The Landscape of RUNX1 Mutations in Acute Myeloid Leukemia: Investigations On Stability of Mutations At Relapse and Utility As a Marker for Minimal Residual Disease

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 657-657
Author(s):  
Alexander Kohlmann ◽  
Niroshan Nadarajah ◽  
Vera Grossmann ◽  
Tamara Alpermann ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
De Novo ◽  
Residual Disease ◽  
Normal Karyotype ◽  
Next Generation ◽  
Equity Ownership ◽  
The Stability ◽  
Runx1 Mutation ◽  
Minimal Residual ◽  
Acute Myeloid

Abstract Abstract 657 Introduction: RUNX1 mutations constitute disease-defining aberrations in acute myeloid leukemia (AML) and were demonstrated to be particularly frequent in secondary and de novo AML with normal karyotype or non-complex alterations and to confer an unfavorable prognosis. Monitoring minimal residual disease (MRD) in AML has been shown to provide prognostic information and is increasingly used for treatment decisions. A variety of molecular markers has been identified suitable for MRD assessment, yet there still is a lack of such markers in a significant number of patients. The use of RUNX1 mutations may bridge a gap. Aims: Patients and Methods: RUNX1 mutation screening was prospectively performed in 814 patients with AML at diagnosis (645 de novo, 109 s-AML, and 60 t-AML). The median age of the patients was 69.6 years (range: 1 – 93 years), including 375 female and 439 male patients, respectively. 50.5% (411/814) of cases presented with a normal karyotype, 38.8% (316/814) with non-complex cytogenetic alterations, 9.6% (78/814) with a complex aberrant karyotype, and 1.1% (9/814) with prognostically favorable cytogenetics. Mutation analysis was performed using a sensitive next-generation amplicon deep-sequencing assay (454 Life Sciences, Branford, CT). Moreover, in a subset of 44 AML patients and additional 59 retrospectively analyzed cases the prognostic impact of MRD levels of RUNX1 mutations was studied at a second time point after completion of intensive induction therapy (median sampling interval: 128 days after diagnosis; range 60 – 180 days). In these follow-up samples the RUNX1 mutations already detected at diagnosis were investigated with a higher coverage (835-fold median coverage) as compared to the diagnostic assessment (759-fold median coverage) resulting in a sensitivity level of 1%. Furthermore, in 57 patients paired samples from diagnosis and relapse were analyzed to assess the stability of RUNX1 mutations. Results: 211/814 patients (25.9%) were detected to carry RUNX1 mutations. The median clone size was 39% and revealed a significant heterogeneity ranging from 2% to 96%. 73.9% (156/211) of mutated patients carried one mutation only, whereas 26.1% (55/211) harbored two (n=46) or more (n=9) mutations. In detail, the 211 patients harbored a total number of 275 alterations in RUNX1: 42.5% (117/275) frame-shift mutations, 34.9% (96/275) missense, 14.2% (39/275) nonsense, 4.4% (12/275) exon-skipping/splicing, and 4.0% (11/275) in-frame insertion/deletion alterations, respectively. Regarding MRD assessment, patients were separated according to the median MRD level (3.92%; range 0.03% - 48.00%) into “good responders” (n=78) with MRD levels below 3.92% and “poor responders” (n=25) with MRD levels above 3.92%. This resulted in significant differences in both event-free survival (median 21.4 vs 5.7 months, p<0.001) and overall survival (73.3% vs 66.1% at 2 years, p=0.016). Moreover, in 57 cases the stability of individual RUNX1 mutations was studied at the time of relapse. In 46/57 (80.7%) cases the same alterations detected at diagnosis were present at relapse, whilst in 2/57 (3.5%) cases the RUNX1 mutation from the diagnostic sample was no longer detectable at relapse. Importantly, in 7/57 (12.3%) patients novel RUNX1 mutations were detected in regions different from those affected at diagnosis. Conclusion: Next-generation deep-sequencing accurately detects and quantifies RUNX1 mutations in AML with high sensitivity. RUNX1 mutations qualify as patient-specific markers for individualized disease monitoring. Thus, the measurement of mutation load by next-generation sequencing may contribute to refine the assignment into distinct risk categories in AML. Analysis of RUNX1 mutations should be considered for the complete coding region at relapse to detect new RUNX1 mutations. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership.

scholarly journals High NPM1 mutant allele burden at diagnosis correlates with minimal residual disease at first remission in de novo acute myeloid leukemia

