracy, with 89% for 785 nm and 72% for 1064 nm. These findings suggest that FT-Raman spectroscopy, paired with multivariate and machine learning analyses, offers a promising method for diagnosing ET with high accuracy by detecting specific molecular changes in serum. Principal Component Analysis (PCA) confirmed that both wavelengths could distinguish ET from healthy subjects. Support Vector Machine (SVM) analysis revealed that the 800-1800 cm-1 range provided the highest diagnostic accuracy, with 89% for 785 nm and 72% for 1064 nm. These findings suggest that FT-Raman spectroscopy, paired with multivariate and machine learning analyses, offers a promising method for diagnosing ET with high accuracy by detecting specific molecular changes in serum.

Detection of primary myelofibrosis in blood serum via Raman spectroscopy assisted by machine learning approaches; correlation with clinical diagnosis.

Primary myelofibrosis (PM) is one of the myeloproliferative neoplasm, where stem cell-derived clonal neoplasms was noticed. Diagnosis of this disease is based on: physical examination, peripheral blood findings, bone marrow morphology, cytogenetics, and molecular markers. However, the molecular marker of PM, which is a mutation in the JAK2V617F gene, was observed also in other myeloproliferative neoplasms such as polycythemia vera and essential thrombocythemia. Therefore, there is a need to find methods that provide a marker unique to PM and allow for higher accuracy of PM diagnosis and consequently the treatment of the disease. Continuing, in this study, we used Raman spectroscopy, Principal Components Analysis (PCA), and Partial Least Squares (PLS) analysis as helpful diagnostic tools for PM. Consequently, we used serum collected from PM patients, which were classified using clinical parameters of PM such as the dynamic international prognostic scoring system (DIPSS) for primary myelofibrosis plus score, the JAK2V617F mutation, spleen size, bone marrow reticulin fibrosis degree and use of hydroxyurea drug features. Raman spectra showed higher amounts of C-H, C-C and C-C/C-N and amide II and lower amounts of amide I and vibrations of CH3 groups in PM patients than in healthy ones. Furthermore, shifts of amides II and I vibrations in PM patients were noticed. Machine learning methods were used to analyze Raman regions: (i) 800 cm-1 and 1800 cm-1, (ii) 1600 cm-1-1700 cm-1, and (iii) 2700 cm-1-3000 cm-1 showed 100 % accuracy, sensitivity, and specificity. Differences in the spectral dynamic showed that differences in the amide II and amide I regions were the most significant in distinguishing between PM and healthy subjects. Importantly, until now, the efficacy of Raman spectroscopy has not been established in clinical diagnostics of PM disease using the correlation between Raman spectra and PM clinical prognostic scoring. Continuing, our results showed the correlation between Raman signals and bone marrow fibrosis, as well as JAKV617F. Consequently, the results revealed that Raman spectroscopy has a high potential for use in medical laboratory diagnostics to quantify multiple biomarkers simultaneously, especially in the selected Raman regions.

Clinical Impact of JAK2V617F Allele Burden in Philadelphia-Negative Myeloproliferative Neoplasms

Objective: The impact of JAK2V617F allele burden on clinical course in Philadelphia-negative (Ph-negative) myeloproliferative neoplasms (MPNs) is not clear. We analyzed the clinical impact of JAK2V617F allele burden in a relatively large series of patients with Ph-negative MPNs and long-term follow-up. Materials and Methods: A total of 228 patients with Ph-negative MPNs, including 118 with essential thrombocythemia (ET), 84 with primary myelofibrosis (PMF), and 26 with polycythemia vera (PV), were analyzed. The JAK2 MutaScreen assay was used to quantify JAK2V617F allele burden in genomic DNA. Results: In PV cases, high JAK2V617F allele burden was associated with a trend towards inferior overall survival. In ET, high JAK2V617F allele burden was associated with lower hemoglobin and hematocrit levels, higher lactate dehydrogenase (LDH) levels, larger spleen size, and increased bleeding and mortality rates. In PMF, high JAK2V617F allele burden was associated with higher leukocyte counts and larger spleen size. In the entire cohort, high allele burden was associated with higher leukocyte and lower platelet counts, higher LDH levels, larger spleen size, higher percentage of bleeding events, higher death rate, and inferior overall survival. Conclusion: Our results suggest that high JAK2V617F allele burdens are associated with more severe disease in PV and ET. In PMF, high JAK2V617F allele burdens were associated with more pronounced myeloproliferative phenotypes. In Ph-negative MPNs, high allele burdens were associated with more aggressive phenotypes. Our data with a long follow-up period support the possibility of JAK2V617F allele burden being used as a marker for predicting clinical phenotype in cases of Ph-negative MPNs.

