Focal adhesion genes refine the intermediate-risk cytogenetic classification of acute myeloid leukemia

© 2018 by the authors.In recent years, several attempts have been made to identify novel prognostic markers in patients with intermediate-risk acute myeloid leukemia (IR-AML), to implement risk-adapted strategies. The non-receptor tyrosine kinases are proteins involved in regulation of cell growth, adhesion, migration and apoptosis. They associate with metastatic dissemination in solid tumors and poor prognosis. However, their role in haematological malignancies has been scarcely studied. We hypothesized that PTK2/FAK, PTK2B/PYK2, LYN or SRC could be new prognostic markers in IR-AML. We assessed PTK2, PTK2B, LYN and SRC gene expression in a cohort of 324 patients, adults up to the age of 70, classified in the IR-AML cytogenetic group. Univariate and multivariate analyses showed that PTK2B, LYN and PTK2 gene expression are independent prognostic factors in IR-AML patients. PTK2B and LYN identify a patient subgroup with good prognosis within the cohort with non-favorable FLT3/NPM1 combined mutations. In contrast, PTK2 identifies a patient subgroup with poor prognosis within the worst prognosis cohort who display non-favorable FLT3/NPM1 combined mutations and underexpression of PTK2B or LYN. The combined use of these markers can refine the highly heterogeneous intermediate-risk subgroup of AML patients, and allow the development of risk-adapted post-remission chemotherapy protocols to improve their response to treatment.Pallarès, VictorThis work was supported by Instituto de Salud Carlos III (Co-funding from FEDER) [CD13/00074 to V.P., PI15/00378 and PIE15/00028 to R.M., FIS PI17/01246, RD12/0036/0071 and FIS PI14/00450 to J.S., RD12/0036/0069 to M.G.., ISCIII-PS13/1640 and PI16/0665 to E.B., and RD12/0036/0014 and PIE13/00046 to M.A.S.] CIBERONC [CB16/12/00284 to M.A.S.]; CIBER-BBN [CBV6/01/1031 and Nanomets3 to R.M.]; AGAUR [2017 FI_B 00680 to A.F.; 2017-SGR-865 and 2014PROD0005 to R.M. and 2014-SGR-1281 to J.S.]; Fundació La Marató TV3 [416/C/2013-2030 to R.M., 100830/31/32 to J.S., M.G.. and M.A.S.]; Josep Carreras Leukemia Research Institute [P/AG 2017 to R.M.]; Spanish Health Research Program [PI12/02321 to M.G..]; Spanish Association Against Cancer (AECC) [to M.C.C.]; a grant from the Cellex Foundation, Barcelona [to J.S.]; a grant from La Generalitat de Catalunya (PERIS) [SLT002/16/00433 to J.S.]; and a grant from Fundación Española de Hematología y Hemoterapia (FEHH) [to V.P.]

Frequency and clinical impact of CDKN2A/ARF/CDKN2B gene deletions as assessed by in-depth genetic analyses in adult T cell acute lymphoblastic leukemia

© The Author(s).Recurrent deletions of the CDKN2A/ARF/CDKN2B genes encoded at chromosome 9p21 have been described in both pediatric and adult acute lymphoblastic leukemia (ALL), but their prognostic value remains controversial, with limited data on adult T-ALL. Here, we investigated the presence of homozygous and heterozygous deletions of the CDKN2A/ARF and CDKN2B genes in 64 adult T-ALL patients enrolled in two consecutive trials from the Spanish PETHEMA group. Alterations in CDKN2A/ARF/CDKN2B were detected in 35/64 patients (55%). Most of them consisted of 9p21 losses involving homozygous deletions of the CDKNA/ARF gene (26/64), as confirmed by single nucleotide polymorphism (SNP) arrays and interphase fluorescence in situ hybridization (iFISH). Deletions involving the CDKN2A/ARF/CDKN2B locus correlated with a higher frequency of cortical T cell phenotype and a better clearance of minimal residual disease (MRD) after induction therapy. Moreover, the combination of an altered copy-number-value (CNV) involving the CDKN2A/ARF/CDKN2B gene locus and undetectable MRD (≤ 0.01%) values allowed the identification of a subset of T-ALL with better overall survival in the absence of hematopoietic stem cell transplantation.This project was supported by the Asociación Española Contra el Cáncer, AECC (project ref.: GC16173697BIGA), by CERCA Program/Generalitat de Catalunya, the Catalan Government: 2014-SGR225 (GRE), Obra Social “La Caixa” and by Celgene Spain. E. Genescà is the recipient of agrant from the Spanish Health Ministry (ISCIII, CA12/00468) and an unrestricted grant from Gilead.A. Gonzalez-Perez is supported by a Ramon y Cajal fellowship (RYC-2013-14554) of the Educational Ministry (Madrid, Spain). This work was also partially supported by FEDER funds from the ISCIII (PT13/0010/0026, CIBERONC (CB16/12/00284 and CB16/12/00400), Madrid, Spain)