2019 ◽  
Vol 94 (8) ◽  
pp. 921-928 ◽  
Author(s):  
Sanjay S. Patel ◽  
Geraldine S. Pinkus ◽  
Lauren L. Ritterhouse ◽  
Jeremy P. Segal ◽  
Paola Dal Cin ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Minimal Residual Disease ◽  
Myeloid Leukemia ◽  
Mutant Allele ◽  
De Novo ◽  
Residual Disease ◽  
Allele Burden ◽  
First Remission ◽  
Minimal Residual ◽  
Acute Myeloid

First Results of a 31-Gene Panel Targeted to Investigate Myeloid Malignancies by Next-Generation Amplicon Deep-Sequencing

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 883-883 ◽  
Author(s):  
Alexander Kohlmann ◽  
Sandra Weissmann ◽  
Ulrike Schoeck ◽  
Vera Grossmann ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Myeloid Leukemia ◽  
Sanger Sequencing ◽  
Normal Karyotype ◽  
Next Generation ◽  
Sequencing Data ◽  
Myeloid Malignancies ◽  
Equity Ownership ◽  
Generation Sequencing ◽  
Acute Myeloid

Abstract Abstract 883 Introduction: Massively parallel next-generation sequencing data have changed the landscape of molecular mutations in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). The number of molecular markers used to characterize myeloid malignancies continues to constantly increase. As such, physicians and laboratories face a great unmet need to test panels of genes at a high level of sensitivity and throughput. Methods: We developed a sensitive next-generation deep-sequencing assay for routine diagnostics. In total, 31 genes with relevance in myeloid malignancies providing both favorable and adverse molecular prognostic information were chosen: ASXL1, BCOR, BCORL1, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3, IDH1, IDH2, JAK2, KDM6A, KIT, KRAS, NOTCH1, NPM1, NRAS, PHF6, PRPF40B, PTPN11, RUNX1, SF1, SF3A1, SF3B1, SRSF2, TET2, TP53, U2AF1, U2AF2, and ZRSR2. Targets of interest comprised either complete coding gene regions or hotspots. In summary, 1,375 amplicons were designed with a median length of 175 bp (range 109–194 bp), representing a total target sequence of 140.35 kb. The sequencing library was constructed starting off 2.2 μg genomic DNA per patient using a singleplex microdroplet-based assay (RainDance, Lexington, MA). Sequencing data was generated using the MiSeq instrument (Illumina, San Diego, CA) loading 4 patients per run. Using the 300 cycles sequencing-by-synthesis chemistry in median 6.099 millions of paired-end reads were generated per run. This resulted in a median coverage per gene of 1,766 reads (range 992-2,432). The total turn-around time of the analysis with this assay was less than 4 days. 49 clinically well-annotated patients harboring myeloid malignancies were analyzed during the evaluation phase. These included 9 acute myeloid leukemia (AML), 9 myelodysplastic syndrome (MDS), 13 chronic myelomonocytic leukemia (CMML), and 18 mixed phenotype acute leukemia, T/myeloid (MPAL-TM) cases. The median age was 69 years (range: 23 – 90 years). Results: In median, the coverage per amplicon harboring a mutation was 2,095-fold, thus enabling a sensitive detection of variants. In total, 146 mutations in 28 of the 31 genes were detected in 47/49 patients with a range of 1–7 mutations per case (median: 3). According to chromosome banding analysis 31/49 cases presented with a normal karyotype. In 30/31 cases with a normal karyotype at least one molecular mutation was observed using this screening panel. 42/146 mutations were detected with a clone size <20%, thus being detected only due to the higher sensitivity of this technique in comparison to direct capillary Sanger sequencing. In this cohort, the most frequently mutated genes were RUNX1 (14/49), DNMT3A (14/49), SRSF2 (11/49), ASXL1 (9/49), and TET2 (9/49). The mutation types comprised 97 missense, 17 duplications, 24 deletions, 5 insertions and 3 insertion/deletions alterations. Novel variants were verified using direct capillary Sanger sequencing (n=19) or sensitive amplicon deep-sequencing (n=65) (454 Life Sciences, Branford, CT). With respect to the technical limit of detecting larger insertions or deletions both a 27-bp insertion (RUNX1, p.Thr121delins9) and a 23-bp deletion variant (ASXL1, p.Glu635ArgfsX15) were successfully sequenced. The highest number of mutations was observed for CMML patients (mean of 3.6 per case; CMML vs remainder: P=0.201). Also, in CMML patients we observed the highest frequency of mutations in major splicing machinery genes such as SF1, SF3A1, SF3B1, SRSF2, U2AF1, U2AF2, and ZRSR2 (11/13 CMML, 84.6% vs 14/36 remainder cases, 38.9%; P<0.001). Importantly, a number of patients (39/49) was detected to harbor mutations in genes reported to be associated with decreased overall survival, both in AML (e.g. TP53, RUNX1, ASXL1, DNMT3A, IDH1, or TET2) and low- or intermediate-1 IPSS risk categories in MDS (e.g. ASXL1, EZH2, ETV6, RUNX1, TP53). As such, detecting these adverse somatic alterations may influence the course of therapy for these patients. Conclusion: We here demonstrated that microdroplet-based sample preparation enabled to target 31 candidate genes for next-generation sequencing in myeloid malignancies in a routine diagnostic environment. This approach provides the potential to screen for prognostically relevant mutations in a fast and comprehensive way providing actionable information suitable to guide therapy. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Schoeck:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