Application of Fourier Transform InfraRed spectroscopy of machine learning with Support Vector Machine and principal components analysis to detect biochemical changes in dried serum of patients with primary myelofibrosis.

Primary myelofibrosis (PM) is a myeloproliferative neoplasm characterized by stem cell-derived clonal neoplasms. Several factors are involved in diagnosing PM, including physical examination, peripheral blood findings, bone marrow morphology, cytogenetics, and molecular markers. Commonly gene mutations are used. Also, these gene mutations exist in other diseases, such as polycythemia vera and essential thrombocythemia. Hence, understanding the molecular mechanism and finding disease-related biomarker characteristics only for PM is crucial for the treatment and survival rate. For this purpose, blood samples of PM (n = 85) vs. healthy controls (n = 45) were collected for biochemical analysis, and, for the first time, Fourier Transform InfraRed (FTIR) spectroscopy measurement of dried PM and healthy patients' blood serum was analyzed. A Support Vector Machine (SVM) model with optimized hyperparameters was constructed using the grid search (GS) method. Then, the FTIR spectra of the biomolecular components of blood serum from PM patients were compared to those from healthy individuals using Principal Components Analysis (PCA). Also, an analysis of the rate of change of FTIR spectra absorption was studied. The results showed that PM patients have higher amounts of phospholipids and proteins and a lower amount of H-O=H vibrations which was visible. The PCA results indicated that it is possible to differentiate between dried blood serum samples collected from PM patients and healthy individuals. The Grid Search Support Vector Machine (GS-SVM) model showed that the prediction accuracy ranged from 0.923 to 1.00 depending on the FTIR range analyzed. Furthermore, it was shown that the ratio between α-helix and ß-sheet structures in proteins is 1.5 times higher in PM than in control people. The vibrations associated with the CO bond and the amide III region of proteins showed the highest probability value, indicating that these spectral features were significantly altered in PM patients compared to healthy ones' spectra. The results indicate that the FTIR spectroscope may be used as a technique helpful in PM diagnostics. The study also presents preliminary results from the first prospective clinical validation study.

FTIR- based serum structure analysis in molecular diagnostics of essential thrombocythemia disease.

Essential thrombocythemia (ET) reflects the transformation of a multipotent hematopoietic stem cell, but its molecular pathogenesis remains obscure. Nevertheless, tyrosine kinase, especially Janus kinase 2 (JAK2), has been implicated in myeloproliferative disorders other than chronic myeloid leukaemia. FTIR analysis was performed on the blood serum of 86 patients and 45 healthy volunteers as control with FTIR spectra-based machine learning methods and chemometrics. Thus, the study aimed to determine biomolecular changes and separation of ET and healthy control groups illustration by applying chemometrics and ML techniques to spectral data. The FTIR-based results showed that in ET disease with JAK2 mutation, there are alterations in functional groups associated with lipids, proteins and nucleic acids significantly. Moreover, in ET patients the lower amount of proteins with simultaneously higher amount of lipids was noted in comparison with the control one. Furthermore, the SVM-DA model showed 100% accuracy in calibration sets in both spectral regions and 100.0% and 96.43% accuracy in prediction sets for the 800-1800 cm-1 and 2700-3000 cm-1 spectral regions, respectively. While changes in the dynamic spectra showed that CH2 bending, amide II and CO vibrations could be used as a spectroscopy marker of ET. Finally, it was found a positive correlation between FTIR peaks and first bone marrow fibrosis degree, as well as the absence of JAK2 V617F mutation. The findings of this study contribute to a better understanding of the molecular pathogenesis of ET and identifying biomolecular changes and may have implications for early diagnosis and treatment of this disease.

