Modulation of the Clonal Composition in Relapsed CLL: A Study Based on Targeted Deep-Sequencing of ATM, BIRC3, NOTCH1, POT1, SF3B1, SAMHD1 and TP53

Abstract Background: Relapse or refractory disease is a challenging clinical problem in the majority of chronic lymphocytic leukemia (CLL) patients. Treatment influences the clonal composition by selection and eventually induction of additional genetic abnormalities. Aim: To characterize the clonal evolution in relapsed CLL patients by deep-sequencing analysis of mutations in ATM, BIRC3, NOTCH1, POT1, SF3B1, SAMHD1 and TP53. Patients and Methods: Sequential samples of 20 relapsed CLL patients at three time-points were evaluated: A: at diagnosis (n=16) or in untreated state (n=4), B: at first relapse (n=20) and C: at second relapse (n=2). Patients were treated with diverse treatment schemes and had temporarily achieved either complete or partial remission during the course of the disease. The median time from diagnosis to first-line treatment was 13 months (1 - 169 months). All geneswere sequenced by a deep sequencing approach (Illumina, San Diego, CA). IGHV mutational status was determined by direct Sanger sequencing at time-point A. Chromosome banding analysis (CBA) and FISH data on del(17p), del(11q), trisomy 12 (+12), and del(13q) were available in 33/42 and 36/42 samples, respectively. Results: Initially, samples at first relapse were sequenced. Mutations in SF3B1 (6/20, 30%), TP53 (5/20, 25%), ATM (5/20, 25%), NOTCH1 (4/20, 20%), and SAMHD1 (3/20, 15%) were detected at high frequencies. No mutations were detected in BIRC3 and POT1. In total, 75% of cases presented with at least one mutation (Figure 1): 8 (40%) cases had one, 6 (30%) cases had two and one patient had three genes concomitantly mutated (mut). Patients were also analyzed for IGHV mutational status at diagnosis and presented with unmutated status at a frequency of 85% (17/20). Subsequently, samples from cases with mutations were analyzed at time-point A. In 12/15 (80%) cases the mutations at relapse were already detectable at time-point A with a similar load indicating presence of the main clone before and after chemotherapy. However, in 7/15 (47%) patients new gene mutations were acquired either additionally to existing mutations (n=4) or in previously wild-type cases (n=3). In 5/7 (71%) cases mutations were located in TP53. TP53 mut were the only mutations that were not detected in samples before treatment (sensitivity of 3%). Thus, TP53 mutations might have been initiated by chemotherapy or exist in a minor subclone subsequently selected by chemotherapy. Furthermore, only 4 cases had low-level mutations (3-6% mutation load) at diagnosis in either SAMHD1 or SF3B1 that eventually increased in their burden during disease course. Of note, in two patients a multibranching clonal evolution could be identified (#2, #9). For patient #2 three time-points were analyzed. At diagnosis 2 ATM mutations were detected with mutation loads of about 20%, each. In the course of the disease these mutations were lost, whereas SF3B1 mut showed a stable mutation load in all three time-points of about 40%. In contrast, mutation load of SAMHD1 increased over time from 4% to 87%. CBA was performed at diagnosis and detected independent clones with del(11q) and del(13q). Accordingly, del(11q) detected by FISH at diagnosis was lost and the percentage of cells with del(13q) increased from diagnosis to time-point C. Therefore, patient #2 shows different genetic subclones in parallel that were eradicated or selected by chemotherapy. In patient #9 two SF3B1 mutations were initially detected with the same mutation load of 10%. After treatment one mutation was lost, whereas the load of the second mutation increased indicating at least two different subclones with only one of them being sensitive to chemotherapy. This might be due to different additional aberrations. Indeed, CBA identified two clones: one with +12 alone and one in combination with del(13q). FISH revealed unchanged percentage of +12 at time-point B, whereas del(13q) positive cells were diminished. Conclusions: In 75% of relapsed CLL cases mutations in SF3B1, TP53, ATM, NOTCH1, and SAMHD1 are present at high frequencies. 80% of these mutations are already detectable before treatment initiation representing the main clone. Remarkably, TP53 mutations were the only mutations that were not detected before but only after chemotherapy. Figure 1. Distribution of gene mutations in 15 CLL cases with mutations at diagnosis or before treatment (D) and at relapse (R). Red = mutated, grey = wild-type, white = not analyzed. Figure 1. Distribution of gene mutations in 15 CLL cases with mutations at diagnosis or before treatment (D) and at relapse (R). Red = mutated, grey = wild-type, white = not analyzed. Disclosures Jeromin: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schabath:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Ultra-Deep Sequencing Leads to Earlier and More Sensitive Detection of the TKI Resistance Mutation p.T315I in CML

Blood ◽

10.1182/blood.v124.21.4531.4531 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4531-4531

Author(s):

Constance Baer ◽

Wolfgang Kern ◽

Sarah Mariathas ◽

Claudia Haferlach ◽

Susanne Schnittger ◽

...

