Open Access
Impact of FLT3-ITD mutation status and its ratio in a cohort of 2901 patients undergoing upfront intensive chemotherapy: a PETHEMA registry study
(MDPI, 2022-11-24) Ayala, Rosa; Carreño-Tarragona, Gonzalo; Barragán, Eva; Boluda, Blanca; Larráyoz, María José; Chillón, María Carmen; Carrillo-Cruz, Estrella; Bilbao, Cristina; Sánchez-García, Joaquín; Bernal, Teresa; Martínez-Cuadrón, David; Gil, Cristina; Serrano, Josefina; Rodríguez-Medina, Carlos; Bergua, Juan; Pérez-Simón, José A.; Calbacho, María; Alonso-Domínguez, Juan M.; Labrador, Jorge; Tormo, Mar; Amigo, María Luz; Herrera-Puente, Pilar; Rapado, Inmaculada; Sargas, Claudia; Vázquez Urio, Iria; Calasanz, María José; Gómez-Casares, Teresa; García-Sanz, Ramón; Sanz, Miguel A.; Martínez-López, Joaquín; Montesinos, Pau; Ciencias; Zientziak
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.
Open Access
NGS-based molecular karyotyping of multiple myeloma: results from the GEM12 clinical trial
(MDPI, 2022-10-21) Rosa-Rosa, Juan Manuel; Cuenca, Isabel; Medina, Alejandro; Vázquez Urio, Iria; Sánchez-delaCruz, Andrea; Buenache, Natalia; Sánchez Pérez, Ricardo; Jiménez, Cristina; Rosiñol, Laura; Gutiérrez, Norma C.; Ruiz-Heredia, Yanira; Barrio Santiago; Oriol, Albert; Martín-Ramos, María-Luisa; Blanchard, María-Jesús; Ayala, Rosa; Ríos-Tamayo, Rafael; Sureda, Anna; Hernández, Miguel-Teodoro; Rubia, Javier de la; Alkorta Aranburu, Gorka; Agirre, Xabier; Bladé, Joan; Mateos, María-Victoria; Lahuerta, Juan-José; San-Miguel, Jesús F.; Calasanz, María José; García-Sanz, Ramón; Martínez-López, Joaquín; Ciencias; Zientziak
Next-generation sequencing (NGS) has greatly improved our ability to detect the genomic aberrations occurring in multiple myeloma (MM); however, its transfer to routine clinical labs and its validation in clinical trials remains to be established. We designed a capture-based NGS targeted panel to identify, in a single assay, known genetic alterations for the prognostic stratification of MM. The NGS panel was designed for the simultaneous study of single nucleotide and copy number variations, insertions and deletions, chromosomal translocations and V(D)J rearrangements. The panel was validated using a cohort of 149 MM patients enrolled in the GEM2012MENOS65 clinical trial. The results showed great global accuracy, with positive and negative predictive values close to 90% when compared with available data from fluorescence in situ hybridization and whole-exome sequencing. While the treatments used in the clinical trial showed high efficacy, patients defined as high-risk by the panel had shorter progression-free survival (p = 0.0015). As expected, the mutational status of TP53 was significant in predicting patient outcomes (p = 0.021). The NGS panel also efficiently detected clonal IGH rearrangements in 81% of patients. In conclusion, molecular karyotyping using a targeted NGS panel can identify relevant prognostic chromosomal abnormalities and translocations for the clinical management of MM patients.
