Impact of FLT3–ITD mutation status and its ratio in a cohort of 2901 patients undergoing upfront intensive chemotherapy: a PETHEMA registry study

FLT3–ITD results in a poor prognosis in terms of overall survival (OS) and relapse-free survival (RFS) in acute myeloid leukemia (AML). However, the prognostic usefulness of the allelic ratio (AR) to select post-remission therapy remains controversial. Our study focuses on the prognostic impact of FLT3–ITD and its ratio in a series of 2901 adult patients treated intensively in the pre-FLT3 inhibitor era and reported in the PETHEMA registry. A total of 579 of these patients (20%) harbored FLT3–ITD mutations. In multivariate analyses, patients with an FLT3–ITD allele ratio (AR) of >0.5 showed a lower complete remission (CR rate) and OS (HR 1.47, p = 0.009), while AR > 0.8 was associated with poorer RFS (HR 2.1; p < 0.001). Among NPM1/FLT3–ITD-mutated patients, median OS gradually decreased according to FLT3–ITD status and ratio (34.3 months FLT3–ITD-negative, 25.3 months up to 0.25, 14.5 months up to 0.5, and 10 months ≥ 0.5, p < 0.001). Post-remission allogeneic transplant (allo-HSCT) resulted in better OS and RFS as compared to auto-HSCT in NPM1/FLT3–ITD-mutated AML regardless of pre-established AR cutoff (≤0.5 vs. >0.5). Using the maximally selected log-rank statistics, we established an optimal cutoff of FLT3–ITD AR of 0.44 for OS, and 0.8 for RFS. We analyzed the OS and RFS according to FLT3–ITD status in all patients, and we found that the group of FLT3–ITD-positive patients with AR < 0.44 had similar 5-year OS after allo-HSCT or auto-HSCT (52% and 41%, respectively, p = 0.86), but worse RFS after auto-HSCT (p = 0.01). Among patients with FLT3–ITD AR > 0.44, allo-HSCT was superior to auto-HSCT in terms of OS and RFS. This study provides more evidence for a better characterization of patients with AML harboring FLT3–ITD mutations

Networking for advanced molecular diagnosis in acute myeloid leukemia patients is possible: the PETHEMA NGS-AML project

Next-generation sequencing (NGS) has recently been introduced to efficiently and simultaneously detect genetic variations in acute myeloid leukemia (AML). However, its implementation in the clinical routine raises new challenges focused on the diversity of assays and variant reporting criteria. In order to overcome this challenge, the PETHEMA group established a nationwide network of reference laboratories aimed to deliver molecular results in the clinics. We report the technical cross-validation results for NGS panel genes during the standardization process and the clinical validation in 823 samples of 751 patients with newly diagnosed or refractory/relapse AML. Two cross-validation rounds were performed in seven nationwide reference laboratories in order to reach a consensus regarding quality metrics criteria and variant reporting. In the pre-standardization cross-validation round, an overall concordance of 60.98% was obtained with a great variability in selected genes and conditions across laboratories. After consensus of relevant genes and optimization of quality parameters the overall concordance rose to 85.57% in the second cross-validation round. We show that a diagnostic network with harmonized NGS analysis and reporting in seven experienced laboratories is feasible in the context of a scientific group. This cooperative nationwide strategy provides advanced molecular diagnostic for AML patients of the PETHEMA group (clinicaltrials gov. Identifier: NCT03311815)

Expression of Sirtuin 1 and 2 Is Associated with Poor Prognosis in Non-Small Cell Lung Cancer Patients

Sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2) are NAD+-dependent protein deacetylases involved in the regulation of key cancer-associated genes. In this study we evaluated the relevance of these deacetylases in lung cancer biology

Impact of FLT3–ITD Mutation Status and Its Ratio in a Cohort of 2901 Patients Undergoing Upfront Intensive Chemotherapy: A PETHEMA Registry Study