Keyword(s):

Deep Sequencing ◽

Research Funding ◽

Sanger Sequencing ◽

Read Length ◽

Strong Increase ◽

Mutation Load ◽

Employment Equity ◽

Time Points ◽

Equity Ownership ◽

Time Point

Abstract Background: Chronic myeloid leukemia (CML) cells can acquire resistance to tyrosine kinase inhibitors (TKI) that in ~40% of cases is due to acquisition of mutations in the ABL1 kinase domain of the BCR-ABL1 transcript. The p.T315I (c.944C>T) mutation (mut) mediates resistance to most BCR-ABL1 TKIs (Imatinib, Dasatinib, Nilotinib and Bosutinib), whereas sensitivity to ponatinib has been demonstrated. Patients with p.T315Imut show a rapid increase in malignant cell burden and can progress to blast crisis. An earlier detection of the p.T315Imut may allow TKI treatment intervention ahead of disease progression. However, the sensitivity of conventional Sanger sequencing for detection of mutations is not less than 10-20%. Aim: To study the dynamics of evolution and progression of the p.T315Imut using ultra-deep sequencing (UDS) in comparison with Sanger sequencing. Patients and Methods: We selected 18 CML patients with high p.T315Imut levels originally detected by Sanger sequencing for routine diagnostics. Subsequently, we backtracked prior blood samples of all patients for a mean period of eight months (2-15 months) before detection of p.T315Imut by Sanger sequencing, analyzing 3-7 time points per patient. Patients (4 female and 14 male) had a median age of 60 years (18-84 years) and received treatment as follows: only Imatinib (n=3), only Nilotinib (n=3), only Dasatinib (n=1), treated with two prior (n=6) or three prior TKIs (n=5) by the time of p.T315Imut detection by Sanger sequencing. For more sensitive mutation detection, we amplified the BCR-ABL1 fusion transcript and designed two sequencing amplicons (550 bp and 575 bp) for UDS with the XL+ Kit for extended read length (Roche/454, Branford, CT). A minimal read coverage of 1,000 per base was reached. Our backtracking study by UDS was performed on samples sent in at intervals of approximately 3 months. Results: To prove high sensitivity of UDS with the 454 XL+ protocol we performed dilution experiments for three sequence variants and replicated sequencing experiments with low level mutations. The detection limit was at 1-2% mutation level and thus is 10-fold better than the sensitivity reached by Sanger sequencing. At the time point of initial routine diagnosis of p.T315Imut the median mutation load was 87.5% (30-100%) by Sanger sequencing and very similar by UDS (median: 84%; range: 40-99%; R2=0.7). In 6/18 patients backtracking identified a sample with a low p.T315I mutation level of <5% (1.9-13.6 months, median: 3.2 months) before a mutation load of >10% (Sanger sequencing detection level) was reached. Thus, in 33.3% of all cases a small, early clone of CML with p.T315Imut was identified. At subsequent time points, all 6 patients experienced a strong increase of the p.T315Imut level (>50%), which represents the very fast expansion of the mutated clone. In a second subset of 10 patients, the p.T315Imut load was already >30% when first detected by UDS. The median interval to the last p.T315I negative time point was 2.4 months (0.9-3.5) and no sample between the p.T315I negativity and high positivity was available. This subset confirms the fast outgrowth of the p.T315Imut positive clone. The p.T315Imut load had a median increase of 0.9% (0.2-3.1%) per day, when calculated as average increase from the last negative sample to the time point with maximum mutation load. The other 2 patients had high p.T315Imut levels (>40%) for our entire monitoring period. At the time of p.T315I detection by UDS, we observed eight patients with additional resistance mutations. The accumulation of mutations in one clone results in an extremely resistant CML. This was detected in one patient, where a p.T253H clone (Imatinib and Dasatinib resistant) gained the p.T315Imut. This clone expanded to 73% within 79 days. In contrast, we identified five cases with multiple CML clones carrying different mutations. However, the p.T315Imut clone was able to overgrow up to six other resistant clones. Conclusions: We showed: 1) the p.T315Imut rapidly increases upon occurrence, supporting the relevance of regular mutation monitoring in CML patients, when resistance to TKIs is suspected. 2) that small p.T315Imut clones in the 1-2% range can be sensitively detected by UDS in 33% of all samples if sampling intervals are within the 3 months range. 3) earlier detection of the p.T315Imut by UDS is a potentially valid method to allow a prompt change of TKIs before clonal expansion of the p.T315Imut cells. Disclosures Baer: MLL Munich Leukemia Laboratory: Employment; ARIAD Pharmaceuticals: Research Funding. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Mariathas:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; ARIAD Pharmaceuticals: Research Funding.