Raman spectroscopy-based biomarker screening by studying the fingerprint and lipid characteristics of Polycythem..a Vera cases blood serum.

This study aimed to develop a novel approach for diagnosing Polycythemia Vera (PV), a stem cell-derived neoplasm of the myeloid lineage. The approach utilized Raman spectroscopy coupled with multivariate analysis to analyze blood serum samples collected from PV patients. The results showed that PV serum exhibited lower protein and lipid levels and structural changes in the functional groups that comprise proteins and lipids. The study also demonstrated differences in lipid biosynthesis and protein levels in PV serum. Using the Partial Least Square Discriminant Analysis (PLS-DA) model, Raman-based multivariate analysis achieved high accuracy rates of 96.49 and 93.04% in the training sets and 93.10% and 89.66% in the test sets for the 800-1800 cm-1 and 2700-3000 cm-1 ranges, respectively. The area under the curve (AUC) values of the test datasets were calculated as 0.92 and 0.89 in the 800-1800 cm-1 and 2700-3000 cm-1 spectral regions, respectively, demonstrating the effectiveness of the PLS-DA models for the diagnosis of PV. This study highlights the potential of Raman spectroscopy-based analysis in the early and accurate diagnosis of PV, enabling the application of effective treatment strategies.

Comprehensive mastocytosis data analysis from a single center.

Mastocytosis is a very rare disorder and is divided into three prognostically distinct variants by World Health Organization: Cutaneous mastocytosis (CM), systemic mastocytosis (SM), and mast cell sarcoma or localized mast cell (MC) tumors. The wide range of complaints may cause patients to consult various clinics, with resulting mis- or underdiagnosis. Therefore, cooperation between different subspecialties is of paramount importance. In this article, we have compiled 104 adult mastocytosis cases diagnosed and followed in our Hematology and other clinics. 86 (82.7%) of 104 patients had systemic mastocytosis. Osteoporosis, disease-related complications, and secondary malignancies are important topics in this group. We know that indolent form has great survival. But smoldering or aggressive mastocytosis has a poor prognosis. CM and indolent SM have a significantly better prognosis compared to aggressive SM (p < 0.001). We found that the presence of more than 25% of mast cells in the bone marrow, the presence of concomitant marrow dysplasia, and the presence of disease-related complications affect survival (p < 0.001). In addition to the WHO classification, the IPSM scoring system is indicative of the prognosis in this rare disease.

OCT-1 Expression in Patients with Chronic Myeloid Leukemia: A Comparative Analysis with Respect to Response to Imatinib Treatment.

The introduction of tyrosine kinase inhibitors (TKI) has resulted in a significant improvement in the treatment of CML patients. However, some CML patients are resistant to imatinib therapy, the initial TKI therapy in the CML. Therefore, it is important to find prognostic markers for resistance. The OCT-1 gene involved in imatinib uptake is also suspected to cause imatinib resistance. The aim of this study was to investigate the role of OCT-1 in imatinib resistance by comparing OCT-1 expression levels in imatinib resistant and imatinib sensitive patients with chronic myeloid leukemia (CML). This study was conducted on 101 patients with CML [imatinib sensitive (n = 51) and imatinib resistant (n = 50)] who were treated with imatinib. Gene expression analysis was done using QRT-PCR. The relative expression levels of OCT-1 were calculated using 2(-ΔΔCT) method. OCT1 mRNA expression levels were 0.149 (0.011-2.532) and 0.119 (0.008-2.868) in imatinib-sensitive group and imatinib-resistant group, respectively. OCT-1 expression levels were not significantly different in the imatinib-sensitive group when compared to imatinib resistant group (p > 0.05). OCT-1 expression was also similar in BCR-ABL1 kinase domain mutation positive and negative cases (p > 0.05). The imatinib-resistant group had a higher rate of hydroxyurea or interferon-alpha treatment prior to imatinib therapy and a lower rate for first-line imatinib as the only treatment than the imatinib-sensitive group (p = 0.002 and p = 0.002, respectively). According to the results of our study, OCT-1 does not have a biomarker feature in the evaluation of imatinib response. In addition, the study should be performed in larger patient groups.

