<i>JAK2</i> MT:WT 1∶1 reference constructs.

<p>Upper diagram: reference construct from genomic DNA. Bottom diagram: reference construct from complementary DNA. These diagrams show the allele-specific primers used to amplify the mutated allele (MT) and the wild-type allele (WT). Indicated are the GenBank files and the molecular sizes of each segment and of each amplification product.</p

Comparison of ARMS-PCR and qPCR assays.

<p>Ten cases shown to be positive for the JAK2^V617F mutation using ARMS-PCR exhibited an allele burden of 55% ±9% (mean ± SE) according to qPCR. Ten negative cases according to ARMS-PCR showed an allele burden of 1.9% ±0.6%, including two cases that were negative by ARMS-PCR and positive by qPCR with a value above our cutoff (>3.65%, estimated from a healthy population).</p

Validation of the one-plus-one reference system qPCR assay.

<p>A. Twelve MPN cases (n = 10) and negative controls (n = 2) shown highly significant (p<0.0001) correlation (r = 0.967 95%CI = 0.88–0.99) between <i>JAK2^V617F</i> ABg measured by an allele-specific Taqman probe-based qPCR method (16) and by the one-plus-one reference system. B. Other group of 12 MPN cases shown highly significant (p<0.0001) correlation (r = 0.968 95%CI = 0.88–0.99) between <i>JAK2^V617F</i> ABg measured by a MPN-patient/healthy-control DNA-standardized allele-specific qPCR amplification method (17) and by the one-plus-one reference system. Linear regressions are shown in both graphs.</p

Graphics of deviation from the 1∶1 ratio in the dynamic range of dilutions of the reference plasmids.

<p>Left panel, gAB values for each plasmid dilution of gDNA; right panel, gAB values for plasmid cDNA.</p

Allele burden and expression of <i>JAK2</i>^V617F mutation.

*<p>The propagated error (SD) of the AB from individual values of MT and WT measurements was negligible; therefore, it was not considered (range 6.37×10⁻⁸–1.5310⁻⁵).</p>†<p>Case N° 18 was negative for the <i>JAK2</i>^V617F mutation.</p

DataSheet_1_In-depth characterization of NK cell markers from CML patients who discontinued tyrosine kinase inhibitor therapy.docx

IntroductionTreatment-free remission (TFR) in patients with chronic myeloid leukemia in chronic phase is considered a safe option if suitable molecular monitoring is available. However, the question arises as to which factors can contribute to the maintenance of TFR, and immunologic surveillance of the remaining leukemic cells is believed to be one of them. Argentina Stop Trial is an open-label, single-arm, multicenter trial assessing TFR after tyrosine kinase inhibitors interruption, that after more than 4 years showed a successful TFR rate of 63%.MethodsIn this context, we set up an immunological study by flow cytometry in order to analyze specific NK cell subsets from peripheral blood patient samples both at the time of discontinuation as well as during the subsequent months.ResultsAt the time of discontinuation, patients show a mature NK cell phenotype, probably associated to TKI treatment. However, 3 months after discontinuation, significant changes in several NK cell receptors occurred. Patients with a higher proportion of CD56dim NK and PD-1+ NK cells showed better chances of survival. More interestingly, non-relapsing patients also presented a subpopulation of NK cells with features associated with the expansion after cytomegalovirus infection (expression of CD57+NKG2C+), and higher proportion of NKp30 and NKp46 natural cytotoxicity receptors, which resulted in greater degranulation and associated with better survival (pDiscussionThis NK cell subset could have a protective role in patients who do not relapse, thus further characterization could be useful for patients in sustained deep molecular response.</p