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17p Deletion Is the Most Frequent Abnormality Acquired During Clonal Evolution in Chronic Lymphocytic Leukemia (CLL): 429 Cases Analyzed by Interphase FISH and Chromosome Banding Analysis,

Blood ◽

10.1182/blood.v118.21.3870.3870 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3870-3870

Author(s):

Claudia Haferlach ◽

Melanie Zenger ◽

Susanne Schnittger ◽

Wolfgang Kern ◽

Torsten Haferlach

Keyword(s):

Median Time ◽

Chromosome Banding ◽

Clonal Evolution ◽

Complex Karyotype ◽

Employment Equity ◽

Mutation Status ◽

Time Points ◽

Banding Analysis ◽

Chromosome Banding Analysis ◽

Equity Ownership

Abstract Abstract 3870 Background and Aim: CLL is a chronic disease with heterogeneous clinical course. While a subset of patients requires early treatment others are followed without treatment for many years. Cytogenetic aberrations have major impact on the prognosis. The aim of this study was to evaluate 1) the frequency of gain of additional chromosome aberrations during the course of the disease (clonal evolution,CE) 2) the pattern of genetic abnormalities acquired during the CE 3) the association between genetic parameters at diagnosis and CE and 4) the impact of CE on clinical outcome. An additional aim was to compare monitoring by interphase FISH (IP-FISH) or chromosome banding analysis (CBA). Patients and Methods: Two different cohorts were evaluated: A) 363 CLL patients who were analyzed during the course of their disease at least at 2 time points by IP-FISH. In this cohort only patients were enrolled who were analyzed at each time point with the complete FISH panel using probes for 13q14 (D13S25, D13S319), 11q22 (ATM), 17p (TP53), 6q21/6q23, chromosome 12 centromer and IGH -CCND1. B) 245 CLL patients who were evaluated by CBA at least at 2 time points. 179 cases were included in both cohorts. Results: In cohort A 954 FISH analyses were performed in 363 cases (mean: 2.6, range: 2–14). The median time between the first and the last evaluation was 21.1 months (range 1.0–68.9 months). Overall, in 42 of 363 patients (11.6%) clonal evolution was observed, 9.3% of untreated and 16.8% of treated patients showed clonal evolution (p=0.05). The most frequently acquired abnormality was a 17p deletion detected in 12/42 (28.6%) cases, followed by deletion of 13q14 and 11q22 (9 cases each, 21.5%). In 6/131 (4.6%) cases with heterozygous 13q14 deletion at first analysis a homozygous 13q14 deletion was observed during follow up. In 290 of 363 the IGHV mutation status was available. An unmutated IGHV status tended to be associated with clonal evolution, 26/35 (74.3%) cases with and 147/255 (57.6%) patients without clonal evolution showed an unmutated IGHV status (p=0.067). No association between any specific abnormality detected by FISH and clonal evolution was observed. The median time between first FISH analysis and the first detection of clonal evolution was 25 months (range 2–65 months). In cohort B 618 CBA were performed in 245 cases (mean: 2.5, range: 2–8). The median time between the first and the last evaluation was 18.8 months (range 1.0–68.9 months). In 73 patients (30.0%) clonal evolution was observed. The most frequently acquired abnormality was loss of 17p detected in 26 cases, followed by deletion of 13q (n=21), and 11q (n=8). Other recurrent aberrations occurring during CE were gains of 8q (n=14), 13q (n=11), 17q (n=8), 1q (n=7), 3q (n=6), 16q (n=6), 4q (n=5), 1p (n=5), 9q (n=4), 15q (n=4), losses of 8p (n=10), 9q (n=8), 8q (n=7), 9p (n=7), 6q (n=7), 1q (n=6), 6p (n=5), 1p (n=5), 10q (n=4), 7q (n=3) and 14q32-rearrangement (n=6) with different partners (2p11, 4p16, 10p11, 2x 8q24, 19q13). In 202 of 245 patients the IGHV mutation status was available. An unmutated IGHV status was significantly more frequent in cases with as compared to patients without CE (44/62 (71.0%) vs 75/140 (53.6%), p=0.021). The median time between first CBA and the first detection of clonal evolution was 21 months (range 1–65 months). Clonal evolution was observed in 7/48 (14.6%) patients with normal karyotype, in 48/159 (30.2%) cases with non-complex aberrant karyotype and in 18/38 (47.4%) patients with complex karyotype (≥ 3 abnormalities) (p=0.04 for normal vs non-complex aberrant and p=0.056 for non-complex aberrant vs complex). For 135 of 245 cases clinical data with respect to treatment was available (45 cases with and 90 without CE). 33/45 (73%) patients with and 52/90 (57.8%) without clonal evolution had received treatment. A tendency towards a shorter overall survival was observed in patients with as compared to patients without CE detected by CBA (alive at 10 yrs 75.4% vs 93.5%). Conclusions: 1. Chromosome banding analysis detects clonal evolution more frequently than IP-FISH (30.0% vs 11.6%). 2. Clonal evolution occurs more frequently in patients with an unmutated IGHV status and an aberrant karyotype with the highest frequency in patients with complex karyotype. 3. Sequential analyses by FISH and CBA seem reasonable as especially 17p abnormalities occur frequently during the course of the disease, which impacts on treatment decisions. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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AML with RUNX1 Mutations and Loss of RUNX1 Wild-Type - a Distinct Subset?

