Summary
Refractoriness to induction therapy and relapse after complete remission are the leading causes of death in patients with acute myeloid leukaemia (AML). This study focussed on the prediction ...of response to standard induction therapy and outcome of patients with AML using a combined strategy of mutational profiling by next‐generation sequencing (NGS, n = 190) and ex vivo PharmaFlow testing (n = 74) for the 10 most widely used drugs for AML induction therapy, in a cohort of adult patients uniformly treated according to Spanish PETHEMA guidelines. We identified an adverse mutational profile (EZH2, KMT2A, U2AF1 and/or TP53 mutations) that carries a greater risk of death hazard ratio (HR): 3·29, P < 0·0001. A high correlation was found between the ex vivo PharmaFlow results and clinical induction response (69%). Clinical correlation analysis showed that the pattern of multiresistance revealed by ex vivo PharmaFlow identified patients with a high risk of death (HR: 2·58). Patients with mutation status also ran a high risk (HR 4·19), and the risk was increased further in patients with both adverse profiles (HR 4·82). We have developed a new score based on NGS and ex vivo drug testing for AML patients that improves upon current prognostic risk stratification and allows clinicians to tailor treatments to minimise drug resistance.
Next-generation sequencing (NGS) has redefined the genetic landscape of acute myeloid leukemia (AML), providing new molecular markers for diagnostic and prognostic classifications. However, its ...application in the clinical setting is still challenging. We hypothesized that a 19-gene AML-targeted NGS panel could be a valid approach to obtain clinically relevant information. Thus, we assessed the ability of this panel to classify AML patients according to diagnostic and prognostic indexes in a cohort of 162 patients. The assay yielded a median read depth >2000×, with 88% of on-target reads and a mean uniformity >93% without significant global strand bias. The method was sensitive and specific, with a valid performance at the clinical variant allele frequency cutoff of 3% for point mutations and 5% for insertions or deletions (INDELs). Three-hundred thirty-nine variants were found (36% INDELs and 64% single nucleotide variants). Concordance between NGS and other conventional techniques was 100%, but the NGS approach was able to identify more clinically relevant mutations. Finally, all patients could be classified into one of the 2016 World Health Organization diagnostic categories and virtually all into the recently proposed prognostic indexes (2017 European LeukemiaNet and Genomic classification). To sum up, we validate a reliable and reproducible method for AML diagnosis and demonstrate that small, well-designed NGS panels are sufficient to guide clinical decisions according to the current standards.
Abstract The MYBL2 gene encodes a transcription factor implicated in cell proliferation and maturation whose amplification or overexpression has been associated with different human malignancies, ...suggesting that it could be implicated in tumorigenesis. We analyzed MYBL2 expression and its prognostic value in 291 patients with de novo acute myeloid leukemia (AML) and we also evaluated its association with microRNAs 29 and 30 families. MYBL2 expression in AML patients was increased relative to CD34+ cells. Moreover, MYBL2 overexpression was associated with lower expression of miR-30a ( P = 0.024), miR-30b ( P = 0.021) and miR-30c ( P = 0.009). Multivariate analysis showed that MYBL2 expression was an independent factor for disease-free survival (HR 3.0, 95% CI 1.5–6.0, P = 0.002) and cumulative incidence of relapse (HR 2.6, 95% CI 1.2–5.6, P = 0.015) in patients with an intermediate-risk karyotype. In conclusion, our data showed that MYBL2 expression analysis could be useful to define subgroups of patients with poor prognosis.
Older patients with acute myeloid leukaemia (AML) who are unsuitable for standard induction therapy have limited treatment options. While DNMT3A, TET2, IDH1/2 and TP53 mutations have been previously ...associated to better response to hypomethylating agents, there are no molecular biomarkers for low-dose cytarabine (LDAC)-based regimens.
To predict outcome in AML older patients at diagnosis based on mutation status in the context of FLUGAZA trial. FLUGAZA trial was focus on >65 years AML de novo patients comparing azacytidine vs. fludarabine and LDAC (FLUGA Scheme).