The impact of JAK2V617F mutation on Philadelphia-negative myeloproliferative neoplasms.

BACKGROUND: JAK2V617F mutation is expressed in almost all polycthemia vera (PV), 55% of essential thrombocythemia (ET), and 65% of primary myelofibrosis (PMF) patients. Studies investigating phenotypic effects of JAK2V617F mutation on Philadelphianegative myeloproliferative neoplasms (Ph-negative MPNs) have reported controversial results. This study aims to determine the impact of JAK2V617F mutation on clinical phenotype and outcome in Ph-negative MPNs. METHODS: Clinical correlates and long-term prognostic relevance of the JAK2V617F mutation were analyzed in 410 Phnegative MPNs-170 ET, 135 PV, 105 PMF- from two institutions and followed for a mean of 76.7 months (SD 62.1) (mean 87 months (SD 67.8), 70.4 months (SD 56.4), 68 months (SD 57.4), respectively for ET, PV, and PMF). Two hundred and twenty-eight patients were genotyped for JAK2V617F mutation using the JAK2 Ipsogen MutaScreen assay, which involves allele-specific polymerase chain reaction (PCR), and 182 patients were genotyped using melting curve analysis. RESULTS: In PV patients, JAK2V617F mutation was associated with higher rate in females, lower hemoglobin (Hgb) level, higher leukocyte and platelet count and higher prevalence of thrombosis (p = 0.008, p = 0.018, p = 0.001, p = 0.001, and p = 0.035, respectively). In ET patients, JAK2V617F mutation was associated with higher Hgb and hematocrit (Hct) levels and lower platelet count (p = 0.001, p = 0.001, and p = 0.001, respectively). JAK2V617F-negative ET patients showed a trend towards higher rate of leukemic transformation (p = 0.061). JAK2V617F mutation-positive PMF patients had higher leukocyte count, greater spleen size and showed a trend towards higher Hgb level (p = 0.019, p = 0.042, and p = 0.056, respectively). Among PMF patients with JAK2V617F mutation, the rate of female patients was lower (p = 0.001). Overall survival (OS) in Dynamic International Prognostic Scoring System (DIPSS) - plus high risk PMF patients was shorter compared to the other risk groups (p = 0.001). Leukemia-free survival (LFS) was shorter in DIPSS - plus high risk PMF patients than the other risk groups (p = 0.005). In the entire cohort of Ph-negative MPN patients, JAK2V617F mutation was associated with higher leukocyte count, higher Hgb and Hct levels and lower platelet count, higher frequency of phlebotomies, a trend towards older age, a trend towards greater spleen size, a trend towards a higher prevalence of risk factors for cardiovascular diseases and thrombosis (p = 0.001, p = 0.005, p = 0.001, p = 0.003, p = 0.004, p =0.052, p = 0.056, p = 0.052, and p = 0.059, respectively).

Overview of clinical and genetic features of CML patients with variant Philadelphia translocations involving chromosome 7: A case series.

Variant Philadelphia (Ph) translocations involving chromosome 7 are rarely seen in Chronic Myeloid Leukemia (CML) patients. It is aimed to contribute new cases to the literature by reviewing the cases in our archive and shed light into the understanding of the role of chromosome 7 in CML. This study was carried out in 237 newly diagnosed CML patients with variant Ph translocations. Among the patients, those with variant Ph translocation involving chromosome 7 were evaluated in terms of clinical and genetic characteristics. Chromosome analysis was performed on 24 and 48 h of bone marrow cultures. FISH analysis was performed with BCR-ABL1 dual color dual fusion translocation probes. BCR-ABL1 transcript levels were analysed by QRT-PCR and results were reported as BCR-ABL1/ABL1 (BCR-ABL1 (IS) %) according to international scale. Four of the patients had variant Ph translocations including chromosome 7. The karyotypes were 46,XX,t(7;9;22)(p13;q34;q11); 46,XX,t(7;9;22)(p21;q34;q11); 46,XX,t(7;9;22)(q22;q34;q11) and 46,XY,t(7;9;22)(q22;q34;q11). The breakpoints demonstrated by cytogenetic analysis were confirmed by FISH analysis. Monitoring by QRT-PCR showed that patients with variant Ph translocation including 7p13 and 7p21 had a dramatic decrease in BCR-ABL1 levels resulting in complete hematological, complete cytogenetic and deep molecular responses. Despite achieving complete hematological, complete cytogenetic response in two patients with variant Philadelphia translocation, including 7q22, no major molecular response was achieved and both patients are still in the warning category. Response to tyrosine kinase inhibitör therapy may be associated with both the variant translocation mechanism and new gene interactions that occur due to the breakpoints of additional chromosomes involved in translocation.