2001; 12

acute leukemia. CD123 is expressed with a characteristic pattern in cases of hairy cell leukemia. ©2001, Ferrata Storti Foundation Key words: acute leukemia, B-cell lymphoproliferative disorders, CD123 expression, IL-3 receptor α chain, hairy cell leukemia, minimal residual disease. © F e r r a t a S t o r t i F o u n d a t i o n er signal-regulatory proteins such as CD90, AC133 and CD117. Antigen expression is probably a gradually increasing or decreasing process reflecting the fluent maturation progress and at the end of this process differentiated cells display distinct patterns of antigen expression. 11 Acute myeloid leukemias (AMLs) are considered to be clonal disorders involving early hematopoietic progenitor cells. Differences and similarities in phenotype, genotype and biology are described for leukemic cells and normal hematologic progenitors. One potential difference between normal and leukemic cells lies in their response to hematopoietic growth factors. Design and Methods Patients Bone marrow (BM) n=12 , peripheral blood (PB) n=7 and non-neoplastic lymph nodes (LN) n=3, from healthy donors were used as controls. Sixtyfour BM samples from patients with acute leukemia (AL) were analyzed for the expression of the IL-3α chain receptor (IL-3αR). The patients were categorized as follows: 45 with acute myeloid leukemia (AML) and 19 with acute lymphoblastic leukemia (ALL), 13 with B-cell lineage ALL and 6 with T-cell lineage ALL. The diagnosis of AL was based on standard morphologic and immunophenotypic criteria. Flow cytometry analysis Sample preparation. The number of cells was quantified by microscopy and adjusted to 2×10 6 in each tube. The immunophenotypic analysis was performed on lysed whole BM samples with direct conjugated monoclonal antibodies (MoAbs). Antigen expression was analyzed using triple combinations of the following MoAbs conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinine chlorophyll protein (PerCp) or phycoerythrin-cyanine 5 (PE/Cy 5) fluorochrome tandem. The MoAbs used in the study were: CD22 (4KB128 FITC), glycophorin A (JC 159 PE), CD41 (5B 12 PE), IgM (rabbit anti-human, PE), CD79a (HM57 PE) and TDT (HT-6 FITC) from DAKO, Glostrup, Denmark; CD15 (MMA-FITC), CD34 (8G12-FITC, PE), HLA-Dr (L243 PetCp), CD10 (W8E7 FITC), CD 20 (L27 PE), CD2 (S5.2 FITC), CD33 (67.6 PE), CD7 (4H9 FITC), CD45 (2D1 PerCp), CD13 (L138 PE), CD14 (M0P9 FITC), CD3 (SK7 PerCp), CD4 (Leu 3 FITC), CD5 (Leu 1 FITC), CD8 (Leu 2 PE) purchased from Becton Dickinson, San José, CA, USA (BDIS); CD19 (SJ25-C1 PE/Cy 5) and MPO (H-43-5 FITC) from Caltag Laboratories, Burlingame, CA, USA; CD 123 (9F5 PE), CD10 (HI10a, Cy-Chrome) from Pharmingen, San Diego, CA, USA; CD36 (FAG-52 FITC) from Immunotech, Marseille, France. In B-CLPD samples a different panel of monoclonal antibodies was used to study the complete immunophenotype of each clonal sample: FMC7-FITC (Harlan SeraLab, Sussex), CD103-FITC (Immunoquality, Groningen), CD-10 PE-Cy5, CD123 (9F5 PE), (Pharmingen, San Diego, CA, USA), CD19-RPe/Cy5, CD22-FITC and CD79b-FITC (Dako), CD5-PE, CD23-PE, CD25-PE, CD20-PE, CD10-FITC, CD11c-PE, CD10-FITC, CD11c-PE (Becton Dickinson, San José, CA, USA). The clonality study of B-lymphocytes was undertaken using a triple reagent consisting of a combination of κ-FITC, λ-PE and CD19 PE/Cy5 in a single tube (K/L, Simultest® purchased from Becton Dickinson, San José, CA, USA and CD19-PE/Cy5 from Caltag, San Francisco, USA). Direct immunofluorescence was performed by incubating 2×10 6 cells with the specific MoAb for 15 minutes in the dark at room temperature. An isotype-matched negative control (BDIS) was used in all cases to assess background