We analyzed bone marrow (BM) samples at diagnosis from 209 out of 285 AML patients treated according Flugaza trial (NCT02319135), azacytidine-arm (n=97) and FLUGA-arm (n=112). In this trial, patients were randomized to receive 3 induction cycles with fludarabine and cytarabine (FLUGA) followed by 6 consolidation cycles with reduced intensity FLUGA, vs 3 induction cycles with 5-azacytidine (AZA) followed by 6 consolidation cycles with AZA. Median age at diagnosis was 75 years (65-90). Both treatment groups were balanced for age, leucocytes count, baseline BM blasts, karyotype risk (ELN), and FLT3-internal tandem duplication and NPM1 gene mutations. Mutational profile analysis was carried out by NGS targeted gene sequencing (Ion Torrent S5XL System-Thermo Fisher Scientific) using a 43 genes custom panel implicated in leukemia prognosis.
We detected 893 variants, 247 Indels and 646 SNVs. 206 (23.1%) of them were included as pathogenic or like-pathogenic by clinvar database. Ninety-five percent of patients (n=203) had at least one detectable mutation, and the median number of mutations was 4 (range = 0-8 mutations). The most common gene mutations were TET2 (N=55), FLT3 (n=52), SRSF2 (n=49), TP53 (n=45), DNMT3A (n=45), ASXL1 (n=45), RUNX1 (n=43), IDH2 (n=36), IDH1 (n=34), NPM1, (n=33) and NRAS (n=23). This mutational landscape is different to previous published in younger patients (Grimwade, Blood 2016), with higher number of patients with mutations in TP53 (21.5 vs 8%), SRSF2 (23.9 vs 2%), IDH1 (16.3 vs 7%) and IDH2 (17.2 vs 9%) and lower number of patients with mutation in NMP1 (15.8 vs 33%).
The median OS of global series was 6 months (range 0-40). Multivariate Cox regression in the global series showed that NRAS and TP53 mutations predict reduced OS (Table 1). Distribution of mutations between both arms was not homogeneous (Figure 1) and NRAS (p=0.012) was more frequent among patients randomized to the FLUGA-arm. However, TP53 mutation frequency distribution was homogeneous: 23.7% in AZA-arm and 19.6% in FLUGA-arm (p=NS).
In the AZA-arm, patient´s age was the only variable associated with not achieving composite complete remission (CR plus CR with incomplete recovery) and TET2 and EZH2 mutations were predictors to achieve composite CR. In the FLUGA-arm, TP53 and NRAS mutations were associated with not reaching composite CR (table 2).
In the AZA-arm, cytogenetic was the only variable associated with risk of early death. In the FLUGA-arm, leucocyte count, TP53 and NRAS mutations were associated with risk of early death (table 3).
In the AZA-arm, BCORL1 mutations (4.1%) were the only variable associated with high risk of relapse. In the FLUGA-arm, BCOR (7.1%) and TP53 (19.6%) mutations were associated with high risk of relapse (table 4).
CONCLUSION
The mutational profile of AML in elderly patients is different from the previously published in young patients. We have confirmed that a molecular pattern can identify patients with poor prognosis in elderly AML patients. NRAS and TP53 mutations confer a poor prognosis in LDAC (FLUGA-arm) patients, but this effect disappeared in the AZA-arm. BCOR and BCORL1 mutations were associated to a reduced DFS.
These results confirm that azacytidine could be more efficacious than LDAC treatment for older patients with AML and mutations in TP53, NRAS, TET2 and EZH2. The percentage of patients who presented mutations in these genes amounted to 77% in this AML series.
The study is registered at www.ClinicalTrials.gov as NCT02319135.
This study was supported by the Subdirección General de Investigación Sanitaria (ISCIII, Spain) grants PI13/02387 and PI16/01530.
Display omitted
Salamero:Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Daichii Sankyo: Honoraria. Paiva:Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau. Fernandez:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.
To characterize and describe clinical experience with childhood-onset non-infectious uveitis.
A multicenter retrospective multidisciplinary national web-based registry of 507 patients from 21 ...hospitals was analyzed. Cases were grouped as immune disease-associated (IMDu), idiopathic (IDIu) or ophthalmologically distinct. Characteristics of juvenile idiopathic arthritis-associated (non-HLA-B27-related) uveitis (JIAu), IDIu, and pars planitis (PP) were compared.