Activation-induced cytidine deaminase expression in patients with myelodysplastic syndrome and its relationship with prognosis and treatment

Background/aim: Activation-induced cytidine deaminase (AID) enables antibody diversity in B lymphocytes. It may also have an effect on MDS pathogenesis by causing somatic mutations and by inducing epigenetic changes in myeloid cells. This study aimed to compare AID expression of MDS patients with healthy controls, of MDS patients in different risk groups, and of MDS patients according to their treatment. Materials and methods: Total RNA was isolated and complementary DNA (cDNA) was transcribed from the peripheral blood samples of MDS patients and healthy controls. AID and the reference gene HPRT1 were analyzed using quantitative real-time PCR (QRT-PCR). AID expression relative to HPRT1 was calculated. Patients were classified into "lower risk" and "higher risk" subgroups according to their initial IPSS and IPSS-R scores and their MDS subtypes at the time of study. Patients were also divided into two groups based on receiving treatment with hypomethylating agents. AID expressions of different groups were compared using the Mann­Whitney U test. Results: Thirty MDS patients and thirty healthy controls were included. AID expression in MDS patients was significantly higher compared to healthy controls (p < 0.001). There was no significant difference in AID expression of "lower risk" and "higher risk" subgroups of patients. Patients that received hypomethylating agents did not have a significant difference in AID expression compared with patients that did not receive hypomethylating agents. Conclusion: AID expression is increased in the peripheral blood of MDS patients compared to healthy controls. However, AID expression is not significantly different in "lower risk" and "higher risk" subgroups and in patients treated with hypomethylating agents. Increased AID expression may be an early step in MDS pathogenesis.

Interactive effects of interferon-gamma functional single nucleotid polymorphism (+874 T/A) with cardiovascular risk factors in coronary heart disease and early myocardial infarction risk.

Atherosclerosis is an inflammatory disease characterized by extensive lipid accumulation in the artery wall. Throughout the atherosclerotic process, interferon-gamma (IFN-γ), which is an important pro-inflammatory cytokine, plays a central role in atherosclerotic plaque instability and the occurrence of myocardial infarction (MI). In this study, we aimed to investigate the relationship between IFN-γ +874 T/A (rs2430561) polymorphism and coronary heart disease (CHD) as well as its effects on MI and CHD. Three hundred and ninety patients with CHD (229 with MI, 161 without MI) and 233 healthy controls were screened by the amplification refractory mutation system (ARMS) PCR method for IFN-γ +874 T/A polymorphism. For MI risk, early adult age was important risk factors and the risk was increased with IFN-γ +874 T/A polymorphism. IFN-γ T allele was significantly increased in the CHD patients with age≤45 (p = 0.048) and patients with history of MI (p = 0.007). As IFN-γ is an inflammatory cytokine with an emerging role in the atherosclerotic process, it was suggested that inhibition of IFN-γ activity could be a therapeutic strategy to stabilize human atherosclerotic plaque. Our findings support the association between MI risk and IFN-γ +874 T/A polymorphism in the Turkish population, particularly by increasing the level of IFN-γ in young patients, thereby causing rupture of vulnerable plaques in atherosclerotic lesions. Identification of the IFN-γ +874 T/A gene variants as risk factors for early CHD and MI development may be a practical biomarker to guide the MI risk process and determine the ideal therapeutic approach.

The impact of CYP2D6*4 and GSTP1 Ile105Val polymorphisms on the susceptibility to develop BCR-ABL1 negative myeloproliferative neoplasms.