fluorescence intensity. Cells were lysed (FACS Lysis solution, BDIS) for 3 to 5 minutes and centrifuged at 250 g for 5 min. The cells were washed twice with phosphate buffered saline (PBS) before being resuspended in PBS and examined. Measurements were performed on a FACScan flow cytometer (BDIS). For data acquisition the LYSIS-II (BD) software program (BDIS) was used. At least 10,000 events/tube were measured. The PAINT-A-GATE PRO software program (BDIS) was employed for further data analysis. Thresholds for positivity were based on isotype negative controls. Analytical gates were set on desired viable cells based on forward light scatter and side light scatter. The positivity threshold was 20% for all markers except for cytoplasmic or intranuclear antigens for which a 10% threshold was used. The normal precursor cells were analyzed using a two-step acquisition procedure. In the first step, acquisition of 10,000 cells was performed and information stored for all these events. In the second step, a minimum of 300,000 cells was measured, information being stored only for the precursor cells, which were acquired employing a preestablished CD34 live gate. The level of fluorescence in cells which expressed CD123 was measured in arbitrary units as the mean fluorescence intensity (MFI). Results Normal expression of the IL-3 αR in bone marrow, peripheral blood and lymph nodes In control BM from healthy volunteers, CD123 was expressed in 0.27% (0.1%-0.6%) of the total nucleated cells. In the precursor cell compartment, the percentage of CD123 expression was 53%, ranging between 29% and 78% of the CD34 + cells. We studied the expression of this interleukin receptor in myeloid and lymphoid progenitor subpopulations. In the myeloid compartment (CD34 + CD33 + CD19 -) CD123 was positive in 63% (38%-100%) of cells with a dim-moderate intensity (MFI= 33 ± 21). In contrast, CD123 was negative in normal lymphoid progenitors (CD34 + CD33 -CD19 + CD10 + ) We also analyzed CD123 expression in PB. In our samples, cells with the strongest CD123 expression corresponded to dendritic cells, monocytes were positive for CD123 but showed a very faint reactivity and there was a minor population of B-cells expressing CD123. Granulocytes appeared to be CD123 negative. We tested the CD123 expression in 3 normal lymph nodes. In all the cases the CD19 + cells showed dim CD123 expression (MFI= 16± 12) and T-cells were CD123 negative. IL-3 αR expression in AML CD123 was expressed in 42 out of 45 AML samples assayed (93%). Regarding the FAB classification, CD123 expression was detected in all subtypes except in patients with a megakaryoblastic phenotype (n=2) The IL-3 αR pattern was similar in all the AML cases. CD123 was expressed in the totality of the blast cell population with a homogeneous pattern. The intensity of the expression was moderate (MFI= 69±40) ( CD34 expression was analyzed in the blast cells of AML patients. We found no differences in CD123 expression between CD34 + and CD34 -cases. Specific molecular lesions in AML were studied in order to detect differences in IL-3 αR. Six patients were PML/RARα positive, two patients had MLL rearrangements, and two patients were CBFβ/MYH11 positive. No differences in the CD123 pattern expression were found in patients with these molecular lesions. © F e r r a t a S t o r t i F o u n d a t i o n IL-3 αR expression in ALL In ALL samples, IL-3 αR was restricted to the Bcell lineage. Molecular lesions were studied in order to compare the CD123 expression in different biological subgroups. Three patients were bcr/abl positive and one patient had c-myc rearrangements. No differences in CD123 expression were found in these patients