Frequent Mutations and Their Influence on Prognosis in Patients with Acute Myeloid Leukemia

Abstract Background. Several genetic alterations such as translocations, gene mutations and deletions play an important role in myeloid leukemogenesis. The cytogenetic information is a very significant tool to classify pts at their initial diagnosis into prognostic categories. For pts with cytogenetically normal AML, prognosis can be specified by mutational status of the genes NPM1, FLT3, CKIT, NRAS and DNMT3A. The aim of the research was to investigate the frequency and prognostic impact of FLT3, NPM1, CKIT, NRAS and DNMT3A mutations in AML pts and to analyze their interaction with other prognostic markers. Methods. This study was performed in 200 adult pts (190 pts with de novo and 10 pts with secondary AML), previously untreated, median age 55 years (18-86). According to the results of cytogenetic analyses pts were separated in four groups: with favourable (9,0%), unfavourable (14,0%) prognosis, with normal karyotype (NK) (48,5%) and other aberrations (28,5%). Mutations in FLT3, CKIT and NPM1 were analysed by PCR and in NRAS by sequencing. Mutation analysis of DNMT3A R882 was performed by high-resolution melting curve analysis. Cytogenetic studies were analysed on bone marrow samples using standard GTG-method. Results. Mutations in FLT3, CKIT, NRAS and NPM1 genes were detected in 105/200 (52,5%) pts. A total of 128 mutations were revealed in this group: 24,0% - FLT3-ITD, 6,5% - FLT3-TKD, 20,5% - in NPM1, 10,0% - in NRAS and 3,0% - in CKIT. 82 pts had single mutations and in 23 pts mutations occurred simultaneously: 17 with FLT3-ITD and in NPM1, 2 with FLT3-ITD and FLT3-TKD, 1 with FLT3-TKD and in NPM1, 3 with NPM1 and NRAS mutations. We found that mutations with the significantly higher incidence (p=0,001) were observed in the group of pts with NK (80/97), whereas there were only 8/28 pts with mutations in the group with complex karyotype. When analyzing the age-related features, it was shown that the majority of mutations were detected in the group of pts at the age from 60 to 69 years. Mutations FLT3-ITD and FLT3-TKD were associated with higher WBC count comparing with pts without mutations (p=0,001 and p=0,014, respectively). The median follow-up for overall (OS) and relapse-free (RFS) survival for pts with FLT3-ITD against ptswith FLT3-ITD- was: 5,4 vs 12,8 months and 4,9 vs 10,0 months (p=0,001 and p=0,001), respectively. Mutation FLT3-TKD was also found to be prognostically unfavorable, but only comparing with pts with FLT3-ITD- genotype. As the result of OS and RFS analyses in pts with and without NPM1 mutations we revealed the significant favorable influence of NPM1 mut on the prognosis (p=0,002 and p=0,020, respectively). However pts with genotype FLT3-ITD+/NPM1+ were found to get to the group with an intermediate risk. We detected the significant adverse influence of CKIT mut on RFS (p=0,041). Mutations in NRAS didn't impact on prognosis; we only showed the tendency towards worsening of OS and RFS in group of pts with favorable cytogenetics (p=0,214 and p=0,160, respectively). Mutations DNMT3A R882 were investigated in group of 143 AML pts and were detected in 23 (16,1%) pts. Pts with DNMT3A R882 had higher WBC (p=0,001) and platelets (p=0,020) count at diagnosis and more frequently belonged to FAB groups M5 (p=0,003), as compared with DNMT3A wt. Of 23 pts who had AML with DNMT3A mutations, 17 had tumors with NK profiles (24,3% of a total of 70 cytogenetically normal samples) (p=0,009). Pts with isolated DNMT3A mutations were seen in 4 cases, whereas in the rest of pts they were detected simultaneously with mutations in genes FLT3, NPM1, NRAS and CKIT. DNTM3A mutations were significantly more prevalent in NPM1 mut (p=0,005) and FLT3-ITD (p=0,005) positive cases than wild type. DNMT3A mutations were associated with negative influence on pts OS and risk of relapse, compared with DNMT3A wt (р = 0,031 and р = 0,045, respectively). Summary. Mutations in FLT3 and NPM1 had a significantly higher incidence in the group of pts with a normal karyotype. FLT3 mutations showed the adverse prognostic value. Insertions in NPM1 were shownto be the favorable factor, correlating with prolonged RFS in all pts excepting pts with FLT3-ITD+/ NPM1+ genotype. CKIT mut was associated with higher relapse incidence in AML pts, while NRAS mut showed lack of prognostic significance. AML with DNMT3A mut represent the group, homogeneous on a number of clinical and laboratory parameters, associated with adverse prognosis and a high risk of the relapse. Disclosures No relevant conflicts of interest to declare.

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Prognostic significance of concurrent gene mutations in intensively treated patients with IDH-mutated AML, an ALFA study

Blood ◽

10.1182/blood.2020010165 ◽

2021 ◽

Author(s):

Matthieu Duchmann ◽

Jean-Baptiste Micol ◽

Nicolas Duployez ◽

Emmanuel Raffoux ◽

Xavier Thomas ◽

...

