Prognostic assessment for chronic myelomonocytic leukemia in the context of the World Health Organization 2016 proposal: a multicenter study of 280 patients.

Knowledge on chronic myelomonocytic leukemia (CMML) patients from Argentina and Brazil is limited. Our series of 280 patients depicted an older age at diagnosis (median 72 years old), 26% of aberrant karyotypes, and a prevalence of myelodysplastic (60%) and CMML-0 subtypes (56%). The median overall survival (OS) was 48.2 months for patients in CMML-0 (Ref.), 24.7 months for those in CMML-1 (HR = 2.0, p = 0.001), and 8.8 months for patients in CMML-2 (HR = 4.6, p < 0.001). In the CMML-0 category, median OS were different between myelodysplastic and myeloproliferative subtypes (63.7 vs 21.2 months, p < 0.001); however, no differences were observed within CMML-1 and CMML-2 subtypes (24.7 vs 23.7 months, p = 0.540, and 9.1 vs 8.2 months, p = 0.160). The prognostic impact of 24 variables and 7 prognostic systems was adjusted to the WHO 2016 after validating their usefulness. Multivariate analysis were performed, and the final model revealed Hb ≥ 8 -< 10g/dL (HR 1.7), Hb < 8g/dL (HR 2.8), poor karyotypes (HR 2.1), WHO 2016-CMML-1 (HR 2.1), and CMML-2 (HR 3.5) as independent adverse clinical parameters in our cohort with a borderline influence of platelets count < 50 × 109/L (HR 1.4). We could validate several scoring systems, the WHO 2016 proposal and its prognostic capability, along with accessible covariates, on predicting the outcome in our series of CMML patients from Latin America.

Programme for Harmonization to the International Scale in Latin America for BCR-ABL1 quantification in CML patients: findings and recommendations.

Objectives The quantitation of BCR-ABL1 mRNA is mandatory for chronic myeloid leukemia (CML) patients, and RT-qPCR is the most extensively used method in testing laboratories worldwide. Nevertheless, substantial variation in RT-qPCR results makes inter-laboratory comparability hard. To facilitate inter-laboratory comparative assessment, an international scale (IS) for BCR-ABL1 was proposed. Methods The laboratory-specific conversion factor (CF) to the IS can be derived from the World Health Organization (WHO) genetic reference panel; however, this material is limited to the manufacturers to produce and calibrate secondary reference reagents. Therefore, we developed secondary reference calibrators, as lyophilized cellular material, aligned to the IS. Our purpose was both to re-evaluate the CF in 18 previously harmonized laboratories and to propagate the IS to new laboratories. Results Our field trial including 30 laboratories across Latin America showed that, after correction of raw BCR-ABL1/ABL1 ratios using CF, the relative mean bias was significantly reduced. We also performed a follow-up of participating laboratories by annually revalidating the process; our results support the need for continuous revalidation of CFs. All participating laboratories also received a calibrator to determine the limit of quantification (LOQ); 90% of them could reproducibly detect BCR-ABL1, indicating that these laboratories can report a consistent deep molecular response. In addition, aiming to investigate the variability of BCR-ABL1 measurements across different RNA inputs, we calculated PCR efficiency for each individual assay by using different amounts of RNA. Conclusions In conclusion, for the first time in Latin America, we have successfully organized a harmonization platform for BCR-ABL1 measurement that could be of immediate clinical benefit for monitoring the molecular response of patients in low-resource regions.

Influence of TNF and IL6 gene polymorphisms on the severity of cytopenias in Argentine patients with myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) represent a heterogeneous group of hematologic disorders characterized by cytopenia(s) and predisposition to leukemic progression. An immune dysregulation and an aberrant bone marrow microenvironment seem to be key elements in the physiopathological process of MDS. In order to evaluate a possible association between susceptibility and clinic-pathologic features, we genotyped 153 MDS patients for functional cytokine polymorphisms: TNF (-308 G/A), IFNG (+874 A/T and +875 CAn), IL6 (-174 G/C), and TGFB1 (+869 C/T and +915 G/C). The frequency of TNF and IL6 polymorphisms was different between patients and healthy controls (n = 131), suggesting a relatedness to MDS susceptibility in our population. Furthermore, the presence of each or both high-producing genotypes [TNF: p = 0.048, odds ratio (OR): 3.979; IL6: p = 0.001, OR: 6.835; both: p = 0.010, OR: 6.068] and thrombocytopenia at platelet counts of <50,000/µL (p = 0.004, OR: 4.857) were independently associated with an increased risk of manifesting a hemoglobin level of <8 g/dL at diagnosis. In particular, a severe bicytopenia was more frequently observed in patients with the TNF (high)_IL6 (high) combined genotype (p = 0.004, OR: 8.357), who consistently became transfusion dependent earlier (2.9 vs. 34.6 months; p = 0.001); and this likelihood was more evident in patients with lower bone marrow blast counts. The contribution of the remaining functional polymorphisms to the disease phenotype was less relevant. Our results demonstrate that TNF and IL6 gene polymorphisms, as underlying host features, are likely to play a key role in influencing the severity of the cytopenias in MDS and they may be instrumental for tailoring cytokine-target therapies.