Blood ◽

10.1182/blood.v126.23.2578.2578 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2578-2578

Author(s):

Claudia Haferlach ◽

Niroshan Nadarajah ◽

Annette Fasan ◽

Karolína Perglerová ◽

Wolfgang Kern ◽

...

Keyword(s):

De Novo ◽

Chromosome 21 ◽

Prognostic Impact ◽

Wild Type ◽

Mutation Load ◽

Employment Equity ◽

Runx1 Gene ◽

Equity Ownership ◽

De Novo Aml ◽

Runx1 Mutation

Abstract Background: Mutations in RUNX1 have been reported in 5 to 20% of AML. RUNX1 mutated AML is associated with a myeloid rather than monocytic differentiation, shows a typical pattern of cytogenetic abnormalities with a high frequency of trisomy 8 or 13, has a typical pattern of additional molecular mutations with a high frequency of accompanying ASXL1 and SF3B1 mutations and is nearly mutually exclusive of NPM1 and CEBPA double mutations and other entity-defining genetic abnormalities. In a subset of patients with RUNX1 mutations loss of the wild-type allele can be assumed due to a high mutation load. The aim of this study was the detailed analysis of a subset of RUNX1 mutated AML with RUNX1 wild-type loss with respect to accompanying cytogenetic and molecular genetic abnormalities and prognostic impact. Patients and Methods: A cohort of 467 AML with RUNX1 mutations (mut) at diagnosis identified during diagnostic work-up in our laboratory were the basis of this study. Median age was 72 years (yrs) (range 18-91 yrs), and male:female ratio 296: 171. 366 patients had de novo AML, 77 s-AML following MDS, 24 t-AML. For all patients (pts) cytogenetics and for 341 data on FAB subtype was available. Mutation data was available for NPM1 (n=456), MLL-PTD (n=453), CEBPA (n=449), FLT3-ITD (n=457), FLT3-TKD (n=457), WT1 (n=398), ASXL1 (n=313), TP53 (n=231), DNMT3A (n=177), TET2 (n=174), NRAS (n=305), KRAS (n=213) and SF3B1 (n=119). 64 patients with a mutation load of RUNX1 mutation >70% evaluated by sequencing analysis were selected for further analysis. All 64 cases were analysed by genomic arrays (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany) to determine the copy number state and copy neutral loss of heterozygosity (CN-LOH). Median age was 73 yrs (range 24-87 yrs), and male:female ratio was 27: 37. 50 patients had de novo AML, 11 s-AML following MDS, 3 t-AML. Results: Array CGH revealed a cytogenetically cryptic deletion on the long arm of chromosome 21 encompassing the RUNX1 gene in 5/64 (8%) patients while a CN-LOH on 21q including the RUNX1 gene was observed in 45 cases (70%). Thus in 50 cases (78%) with a high RUNX1 mutation load a RUNX1 wild-type loss (wt-loss) was detected by array CGH. In 43% (6/14) of the remaining cases the high RUNX1 mutation load was caused by amplification of the long arm of chromosome 21 either due to gain of whole chromosomes 21 or to an isochromosome 21q. First we focused on the characterization of RUNX1 mutated cases with RUNX1 wt-loss. In 22/50 cases (44%) an aberrant karyotype was observed with a distinct aberration pattern. 11 cases harbored +13, 5 had +8 and 6 cases a loss of 7q. No other recurrent abnormalities were observed. With respect to concurrent mutations the following frequencies were found: ASXL1 (42%), FLT3 -ITD (34%), TET2 (21%), KRAS (11%), MLL-PTD (8%), NRAS (7%), and FLT3-TKD (6%). No NPM1 mutation or CEBPA double mutations were identified. Comparison of those cases with RUNX1 wt-loss to all other RUNX1 mutated AML (n=417) revealed a significantly higher frequency of +13 (22% vs 9%, p=0.01) and FLT3 -ITD (34% vs 19%, p=0.015). FAB subtypes M0 and M1 were more frequent (46% vs 12%, p<0.001; 35% vs 22%, n.s.) and M2 and M4 less frequent (14% vs 46%, p<0.0001; 5% vs 17%, n.s.). Survival analyses were restricted to 212 de novo AML pts with RUNX1 mut who received intensive chemotherapy (median overall survival (OS): 20 months (mo), median event-free survival (EFS): 12 mo). Median OS and EFS was shorter in patients with RUNX1 wt-loss compared to those without (15 vs 20 mo, n.s., 10 vs 12 mo, p=0.04). In univariate Cox regression analysis a negative impact on OS was observed for RAS mut (relative risk (RR): 2.2, p=0.005), male gender (RR: 1.6, p=0.02), and age (RR: 1.3 per decade, p<0.001). Shorter EFS was associated with RUNX1 wt-loss (RR: 1.7, p=0.04), RAS mut (RR: 1.9, p=0.02) and age (RR: 1.2 per decade, p<0.001). In multivariate analysis RAS mut (OS: RR: 2.4, p=0.002; EFS: RR: 2.0, p=0.008) and age (OS: RR: 1.3 per decade, p<0.001; EFS: RR: 1.2 per decade, p<0.001) had independent prognostic impact. Conclusions: RUNX1 mutated AML with wild-type loss is a distinct AML subset that does not overlap with any of the genetically defined WHO categories and is characterized by an immature phenotype (81% FAB Subtype M0 and M1) and a higher frequency of +13 and FLT3-ITD as compared to RUNX1 mutated AML without wild-type loss. Wild-type loss and RAS mutations are associated with inferior outcome in RUNX1 mutated AML. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Landscape Of Genetic Lesions In 944 Patients With Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v122.21.521.521 ◽