Keyword(s):

Prognostic Significance ◽

Gene Mutations ◽

Normal Karyotype ◽

Genetic Alterations ◽

Prognostic Impact ◽

Hematopoietic Stem ◽

Fully Integrated ◽

Mutational Status ◽

Trial Testing ◽

Prospective Trials

IDH inhibitors are effective in AML, and trials evaluating frontline combinations with intensive chemotherapy (IC) are ongoing. Data on the prognostic significance of co-occurring genetic alterations and allogeneic hematopoietic stem cell transplantation (HSCT) are conflicting in each IDH-mutated subgroup treated by IC, while this information is important for trial design and results interpretation. We retrospectively analyzed 127 IDH1, 135 IDH2R140 and 57 IDH2R172 newly diagnosed AML patients treated with IC in three Acute Leukemia French Association (ALFA) prospective trials. We addressed in each IDH subgroup the prognostic impact of clinical and genetic covariates, and the role of HSCT in eligible patients. In IDH1 patients, presence of NPM1 mutations was the only variable predicting improved OS in multivariate analysis (p < 0.0001). In IDH2R140, normal karyotype (p= 0.008) and NPM1 mutations (p = 0.01) predicted better OS. NPM1 mutations were associated with better DFS (p = 0.0009) whereas presence of DNMT3A mutations was associated with shorter DFS (p = 0.0006). In IDH2R172, platelet count was the only variable retained in the multivariate model for OS (p = 0.002). Among non-favorable ELN-2010 eligible patients, 71 (36%) achieved an HSCT in first complete remission (CR1) and had longer OS (p = 0.03) and DFS (p = 0.02) than not-transplanted patients. Future clinical trial testing frontline IDH inhibitors combined with IC may consider stratification on NPM1 mutational status, the main prognostic factor in IDH1 and IDH2R140 mutated AML. HSCT improve OS of non-favorable IDH1/2-mutated AML and should be fully integrated in the treatment strategy.

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IDH1/2, TET2 and DNMT3A Mutations Are Not Mutually Exclusive in Secondary Acute Myeloid Leukemias,

Blood ◽

10.1182/blood.v118.21.3556.3556 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3556-3556

Author(s):

Olivier Kosmider ◽

Olivier LaRochelle ◽

Marie-Magdelaine Coude ◽

Veronique Mansat-De Mas ◽

Eric Delabesse ◽

...

Keyword(s):

Bone Marrow ◽

De Novo ◽

Conflicts Of Interest ◽

Remission Rate ◽

Normal Karyotype ◽

Prognostic Impact ◽

Mutational Status ◽

Myeloid Leukemias ◽

Melting Analysis ◽

De Novo Aml

Abstract Abstract 3556 IDH1/2, TET2 and DNMT3A mutations have been reported in myeloid malignancies including de novo AML. In this study, we have analyzed the frequency and prognostic impact of these mutations in a large retrospective cohort of patients (pts) with secondary AML (SA) which encompass myelodysplasia-related changes (MRC) AML and therapy-related (TR) AML according to the WHO classification. Bone marrow samples were collected from 247 pts at diagnosis with SA and the mutational status of IDH1/2, TET2 and DNMT3A genes together with other genes frequently mutated in AML (NPM1, FLT-3, N and K-RAS, WT1) was determined by Sanger sequencing or high resolution melting analysis. The cohort of 247 pts consisted in 201 MRC AML and in 46 TR AML, 39.5% of which with a normal karyotype (NK). The frequency of IDH1/2, TET2 and DNMT3A mutations was 12.6, 19.8 and 4.5%, respectively. Two pts had both TET2 and IDH1/2 mutations, 2 pts had TET2 and DNMT3A mutations and 5 pts had both IDH1/2 and DNMT3A mutations showing that these mutations were not mutually exclusive in SA. IDH1/2 and TET2 mutations were significantly more frequent in MRC AML (14.1 and 22.3%) than in TR AML (6.4 and 8.7%) (P =0.04 and P =0.03) while the frequency of DNMT3A mutations was identical in the two subgroups. The SA pts harbouring at least one IDH1/2 or TET2 or DNMT3A mutation were significantly older (P <0.0001) and presented higher leukocyte count and lower MCV (P <0.05) than unmutated pts. Percentage of blasts in the bone marrow was similar in the two groups. Karyotype was normal in 48% of the IDH1/2 or TET2 or DNMT3A mutated pts and 18% of the unmutated patients, indicating that these mutations were strongly associated with NK (P < 0.001). A statistically significant link was found between TET2 or IDH1/2 or DNMT3A mutations and NPM1 mutations, but not with FLT-3, N/K-RAS or WT1 mutations. Complete remission rate and overall survival were evaluated in a group of 158 pts which had received intensive chemotherapy at diagnosis, and were identical in the IDH1/2 or TET2 or DNMT3A mutated and unmutated groups. These mutations did significantly influence survival neither in the subgroup of pts with normal karyotype, nor in the subgroup of MRC-AML, or TR-AML which were of very poor prognosis. These data show that IDH1/2, TET2 or DNMT3A mutations could modify the clinical presentation without impact on prognosis. Disclosures: No relevant conflicts of interest to declare.

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AML with Recurrent Genetic Abnormalities and AML with Myelodysplasia-Related Changes Account for Three-Quarters of Previously Untreated Pediatric AML-the Results of a Nation-Wide Central Review in Japan-

Blood ◽

10.1182/blood.v118.21.4899.4899 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4899-4899

Author(s):

Akitoshi Kinoshita ◽

Hayato Miyachi ◽

Hiromichi Matsushita ◽

Tomohiko Taki ◽

Miharu Yabe ◽

...

Keyword(s):