IMDu (62.3%) and JIAu (51.9%) predominated in young females; and IDIu (22.7%) and PP (13.6%) in older children, without sex imbalance. Ocular complications occurred in 45.3% of cases (posterior synechiae 28%, cataracts 16%, band keratopathy 14%, ocular hypertension 11% and cystoid macular edema 10%) and were associated with synthetic (86%) and biologic (65%) disease-modifying antirheumatic drug (DMARD) use. Subgroups were significantly associated (
< 0.05) with different characteristics. JIAu was typically anterior (98%), insidious (75%), in ANA-positive (69%), young females (82%) with fewer complications (31%), better visual outcomes, and later use of uveitis-effective biologics. In contrast, IDIu was characteristically anterior (87%) or panuveitic (12.1%), with acute onset (60%) and more complications at onset (59%: synechiae 31% and cataracts 9.6%) and less DMARD use, while PP is intermediate, and was mostly bilateral (72.5%), persistent (86.5%) and chronic (86.8%), with more complications (70%; mainly posterior segment and cataracts at last visit), impaired visual acuity at onset, and greater systemic (81.2%), subtenon (29.1%) and intravitreal (10.1%) steroid use.
Prognosis of childhood uveitis has improved in the "biologic era," particularly in JIAu. Early referral and DMARD therapy may reduce steroid use and improve outcomes, especially in PP and IDIu.
Background.
The AML is a heterogeneous disease with a high variety of subtypes stratified by cytogenetic and molecular markers. To date, the major prognosis criteria is cytogenetic, but 40-50% of de ...novo AML patients have normal karyotype; AML treatment is based on risk profiles depending on age and genetic molecular factor. New methods such as Next Generation Sequencing (NGS) and ex vivo sensitivity drug tests might be able to improve this classification and to individualize the treatment for AML patients.
Aims.
We performed targeted massively parallel sequencing on a 32 myeloid genes custom panel to identify a mutational profile of AML which predicts ex vivo pharmacological and clinical response.
Methods.
We analyzed bone marrow (BM) samples at diagnosis from 39 AML patients, 34 were treated with chemotherapy; 28 AML patients received one cycle of Idarubicin (Ida)/ Cytarabine(Cyt) induction treatment (3+7) and 8 received one cycle of Fludarabine/Cyt. Median age at diagnosis was 53 years (24-81), male:female ratio 19:16.
We carried out targeted gene sequencing by NGS (Ion Torrent Proton System-Life Technologies) using a 32 genes custom panel (all coding regions) implicated in leukemia prognosis, including ASXL1, CBL, DNMT3A, EPOR, ETV6, EZH2, FLT3, HRAS, IDH1, IDH2, JAK2, KDM6A, KIT, KRAS, LNK, MLL, MPL, NRAS, PHF6, PRPF40B, PTEN, RUNX1, SF1, SF3A1, SF3B1, SRSF2, TET2, TP53, U2AF35, VHL, ZRSR2 and CALR.
Ex vivo pharmacological studies were performed using an innovative automated flow cytometry method based platform (Ex-viTech) is able to evaluate effect of drugs used to treat AML, Cyt and Ida, using 4 parameter: Emax (Effective Maximum Response) measures potency, EC50 (Effective Concentration inducing 50% cell death) measures efficacy, and the activity was quantified by AUC (Area Under Curve) and VUS (Volume Under Superficies).
Clinical response was evaluated after induction treat. Complete remission was defined according to hematologic recovery and blast count fewer than 5%.
Analysis of NGS results was performed by Ion Report software. Differences measured by Exvitech were determined using t-test or Kruskal-Wallis test. Discrete variables of patients with and without gene variants were compared using the X2 test. For survival analysis we used Kaplan-Meier analysis (log rank) using SPSS 15. Two sided p values below 0.05 were considered statistically significant.
Results.
We found 94 non-synonymous variants (SNVs and small Indels) in coding regions. On average, 90.8% of the target sequence showed mean depth coverage ~ 1100x. See Figure 1.
We analyzed results of pharmacological test by Ex-viTech plataform in function of presence of gene variants. Only significant favorable differences were detected: KRAS (Cyt-Emax p=0,000, Ida-EC50 p=0.001, VUS p=0.003, Cyt-AUC p=0.036, Ida-AUC p=0.000), KIT (VUS p=0,010) and FLT3-TKD (Cyt-Emax p=0.002, Cyt-AUC p=0.001, VUS p=0.011).
Mutational positive score, found it in 40% of patients, was defined by the presence of variants in KRAS, KIT and/or FLT3-TKD; it was correlated with pharmacological results and clinical response. All cases with positive score (n=13) achieve complete remission (CR); only 11 cases without favorable score (n=21) achieve RC (p=0.003) after induction treatment (See Table 2). In addition, response obtained for Ida Emax and Ida EC50 by Ex-viTech was able to detect patients who reached CR after induction treatment (p=0.042, p=0,039 respectively).