harboring specific molecular lesions. CD123 was expressed in all B-cell ALL samples (n=13) and this high expression was in striking contrast to lack of expression of CD123 on normal lymphoid precursors. The intensity of the expression in ALL was higher than in AML samples (MFI= 119±84) IL-3 αR expression in B-cell chronic lymphoproliferative disorders We analyzed IL-3 αR expression in 122 samples of BM, PB or lymph nodes from patients with B-CLPD. In the CLL group (n=77), 7 patients showed CD123 reactivity MFI= 41±42) ( Interestingly, 3 out of 4 patients with aggressive B-cell CLPD (two patients with transformed NHL from mantle and follicular lymphoma and one patient with Burkitt's lymphoma) expressed CD123 with a moderate intensity. The results in the hairy leukemia group were striking. In 6 out of 7 patients studied CD123 was positive with strong intensity, (MFI=120±288) ( © F e r r a t a S t o r t i F o u n d a t i o n Discussion Interleukin-3 is a regulatory glycoprotein known to support the survival, proliferation, and development of progenitor cells from multiple hematopoietic lineages. We investigated IL-3 αR expression in bone marrow and in peripheral blood from normal samples in order to establish normality patterns. In our study, normal lymphoid precursors did not show CD123 reactivity and only a proportion of myeloid progenitors expressed CD123 with a moderate intensity. These findings are in agreement with results from previous authors 13 who found a small CD123 expression in the normal CD34 + population while CD123 was negative in the more primitive CD34 + /CD38 -compartment. In contrast with the small expression of receptor in normal hematopoietic precursors, we found IL-3 αR expression in the majority of myeloid and B-cell leukemic cells. This feature may be useful in MRD studies. 14 Our data show that the combined use of CD123/CD34 with B-cell associated antigens such as CD19/CD10 may be a great help for identifying residual leukemic cells in B-cell leukemia cases. In addition, the CD123 + /CD34 + /CD33 + is a minor phenotype in normal BM. The detection of cells with this phenotype in a very superior number to the standard range could suggest the presence of myeloid leukemic cells. The wide expression of this marker in the majority of AML and B-cell lineage ALL cases analyzed and the low CD123 expression in normal hematopoietic stem cells suggest that IL-3 αR could be a suitable target for immunotherapeutic intervention. © F e r r a t a S t o r t i F o u n d a t i o n expressed in the majority of aggressive or transformed B-CLPD analyzed. We observed the acquisition of CD123 simultaneously with blast transformation in one patient with a mantle-cell lymphoma ( In conclusion, we have shown that CD123 is a good marker of myeloid and B-cell lymphoid leukemic cells and this feature could be used in MRD analysis. CD123 is expressed with a characteristic pattern in HCL cases and this antigen could be important in the differential diagnosis of B-CLPD. Nevertheless, further analysis is necessary to understand better the biological function of the IL-3 αR and the mechanisms by which this receptor transduces possible aberrant regulatory signals in malignant cells. If the biological role of the CD123 is confirmed, this antigen could be used as a target in the selective destruction of malignant cells in the majority of myeloid and B-cell leukemia patients. © F e r r a t a S t o r t i F o u n d a t i o n Contributions and Acknowledgments LM was primarily responsible for the article, was responsible for the analysis and interpretation of data and drafted the article. JFN was responsible for its conception and revised it critically for impo