Who Classification ◽

De Novo ◽

Conflicts Of Interest ◽

Prognostic Significance ◽

Prognostic Impact ◽

Genetic Abnormalities ◽

Precise Diagnosis ◽

De Novo Aml ◽

Pediatric Aml ◽

Mixed Phenotype

Abstract Abstract 4899 [Background] The WHO classification has been widely accepted among physicians who are engaged in treating pediatric AML patients. In 2008, the revised WHO classification has expanded the two categories in AML; AML with recurrent genetic abnormalities and AML with myelodysplasia-related changes. The epidemiology and prognostic significance of these refined categories remains to be explored in children. [Methods] JPLSG AML-05 is a nationwide clinical trial for children with de novo AML, excluding acute promyelocytic leukemia and myeloid leukemia with Down syndrome, which was conducted between November 2006 and December 2010 in Japan. A central review of diagnosis based on the WHO classification was prospectively performed on each case soon after morphological, cytogenetical and immunological data were submitted to data center. Regarding the cases with discrepant results among these parameters, further diagnostic tests including FISH and chimera gene analyses were underwent to confirm the diagnoses. [Results] Four hundred and eighty four patients were enrolled in the study. Thirty patients did not meet the criteria of AML. We could not collected suitable data for diagnosis in 6 patients. Regarding the rest 448 patients, diagnoses based on the WHO classification 2001 and 2008 were determined. According to the 2001 version, 227 (50.6%) had AML with recurrent genetic abnormalities:124 (27.7%) of AML with t(8;21)(q22;q22);(AML1/ETO ), 32 (7.1%) of AML with inv(16)(p13q22); (CBFβ/MYH11), 38 (8.5%) of AML with t(9;11)(p22;q23), and 33 (7.4%) of AML with the other11q23 (MLL) abnormalities, 36 (8.0%) had AML with multilineage dysplasia, and 185 (41.3%) had AML, not otherwise categorized. According to 2008 version, 235 (52.5%) had AML with recurrent genetic abnormalities: 124 (27.7%) of t(8;21)(q22;q22);(AML1/ETO ), 32 (7.1%) of AML with inv(16)(p13q22); (CBFβ/MYH11), 38 (8.5%) of AML with t(9;11)(p22;q23), 33 (7.4%) of AML with the other11q23 (MLL) abnormalities,4 of AML with t(6;9)(p23;q34);DEK-NUP214,2 of AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2);RPN1-EVI13, and 2 of AML with t(1;22)(p13;q13);RBM15-MKL, 88 (19.6.7%) had AML with myelodysplasia-related changes (29 from morphological features of myelodysplasia and 59 from myelodysplasia-related cytogenetic abnormalities), 119 (26.6%) had AML, not otherwise categorized and 7(1.6%) had mixed phenotype acute leukemia (6 of T/myeloid and 1 of B/myeloid). [Discussion] Our comprehensive approach for diagnosis was a useful modality for precise diagnosis of uncertain cases, which might have been assigned to the category of AML, with not otherwise categorized, previously. As a result, the present study shows an increased prevalence of AML with recurrent genetic abnormalities or AML with myeloid dysplasia-related changes among pediatric patients with previously untreated AML. Analysis of the AML-05 trial will elucidate the prognostic impact of these categories. Disclosures: No relevant conflicts of interest to declare.

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A 17-Gene Leukemia Stem Cell (LSC) Score in Adult Patients (Pts) with Acute Myeloid Leukemia (AML) Reveals a Distinct Mutational Landscape and Refines Current European Leukemianet (ELN) Genetic Risk Stratification

Blood ◽

10.1182/blood-2018-99-115114 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 289-289 ◽

Cited By ~ 1

Author(s):

Marius Bill ◽

Deedra Nicolet ◽

Ann-Kathrin Eisfeld ◽

Krzysztof Mrózek ◽

Christopher J. Walker ◽

...

Keyword(s):

De Novo ◽

Gene Mutations ◽

Disease Free Survival ◽

Prognostic Impact ◽

Expression Data ◽

Age Related ◽

Mutational Status ◽

Multivariable Analyses ◽

De Novo Aml ◽

Favorable Group

Abstract Introduction: Prognosis of AML pts is still poor mainly because of refractoriness to or relapse after intensive chemotherapy. High rates of relapse are also attributed to LSCs, which are a small subset of cells with acquired abnormal self-renewal capacity and increased resistance to chemotherapy. A better understanding of LSCs is critical to improve outcomes of pts with AML. Ng et al. (Nature 2016;540:433) defined a 17 stemness-associated gene score that was highly prognostic. Aims: The aim of this study was to validate the prognostic relevance of the 17-gene LSC score and explore its utility in the context of the ELN classification. We also examined gene mutations associated with the 17-gene LSC score. Methods: We analyzed a total of 934 pts [729 aged <60 years (y) and 205 aged ≥60 y] with de novo AML. We used whole transcriptome expression data (RNAseq) to calculate the aforementioned 17-gene LSC score for each pt in our cohort. Similar to Ng et al., we used the median of the whole cohort to discriminate between pts with LSChigh and LSClow scores. The mutational status of 80 cancer- and leukemia-associated genes (Eisfeld et al. Leukemia 2017;31:2211) were determined using a targeted next-generation sequencing panel, CEBPA mutations using Sanger sequencing, and an internal tandem duplication (ITD) of the FLT3 gene using fragment analysis in pretreatment bone marrow or blood samples. All pts were treated on frontline Cancer and Leukemia Group B/Alliance protocols. Results: A comparison of pretreatment clinical and genetic features revealed that LSChigh pts were older (P<.001; median age, 53 vs 46 y) and had higher platelet counts (P<.001; median, 63 vs 50x109/L) than LSClow pts. Pts with a LSChigh score more frequently had FLT3-ITD (P<.001) and mutations in the ASXL1 (P=.001), DNMT3A (P<.001), RUNX1 (P=.002), SRSF2 (P=.02), STAG2 (P=.009), TET2 (P=.008) and TP53 (P<.001) genes. Conversely, these pts had a lower frequency of biallelic CEBPA (P<.001), GATA2 (P=.008) and KIT (P<.001) mutations. Because of differences in treatment intensity, we analyzed outcomes of younger and older pts separately. Younger pts with a LSChigh score had a lower complete remission (CR) rate (P<.001; 63% vs 87%), shorter disease-free survival (DFS; P<.001; 3-y rates, 26% vs 48%; Figure 1A) and overall survival (OS; P<.001; 3-y rates, 27% vs 59%; Figure 1B) compared to those of LSClow pts. In multivariable analyses including clinical and genetic factors that impact on outcome, a LSChigh score associated with lower remission rates (P<.001; HR: 0.36), shorter DFS (P<.001; HR: 1.67) and OS (P<.001; HR: 1.88) after adjusting for other co-variates. We also analyzed the prognostic impact of the LSC score with respect to the 2017 ELN classification. We found that LSC score associated with different ELN groups (P<.001), with LSChigh pts being more often classified in the Adverse or Intermediate group and less often in the Favorable group. Within the ELN Favorable and Adverse groups, LSChigh score retained its prognostic impact and identified pts with a lower CR rate and shorter DFS and OS (Table1). In older pts, a LSChigh score also associated with lower CR rate (P=.004; 50% vs 72%), shorter DFS (P=.04; 3-y rates, 6% vs 17%; Figure 1C) and OS (P<.001; 3-y rates, 9% vs 27%; Figure 1D). In multivariable analyses, LSC score remained significant only for OS (P<.003; HR: 1.70) after adjusting for other co-variates. Regarding the ELN classification, pts with LSChigh score in the Favorable group had shorter OS (P=.05; 3-y rates, 17% vs 50%) and, by trend, shorter DFS (P=.09; 3-y rates, 17% vs 39%); no significant differences were found in Intermediate or Adverse groups. Conclusions: We used RNAseq expression data and applied the previously established 17-gene LSC signature to score 934 de novo AML pts. We detected distinct mutational differences between LSChigh and LSClow pts, with LSChigh pts more often carrying gene mutations associated with age-related clonal hematopoiesis (i.e., ASXL1, DNMT3A, TET2, SRSF2 and TP53 mutations). Moreover, this score, derived from the expression of stemness-associated genes, has not only a prognostic impact on its own but also in the context of the current 2017 ELN classification. Disclosures Kolitz: Magellan Health: Consultancy, Honoraria. Powell:Rafael Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