Inter-individual variations in the genes encoding xenobiotic-metabolizing enzymes have been reported to alter susceptibility to various diseases involving hematological disorders. The purpose of this case-control study was to investigate the relationship between CYP2D6*4 and GSTP1 Ile105Val polymorphisms and the risk of developing BCR-ABL1 negative myeloproliferative neoplasms (MPN). PCR-RFLP was used for genotyping single nucleotide polymorphisms (SNP) in CYP2D6 and GSTP1 in 139 patients with MPN and 126 controls. There was a significantly increased risk for developing BCR-ABL1 negative MPN for the group bearing the CYP2D6*4 variant allele (X2: 4.487; OR 1.738; 95% CI 1.040-2.904; p = 0.034). The platelet count was higher in CYP2D6*4 allele carriers (p = 0.047). There was no association between the GSTP1 Ile105Val polymorphism and the risk of developing MPNs. MPN patients bearing the GSTP1 Ile105Val variant allele had a higher prevalence of bleeding complications (X2: 7.510; OR 4.635; 95% CI 1.466-14.650; p = 0.006). Our study provides new data that the CYP2D6*4 polymorphism may be associated with an increased risk to develop MPNs while the GSTP1 Ile105Val polymorphism does not show such an association. To our knowledge, the current study is the first to investigate the relationship between CYP2D6*4 and GSTP1 Ile105Val polymorphisms and the risk of developing MPNs in the Turkish population. Further studies with more patients and controls are needed to support our data.

The Clinical Significance of IDH Mutations in Essential Thrombocythemia and Primary Myelofibrosis.

BACKGROUND: Limited data exist regarding impact of IDH mutations in Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs). Prognostic significance of IDH mutations was asessed in 184 Ph-negative MPN patients - 107 essential thrombocythemia (ET) and 77 primary myelofibrosis (PMF). METHODS: High-resolution melting (HRM) analysis was used to detect IDH1 and IDH2 mutations. RESULTS: PMF and ET patients showed no significant difference for prevalence of IDH mutations. Mutant IDH (IDH1 or IDH2) was documented in five of PMF (6.5%) and two of ET patients (1.9%). IDH mutations in ET patients included one IDH1 R132C and one IDH2 R140Q. Of the five IDH-mutated PMF patients, four (80%) displayed IDH1 (three IDH1 R132C and one IDH1 R132S) and one (20%) carried IDH2 (IDH2 R140Q) mutation. Sixty percent (three in five) of IDH-mutated PMF patients carried JAK2V617F with following allele burdens: 31-50%, 5-12.5% and 31-50%, respectively. Three of 77 PMF patients (3.9%) simultaneously harbored IDH and JAK2V617F mutations. IDH mutations in PMF showed a trend towards higher rate in females (100% and 52.8%, respectively). Bleeding complications were significantly higher in IDH-mutated PMF patients compared to IDH wild-type counterparts. Trend towards a lower prevalance of acetylsalicylic acid (ASA) use was present in IDH mutant PMF patients compared to wild-type counterparts (20% and 63.9%, respectively). Death rate was higher in IDH-mutated PMF patients compared to IDH wild-type PMF patients (60% and 15.3%). In univariate analysis, a significantly shorter leukemia-free survival (LFS) was observed in IDH-mutated PMF patients. CONCLUSIONS: We conclude that IDH mutations indicate a risk for leukemic transformation in PMF.

Impact of JAK2V617F Mutational Status on Phenotypic Features in Essential Thrombocythemia and Primary Myelofibrosis.