Gene polymorphism profiles of drug-metabolising enzymes GSTM1, GSTT1 and GSTP1 in an Argentinian population.

BACKGROUND: Glutathione S-transferases (GSTs) are drug-metabolising enzymes involved in biotransformation of carcinogens, drugs, xenobiotics and oxygen free radicals. Polymorphisms of GST genes contribute to inter-individual and population variability in the susceptibility to environmental risk factors, cancer predisposition and pharmacotherapy responses. However, data about GST variability in Argentina are lacking. AIM: The purpose was to determine the prevalence of GSTM1, GSTT1 and GSTP1 polymorphisms in the general population from a central region of Argentina and to perform inter-population comparisons. SUBJECTS AND METHODS: GSTM1 and GSTT1 gene deletions and GSTP1 c.313A > G were genotyped by PCR assays in 609 healthy and unrelated Argentinians. RESULTS: The frequencies of variant genotypes in Argentinians were GSTM1-null (45%), GSTT1-null (17%) and GSTP1-GG (11%). GSTM1-present genotype was significantly associated with GSTP1-AG or GSTP1-GG variants (p = 0.037; p = 0.034, respectively). Comparison with worldwide populations demonstrated that the GST distributions in Argentina are similar to those reported for Italy and Spain, whereas significant differences were observed regarding Asian and African populations (p < 0.001). CONCLUSION: This study has determined, for the first time, the normative profile of three pharmacogenetically relevant polymorphisms (GSTM1, GSTT1 and GSTP1) in the largest Argentinian cohort described to date, providing the basis for further epidemiological and pharmacogenetic studies in this country.

[Guidelines for molecular monitoring of BCR-ABL1 in chronic myeloid leukemia patients by RT-qPCR]. / Recomendaciones metodológicas para el monitoreo molecular de BCR-ABL1 en pacientes con leucemia mieloide crónica por PCR cuantitativa en tiempo real.

Current clinical guidelines for managing chronic myeloid leukemia include molecular monitoring of BCR-ABL1 transcript quantitative reverse-transcription PCR. Despite the proven prognostic significance of molecular response, it is not widely appreciated that quantitative reverse-transcription PCR potentially produces highly variable data, which may affect the validity of results, making comparability between different laboratories difficult. Therefore, standardized reporting of BCR-ABL1 measurements is needed for optimal clinical management. An approach to achieve comparable BCR-ABL1 values is the use of an international reporting scale. Conversion to the international scale is achieved by the application of laboratory specific conversion factor that is obtained by using validated secondary reference calibrators. Moreover, with the aim to mitigate the interlaboratory imprecision of quantitative BCR-ABL1 measurements and to facilitate local laboratory results interpretation and reporting, we decide to prepare laboratory guidelines that will further facilitate interlaboratory comparative studies and independent quality-assessment programs, which are of paramount importance for worldwide standardization of BCR-ABL1 monitoring results, in particular for those most isolated laboratories, with not easy access to commercial kits or sample interchange programs.

TP53 codon 72 polymorphism predicts chronic myeloid leukemia susceptibility and treatment outcome.