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Microarray Analysis Detects Unique Expression Pattern for NPM1-Mutated AML with Normal Karyotype and Reveals Pathobiological Insights.

Blood ◽

10.1182/blood.v110.11.2382.2382 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2382-2382

Author(s):

Torsten Haferlach ◽

Claudia Haferlach ◽

Alexander Kohlmann ◽

Lothar Wieczorek ◽

Martin Dugas ◽

...

Keyword(s):

Gene Expression ◽

Melting Curve ◽

Normal Karyotype ◽

Support Vector ◽

Melting Curve Analysis ◽

Study Cohort ◽

Data Set ◽

Npm1 Mutation ◽

Mutation Status ◽

Mutational Status

Abstract Recent data indicate that mutations in exon 12 of the nucleophosmin (NPM1) gene characterize a distinct subgroup of adult and pediatric acute myeloid leukemia (AML). AML carrying NPM1 mutations account for about one-third of all adult AML, exhibit distinctive biological and clinical features and show a strong association to AML with normal karyotype (55% mutated). However, the role of NPM1 in leukemogenesis still remains elusive. Here we present data on a cohort of n=66 AML cases with normal karyotype analyzed by high-density whole genome expression microarrays (Affymetrix HG-U133 Plus 2.0). In parallel melting curve analysis was used to assess NPM1 mutational status: 41 cases were characterized as mutated (NPM1+) and 25 cases were unmutated (NPM1−). We first investigated the gene signature that discriminated NPM1+ from NPM1− cases. Genes that were significantly overexpressed comparing NPM1+ against NPM1– cases included a strong homeobox genes signature (HOXA1, HOXA5, HOXA7, HOXA9, HOXA10, HOXA11, HOXB2, HOXB4, HOXB5, HOXB6, HOXB7, MEIS1, and PBX3). A functional analysis (Gene Ontology) revealed a clear association of the group of overexpressed genes with the cell components nucleosome, chromatin, and the nuclear envelope-endoplasmatic reticulum network as well as involvement in the biological processes of nucleosome and chromatin assembly, establishment of protein transport and localization, and Notch signaling pathway. In contrast, the cellular processes completely differed when genes were investigated that were significantly underexpressed in NPM1+ cases compared to NPM1− cases. This group of genes encoded membrane-related proteins (gap junction, intercellular junction, signalosome complex) and proteins involved in cellular morphogenesis and cell communication. The differences in gene expression signatures between NPM1+ and NPM1− cases permit a robust classification approach by gene expression profiling. Support Vector Machine analysis resulted in >92% prediction accuracy of NPM1 mutation status (10-fold cross-validation). The sensitivity was very high for the positive detection of NPM1+ cases (>97%). Using a 100-fold re-sampling approach and splitting the complete data set into a training set (n=44) and testing set (n=22) the following genes were most frequently selected as top discriminatory genes: HOXA5, HOXB4, HOXB5, HOXB6, MEIS1, PBX3, FGFR1, ADAM17, PRICKLE1, and TMPO. Interestingly, the classification was less accurate when also FLT3 internal tandem duplication mutation status was taken into account. The study cohort (n=66) then was distributed as follows: 19 NPM1+/FLT3+, 22 NPM1+/FLT3−, 4 NPM1−/FLT3+, and 21 NPM1−/FLT3− negative cases. Only 14 of 22 (64%) NPM1+/FLT3– cases were correctly predicted, with miscalls falling both into the group of NPM1+/FLT3+ and NPM1−/FLT3− cases. In conclusion, NPM1 mutations are the most frequent mutations in adult AML to date and their central prognostic role is increasingly recognized. Given the fact that they are nearly mutually exclusive with major recurrent genetic abnormalities and that they can be characterized by a distinctive gene expression program these data especially for of NPM1+/FLT3− AML with better outcome may support to classify this as a separate biological subgroup of AML with normal karyotype.

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Differential Prognostic Impact Of Peripheral Blood Blast Clearance In AML Based On Type Of Therapy and FLT3 Mutation Status

Blood ◽

10.1182/blood.v122.21.2584.2584 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2584-2584

Author(s):

Christopher B. Benton ◽

Lisa Gu ◽

Hagop M. Kantarjian ◽

Peng Qiu ◽

Farhad Ravandi ◽

...