2013 ◽

Vol 122 (21) ◽

pp. 521-521 ◽

Cited By ~ 3

Author(s):

Yasunobu Nagata ◽

Vera Grossmann ◽

Yusuke Okuno ◽

Ulrike Bacher ◽

Genta Nagae ◽

...

Keyword(s):

Myelodysplastic Syndromes ◽

Cox Regression ◽

Molecular Model ◽

Clonal Evolution ◽

Univariate Analysis ◽

Gene Mutations ◽

Risk Groups ◽

Employment Equity ◽

Linear Predictor ◽

Equity Ownership

Abstract Background Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms characterized by varying degrees of cytopenias and a predisposition to acute myeloid leukemia (AML). With conspicuous clinical and biological heterogeneity in MDS, an optimized choice of treatment based on accurate diagnosis and risk stratification in individual patients is central to the current therapeutic strategy. Diagnosis and prognostication in patients with myelodysplastic syndromes (MDS) may be improved by high-throughput mutation/copy number profiling. Methods A total of 944 patients with various MDS subtypes were screened for gene mutations and deletions in 104 known/putative genes relevant to MDS using targeted deep-sequencing and/or array-based genomic hybridization. Impact of genetic lesions on overall survival (OS) was investigated by univariate analysis and a conventional Cox regression, in which the Least Absolute Shrinkage and Selection Operator (lasso) was used for selecting variables. The linear predictor from the Cox regression was then used to assign the patients into discrete risk groups. Prognostic models were constructed in a training set (n=611) and confirmed using an independent validation cohort (n=175). Results After excluding sequencing/mapping errors and known or possible polymorphisms, a total of 2,764 single nucleotide variants (SNVs) and insertions/deletions (indels) were called in 96 genes as high-probability somatic changes. A total of 47 genes were considered as statistically significantly mutated (p<0.01). Only 6 genes (TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and RUNX1) were mutated in >10% of the cases. Less common mutations (2−10%) involved U2AF1, ZRSR2, STAG2, TP53, EZH2, CBL, JAK2, BCOR, IDH2, NRAS, MPL, NF1, ATM, IDH1, KRAS, PHF6, BRCC3, ETV6, and LAMB4. Intratumoral heterogeneity was evident in as many as 456 cases (48.3%), even though the small number of gene mutations available for evaluation was thought substantially to underestimate the real frequency. The number of observed intratumoral subpopulations tended to correlate with the number of detected mutations and therefore, advanced WHO subtypes and risk groups with poorer prognosis. Mean variant allele frequencies (VAFs) showed significant variations among major gene targets, suggesting the presence of clonogenic hierarchy among these common mutations during clonal evolution in MDS. The impact of these genetic lesions on clinical outcomes was initially investigated in 875 patients. In univariate analysis, 25 out of 48 genes tested significantly affected overall survival negatively (P<0.05), and only SF3B1mutations were associated with a significantly better clinical outcome. Next, to evaluate the combined effect of these multiple gene mutations/deletions, together with common clinical/cytogenetic variables used for IPSS-R, OS was modeled by a conventional Cox regression. A total of 14 genes, together with age, gender, white blood cell counts, hemoglobin, platelet counts, cytogenetic score in IPSS-R, were finally selected for the Cox regression in a proportional hazard model and based on the linear predictor of the regression model, we constructed a prognostic model (novel molecular model), in which patients were classified into 4 risk groups showing significantly different OS (“low”, “intermediate”, “high”, and “very high risk”) with 3-year survival of 95.2%, 69.3%, 32.8%, and 5.3%, respectively (P<0.001). These results demonstrated that the mutation/deletion status of a set of genes could be used as variables independent of clinical parameters to build a clinically relevant prognostic score. When applied to the validation cohort, the novel molecular model was even shown to outperform the IPSS-R. Conclusions Large-scale genetic and molecular profiling by cytogenetics, NGS and array-CGH not only provided novel insights into the pathogenesis and clonal evolution of MDS, but also helped to develop a powerful prognostic model based on gene mutations and other clinical variables that could be used for risk prediction. Molecular profiling of multiple target genes in MDS is feasible and provides an invaluable tool for improved diagnosis, biologic subclassification and especially prognostication for patients with MDS. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Bacher:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Monitoring of Minimal Residual Disease Using Next-Generation Deep-Sequencing in 460 Acute Myeloid Leukemia Cases identifies RUNX1 Mutated Patients with Resistant Disease