BCR-ABL1 gene is a key molecular marker of chronic myeloid leukemia (CML), but it is still unclear which molecular factors may influence CML risk or lead to variable responses to tyrosine kinase inhibitors (TKIs). The aim of this study was to investigate the impact of TP53 c.213 G>C(Arg72Pro; rs1042522) polymorphism on CML risk and its correlation with clinical outcome. Peripheral blood samples from 141 treated CML patients and 141 sex- and age-matched healthy individuals were genotyped by PCR-RFLP. Standard genetic models for disease penetrance were evaluated by logistic regression analysis and Kaplan-Meier method was performed to estimate survival curves. Our study suggests that TP53 c.213 G>C polymorphism may be involved in CML development considering a recessive model (p=0.01; OR: 0.19; CI: 0.06-0.68). In addition, a non-homogenous distribution was found for this polymorphism in males and patients youngers than 50years (p=0.02). According to clinical response, TP53-GG genotype was associated with higher levels of BCR-ABL1 transcripts (p=0.04) and shorter event free survival (p=0.04). Moreover, a trend toward significance was found for failure free survival (p=0.06) and time to imatinib failure (p=0.08). In conclusion, our data suggest that a;TP53 c.213 G>C may be a potential biomarker of CML susceptibility and clinical outcome.

BCR-ABL Mutations in Chronic Myeloid Leukemia Treated With Tyrosine Kinase Inhibitors and Impact on Survival.

This is the largest Latin American study of BCR-ABL mutations in chronic myeloid leukemia (CML) patients, resistant to imatinib (IM). In 195/467 (41%) patients, mutations were detected. The most frequent mutation was T315I (n = 31, 16%). Progression-free (PFS) and overall survival (OS) at 5 years were lower in patients with BCR-ABL mutations (43% vs. 65%, p = 0.07 and 47% vs. 72%, p = 0.03, respectively) and in those with the T315I mutation (p = 0.003 and p = 0.03). OS and PFS were superior in subgroup who switched to second generation inhibitors (SGIs) after IM failure (OS: 50% vs. 39% p = 0.01; PFS: 48% vs. 30% p = 0.02). BCR-ABL mutations conferred a significant poor prognosis in CML patients.

BCR-ABL mutations in chronic myeloid leukemia treated with tyrosine kinase inhibitors and impact on survival.

This is the largest Latin American study of BCR-ABL mutations in chronic myeloid leukemia (CML) patients, resistant to imatinib (IM). In 195/467 (41%) patients, mutations were detected. The most frequent mutation was T315I (n = 31, 16%). Progression-free (PFS) and overall survival (OS) at 5 years were lower in patients with BCR-ABL mutations (43% vs. 65%, p = 0.07 and 47% vs. 72%, p = 0.03, respectively) and in those with the T315I mutation (p = 0.003 and p = 0.03). OS and PFS were superior in subgroup who switched to second generation inhibitors (SGIs) after IM failure (OS: 50% vs. 39% p = 0.01; PFS: 48% vs. 30% p = 0.02). BCR-ABL mutations conferred a significant poor prognosis in CML patients.

Clinical activity of ponatinib in one patient with chronic myeloid leukemia in chronic phase with e19a2 transcript and T315I mutation.

BACKGROUND: Chronic myeloid leukemia (CML) is a hematological disorder that in rare cases, mainly in CML neutrophilic, presents the e19a2 rearrangement. The encoded product is a 230-KDa protein. Despite the remarkable responses to treatment of most patients, a small but significant fraction of them develop clinical resistance to the tyrosine kinase inhibitors (TKIs). The most common mechanism of resistance is point mutations in the ABL1 kinase domain. The recently approved third-generation TKI ponatinib demonstrated remarkable activity in patients with multi-TKI-resistant disease. Particularly impressive was its efficacy in patients with T315I mutation that is resistant to all other TKIs. METHODS: Qualitative PCR was carried out by multiplex approach. Relative transcripts quantification was performed by one-step real-time PCR, with a specific Taqman probe and primers for the e19a2 rearrangement. We carried out a mutational screening by high-resolution melting, and the mutation was identified by Sanger method. The mutation burden was quantified by quantitative PCR using allele-specific primers. RESULTS: In a patient with CML, we identified a PCR product corresponding to e19a2 rearrangement harboring T315I mutation. At the time of mutational analysis, during dasatinib treatment, the T315I clone was 100% and the quantification of BCR-ABL1 was 18%. After ponatinib therapy, the T315I mutation burden decreased down to undetectable levels and the BCR-ABL1 transcripts showed a very low value (0.011%). CONCLUSIONS: Here, we report the hematological, cytogenetic, and molecular response of a patient with refractory CML in chronic phase with e19a2 transcripts, carrying T315I mutation that was successfully treated with ponatinib.