Keyword(s):

Induction Chemotherapy ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Prognostic Significance ◽

Specific Treatment ◽

Rank Test ◽

P Value ◽

Prognostic Impact ◽

Mutation Status ◽

Mutational Status

Abstract Background The day of clearance of peripheral blood blast cells (PBBC) is a prognostic marker in patients with acute myeloid leukemia (AML) treated with induction chemotherapy with cytarabine (ara-C) plus idarubicin (AI regimen: ara-C 1.5g/m2 x 4 days, idarubicin 12mg/m2 x 3 days). Earlier PBBC clearance correlates with improved overall survival (OS). We extended this observation to subsets of patients receiving the AI regimen in combination with a targeted agent such as the FLT3 inhibitor sorafenib (AI regimen plus sorafenib 400mg orally twice daily x 7 days) and vorinostat (vorinostat 500mg three times daily x 3 days followed by AI regimen), and examined the interaction of FLT3 mutational status and PBBC clearance. Patients and Methods PBBC clearance (defined as PBBC=0% by CBC differential) was examined for patients with non-APL AML undergoing AI alone (n=168), AI+sorafenib (n=75), and AI+vorinostat (n=102). Patient characteristics for all patients (n=345) were as follows: median age 54 years (range, 18-72), FLT3-mutated (FLT3+, includes FLT3-ITD and FLT3-D385) (95, 28%), wild-type FLT3 (FLT3-neg) (207, 60%), median WBC 5.0x109/dL (range, 0-229), median PBBC 17% (range, 0-99), median BM blasts 46% (range, 1-98). Patients for each cohort were divided based on day of PBBC clearance and survival evaluated by Kaplan-Meier curves. Comparisons of curves were carried out using a log-rank test. Results The overall response rates (ORR=CR+CRp) for the AI, AI+sorafenib, and AI+vorinostat induction cohorts were 63%, 79%, and 76% respectively. We evaluated OS for each of the three cohorts individually. In our first analysis, we divided patients into 5 groups according to PBBC clearance day: group A (0-1 days), B (2-3 days), C (4-5 days), D (6-8 days), and E (>8 days). We found that OS for patients in groups A-E was significantly different only in the AI therapy cohort (p-value<0.01), and not in the AI+sorafenib (p-value=0.15) or AI+vorinostat (p-value=0.1) cohorts. Noting that earlier blast clearance generally correlated with improved OS, we simplified our analysis by dividing patients into only two groups based on blast clearance. We performed two separate analyses using a 3-day cutoff (0-3 days vs. >3 days) or a 5-day cutoff (0-5 days vs. >5 days). For a blast clearance cutoff of 3 days, OS was significantly different in the AI+vorinostat cohort (p-value=0.02) and not in the AI alone (p-value=0.27) or AI+sorafenib (p-value=0.1) cohorts. For a 5-day cutoff, OS was significantly different in the AI (p-value<0.001) and AI+vorinostat (p-value=0.04) cohorts, but not the AI+sorafenib (p-value=0.13) cohort. We next evaluated FLT3+ and FLT3-neg patients individually for all three cohorts. Using a 3-day cutoff for blast clearance, the prognosis of FLT3+ patients could be significantly differentiated for patients treated with the FLT3 targeted regimen AI+sorafenib (p-value=0.04), but not AI alone (p-value=0.64) or AI+vorinostat (p-value=0.48). In distinction, FLT3-neg patients receiving AI+vorinostat could be well differentiated (p-value=0.06), but not FLT3-neg patients receiving AI alone (p-value=0.21) or AI+sorafenib (p-value=0.6). Conclusions The prognostic significance of the day of PBBC clearance during induction chemotherapy in AML is dependent on the specific treatment used and the mutation status of the patients being treated. For patients treated with AI alone, disappearance of blasts within 5 days of induction strongly predicts superior survival compared to patients who clear blasts after 5 days. For patients treated with AI+sorafenib, there is not a strong correlation between day of blast clearance and prognosis, unless FLT3+ patients are investigated alone, where there is a significant correlation between clearing blasts within 3 days of induction and prognosis. For patients treated with AI+vorinostat, disappearance of blasts within 3 days suggests a better survival in FLT3-neg but not FLT3+ patients. Additional approaches are needed to evaluate the prognostic value of clearance of PBBC in AML in the context of targeted therapy and mutational status of disease. Disclosures: No relevant conflicts of interest to declare.

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FLT3, NPM1 and CEBPA Mutations in 195 Children with Acute Myeloid Leukemia in Argentina

Blood ◽

10.1182/blood.v124.21.5318.5318 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5318-5318

Author(s):

Cristina N. Alonso ◽

Patricia L. Rubio ◽

Adriana Medina ◽

Silvia Eandi Eberle ◽

Andrea Bernasconi ◽

...

Keyword(s):