Open Access
The genomic profiling of high-risk smoldering myeloma patients treated with an intensive strategy unveils potential markers of resistance and progression
(Springer Nature, 2024-04-29) Medina-Herrera, Alejandro; Vázquez Urio, Iria; Cuenca, Isabel ; Rosa-Rosa, Juan Manuel; Ariceta, Beñat; Jiménez, Cristina; Fernández-Mercado, Marta; Larráyoz, María José; Gutiérrez, Norma C.; Fernández-Guijarro, M.; González-Calle, Verónica; Rodríguez-Otero, Paula; Oriol, Albert; Rosiñol, Laura; Alegre, Adrián; Escalante, Fernando; Rubia, Javier de la; Teruel, A. I.; Arriba, Felipe de; Hernández, Miguel-Teodoro; López-Jiménez, Javier; Ocio, Enrique María; Puig, Noemí; Paiva, Bruno David Lourenço; Lahuerta, Juan-José; Bladé, Joan; San-Miguel, Jesús F.; Mateos, María-Victoria; Martínez-López, Joaquín; Calasanz, María José; García-Sanz, Ramón; GEM/PETHEMA (Grupo Español de Mieloma/Programa para el Estudio de la Terapéutica en Hemopatías Malignas) cooperative study group; Ciencias; Zientziak
Smoldering multiple myeloma (SMM) precedes multiple myeloma (MM). The risk of progression of SMM patients is not uniform, thus different progression-risk models have been developed, although they are mainly based on clinical parameters. Recently, genomic predictors of progression have been defined for untreated SMM. However, the usefulness of such markers in the context of clinical trials evaluating upfront treatment in high-risk SMM (HR SMM) has not been explored yet, precluding the identification of baseline genomic alterations leading to drug resistance. For this reason, we carried out next-generation sequencing and fluorescent in-situ hybridization studies on 57 HR and ultra-high risk (UHR) SMM patients treated in the phase II GEM-CESAR clinical trial (NCT02415413). DIS3, FAM46C, and FGFR3 mutations, as well as t(4;14) and 1q alterations, were enriched in HR SMM. TRAF3 mutations were specifically associated with UHR SMM but identified cases with improved outcomes. Importantly, novel potential predictors of treatment resistance were identified: NRAS mutations and the co-occurrence of t(4;14) plus FGFR3 mutations were associated with an increased risk of biological progression. In conclusion, we have carried out for the first time a molecular characterization of HR SMM patients treated with an intensive regimen, identifying genomic predictors of poor outcomes in this setting.
Open Access
Integrated flow cytometry and sequencing to reconstruct evolutionary patterns from dysplasia to acute myeloid leukemia
(American Society of Hematology, 2023-01-10) Simoes, Catia; Chillón, María Carmen; Martínez-Cuadrón, David; Calasanz, María José; Vridiales, María-Belén; Vázquez Urio, Iria; Hernández-Ruano, Montserrat; Ariceta, Beñat; Aguirre-Ruiz, Paula; Burgos, Leire; Alignani, Diego; Sarvide, Sarai; Villar, Sara; Alfonso Pierola, Ana; Prósper, Felipe; Ayala, Rosa; Martínez-López, Joaquín; Bergua Burgués, Juan Miguel; Vives, Susana; Pérez-Simón, José A.; García-Fortes, María; Bernal del Castillo, Teresa; Colorado, Mercedes; Olave, Mayte; Rodríguez-Gutiérrez, Juan I.; Labrador, Jorge; Gonzalez Díaz, Marcos; San-Miguel, Jesús F.; Sanz, Miguel A.; Montesinos, Pau; Paiva, Bruno David Lourenço; Ciencias; Zientziak
Clonal evolution in acute myeloid leukemia (AML) originates long before diagnosis and is a dynamic process that may affect survival. However, it remains uninvestigated during routine diagnostic workups. We hypothesized that the mutational status of bone marrow dysplastic cells and leukemic blasts, analyzed at the onset of AML using integrated multidimensional flow cytometry (MFC) immunophenotyping and fluorescence-activated cell sorting (FACS) with next-generation sequencing (NGS), could reconstruct leukemogenesis. Dysplastic cells were detected by MFC in 285 of 348 (82%) newly diagnosed patients with AML. Presence of dysplasia according to MFC and World Health Organization criteria had no prognostic value in older adults. NGS of dysplastic cells and blasts isolated at diagnosis identified 3 evolutionary patterns: stable (n = 12 of 21), branching (n = 4 of 21), and clonal evolution (n = 5 of 21). In patients achieving complete response (CR), integrated MFC and FACS with NGS showed persistent measurable residual disease (MRD) in phenotypically normal cell types, as well as the acquisition of genetic traits associated with treatment resistance. Furthermore, whole-exome sequencing of dysplastic and leukemic cells at diagnosis and of MRD uncovered different clonal involvement in dysplastic myelo-erythropoiesis, leukemic transformation, and chemoresistance. Altogether, we showed that it is possible to reconstruct leukemogenesis in ~80% of patients with newly diagnosed AML, using techniques other than single-cell multiomics.