Myelodysplastic syndromes in South America: a multinational study of 1080 patients.

There are previously reported data describing differences between Asian and European patients with Myelodysplastic Syndromes (MDS), few direct comparisons based on cancer registration characteristics or using cohorts to validate scoring systems. This is the first study from South-America, which attempts to describe demographic, clinical features, and outcome of MDS patients. We retrospectively analyzed 1,080 patients with de novo MDS from Argentina (635), Brazil (345), and Chile (100). Chilean patients were younger (P = 0.001) with female preponderance (P = 0.071). Brazilian series showed a higher predominance of RARS subtype regarding FAB and WHO classifications (P < 0.001). Hemoglobin levels were significantly lower in Brazilian and Chilean series (P < 0.001), and Chilean series also showed a lower platelet count (P = 0.028), with no differences concerning the neutrophil count, % BM blast, and the distribution of cytogenetic risk groups (P > 0.05). Chilean series depicted a lower overall survival (OS; 35 months vs. 56 months-Argentine; 55 months-Brazil, P = 0.030), which was consistent with a higher predominance of the high-risk group according both to the IPSS and IPSS-R (P = 0.046 and P < 0.001). The IPSS-R system and its variables showed a good reproducibility to predict clinical outcome for the whole South-American population. Epidemiological and clinical characteristics, distribution among prognostic subgroups, the OS, and the access to disease modifying therapies were more similar between Argentinean and Brazilian compared with Chilean MDS series. This will need further analysis in a larger group of patients. Descriptive and comparative studies are necessary to establish epidemiological features useful for public health attitudes to generate suitable therapeutic schemes.

Expression of LYN and PTEN genes in chronic myeloid leukemia and their importance in therapeutic strategy.

Tyrosine kinase inhibitors (TKIs), imatinib, nilotinib and dasatinib, are the current treatment of chronic myeloid leukemia (CML). BCR-ABL1 point mutations are the principal cause of resistance to treatment; however other mechanisms could be involved in failure to TKI therapy. LYN is a src kinase protein that regulates survival and responsiveness of tumor cells by a BCR-ABL1 independent mechanism. PTEN tumor suppressor gene is downregulated by BCR-ABL1 in CML stem cells and its deletion is associated with acceleration of disease. In this study we evaluated the expression of LYN, PTEN and the ratio of both genes in 40 healthy donors (HD) and in 139 CML patients; 88 of them resistant to TKI in different phases of disease and 51 in chronic phase classified as optimal responders (OR) to TKI [40 treated with imatinib or nilotinib (OR-IN) and 11 treated with dasatinib (OR-D) therapy]. When we analyzed the gene expression values of LYN, an increase was observed only in advanced stages of the disease, however, when we analyzed the ratio between LYN and PTEN genes, the group of resistant patients in chronic phase in imatinib or nilotinib treatment (CP-IN) also showed a significant increase. Resistant patients treated with dasatinib, a src kinase inhibitor, presented a similar ratio to the observed in HD. In addition, the LYN/PTEN ratio and the LYN expression showed a direct significant correlation with BCR-ABL1 transcript levels in unmutated resistant patients treated with non-src kinase inhibitors. We were able to identify 8/35 (23%) of cases in CP-IN and 4/12 (33%) in accelerated phase and blast phase (AP/BC-IN), in which resistance could be associated with an increase in the ratio of the LYN/PTEN. Our data suggest that the LYN/PTEN expression ratio may be a sensitive monitor of disease progression in unmutated CML patients under imatinib or nilotinib treatment. This ratio could detect cases when resistance is related to altered LYN expression, suggesting that the treatment change to a src kinase inhibitor would be most suitable to overcome resistance.

Expression of genes potentially involved in loss of response in patients with chronic myeloid leukemia.