Complex measurements may be required to establish the prognostic impact of immunophenotypic markers in AML

[Objectives]: The prognostic impact of immunophenotypic markers in acute myeloid leukemia (AML) is controversial. [Methods]: We retrospectively analyzed the value of CD34, CD117, CD7, and CD123 expression in a consecutive series of 592 adult patients with de novo AML. [Results]: CD34+ measured as a percentage (≥2.88%) and CD34 mean fluorescence intensity (MFI) (≥146.79, arbitrary units [AU]) expression had a prognostic impact in terms of overall survival (OS; P = .005, P = .003), leukemia-free survival (LFS; P = .011, P <.001), and cumulative incidence of relapse (CIR; P = .014, P =. 001). The percentage of CD117+ cells (61.29%) was associated with shorter LFS (P =. 043), and CD117 MFI (≥284.01 AU) was associated with a shorter OS (P =. 033) and LFS (P =. 028). In the multivariate analysis, high CD34 MFI retained the independent value as predictor of LFS and CIR (P =. 012; hazard ratio [HR], 1.59; 95% confidence interval [CI], 1.11- 2.28 and P =. 045; HR, 1.58; 95% CI, 1.01-2.46). [Conclusions]: CD34 positivity threshold with prognostic relevance is low (3% positive cells). Immunophenotypic findings in AML probably could only be fully exploited after a complex analysis that takes into account unconventional thresholds and the MFI.Peer Reviewe

NEDD 9 an independent good prognostic factor in intermediaterisk acute myeloid leukemia patients

Intermediate-risk acute myeloid leukemia (IR-AML) is the largest subgroup of AML patients and is highly heterogeneous. Whereas adverse and favourable risk patients have well-established treatment protocols, IR-AML patients have not. It is, therefore, crucial to find novel factors that stratify this subgroup to implement risk-adapted strategies. The CAS (Crk-associated substrate) adaptor protein family regulates cell proliferation, survival, migration and adhesion. Despite its association with metastatic dissemination and prognosis of different solid tumors, the role of these proteins in hematological malignancies has been scarcely evaluated. Nevertheless, previous work has established an important role for the CAS family members NEDD9 or BCAR1 in the migratory and dissemination capacities of myeloid cells. On this basis, we hypothesized that NEDD9 or BCAR1 expression levels could associate with survival in IR-AML patients and become new prognostic markers. To that purpose, we assessed BCAR1 and NEDD9 gene expression in a cohort of 73 adult AML patients validating the results in an independent cohort (n = 206). We have identified NEDD9, but not BCAR1, as a new a marker for longer overall and disease-free survival, and for lower cumulative incidence of relapse. In summary, NEDD9 gene expression is an independent prognostic factor for favourable prognosis in IR-AML patients.This work was supported by Instituto de Salud Carlos III (Co-funding from FEDER) [CD13/00074 to V.P., PI15/00378 to R.M., FIS PI11/00872, RD12/0036/0071 and FIS PI14/00450 to J.S., PIE15/00028 to R.M. and J.S., RD12/0036/0069 to M.G.D., ISCIII-PS13/1640 and RD12/0036/0014 to M.A.S.]; CIBER-BBN [CBV6/01/1031 and Nanomets3 to R.M.]; Generalitat de Catalunya [PERIS SLT002/16/00433 to J.S.]; AGAUR [2014-SGR-1041, 2014PROD0005 to R.M. and 2014-SGR-1281 to J.S.]; Fundació La Marató TV3 [416/C/2013-2030 to R.M., 100830/31/32 to J.S., M.G.D. and M.A.S.]; Josep Carreras Leukemia Research Institute [P/AG 2014 to R.M.]; Spanish Health Research Program [PI12/02321 to M.G.D.]; Spanish Association Against Cancer (AECC) [to MCC]; and a grant from the Cellex Foundation, Barcelona [to J.S.].Peer Reviewe

Molecular profiling refines minimal residual disease-based prognostic assessment in adults with Philadelphia chromosome-negative B-cell precursor acute lymphoblastic leukemia

Minimal residual disease (MRD) assessment is an essential tool in contemporary acute lymphoblastic leukemia (ALL) protocols, being used for therapeutic decisions such as hematopoietic stem cell transplantation in high‐risk patients. However, a significant proportion of adult ALL patients with negative MRD still relapse suggesting that other factors (ie, molecular alterations) must be considered in order to identify those patients with high risk of disease progression. We have identified partial IKZF1 gene deletions and CDKN2A/B deletions as markers of disease recurrence and poor survival in a series of uniformly treated adolescent and adult Philadelphia chromosome‐negative B‐cell progenitor ALL patients treated according to the Programa Español de Tratamientos en Hematología protocols. Importantly, CDKN2A/B deletions showed independent significance of MRD at the end of induction, which points out the need for treatment intensification in these patients despite being MRD‐negative after induction therapy.Fundació Internacional Josep Carreras; Generalitat de Catalunya, Grant/Award Number: 2017 SGR 288 GRC; Instituto de Salud Carlos III; Ministerio de Salud Carlos III RTICC‐FEDER, Grant/Award Numbers: RD12/0036/0029, RD/0036/044; Obra Social “La Caixa”; Sociedad Española de Hematología y Hemoterapia; Fondo de Investigaciones Sanitarias, Grant/Award Numbers: PI14/01971, PI10/0141