Conflicts Of Interest ◽

Prognostic Significance ◽

Normal Karyotype ◽

Prognostic Impact ◽

Sequencing Analysis ◽

Rt Pcr ◽

Genetic Abnormalities ◽

Childhood Aml ◽

The Mean ◽

Cebpa Mutations

Abstract Background: Mutations of FLT3, NPM1 and CEBPA are found in 25 to 35% of adult-AML. These mutations correlate with outcome, especially in AML with normal karyotype. There are few reports concerning the incidence and prognostic significance of these mutations in childhood-AML and there is no data from Argentina. Objectives: To describe the prevalence of FLT3, NPM1 and CEBPA mutations and to analyze the prognostic impact in the outcome in our setting. Methods: Samples from 195 children treated with AML protocols were retrospectively analyzed. The mean age at diagnosis was 6.8 [0.0-17.9] years, including 65 patients younger than 2 years of age. FAB subtypes were M2: 18%, M3: 15%, M4: 12%, M5: 34%, M6: 3%, M7: 10%, while 16 cases (8%) disclosed an ambiguous lineage immunophenotype. Genetic abnormalities of AML cases were characterized by cytogenetic analysis (97%) and/or RT-PCR for AML1-ETO, CBFB-MYH11, PML-RARA, MLL-AF4, MLL-AF9, MLL-ENL and MLL-AF10 fusion transcripts (95%). The distribution of the genetic abnormalities was: AML1-ETO: 11%, PML-RARA: 15%, CBFB-MYH11: 6%, MLL/11q23: 23%, other abnormalities: 25% and normal karyotype: 16%. Detection of NPM1 and CEBPA mutations was performed by Gene-scanning; FLT3-ITD and FLT3-TKD were studied by RT-PCR and RFLP respectively. Positive cases were further characterized by sequencing analysis. Results: The prevalences of the studied mutations were: FLT3-ITD: 10.3%, FLT3-TKD: 8.2%, NPM1mut: 4.6% and CEBPAmut: 2.1%. Within the group of AML with normal karyotype the incidences were: FLT3-ITD: 12.5%, FLT3-TKD: 6.3%, NPM1mut: 25.0% and CEBPAmut: 12.5%. The mean age for each subgroup was: FLT3-ITD: 14 years, FLT3-TKD: 9 years, NPM1mut: 12 years and CEBPAmut: 12 years. Simultaneous presence of FLT3-ITD and NPM1 mutations was detected in 2 cases while 1 patient disclosed both FLT3-TKD and CEBPAmut. FLT3-ITD and FLT3-TKD showed significant association with the presence of PML-RARA (p<0.00001 and p=0.055 respectively). Eight out of nine patients with NPM1mut and 4/4 patients with CEBPAmut were AML with normal karyotype. The FAB subtypes more frequently observed for each subgroup were: FLT3-ITDmut: M3 (n:10/20; p<0.00001), FLT3-TKDmut: M5 (n:8/16; p=n.s.), NPM1mut: M2 (n:4/9; p=0.062) and CEBPAmut: M2 (n:3/4; p=0.019). The mean ages of patients with FLT3-ITDmut, NPM1mut and CEBPAmut were significantly higher (p<0.00001, p=0.006 and p=0.033, respectively). FLT3-TKD was the only mutation detected in 5/45 (11%) of patients younger than 1 year of age. The five-years leukemia-free survival probabilities (pLFS) and standard error (SE) were: Total AML: 49 (4)%, FLT3-ITDmut:68 (12)%, FLT3-TKDmut:46 (17)%, NPM1mut: 75 (15)%, CEBPAmut: 100 (0)% and NPM1mut/CEBPAmut/FLT3-ITDneg: 83 (15)% (p<0.00001). The pLFS (SE) of patients with normal karyotype and FLT3-ITDneg and NPM1mut or CEBPAmut was 88 (12)% (p=0.066). Conclusions: This is the first report of the frequencies of FLT3, NPM1 and CEBPA mutations in childhood AML in our country. The incidences of NPM1mut and CEBPAmut were significantly higher in AML with normal karyotype. Our data confirm the favorable prognosis of AML with NPM1mut/FLT3-ITDneg and CEBPAmut/FLT3-ITDneg genotypes, especially in cases with normal karyotype. The present results support the notion that this group should be considered as a new AML subset with better outcome. This group of AML patients with better outcome could be included in the standard risk group, thus avoiding intensive treatments and related toxicity. Disclosures No relevant conflicts of interest to declare.

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Clinical Impact of the Presence of a Diffuse Large B-Cell Lymphoma (DLBCL) Component in the Outcome of Untreated Patients with Follicular Lymphoma (FL)

Blood ◽

10.1182/blood.v128.22.3043.3043 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3043-3043

Author(s):

Laura Magnano ◽

Ivan Dlouhy ◽

Olga Balagué ◽

Alfredo Rivas-Delgado ◽

Jordina Rovira ◽

...

Keyword(s):

De Novo ◽

Conflicts Of Interest ◽

Histological Grade ◽

B Cell Lymphoma ◽

Aggressive Lymphoma ◽

Prognostic Impact ◽

Single Institution ◽

Mutational Status ◽

Biological Studies ◽

Histological Transformation

Abstract Introduction: Histological transformation into an aggressive lymphoma, usually DLBCL, may occur during the follow-up of FL patients and determines a poor outcome. The presence of a DLBCL component in a FL (FL/DLBCL) is observed in some cases at diagnosis, usually being considered a transformation of a FL with a previously undetected indolent course. The clinical implications of this situation in terms of prognosis and treatment remain unclear. The aim of the present study was to analyze the clinicobiological characteristics and outcome of patients diagnosed with FL/DLBCL in the rituximab era, in comparison with patients diagnosed with pure FL or de novo DLBCL at the same period in a single institution. Patients and Methods: Eight hundred seventy eight patients sequentially diagnosed with either FL, DLBCL or FL/DLBCL between 2002 and 2015 ina single institution were included in the study. The histological distribution was as follows: FL grade 1, 2, 3a, 320 cases (140M/180F; median age, 58 years), FL 3b, 8 cases (4M/4F; median age, 66 years), DLBCL, 510 cases (275M/235F; median age, 65 years) and FL/DLBCL, 40 (16M/24F; median age, 65 years).According to institutional guidelines, the latter were treated as aggressive lymphomas, with no maintenance or further intensification. All FL/DLBCL biopsies were reviewed and the DLBCL component semiquantified. Cell of origin (COO) assessment by gene expression-based assay was determined in 127 cases and NOTCH1-2 mutational status in 216. Results: Main clinicobiological characteristics of the patients according to the histology are listed in the table. Compared to DLBCL cases, FL/DLBCL patients showed more frequently ambulatory performance status, primary nodal origin and advanced stage, with these features being closer to those of FL patients. On the contrary, FL/DLBCL patients had an intermediate position between FL and DLBCL cases regarding B-symptoms, bone marrow infiltration, hemoglobin and serum LDH levels. The proportion of DLBCL component in FL/DLBCL cases ranged from 5 to 95%, with no significant differences in the initial characteristics according to the proportion of DLBCL component. COO was determined in 11 FL/DLBCL, with 8 (73%) being GCB, 2 (18%) ABC, and 1 unclassified. Such distribution in 116 DLBCL with COO available was: 50 (43%) GCB, 50 (43%) ABC and 16 unclassified. NOTCH 1-2 was mutated in 4/39 (10%) FL/DLBCL, whereas this proportion was 1/41 (2%) FL and 11/136 (8%) DLBCL. All FL/DLBCL patients were treated as aggressive lymphoma with no intensification after front-line therapy. The proportion of primary refractoriness in FL/DLBCL patients was significantly higher than in FL and similar to that of DLBCL. Progression free survival (PFS) and overall survival (OS) are showed in the table and figure. Among the 40 FL/DLBCL cases, the amount of DLBCL component did not show prognostic impact, whereas the histological grade did (5-year OS FL grades 1-2-3a/DLBCL, 92% vs. FL grade 3b/DLBCL, 38%; p=0.001). In the different cohorts analyzed (FL+FL/DLBCL, FL+FL/DLBCL+DLBCL, and FL/DLBCL+DLBCL), FL/DLBCL histology did not show independent value for OS in multivariate analyses that included standard prognostic variables, namely, serum β2m and FLIPI or IPI scores. Sequential biopsies were available in 120 cases, with the following distribution according to the primary diagnosis: FL, 60 cases (at relapse: 37 FL and 23 DLBCL), FL/DLBCL, 6 cases (at relapse: 3 FL, 2 FL/DLBCL and 1 DLBCL), and DLBCL, 54 cases (at relapse: 4 FL and 50 DLBCL). Conclusion: Patients with FL/DLBCL, although infrequent, exists and have clinicobiological differentiated features compared to pure FL and DLBCL. When treated with standard immunochemotherapy their outcome is not worse than that of de novo DLBCL. Some clinicobiological characteristics suggest FL/DLBCL to be a specific category rather than a transformation from a FL; however, this warrants further biological studies. Disclosures No relevant conflicts of interest to declare.