Chronic Myeloid Leukemia (CML) is a hematological malignancy characterized by the presence of the BCR::ABL1 fusion gene, which leads to uncontrolled cell growth and survival. Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of CML, but a significant proportion of patients develop resistance or lose response to these drugs. Understanding the molecular mechanisms underlying treatment response and resistance is crucial for improving patient outcomes. This study aimed to analyze the expression patterns of genes involved in treatment response and resistance in CML patients receiving TKI therapy. The expression levels of MET, FOXO3, p15, p16, HCK, and FYN genes were examined in CML patients and compared to healthy donors. Gene expression levels were compared between optimal responders (OR) and resistant patients (R) vs. healthy donors (HD). The MET and FOXO3 OR group showed significant differences compared with the HD, (p < 0.0001) and (p = 0.0003), respectively. p15 expression showed significant differences between OR and HD groups (p = 0.0078), while no significant differences were found in p16 expression between the HD groups. FYN showed a statistically significant difference between R vs. HD (p = 0.0157). The results of HCK expression analysis revealed significant differences between OR and HD (p = 0.0041) and between R and HD (p = 0.0026). When we analyzed OR patients with undetectable BCR::ABL1 transcripts, a greater expression of HCK was observed in the R group. These findings suggest that monitoring the expression levels of MET and FOXO3 genes could be valuable in predicting treatment response and relapse in CML patients. Our study provides important insights into the potential use of gene expression analysis as a tool for predicting treatment response and guiding treatment decisions in CML patients. This knowledge may ultimately contribute to the development of personalized treatment strategies to improve patient outcomes in CML.

Functional polymorphisms of DNA repair genes in Latin America reinforces the heterogeneity of Myelodysplastic Syndrome.

Nucleotide excision repair pathway (NER) is an essential mechanism for single-strand breaks (SSB) repair while xeroderma pigmentosum family (XPA to XPG) is the most important system to NER. Myelodysplastic syndrome (MDS) is a heterogeneous hematological cancer characterized by cytopenias and risk of acute myeloid leukemia (AML) transformation. MDS pathogenesis has been associated with problems of DNA repair system. This report aimed to evaluate NER polymorphisms (XPA rs1800975, XPC rs2228000, XPD rs1799793 and XPF rs1800067) in 269 MDS patients of different populations in Latin America (173 Brazilian and 96 Argentinean). Genotypes were identified in DNA samples by RT-qPCR using TaqMan SNP Genotyping Assay. Regarding rs1799793 polymorphism of XPD for Brazilian population, the heterozygous genotype AG presented a high odds ratio (OR) to have a normal karyotype (p = 0.012, OR=3.000) and the mutant homozygous genotype AA was associated to a high OR of AML transformation (p = 0.034, OR=7.4). In Argentine population, the homozygous mutant AA genotype of rs1800975 polymorphism of XPA was associated with an increased odd to have hemoglobin levels below 8g/dL (p = 0.013, OR=10.000) while for the rs1799793 polymorphism of XPD, the heterozygous AG genotype decreased OR to be classified as good (p < 0.001, OR=9.05 × 10-10), and intermediate (p < 0.001, OR=3.08 × 10-10), according to Revised-International Prognostic Scoring System. Regarding the rs1800067 polymorphisms of XPF, the homozygous mutant AA genotype showed a decreased OR to be classified as good (p < 0.001, OR=4.03 × 10-13) and intermediate (p < 0.001, OR=2.54 × 10-13). Our report reinforces the heterogeneity of MDS and demonstrates the importance of ethnic differences and regional influences in pathogenesis and prognosis of MDS.

High cell-free DNA is associated with disease progression, inflammasome activation and elevated levels of inflammasome-related cytokine IL-18 in patients with myelofibrosis.