scholarly journals The Presence of Minimal Residual Disease, As Determined By Highly Sensitive Quantitation of NPM1-Mutatation, Provided Powerful Prognostic Information in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5097-5097
Author(s):  
Atsushi Marumo ◽  
Hiroki Yamaguchi ◽  
Yuho Najima ◽  
Kensuke Usuki ◽  
Shinichi Kako ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Normal Karyotype ◽  
Npm1 Mutation ◽  
Minimal Residual ◽  
Hematological Remission ◽  
Acute Myeloid

Background: As recurrence of acute myeloid leukemia (AML) is difficult to predict, it is important to detect it by measuring minimal residual disease (MRD). PML-RARA, RUNX-RUNX1T1, CBFB-MYH11 are regarded as the reliable MRD markers. However, in AML with normal karyotype and many other forms, no MRD markers have been established. NPM1 mutations, occurring in approximately 30% of adult AML cases, and 50-60% of AML cases with normal karyotype, represent one of the most frequent mutations in AML. Recently, NPM1 mutation is reported to be useful in assessing MRD. We undertook a retrospective and prospective investigation of the usefulness of NPM1 mutation as an MRD marker in Japanese patients with AML. Methods: The subjects were 38 NPM1-mutated AML patients with first hematological remission at several hospitals related to our institution between 2001 and 2018. This study was approved by the ethics committee of Nippon Medical School and the informed consents were obtained from all patients, according to the Declaration of Helsinki. We analyzed peripheral blood cells or bone marrow cells at diagnoses, and evaluated only bone marrow cells after diagnoses. Detection of NPM1 mutation was carried out using allele-specific real time PCR following creation of a complementary primer. After dilution of the samples, sensitivity to TCTG, CATG, and CCTG was found to be 0.001%. The NPM1 mutant copies were qualified only at successful amplification of internal control. Results: The median age of the patients was 58 years (18-79 years). There were 32 cases with intermediate cytogenetic prognosis and 6 cases with unclear chromosomal profile. Of the 38 cases, 14 cases (37%) were FLT3-ITD-positive and allogeneic hematopoietic stem cell transplantation was carried out in 14 cases (37%). The base sequence was TCTG in 36 cases and CCTG in 2 cases. Persistence of NPM1-mutatation was present in 25 patients with first hematological remission (66%). Compared with patients with MRD negative, patients with MRD positive were associated with DNMT3A mutation (MRD positive 12/25 vs MRD negative 0/13, p=0.003). The rate of relapse in patients with MRD positive was significantly higher than those of in patients with MRD negative (MRD positive 76% vs MRD negative 23%, p=0.004). The rates of relapse free survival (RFS) and overall survival (OS) in patients with MRD positive were significantly lower than those in patients with MRD negative (RFS at 2 years: MRD positive 14% vs MRD negative 86% p=0.003; Figure 1, OS at 2 years: MRD positive 25% vs MRD negative 93%, p<0.001). In FLT3-ITD negative group, the rates of RFS in patients with MRD positive were significantly lower than those in patients with MRD negative. (RFS at 2 years: MRD positive 21% vs MRD negative 92% p=0.001; Figure 1). Conclusion: The presence of MRD with NPM1 mutation is significantly associated with relapse and it is useful to decide their treatment strategy. Especially, there is the usefulness of NPM1 mutation as an MRD marker in NPM1 positive Flt3-ITD negative AML patients who are generally classified as favorable risk. According to previous reports, it is known that NPM1-mutated AML sometimes relapse with losing NPM1 mutations. However, in this study, all NPM1-mutated AML relapse without losing NPM1 mutations. We need to collect more patients and are going to confirm whether there are patients who relapse with losing NPM1 mutations or not. We plan to analyze the genetic background of MRD positive and negative patients with next-generation sequencing. We are going to announce the genetic characteristics in addition to this result at ASH. Disclosures Usuki: Astellas Pharma Inc: Research Funding, Speakers Bureau; Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau. Kako:Bristol-Myers Squibb: Honoraria; Pfizer Japan Inc.: Honoraria. Inokuchi:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria; Celgene: Honoraria; Pfizer: Honoraria.

