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ObjectivesThe 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.MethodsThe 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.ResultsOur 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.ConclusionsIn 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.

4832 Background Myeloproliferatives neoplasms (MPN) are clonal stem cell disorders characterized by proliferation of one or more of the myeloid lineages, associated with genetic abnormalities that include translocations or point mutations of genes that encode cytoplasmic or tyrosin quinase (TK) receptor proteins. This produces an abnormal constitutively activation of signal transduction pathways, leading to an unregulated proliferation. According to the WHO criteria, MPN are classified into BCR-ABL+/Philadelfia Ph+: chronic myeloid leukemia (CML) and MPN BCR-ABL fusion-/Ph-. A single acquired point mutation, JAK2V617F, has been described in 95% of Polycytemia vera (PV), in 50 % of essencial thrombocythemia (ET) and idiopathic myelofivrosis (IMF), and generally absent in MPN Ph+. In the last years, it has been described the co-ocurrence of both BCR-ABL and V617F mutation in few cases of CML patients. We report here the rare and concomitant ocurrence of JAK2V617F mutation with BCR-ABL translocation at presentation in atypical CML. Methods Blood samples from six patients with clinical suspicion of MPN diagnosis, were referred to our laboratory to cytogenetic studies and molecular analysis of BCR-ABL fusion gene expression by conventional RT-PCR and JAK2V617F status mutation by ASO-PCR in three of them. All patients showed a slightly elevated white blood cells level (7200-25900), trombocythosis (700 -1036 platelets) and small splenomegaly. Three patients, after CML diagnosis, recieved Imatinib therapy and were monitored by quantitative BCR-ABL real time PCR. Due to persistent thrombocytosis, slightly elevated white blood cells level and small splenomegaly; JAK2 status was analized in these blood specimens, and later retrospectively in the stored initial diagnosis samples. Results BCR-ABL rearrangement (b3a2 isoform) and JAK2V617F mutation were identified in all 6 patients at diagnosis. Three cases showed lack of Ph chromosome, 1 patient showed 15 % Ph+ metaphases and in the remained two patients no data was available. Quantitative PCR for BCR-ABL expression performed in 3 patients during follow-up (8-12 months) showed BCR-ABL/BCR ratio < = 0.0018 % (scored according to the International Scale) and the presence of JAK2V617F mutation. Retrospective assessment of stored bood samples showed that JaK mutation was already present at the time of the diagnosis of CML. Conclusions The coexistence of both genetic defects, BCR-ABL fusion gene and JAK2V617F mutation in NMP patients is a rare and uncommon feature. We found 6 MPN patients hourboring both genetics features in blood dignosis samples. In three cases JAK2V617F mutation was detected in MPN patients BCR-ABL positive after the remission induction with Imatinib. The rapid remission of BCR-ABL transcript, after a short period of Imatinib treatment, led us to think that BCR-ABL fusion gene expression was present in a low burden at diagnosis. The complete reduction of BCR-ABL rearrangement, after the imatinib therapy, and the persistence of JAK 2 mutation suggests two possible mechanisms for this double genetic alteration: 1) a haematopoyetic cell subclone with a pre-existing JAK2V617F acquires the BCR-ABL fusion gene, which confers a selective advantage to double mutant progenitor; 2) two clons, one of them having BCR-ABL rearrangement, and the other one the JAK2V617F mutation (biclonal origin). These cases intend to contribute to the discussion about the onset of the molecular alterations, and their correlation with the differente phenotypes and clinical management. Disclosures: No relevant conflicts of interest to declare.

Acute myeloblastic leukemias (AML) are the most frequent acute leukemias in adults. Recent genomic and candidate gene studies identified novel recurrent somatic mutations in patients with AML with biological, clinical and therapeutic significance. In Uruguay, the characterization of AML is dependent on the integration of cytomorphology, immunophenotype, cytogenetics and molecular biology (mutations in FLT3, NPM1, CEBPA and cKIT). This allows stratification in prognostic risk groups and rationalization of therapeutic resources. However, with this molecular markers, complete stratification is not always succesful in patients with normal karyotype ( NK) . In order to expand the AML genetic markers analysis in Uruguayan patients ( 3.000.0000 persons country ) ; DNA samples from 49 adult patients at the onset of the disease and 3 healthy controls were analyzed by next generation sequencing (NGS, Illumina) using a customized panel of 30 cancer associated genes. After excluding synonymous variants and those with a population frequency >1%, we ended with 493 variants in promoters and intron ends, and 173 in the 28 genes coding regions. Notably, all patients had at least one variant. While none of the non-coding region variants were reported as pathogenic in COSMIC and/or ClinVar databases, 45 of those falling in coding regions were reported as pathogenic. The pathogenic variants were detected in 36/49 patients and 1 healthy control distributed in 16 genes. The most affected genes were TET2, NRAS, DNMT3, FLT3 and cKIT with 7, 6, 5, 4, 4 variants respectively. This preliminar findings in this study, highlight the relevance of detecting gene variability underlying prognostic risk subgroups to deliver patient tailored clinical decision support. The correlation between the genetic abnormalities, associated with the clinicopahtological features and the epigenetic studies have prognostic significance, and are continued to be analyzed. Disclosures No relevant conflicts of interest to declare.