Myelofibrosis (MF) is a clonal hematopoietic stem cell disorder classified among chronic myeloproliferative neoplasms, characterized by exacerbated myeloid and megakaryocytic proliferation and bone marrow fibrosis. It is induced by driver (JAK2/CALR/MPL) and high molecular risk mutations coupled to a sustained inflammatory state that contributes to disease pathogenesis. Patient outcome is determined by stratification into risk groups and refinement of current prognostic systems may help individualize treatment decisions. Circulating cell-free (cf)DNA comprises short fragments of double-stranded DNA, which promotes inflammation by stimulating several pathways, including inflammasome activation, which is responsible for IL-1ß and IL-18 maturation and release. In this work, we assessed the contribution of cfDNA as a marker of disease progression and mediator of inflammation in MF. cfDNA was increased in MF patients and higher levels were associated with adverse clinical outcome, a high-risk molecular profile, advanced disease stages and inferior overall survival, indicating its potential value as a prognostic marker. Cell-free DNA levels correlated with tumor burden parameters and markers of systemic inflammation. To mimic the effects of cfDNA, monocytes were stimulated with poly(dA:dT), a synthetic double-stranded DNA. Following stimulation, patient monocytes released higher amounts of inflammasome-processed cytokine, IL-18 to the culture supernatant, reflecting enhanced inflammasome function. Despite overexpression of cytosolic DNA inflammasome sensor AIM2, IL-18 release from MF monocytes was shown to rely mainly on the NLRP3 inflammasome, as it was prevented by NLRP3-specific inhibitor MCC950. Circulating IL-18 levels were increased in MF plasma, reflecting in vivo inflammasome activation, and highlighting the previously unrecognized involvement of this cytokine in MF cytokine network. Monocyte counts were higher in patients and showed a trend towards correlation with IL-18 levels, suggesting monocytes represent a source of circulating IL-18. The close correlation shown between IL-18 and cfDNA levels, together with the finding of enhanced DNA-triggered IL-18 release from monocytes, suggest that cfDNA promotes inflammation, at least in part, through inflammasome activation. This work highlights cfDNA, the inflammasome and IL-18 as additional players in the complex inflammatory circuit that fosters MF progression, potentially providing new therapeutic targets.

Expression dynamics of the immune mediators ARG1, TBET, CIITA, IL10 and TGFB1 in chronic myeloid leukaemia patients during the first year of imatinib therapy.

The use of tyrosine kinase inhibitors seems to restore the broadly compromised immune system described in chronic myeloid leukaemia (CML) patients at diagnosis leading to a re-activation of the effector-mediated immune surveillance. Here, we describe the expression dynamics of immune factors during the first year on imatinib therapy. Gene expression was evaluated in 132 peripheral blood samples from 79 CML patients, including 34 who were serially followed. An aliquot of the stored sample used to monitor BCR-ABL1 levels was retro-transcribed to cDNA and gene expression was quantified by real-time PCR. An elevated expression of ARG1 was observed at diagnosis, while TBET, CIITA, IL10 and TGFB1 were significantly decreased. Once on therapy, each gene displayed a particular behaviour. ARG1 normalized to control levels at 3 months only in optimal molecular responders and was identified as the major contributor to the difference among patients. TBET reached normal levels after 12 months in optimal responders and non-responders, regardless the Th1-response previously associated, and CIITA continued downregulated. IL10 and TGFB1 achieved normal levels early at 3 months in both groups, afterwards IL10 was sustained while TGFB1 was slightly increased after 1 year in responders. Our findings are in agreement with an immune re-activation after imatinib initiation; however, some immune mediators may require a longer exposition. The follow-up of novel and reliable biomarkers, such as ARG1, one of the principal mechanisms of myeloid-derived-suppressor cells to inhibit immune system, may be useful to deepen the characterization of early responder patients.

The presence of -308A TNFα is associated with anemia and thrombocytopenia in patients with myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal hematological diseases characterized by refractory cytopenia(s). MDS patients show increased levels of tumor necrosis factor alpha (TNFα) which is a multifunctional proinflammatory cytokine. The aim of this work is to examine the presence of -308A/G TNFα variants and to analyze whether it is associated with clinical parameters in a cohort of 101 Argentinean de novo MDS patients. The A/A+A/G genotype at TNFα -308 was overrepresented 2-fold in our population (p=0.0499, odds ratio-OR: 2.107) and these differences were more evident in RA-FAB subtype (p=0.0424, OR: 2.502). The presence of the high expressing -308A allele was associated with lower hemoglobin level (8.3 vs 9.9g/dL; p=0.0206), reduced platelet counts (89,000 vs 130,000/µL; p=0.0381) and younger age (59 vs 68years; p=0.0122) at diagnosis. Also, these patients showed 3.8-fold higher risk of transfusion requirement (76% vs 46%, p=0.0105) during the follow up. In conclusion, the presence of an inherited -308A TNFα, which increases its transcription level, was associated with the MDS phenotype in our cohort of Argentine MDS patients. Also, an overexpression of TNFα may promote an underlying proinflammatory state that cooperates with intrinsic defects within MDS progenitors to increase the severity of certain phenotypic features of the disease.

Partial and total monosomal karyotypes in myelodysplastic syndromes: comparative prognostic relevance among 421 patients.