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FISH Reveals Abnormalities of Unsuspected Regions in Chromosomally Normal De Novo Myelodysplastic Syndromes (MDS).

Blood ◽

10.1182/blood.v114.22.2599.2599 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2599-2599

Author(s):

Paolo Bernasconi ◽

Irene Dambruoso ◽

Marina Boni ◽

Paola Maria Cavigliano ◽

Ilaria Giardini ◽

...

Keyword(s):

De Novo ◽

Chromosomal Abnormalities ◽

Conflicts Of Interest ◽

Prognostic Significance ◽

Normal Karyotype ◽

Low Risk ◽

Fish Analysis ◽

Disease Evolution ◽

Therapeutic Decisions ◽

Conventional Cytogenetics

Abstract Abstract 2599 Poster Board II-575 In de novo MDS the chromosomal pattern is a mandatory step for an accurate diagnosis, predicts overall survival (OS) and the risk of MDS/AML evolution, guides therapeutic decisions. However, conventional cytogenetics (CC) studies show a normal un-informative chromosomal pattern in about half of MDS patients, especially in low-risk disease. FISH with probes pinpointing the chromosomal regions most frequently affected in MDS can increase the incidence of abnormal karyotypes up to 60%, but the percentage of normal karyotypes remains high and makes the search of novel cytogenetic/molecular markers a urgent need. A fundamental contribution to overcome CC and FISH shortcomings, has been recently provided by array CGH (aCGH) studies which have revealed that, independently of the cytogenetic pattern, MDS patients may harbour novel abnormalities involving unsuspected chromosomal regions. Based on this assumption, we decided to investigate whether FISH with probes already employed in aCGH studies can truly unmask cryptic lesions in chromosomally normal MDS patients, whether these defects are either chromosomal gains/losses or balanced rearrangements and whether these chromosomal abnormalities influence OS and disease evolution. FISH analyses were carried out in thirty-five patients examined between January 2005 and June 2008. There were thirteen females and twenty-two males, whose median age was 66 years (range 24–78). According to WHO classification, 6 patients were classified as RA, 13 as RAEB-1 and 16 as RAEB-2. According to IPSS score, 7 patients were considered low-risk, 14 intermediate-1 risk and 14 intermediate-2 risk. Median follow-up was nine months (range 1–46). At the time of the analyses no patients has died; 6 have progressed to RAEB-2 and 3 to AML. Probes for FISH analysis were chosen following two criteria: the frequency of their involvement in chromosomal abnormalities identified by aCGH studies and their Mb position on Human Mar. 2003 assembly according to the UCSC genome browser. All probes, obtained from BACPAC Resources Center at C.H.O.R.I. (Oakland, USA), were labelled and applied as previously reported. The following probes were applied: RP11-912d8 (19q13.2); RP11-196p12 (17q11.2); RP11-269c4 (14q12); RP11-351o1 (10q21.3); RP11-144g6 (10q11.2); RP11-122a11 (7q34); RP11-951k18 (5q13.1); RP11-100m20 (4p14); RP11-544h14 (2q33). The cut-off values for interphase FISH (i-FISH) were obtained from the analysis of 300 nuclei from ten normal samples and were fixed at 10%. An abnormal FISH pattern was revealed in eighteen patients (51.4%). It was observed in 3/6 RA patients, in 7/13 RAEB-1 and in 8/16 RAEB-2 and in 2/7 IPSS low-risk, in 7/14 intermediate-1 risk and in 9/14 intermediate-2 risk MDS patients. Seven presented a 19q13.2 deletion, three a 14q12 deletion, four an amplification of band 4p14, two a defect of band 10q21.3, two a potential amplification and one a deletion of band 10q11.2, two a deletion of band 5q13.1 and one a deletion of band 17q11.2. Cryptic defects were also revealed in six of the nine patients who experienced disease evolution on FISH analyses. This event occurred in 2/3 RA, in 2/7 RAEB-1 and in 2/8 RAEB-2 patients with an abnormal FISH pattern. Despite these data, the prognostic significance of an abnormal FISH pattern needs to be assessed on additional patients. In conclusion, our data show that i) FISH can truly reveal novel lesions involving unsuspected chromosomal regions in 51% of MDS patients with a normal karyotype; ii) most of these lesions consist of chromosomal gains/losses; iii) an abnormal FISH pattern seems to correlate with disease progression, but this correlation needs to be tested on additional patients. Disclosures: No relevant conflicts of interest to declare.