Blood ◽

10.1182/blood.v118.21.747.747 ◽

2011 ◽

Vol 118 (21) ◽

pp. 747-747 ◽

Cited By ~ 2

Author(s):

Alexander Kohlmann ◽

Vera Grossmann ◽

Stefan Harbich ◽

Frank Dicker ◽

Tamara Alpermann ◽

...

Keyword(s):

Deep Sequencing ◽

Residual Disease ◽

Normal Karyotype ◽

Refractory Disease ◽

Employment Equity ◽

Clone Size ◽

Equity Ownership ◽

Conventional Methods ◽

Time Point

Abstract Abstract 747 Introduction: RUNX1 (runt-related transcription factor 1) deregulations constitute a disease-defining aberration in AML. RUNX1 mutations were proposed as clinically useful biomarkers to follow disease progression from MDS to s-AML, as well as to monitor minimal residual disease (MRD). Study design: First, a next-generation amplicon deep-sequencing (NGS) assay was developed and a validation study was performed on genomic DNA obtained from mononuclear cells on a longitudinal series of 116 retrospective samples obtained from 25 patients. These samples were collected between 11/2005 and 6/2010 and were characterized for RUNX1 mutations by DHPLC and Sanger sequencing (conventional methods). In median, 3,346 reads per amplicon were generated and in all cases NGS analyses concordantly detected the mutations known from conventional methods. Furthermore, in 2/25 (8%) cases, NGS detected additional low-level mutations with 0.9% and 3.2% of reads mutated that were not observed by standard Sanger technique. Concerning MRD monitoring, in 7/25 (28%) cases an increasing clone size, i.e. mutations as low as 0.2% - 7.0%, was detectable up to 9 months earlier than by conventional methods. This established assay then was applied to characterize an unselected prospectively collected cohort during the subsequent 12-months routine diagnostics period starting 07/2010. Results: In total, 2,705 NGS RUNX1 mutation analyses were performed on a variety of hematological malignancies. We report on analyses on 460 AML cases at diagnosis including 369 de novo AML, 57 s-AML, and 34 t-AML cases (median age: 68 years; females: 204; males 256). 51% of cases presented with a normal karyotype, 38% harbored non-complex cytogenetic alterations, 10% carried a complex aberrant karyotype, and 1% of patients were characterized by favorable cytogenetics. Overall, 141 RUNX1 mutations were observed in 24.3% (112/460) of cases. At diagnosis, the clone size ranged from 2% to 95% (median: 40%). 82% (92/112) of mutated patients carried one, whereas 18% (20/112) harbored two (n=17) or more (n=3) mutations. The 141 mutations were characterized as follows: 43% (60/141) frame-shift mutations, 34% (49/141) missense, 15% (21/141) nonsense, 5% (7/141) exon-skipping, and 3% (4/141) in-frame insertion/deletion alterations, respectively. The mutations were predominantly located in the RHD domain (54%) or TAD domain (20%). In subsequent serial NGS analyses 31/112 evaluable RUNX1 mutated cases were studied and in 88 individual samples the alterations detected at diagnosis were specifically investigated with high coverage. With a median sampling interval of 50 days for the NGS analyses between 2 and 9 samples per patient were analyzed during the first year of treatment. In this cohort, three categories of patients were detectable: (i) 55% (17/31) of patients responded to therapy and were characterized by a total clearance of the mutated clone at the first time point of follow-up (804-fold median sequencing coverage; sensitivity ∼1:800). (ii) A second group consisted of 10% (3/31) of patients with refractory disease that stayed mutated, but were excluded from further analyses since they underwent transplantation. (iii) The third group comprised 35% (11/31) of patients: None of these patients demonstrated a clone size reduction below 0.7% of reads at the first follow-up analysis (reduction to a median of 21% mutated reads; range 0.7% - 41%). Also, at the second time point (in median 108 days after initial diagnosis), mutated clones were still detectable (reduction to a median of 8% mutated reads; range 4% - 15%). Most of these cases (10/11) had refractory disease as assessed by cytomorphology or molecular analyses. 10/11 cases did harbor a normal karyotype; n=1 with del(7q). Further, 6 of these 11 patients with refractory disease, as defined using NGS, were found to carry RUNX1 double mutations. Finally, in all (3/3) cases with double mutations in the same domain and refractory disease a changing antiparallel distribution of the clone size from initial diagnosis to first follow-up was observed. Conclusions: NGS accurately detects and quantifies RUNX1 mutations in AML with high sensitivity. The technique of deep-sequencing was observed to be superior to current routine methods, in particular during follow-up and in detecting MRD and thus has the potential to enable an individualized monitoring of disease progression and treatment efficacy. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Grossmann:MLL Munich Leukemia Laboratory: Employment. Harbich:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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In CML Patients with Good Response to TKIs Other Gene Mutations Are Frequently (37%) Present in Addition to Philadelphia Negative, Cytogenetically Aberrant Clones but Are Rare (4%) in Cases with MMR and Normal Karyotype