Bone marrow mesenchymal stem/stromal cells from risk-stratified acute myeloid leukemia patients are anti-inflammatory in in vivo preclinical models of hematopoietic reconstitution and severe colitis

financial support for this work was obtained from: Health Canada (H4084-112281) to PM and MR-M. The European Research Council (CoG-2014-646903) to PM. The Spanish Ministry of Economy and Competitiveness (SAF-SAF2013-43065), to PM. The Generalitat de Catalunya (SGR330 and PERIS 2017-2019 Program) to P.M. The Asociacion Espanola Contra el Cancer (AECC-CI-2015) and Fero Foundation to CB. The "Fundacion Hay Esperanza'' to E.A. The Health Institute Carlos III (ISCIII/FEDER/PI14-01191) to CB. PM also acknowledges the institutional support from The Obra Social La Caixa-Fundacio Josep Carreras. PM is investigator of the Spanish Cell Therapy cooperative network (TERCEL).Peer reviewe

Survival improvement of patients with FLT3 mutated acute myeloid leukemia: results from a prospective 9 years cohort

Abstract Midostaurin added to intensive chemotherapy is the standard of care for acute myeloid leukemia (AML) with FLT3 mutations (FLT3mut). We analyzed the impact of midostaurin in 227 FLT3mut-AML patients included in the AML-12 prospective trial for fit patients ≤70 years (#NCT04687098). Patients were divided into an early (2012–2015) and late (2016–2020) cohorts. They were uniformly treated except for the addition of midostaurin in 71% of late group patients. No differences were observed in response rates or the number of allotransplants between groups. Outcome was improved in the late period: 2-year relapse incidence decreased from 42% vs 29% in early vs late group (p = 0.024) and 2-year overall survival (OS) improved from 47% vs 61% (p = 0.042), respectively. The effect of midostaurin was evident in NPM1mut patients (n = 151), with 2-yr OS of 72% (exposed) vs 50% (naive) patients (p = 0.011) and mitigated FLT3-ITD allelic ratio prognostic value: 2-yr OS with midostaurin was 85% and 58% in low and high ratio patients (p = 0.049) vs 67% and 39% in naive patients (p = 0.005). In the wild-type NPM1 subset (n = 75), we did not observe significant differences between both study periods. In conclusion, this study highlights the improved outcome of FLT3mut AML fit patients with the incorporation of midostaurin

The poor prognosis of low hypodiploidy in adults with B-cell precursor acute lymphoblastic leukaemia is restricted to older adults and elderly patients

The prognostic significance of low‐hypodiploidy has not been extensively evaluated in minimal residual disease (MRD)‐oriented protocols for adult acute lymphoblastic leukaemia (ALL). We analysed the outcome of hypodiploid adult ALL patients treated within Programa Español de Tratamientos en Hematología (PETHEMA) protocols. The 5‐year cumulative incidence of relapse (CIR) of low‐hypodiploid B‐cell precursor (BCP)‐ALL was significantly higher than that of high‐hypodiploids (52% vs. 12%, P = 0.013). Low‐hypodiploid BCP‐ALL patients aged ≤35 years showed superior survival (71% vs. 21%, P = 0.026) and lower 5‐year CIR (17% vs. 66%, P = 0.090) than low‐hypodiploids aged >35 years. Older adults and elderly low‐hypodiploid BCP‐ALL patients show dismal prognosis although achieving an end‐induction good MRD response.This work was supported in part by grants from Asociación Española Contra el Cáncer, AECC (GC16173697BIGA), Instituto de Salud Carlos III (PI14/01971 FI), 2017‐SGR288 (GRC), CERCA Program from Generalitat de Catalunya and “La Caixa” Foundation