OBJECTIVE: The JAK2V617F mutation is present in the majority of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). The impact of this mutation on disease phenotype in ET and PMF is still a matter of discussion. This study aims to determine whether there are differences in clinical presentation and disease outcome between ET and PMF patients with and without the JAK2V617F mutation. MATERIALS AND METHODS: In this single-center study, a total of 184 consecutive Philadelphia-negative chronic myeloproliferative neoplasms, 107 cases of ET and 77 cases of PMF, were genotyped for JAK2V617F mutation using the JAK2 Ipsogen MutaScreen assay, which involves allele-specific polymerase chain reaction. RESULTS: ET patients positive for JAK2V617F mutation had higher hemoglobin (Hb) and hematocrit (Hct) levels, lower platelet counts, and more prevalent splenomegaly at diagnosis compared to patients negative for the JAK2V617F mutation, but rates of major thrombotic events, arterial thrombosis, and venous thrombosis were comparable between the groups. At presentation, PMF patients with JAK2V617F mutation had significantly higher Hb and Hct levels and leukocyte counts than patients without the mutation. Similar to the findings of ET patients, thromboembolic rates were similar in PMF patients with and without theJAK2V617F mutation. For ET and PMF patients, no difference was observed in rates of death with respect to JAK2V617F mutational status. Moreover, leukemic transformation rate was not different in our PMF patients with and without JAK2V617F mutation. CONCLUSION: We conclude that JAK2V617F-mutated ET patients express a polycythemia vera-like phenotype and JAK2V617F mutation in PMF patients is associated with a more pronounced myeloproliferative phenotype.

Co-existence of BRAF V600E Gene Mutation in Tumor and Non-tumoral Surrounding Tissues in Colorectal Cancer.

BACKGROUND/AIM: The murine sarcoma viral (V-Raf) oncogene homolog B (BRAF) V600E mutation, which increases protein kinase activity in BRAF-mitogen-activated protein kinase kinase (MEK) - extracellular signal-regulated kinases (ERK) (mitogen-activated protein kinase (MAPK)) signaling, is found in 5-40% of all colorectal carcinoma cases. Proteins with this mutation are reported to be 130-fold more active, which results in induced proliferation, differentiation, cellular survival, and angiogenesis. The aim of the present study was to investigate tumor tissues, together with the surrounding non-tumoral tissues, for BRAF mutation presence, which may be an indicator for possible recurrence or prognosis as in the 'field carcinogenesis' model. MATERIALS AND METHODS: The BRAF V600E genotype of 152 colorectal adenocarcinoma paraffin-embedded specimens were determined by mutant-allele-specific amplification-polymerase chain reaction. RESULTS: According to our results, the presence of BRAF mutation increases risk of lymph node invasion by 1.55-fold [χ(2)=3.83, p=0.05, odds ratio (OR)=1.55, 95% confidence interval (CI)=1.00-2.42], histologically medium or high-grade tumor by 1.60-fold (χ(2)=4.34, p=0.030, OR=1.60, 95% CI=1.03-2.48), vascular invasion by 1.55-fold (χ(2)=3.55, p=0.05, OR=1.55, 95% CI=0.99-2.42), perineural invasion by 1.50-fold (χ(2)=3.16, p=0.07, OR=1.5, 95% CI=0.96-2.33) and the combination of these poor prognostic features by 1.54-fold (χ(2)=2.47, p=0.11, OR=1.54, 95% CI=0.93-2.53). We also found that females are more prone to having the mutation and that being female increases the risk of having this mutation by 1.54-fold (χ(2)=3.58, p=0.05, OR=1.54, 95% CI=0.97-2.44). CONCLUSION: BRAF V600E mutation in non-tumoral surrounding tissue in patients with colorectal cancer may be used as a valuable marker to foresee clinical outcome or a possible recurrence. To our knowledge, this was the first study to take into consideration the non-tumoral surrounding tissues in addition to the tumor tissue.

Preliminary Study: Prominent miRNAs of Breast Malignant Tissues Compared to Normal Tissues in Turkish Patients with Breast Cancer.