Blood ◽

10.1182/blood.v124.21.3126.3126 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3126-3126 ◽

Cited By ~ 1

Author(s):

Susanne Schnittger ◽

Manja Meggendorfer ◽

Niroshan Nadarajah ◽

Tamara Alpermann ◽

Wolfgang Kern ◽

...

Keyword(s):

Kinase Inhibitors ◽

Gene Mutations ◽

Clinical Importance ◽

Normal Karyotype ◽

Imatinib Treatment ◽

Mutation Load ◽

Employment Equity ◽

Cytogenetic Aberration ◽

Equity Ownership ◽

Over Time

Abstract Background: In chronic myeloid leukemia (CML) clonal chromosome aberrations in metaphases not carrying a t(9;22)(q34;q11) have been described during treatment with tyrosine kinase inhibitors (TKI), so-called Philadelphia-negative (Ph-) clones. Very rarely transformation to MDS was observed in patients carrying such Ph- clones but mainly restricted to patients harboring -7. Overall, the clinical significance of this phenomenon remains obscure. Aim: 1) Analyze in a large cohort of TKI-treated CML patients who developed Ph- clones the presence and occurrence of molecular mutations over time. 2) Evaluate whether molecular mutations are also present in CML patients who were at least in major molecular remission (MMR) and presented with a normal karyotype. Patients and Methods: First Cohort: 51 CML patients (pts, 23 males, 28 females; median age: 60 yrs, range: 37-84 yrs) with response to TKI (imatinib only: n=32, nilotinib only: n=2, imatinib and dasatinib or nilotinib: n=11, all three TKIs: n=6) who developed Ph- clones. Cytogenetics in these pts were as follows: +8 sole (n=24), -Y (n=8), -7 sole (n=4), +9 (n=2), other trisomies (n=4), 9 had other aberrations including some with combinations of two different clones (n=4). In median these abnormalities were present in 30% (range 8-100%) of analyzed metaphases. BCR-ABL1 levels at the time point of analysis were between 0 and 3.8 (median: 0.023) according to international scale. Second Cohort: 50 CML pts (24 males, 26 females; median age: 56 yrs, range: 21-83 yrs), who were at least in MMR and without development of any cytogenetic aberration after 3 years of imatinib treatment. Median time from start of therapy to analysis was 2.6 years (range 3 months to 14 yrs). All cases were analyzed with a pan-myeloid gene panel of 29 genes: ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3 (TKD), IDH1, IDH2, JAK2, KIT, KRAS, MLL-PTD, NOTCH1, NPM1, NRAS, PRPF40B, PTPN11, SF1, SF3A1, SF3B1, SRSF2, TET2, TP53, U2AF1, U2AF2 and ZRSR2. Either complete coding genes or hotspots were first amplified by a microdroplet-based assay (RainDance, Billerica, MA) and subsequently sequenced with a MiSeq instrument (Illumina, San Diego, CA). In addition, RUNX1 was sequenced on the 454 NGS platform (454 Life Sciences, Branford, CT). Results: In the first cohort 28 mutations were found in 19 patients, as 5 patients had 2 and 2 patients even 3 mutations.Thus,in 19/51 pts (37.3%) ≥1 mutation was detected. Median mutation load was 11.5% (range: 2-56%). In detail, mutations in the following genes were detected: ASXL1 (n=9), DNMT3A (n=7), RUNX1 (n=3), NRAS (n=2), TET2 (n=2) and one each in CBL, EZH2, IHD1, PRPF40B, and TP53. Subsequently, these mutations were evaluated in samples from earlier or later time points (18 pts with a total of 235 samples, range: 3-20 samples/pt). In 12 cases a sample from diagnosis of CML was available. In 2 cases a CBL and an ASXL1 mutation were already detectable at low levels, 1.4% and 2%, respectively, at the time of diagnosis and later increased with decreasing BCR-ABL1 levels. In all other 10 cases the mutations were not detectable at diagnosis and were for the first time detectable during TKI treatment (in median after 24 months after diagnosis, range 2-73 months). In the remaining 6 cases date of occurrence could not be determined