FLT3–ITD results in a poor prognosis in terms of overall survival (OS) and relapse-free survival (RFS) in acute myeloid leukemia (AML). However, the prognostic usefulness of the allelic ratio (AR) to select post-remission therapy remains controversial. Our study focuses on the prognostic impact of FLT3–ITD and its ratio in a series of 2901 adult patients treated intensively in the pre-FLT3 inhibitor era and reported in the PETHEMA registry. A total of 579 of these patients (20%) harbored FLT3–ITD mutations. In multivariate analyses, patients with an FLT3–ITD allele ratio (AR) of >0.5 showed a lower complete remission (CR rate) and OS (HR 1.47, p = 0.009), while AR > 0.8 was associated with poorer RFS (HR 2.1; p 0.5). Using the maximally selected log-rank statistics, we established an optimal cutoff of FLT3–ITD AR of 0.44 for OS, and 0.8 for RFS. We analyzed the OS and RFS according to FLT3–ITD status in all patients, and we found that the group of FLT3–ITD-positive patients with AR 0.44, allo-HSCT was superior to auto-HSCT in terms of OS and RFS. This study provides more evidence for a better characterization of patients with AML harboring FLT3–ITD mutations.Depto. de MedicinaFac. de MedicinaTRUEInstituto de Salud Carlos IIIFundación CRIS Contra el CáncerInstituto de Investigación Hospital 12 de OctubreUnión Europeapu

SIRT1 and SIRT2 are upregulated in NSCLC.

<p>(A, B) Immunoblotting of SIRT1 (A) and SIRT2 (B) in NSCLC cell lines and normal immortalized lung epithelial HBEC-3KT cells. The figure is representative of three different experiments. β-actin was used as control. (C-H) Representative immunohistochemical staining for SIRT1 (C, D, G) and SIRT2 (E, F, H) in normal human lung (C-F) and tumor (G, H) specimens expressing different levels of SIRT1 and SIRT2 proteins. In tumors, SIRT1 expression is predominantly nuclear whereas SIRT2 immunoreactivity is mostly localized in the cytoplasm. Bar = 50 μm.</p

Treatment with tnv-1 decreases growth of NSCLC cell lines.

<p>A, NSCLC cell lines were treated with 10 μM tnv-1. After three (3 d) and five (5 d) days the proliferation was measured by MTT assay. Relative proliferation values were obtained by dividing the absorbance values with those of the DMSO controls. Error bars represent standard deviation (SD). **, P < 0.01. B, Representative image of clonogenic assays (anchorage dependent growth). Cells were seeded and treated with tnv-1 (10 μM). After 10 days, colony formation was determined. C, Quantification of soft agar colony formation (anchorage independent growth) after tnv-1 treatment (10 μM) of NSCLC cell lines. Mean relative colony numbers and SD are shown. **, P < 0.01. D, Cell cycle distribution of NSCLC cells. Cells were treated with tnv-1 (10 μM) for 72 h, collected and analyzed by flow cytometry. E, Effect of tnv-1 (10 μM) on apoptosis. Cells were treated (48 h), collected and the cell death was assessed by Annexin V labeling. Error bars, SD.</p

Combination of high levels of SIRT1 and SIRT2 proteins predicts shorter RFS and OS.

<p>Kaplan-Meier curves of RFS (A, C, E) and OS (B, D, F) for SIRT1 (A, B), SIRT2 (C, D) and the combination of SIRT1 and SIRT2 (E, F) as assessed by immunohistochemical staining.</p

Tnv-1 regulates TP53, SIRT1 and CDKN1A expression levels in TP53 wild-type NSCLC cells.

<p>A, TP53 wild-type A549 and H460 cells were treated with 10 μM tnv-1 for three days (3 d) and five days (5 d). Levels of SIRT1 and TP53 were determined by Western blot analysis using β-actin as loading control. B and C, NSCLC cells were treated with 10 μM tnv-1 for 72 hours. Sirtuin 1 (B) and p21 (CDKN1A) mRNA expression (C) was determined by real-time PCR and normalized to IPO8 mRNA levels. Error bars, SD.</p

Clinicopathological features of 105 NSCLC patients.

<p>ADC, adenocarcinomas; SCC, squamous cell carcinoma; WD, well differentiated; MD, moderately differentiated; PD, poorly differentiated; SD, standard deviation.</p><p>Clinicopathological features of 105 NSCLC patients.</p

Downregulation of SIRT1 and SIRT2 inhibits proliferation of NSCLC cells.

<p>A, immunoblotting of SIRT1, SIRT2 and β-actin in A549 and H1299 cells transfected with scrambled siRNA (scr), siRNA targeting SIRT1 (siSIRT1 #1 or siSIRT1 #2) or SIRT2 (siSIRT2 #1 and siSIRT2 #2). The inhibition was verified after 72 h by Western blotting. B, A549 and H1299 cells were transfected with different siRNAs as indicated. MTT assay was performed 72 h post-transfection. The figure is representative of three different experiments: *, P < 0.05; **, P < 0.01.</p