Open Access
Assessment of minimal residual disease by next generation sequencing in peripheral blood as a complementary tool for personalized transplant monitoring in myeloid neoplasms
(MDPI, 2020) Aguirre-Ruiz, Paula; Ariceta, Beñat; Viguria Alegría, María Cruz; Zudaire, María Teresa; Blasco-Iturri, Zuriñe; Arnedo, Patricia; Aguilera-Diaz, Almudena; Jauregui, Axier; Mañú, Amagoia; Prósper, Felipe; Mateos, María Carmen; Fernández-Mercado, Marta; Larráyoz, María José; Redondo, Margarita; Calasanz, María José; Bandrés Elizalde, Eva; Vázquez Urio, Iria; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua
Patients with myeloid neoplasms who relapsed after allogenic hematopoietic stem cell transplant (HSCT) have poor prognosis. Monitoring of chimerism and specific molecular markers as a surrogate measure of relapse is not always helpful; therefore, improved systems to detect early relapse are needed. We hypothesized that the use of next generation sequencing (NGS) could be a suitable approach for personalized follow-up post-HSCT. To validate our hypothesis, we analyzed by NGS, a retrospective set of peripheral blood (PB) DNA samples previously evaluated by high-sensitive quantitative PCR analysis using insertion/deletion polymorphisms (indel-qPCR) chimerism engraftment. Post-HCST allelic burdens assessed by NGS and chimerism status showed a similar time-course pattern. At time of clinical relapse in 8/12 patients, we detected positive NGS-based minimal residual disease (NGS-MRD). Importantly, in 6/8 patients, we were able to detect NGS-MRD at time points collected prior to clinical relapse. We also confirmed the disappearance of post-HCST allelic burden in non-relapsed patients, indicating true clinical specificity. This study highlights the clinical utility of NGS-based post-HCST monitoring in myeloid neoplasia as a complementary specific analysis to high-sensitive engraftment testing. Overall, NGS-MRD testing in PB is widely applicable for the evaluation of patients following HSCT and highly valuable to personalized early treatment intervention when mixed chimerism is detected.
Open Access
Whole exome sequencing or pan-myeloid NGS gene panel to assess leukemic evolution of myelodysplastic syndromes: advantages and disadvantages
(Biomedical Research Network, 2022-01-12) Aguilera-Diaz, Almudena; Ariceta, Beñat; Vázquez Urio, Iria; Larráyoz, María José; Aguirre-Ruiz, Paula; Mañú, Amagoia; Calasanz, María José; Fernández-Mercado, Marta; Ciencias; Zientziak
NGS gene panels interrogating few genes clinically relevant for a specific disease are being extensively used for diagnosis, prognosis, and treatment. However, when panel results are inconclusive, some laboratories recommend the use of Whole Exome sequencing (WES), or WES is even offered as the first technique to genetically assess patient condition. In this short communication we report a comparison of WES and a myeloid NGS gene panel data from 16 samples of 8 cases with Myelodysplastic Syndromes (MDS) that evolved to Acute Myeloid Leukemia (AML), addressing their advantages and disadvantages from both technical and clinical point of view. On the one hand, our data show a loss of clinically relevant variants sequenced with WES that were indeed called by the NGS panel at a low Variant Allele Frequency (VAF); this finding was not surprising since WES was sequenced at an average depth of 250x, while the NGS panel was sequenced at an average depth of 4500X. On the other hand, WES called a likely pathogenic variant in GNAS p. Arg844Cys, missed by the panel due to design constraints. Therefore, based in our data, both techniques were complementary and therefore potentially clinically valuable: WES for the discovery of new variants, and NGS gene panels for the detection of emerging clones, which gives a more precise image of the tumor clonal heterogeneity.