miRNA involvement has been observed in almost every type of cancer, including breast cancer. The etiology of abnormal expression of miRNAs in cancer is still not clearly understood. In order to obtain insight into miRNA de-regulation in breast cancer, we analyzed expression levels of five breast cancer-related miRNAs, miRNA21, miRNA155, miRNA19a, miRNA17-5p and let7a miRNA, in both malignant and neighboring non-tumoral paraffin-embedded tissues of 47 patients with invasive ductal breast cancer. The targeted miRNAs, and a reference snRNA, U6, were analyzed by real-time polymerase chain reaction. let7a Levels were significantly lower in patients with lymphatic invasion than in those without (p=0.047). miR21 was down-regulated in 93.3% of patients with necrosis [p=0.017 (Fisher's exact test (FE))], while at least one oncogenic miRNA was up-regulated in 87.3% of the patients with invasive ductal carcinoma [p=0.009 (FE)]. In addition, tumor-suppressor miRNA was down-regulated or unaltered in 65.8% of the patients with tumor grade 2 or 3 and in all with grade 1 [p=0.047 (FE)]. Based on this preliminary study, we suggest that these miRNAs, especially let7a and miRNA21, might be useful markers in follow-up of breast cancer and in prognosis.

Prognostic significance of ASXL1, JAK2V617F mutations and JAK2V617F allele burden in Philadelphia-negative myeloproliferative neoplasms.

BACKGROUND: Despite insights into the genetic basis of Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs), a significant proportion of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients present with no known MPN disease alleles. There were no previous studies investigating the impact of ASXL1 mutations in Ph-negative MPNs in Turkey. In the current study, we investigated the prognostic significance of ASXL1 mutations in Turkish MPN patients. We also aimed to determine the prognostic significance of JAK2V617F allele burden and the relationship of JAK2V617F mutation with ASXL1 mutations in Ph-negative MPNs. METHODS: About 184 patients from a single center diagnosed with Ph-negative MPNs were screened for ASXL1, JAK2V617F mutations, and JAK2V617F allele burden: 107 ET and 77 PMF. RESULTS: A total of 29 ASXL1 mutations were detected in 24.7% of PMF and 8.4% of ET patients. ASXL1-mutated ET patients showed a trend toward an increase in the incidence of cerebrovascular events and higher total leukocyte counts. ASXL1-mutation in PMF was associated with older age and a higher prevalence of bleeding complications. In univariate analysis, overall survival (OS) was significantly reduced in ASXL1-mutated PMF patients. In multivariate analysis, Dynamic International Prognostic Scoring System-plus high-risk category and ASXL1 mutation status were independently associated with shorter survival in PMF. In PMF, mutational status and allele burden of JAK2V617F showed no difference in terms of OS and leukemia-free survival. CONCLUSION: We conclude that ASXL1 mutations are molecular predictors of short OS in PMF.

The Burden of JAK2V617F Mutated Allele in Turkish Patients With Myeloproliferative Neoplasms.

BACKGROUND: Studies regarding the impact of JAK2V617F allele burden on phenotypic properties and clinical course in Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs) have reported variable results. We aimed to analyze the association of mutated JAK2V617F allele burden with laboratory characteristics and clinical phenotype in Turkish patients (107 essential thrombocythemia (ET) and 77 primary myelofibrosis (PMF)). METHODS: Peripheral blood samples of 184 patients with Ph-negative MPNs were analyzed for JAK2V617F allele status and burden. JAK2 MutaScreen assay (Ipsogen, Luminy Biotech, Marseille, France) was used to detect the JAK2V617F status and quantitative JAK2V617F allele burdens in genomic DNA using TaqMan allelic discrimination. RESULTS: Frequency of JAK2V617F-positive patients with high mutation load (allele burden > 50%) was higher in PMF compared to ET (23.4% and 4.7%, respectively; P = 0.001). We found significant association between ET patients with high JAK2V617F allele burden and lower hemoglobin (Hgb) and hematocrit (Hct), higher LDH levels and more prevalent massive splenomegaly (P = 0.001, P = 0.001, P = 0.012 and P = 0.015, respectively). ET patients with high mutation load displayed higher prevalence of bleeding compared to low mutation load and wild-type mutational status (P = 0.003). Rate of DVT was significantly higher in ET patients with mutant allele burden in upper half compared to lower half and wild-type (P = 0.029). We observed significant association between PMF patients with high JAK2V617F allele burden and higher Hgb, Hct levels and leukocyte counts (P = 0.003, P = 0.021 and P = 0.001, respectively). CONCLUSIONS: Our study demonstrated JAK2V617F allele burden correlates with clinical features in ET and PMF. We conclude quantification of JAK2V617F mutation contributes to the workup of Ph-negative MPNs.