Ex-viTech results showed that patients with positive score have a significantly more potent and effective cytotoxic effect than patients without it. See table 2.
No differences were observed respect Overall Survival (OS) between cases with mutational favorable score and without it. However, we observed a plateau in OS with favorable mutational score. With a median follow-up of 5.9 months (1.9-25.74), patients having positive score showed better relapse free survival (p = 0.013). See Figure 3.
Conclusion.
Very deep sequencing NGS identifies a mutational profile score (KRAS, KIT or FLT3-TKD) in AML patients, which predicts ex vivo pharmacological and clinical response. This favorable score was found in 40% of AML patients. Merging NGS and ex vivo drug sensibility might be able early to predict response to induction and individualize treatments.
This study was funded by Instituto Carlos III (PI13/02387).
Display omitted Display omitted Display omitted
Ballesteros:Vivia Biotech: Employment.
Introduction: Allogeneic transplantation is the only curative option for patients with high risk leukemias or MDS. Only one third of them have an HLA identical sibling donor and around 60-70% will ...find an unrelated donor, that´s why haploidentical stem cell transplantation (HAPLO-HSCT) offers a therapeutic option to most of these patients. Myeloablative conditioning (MAC) produce better disease control than reduced intensity conditioning regimens (RIC), but with higher toxicity, rendering long term similar results.
Patients and methods: We retrospectively evaluated the results of our MAC-HAPLO regimens in patients diagnosed with high risk leukemias or MDS (Fludarabine 30 mg/m2 x5 days, Cyclophosphamide14,5 mg/kg x2 days, IV Busulfan 3,2 mg/kg x 3 days (BUX3), or Fludarabine 40 mg/m2 x4 days and IV Busulfan 3,2 mg/kg x 4 days (BUX4)) with GVHD prophylaxis based on PT-CY (50 mg/kg on days +3 and +4) and a calcineurin inhibitor plus mycophenolate from day +5, performed in GETH centers (Spanish Group for Hematopoietic Transplantation).
Results: From Feb-2012, 65 MAC-HAPLO HSCT have been reported by 14 centers. Median age was 41 years (15-67), 66% were males and all were in advanced disease phase or presented high risk features (AML 47/ALL 8/MDS 5/ Others 5). Previous HSCT had been employed in 12% (autologous in 5, allogeneic in 3), and in 88% the HAPLO-HSCT was their first transplant. Disease status at HAPLO-HSCT was morphologic CR in 80%, but disease persisted in 52% (MRD+ by flow or molecular markers 32%, morphologic disease 20%). Their disease risk index (DRI) was high or very high in 65%, and the comorbidity index (HCT-CI) was higher than 2 in 18%. PBSC was the graft source in 56 (86%), non T-cell depleted in all cases. The haploidentical donor was the patient´s mother (21.5%), father (12%), siblings (43%) or offspring (23%). MAC regimen was BUX3 in 25 (38.5%) and BUX4 in 40 patients (61.5%). Median infused CD34+ cells were 5.31 x10e6/kg (2.75-11.42). There were no graft failures. Median neutrophils engraftment was reached at day +17 (12-29) and platelets >20K at day +26 (11-150). Complete chimerism was obtained at a median of 28 days (13-135) in 60 evaluated patients. Cumulative incidence (CI) of non-relapse mortality (NRM) was 12.5% at day +100 and 19% at 1 year. CI of grade II-IV acute GVHD was 28.5% at day +100, and grade III-IV was 6.5%. CI of chronic GVHD at 2 years was 28%, being extensive in 8% . No differences in acute or chronic GvHD CI were seen when comparing BUX3 against BUX4. After a median follow-up of 17 months (5-50), estimated 24-months event-free survival (EFS) and overall survival (OS) were 58,5% and 60% respectively. CI of relapse or progression was 21%. No significant differences in NRM, EFS, OS and relapse incidence were detected between BUX3 and BUX4. The impact of CR prior to MAC-HAPLO, the DRI or chronic GvHD in the disease control have not been apropiately demostrated probably due to the limited number of events in our series.
Conclusions: IV Busulfan based MAC-HAPLO with PT-CY in the treatment of high risk leukemias and MDS offers good disease control with manageable toxicity, with either BUX3 or BUX4. These efective MAC regimens combined with peripheral blood HAPLO donors could control high risk hematologic diseases in the long term. Severe acute or chronic GvHD are low frecuency events, but relapses persist as the main problem in this patients population.
No relevant conflicts of interest to declare.
PDF