Myelodysplastic syndromes (MDS) include a group of heterogeneous hematological disorders with a variable risk of leukemic evolution and short survival. Around 40-50% of patients show abnormal karyotypes that are mostly characterized by monosomies or deletions. Cytogenetic findings are an independent prognostic factor and the International prognostic scoring system (IPSS) differentiates three cytogenetic categories, despite the Intermediate one being heterogeneous. The aim of this study, including 421 Argentinean patients with primary MDS, is to characterize the cytogenetic profile, to test its prognostic value and to compare partial and monosomal karyotypes against other cytogenetic findings. An abnormal karyotype (median survival: 26 months) was observed in 176 patients. The presence of complex karyotypes, number of alterations, and the IPSS cytogenetic groups showed significant differences for predicting outcome. Behavior of patients with isolated deletions (median survival: 49 months) did not differ from those with normal karyotype (56 months, P = 0.654) or Good prognostic findings (43 months, P = 0.371). However, a worse prognosis was observed when another alteration was added (31 months, P = 0.043). Karyotypes with autosomal monosomies (median survival: 16 months) had a prognostic impact similar to other Poor cytogenetic findings (17 months, P = 0.626). In our population classified according to French-American-British (FAB) or World Health Organization (WHO), this new categorization of cytogenetic abnormalities, recognizing three different risk groups, showed an independent prognostic impact and a better discriminating power than the IPSS categories. It can be concluded that all isolate deletions (excluding 7q-) are good prognostic findings and all monosomies (excluding Y chromosome loss) are bad indicators.

Eighteen years of molecular genotyping the hemophilia inversion hotspot: from southern blot to inverse shifting-PCR.

The factor VIII gene (F8) intron 22 inversion (Inv22) is a paradigmatic duplicon-mediated rearrangement, found in about one half of patients with severe hemophilia A worldwide. The identification of this prevalent cause of hemophilia was delayed for nine years after the F8 characterization in 1984. The aim of this review is to present the wide diversity of practical approaches that have been developed for genotyping the Inv22 (and related int22h rearrangements) since discovery in 1993. The sequence- Southern blot, long distance-PCR and inverse shifting-PCR-for Inv22 genotyping is an interesting example of scientific ingenuity and evolution in order to resolve challenging molecular diagnostic problems.

BAX/BCL-XL gene expression ratio inversely correlates with disease progression in chronic myeloid leukemia.

BCR-ABL fusion gene is implicated in the pathogenesis of chronic myeloid leukemia (CML), encoding the oncoprotein p210(BCR-ABL) with anti-apoptotic activity. The inability to undergo apoptosis is an important mechanism of drug resistance and neoplastic evolution in CML. The gene transcript expression of mitochondrial apoptotic related genes BAX and BCL-XL was evaluated by quantitative Real Time PCR (qPCR) in vitro in K562 cells and in vivo in peripheral blood of 66 CML patients in different stages of the disease: 13 cases at diagnosis, 34 in chronic phase (CP), 10 in accelerated phase (AP) and 9 in blast crisis (BC). Our results in K562 cells showed that all treatments with different tyrosine kinase inhibitors (TKIs) induced a decreased expression of the antiapoptotic oncogene BCL-XL, whereas the proapoptotic gene BAX remains constant with minor modifications. A significantly lower BAX/BCL-XL expression ratio (mean±SEM) than a group of healthy individuals (4.8±0.59) were observed in CML patients at diagnosis (1.28 ± 0.16), in AP (1.14±0.20), in BC (1.16±0.30) and in 18% of cases of patients in CP (2.71±0.40). Most CP cases (82%) showed a significantly increased ratio (10.03±1.30), indicating that the treatment with TKIs efficiently inhibited the expression of BCL-XL by blocking BCR-ABL oncoprotein. The BAX/BCL-XL ratio showed a significant inverse correlation (Spearman P<0.0001) with BCR-ABL/ABL relative expression indicating that low BAX/BCL-XL was associated with disease progression. Accordingly, the follow up of a cohort of eight cases during 6months from diagnosis showed that while the BAX/BCL-XL ratio rapidly increased after treatment in seven cases with good evolution, it decreased in the single case that showed rapid evolution and short survival. Our data suggest that BAX/BCL-XL expression ratio may be a sensitive monitor of disease progression and an early predictor of TKI therapy responsiveness in CML patients.