NPM Mutations and Not FLT3 Mutations Are a Potential Marker for Monitoring Minimal Residual Disease in Acute Myeloid Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2016-2016
Author(s):  
Michela Palmisano ◽  
Tiziana Grafone ◽  
Emanuela Ottaviani ◽  
Nicoletta Testoni ◽  
Stefania Paolini ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Residual Disease ◽  
Potential Marker ◽  
Flt3 Mutations ◽  
Hplc Profile ◽  
Minimal Residual ◽  
Acute Myeloid

Abstract Nucleophosmin (NPM) is a multifunctional phosphoprotein that acts as nucleocytoplasmic shuttling protein, with tumour suppressor and oncogenic functions. Recently, NPM mutations have been found in a subset of adults with de novo acute myeloid leukemia (AML). These mutations occur in the last coding exon (exon 12), causing a frameshift and the formation of novel C-termini. The abnormal mutated NPM protein shows aberrant cytoplasmic localization and is frequently associated with FLT3 mutation. These observations provide basis for studies of the pathogenesis in AML. We did sequential analysis on patient samples during the clinical course to investigate the stability and pathogenetic role of NPM mutation in AML and the association with FLT3 mutations. The NPM mutations were determined by D-HPLC analysis; samples exhibiting an abnormal D-HPLC profile were confirmed by direct sequencing. We investigated 28 patients of de novo AML. Eleven samples were sequenced since they showed an heteroduplex D-HPLC profile. Type A mutation (960_963dupTCTG) was the commonest observed change, occurring in 9/11 samples, followed by type B mutation (960_963insCATG) in 1 case and type D mutation (960_963insCCTG) in 1 case. Furthermore, we observed that 5/11 patients harboring NPM mutation presented also mutant FLT3 at diagnosis. Analyzing NPM mutations during progression of disease, we observed that NPM mutation disappeared at complete remission and the same mutation reappeared at relapse. No differences were found in wild type NPM. Instead, we found a modification of FLT3 status associated to evolution of disease in 7/28 patients: 2 patients lost the mutation at relapse, 4 patients acquired the mutation at relapse and 1 patient modified the mutation from D835 to ITD (Table 1). Together these results suggest that NPM mutations and not FLT3 mutations may have utility as a potential marker for monitoring minimal residual disease. Studies on the biological effects of NPM mutations will contribute to disclose the role of NPM mutations in the pathogenesis of AML and their interactions with other genetic alterations such as FLT3.

scholarly journals CLINICAL ANALYSIS AND OPTIMIZATION OF POST-REMISSION THERAPY FOR ACUTE MYELOID LEUKEMIA PATIENTS WITH MINIMAL RESIDUAL DISEASE AS DETERMINED BY FLOW CYTOMETRY

2010 ◽  
Vol 2 (2) ◽  
pp. e2010020 ◽  
Author(s):  
Daichi Inoue ◽  
Hayato Maruoka ◽  
Takayuki Takahashi
Keyword(s):  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Minimal Residual Disease ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Residual Disease ◽  
Significant Prognostic Factor ◽  
Group A ◽  
Minimal Residual ◽  
Acute Myeloid

Background: Although several prognostic indicators of de novo acute myeloid leukemia (AML) patients have been identified, the clinical significance of minimal residual disease (MRD) needs to be evaluated further in Japanese adult patients.Methods: Using three color flow cytometry, we identified leukemia-associated phenotypes (LAP) in bone marrow specimens at diagnosis and assessed the relationship between clinical outcomes and the presence of marrow MRD in 33 patients who achieved a morphologic complete remission (CR) and were followed after CR.Results: Of 33 consecutive patients, we detected MRD in 20 patients after achieving CR (Group A) and did not in 13 patients (Group B), with 2-year overall survival (OS) rates of 49.0% and 84.6%, respectively (P =.0317), and relapse-free survival (RFS) rates of 13.7% and 91.7%, respectively (P=.0010). By multivariate analysis, MRD-positivity at post-induction was found to be associated with a shorter duration of RFS (P=.0042). Notably, we achieved MRD negativity in only 2 patients (10%) of Group A in spite of subsequent intensive consolidation therapies and found that the fluctuation of the MRD level during consolidation therapies was not a significant prognostic factor. Four patients in Group A underwent allogeneic hematopoietic stem-cell transplantation (HSCT) when in the CR state and did not experience relapse at a median follow-up period of 20.5 months after HSCT.Conclusions: MRD is critical for predicting de novo AML outcomes. Most MRD-positive patients cannot achieve MRD negativity with conventional chemotherapy. Thus, HSCT may be the primary therapeutic option for these patients.

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