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Rising Percentages of Blasts Are Associated with Continuously Increasing Frequencies of FLT3-ITD and NPM1 Mutations In AML and In Addition Depend on Combinations of These Markers: Analysis of 805 Cases with Normal Karyotype AML.

Blood ◽

10.1182/blood.v116.21.1043.1043 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1043-1043

Author(s):

Torsten Haferlach ◽

Ulrike Bacher ◽

Tamara Alpermann ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

...

Keyword(s):

Survival Rate ◽

De Novo ◽

Univariate Analysis ◽

Prognostic Significance ◽

Melting Curve ◽

Normal Karyotype ◽

Employment Equity ◽

Data Set ◽

Equity Ownership ◽

Wbc Count

Abstract Abstract 1043 Introduction: FLT3-ITD is supposed to function as driver mutation in acute myeloid leukemia (AML), e.g. higher WBC counts are found in FLT3-ITD mutated (FLT3+) cases. Also for NPM1 mutations (NPM1+) higher blast proportions were described. As both mutations can be concomitantly found we investigated correlations and the biological background of these aspects. Patients/Methods: We analyzed 805 pts (m/410; f/395; median, 66.6 yrs; 20.0–93.3 yrs) with de novo normal karyotype AML for FLT3+ and NPM1+ and by bone marrow (BM) cytomorphology. Blast definition and classification was done according to FAB and WHO criteria on May Giemsa Gruenwald, MPO, and NSE stained BM aspirates. The FLT3-ITD status and the FLT3-ITD mutant/wildtype ratio were analyzed by fragment analysis (genescan); the NPM1+ status was investigated by melting-curve based PCR assay. Results: The overall mutation rate for FLT3+ was 219/805 (27.2%), for NPM1+ 391/805 (48.6%). Mean WBC count was higher in FLT3+ cases when compared to the FLT3- (69.0 × 109/l vs. 25.7 × 109/l; p<0.001). The 2-year survival rate was best in the NPM1+/FLT3- pts (n=240; 82.3%) when compared to all other subgroups (NPM1+/FLT3-ITD+: n=151; 55.2%; NPM1-/FLT3+: n=68; 40.5%; NPM1-/FLT3-: n=346; 62.1%; p=0.003). This demonstrates the universal validity of our data set. Frequencies of both FLT3-ITD or NPM1 mutated cases were increasing by blast percentages per decade from 20% to 100%: For the FLT3+: blasts 20–29%: 15/108 (12.2% of cases); 30–39%: 7/91 (7.7%); 40–49: 11/69 (15.9%); 50–59%: 14/80 (17.5%); 60–69%: 25/102 (24.5%); 70–79%: 34/114 (29.8%); 80–89%: 55/123 (44.7%); 90–100%: 58/103 (56.3%) (p<0.001). For NPM1+: blasts 20–29%: 38/123 (30.9% of cases); 30–39%: 31/91 (34.1%); 40–49: 20/69 (29.0%); 50–59%: 36/80 (45.0%); 60–69%: 51/102 (50.0%); 70–79%: 62/114 (54.4%); 80–89%: 83/123 (67.5%); 90–100%: 70/103 (68.0%) (p<0.001). Subsequently, we separated the whole cohort according to the threshold 40% BM blasts: 2 yrs survival rate was significantly lower in pts with ≥40% of blasts (n=591; 63.4%) when compared to those with <40% (n=214; 77.5%; p=0.004). Patients with ≥40% of blasts showed significant higher rates of NPM1+/FLT3+ (blasts <40%: 13/214; 6.1% vs. ≥40%: 138/591; 23.4%; p<0.001) and NPM1-/FLT3+ (blasts <40%: 9/214; 4.2% vs. ≥40%: 59/591; 10%; p=0.009). NPM1-/FLT3- cases were less frequent in cases with ≥40% of blasts (blasts <40%: 136/214 NPM1-/FLT3-; 63.6% vs. blasts ≥40%: 210/591; 35.5%; p<0.001). Focusing on the blast levels in the different molecular subgroups, cases with 90–100% blasts were most frequent in the NPM1+/FLT3+ (double mutated) subgroup (45/151; 29.8%). In the NPM1+/FLT3- and the NPM1-/FLT3+ subgroups, cases with 80–89% of blasts were most frequent (43/240; 17.9%; and 15/68; 22.1%, respectively). In contrast, the NPM1-/FLT3- pts were most frequently from the 20–29% blast category (79/346; 22.8%) (p<0.001 for comparison of all subgroups). A higher FLT3-ITD-mutant/wildtype ratio was correlated with a higher proportion of BM blasts (Spearman; p<0.001). In univariate analysis for prognostically relevant parameters, OS was significantly related to age (p<0.001; RR=1.36 per 10 years of increase), WBC (p<0.001; RR=1.06 per 10×109/l), blasts <40% (p=0.005; RR=2.29), the NPM1+/FLT3- subgroup (p≤0.001; RR=0.40), and a FLT3mut/wildtype ratio ≤0.5 (p=0.024; RR=1.78). No significance was found for gender, Hb, thrombocytes, and FAB subtypes. In multivariate analysis, age (p<0.001), WBC (p=0.003), blasts <40% (p=0.008) and the NPM1+/FLT3- status (p=0.002) retained prognostic significance. Conclusion: The frequencies of the FLT3+ and the NPM1+ cases are continuously increasing in parallel to increasing blast percentages in normal karyotype AML. Thresholds of 40% of blasts are suitable to discriminate different prognostic groups which can be related to significantly higher frequencies of NPM1+/FLT3+ and NPM1-/FLT3+ in patients with higher blast proportions. Therefore, both molecular markers apparently contribute to blast proliferation in normal karyotype AML. Combinations of these markers are also relevant since blasts were highest in the double NPM1/FLT3-mutated cases and lowest in the NPM1-/FLT3- cases in our study. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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