by backtracking as all earlier samples available were positive for the respective mutation. However, the over time mutation levels were inversely related to BCR-ABL1 expression indicating the presence in independent clones. Within the second cohort with cases in MMR that remained cytogenetically normal only in 2 of the 50 pts (4%) mutations were detected. In one patient a DNMT3A mutation was detected that could be monitored for 8 years with constant low mutation load (3-6%). This was not detectable at diagnosis and occurred after 6 months on imatinib. Very similarly, in the second case a TET2 mutation was first detected after 6 months on imatinib with a mutation load of 2% that very slowly increased to 7% within 8 years. Conclusions: 1) In CML patients that develop Ph- clones other mutations occur in 37.3%. 2) In contrast, in randomly selected CML pts with MMR that are cytogenetically normal, molecular mutations can be detected in only 4%. 3) The clinical importance of molecular mutations in CML in MMR remains unclear. 4) However, these results implicate that chromosomal aberrations are an indicator for genomic instability, also at the molecular level. Disclosures Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Next-Generation Sequencing Reveals Clonal Evolution at the Immunoglobulin Loci in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v124.21.3302.3302 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3302-3302 ◽

Cited By ~ 1

Author(s):

Jennifer R. Brown ◽

Stacey M. Fernandes ◽

Siddha Kasar ◽

Kevin Hoang ◽

Martin Moorhead ◽

...

Keyword(s):

B Cell ◽

Peripheral Blood ◽

Clonal Evolution ◽

Post Treatment ◽

Lymphocytic Leukemia ◽

Clinical Relapse ◽

Employment Equity ◽

Time Points ◽

Equity Ownership ◽

Generation Sequencing

Abstract Background: Immunoglobulin (Ig) gene rearrangement is a hallmark of early B-cell development. Chronic lymphocytic leukemia (CLL) is typically considered a malignancy of mature B-cells and is thought to originate from the oncogenic transformation of a single pre- or post-germinal B-cell. Activation-induced deaminase (AID), an enzyme that induces somatic hypermutation (SHM) at the heavy and light chain Ig loci, has been shown to be active in CLL cells in vitro (Patten et al., Blood 2012). Previous studies suggest that multiple CLL-specific Ig clonotypes related by SHM may be present in patients (pts) with dominant CLL clones possessing somatically mutated or unmutated Ig loci (Logan et al., PNAS 2011; Campbell et al., PNAS 2008). To our knowledge, evolution of the dominant CLL-specific Ig clonotype over the course of treatment has not been demonstrated. Here we utilized the LymphoSIGHT™ method, a next-generation sequencing-based method for lymphocyte characterization and quantification, to quantify clonal evolution at the Ig heavy and kappa chain (IGH and IGK) loci in 63 pts with CLL. Methods: Samples were collected at Stanford University and the Dana-Farber Cancer Institute. Peripheral blood mononuclear cells were isolated, and genomic DNA was extracted. Using unbiased universal primer sets, we amplified IGH and IGK variable, diversity, and joining gene segments. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination (Faham et al., Blood 2012). CLL-specific clonotypes were identified for each patient based on their high frequency (>5%) within the B-cell repertoire of a diagnostic (dx) sample. The highest frequency CLL clonotype identified in a dx sample is termed the “index clonotype”. Dx and post-treatment peripheral blood samples were assessed for evidence of evolved CLL clonotypes using LymphoSIGHT. A clonotype was considered “evolved” based on CDR3 sequence homology to the dx “index clonotype.” Results: CLL clonotypes were identified in dx samples from 63 pts (51 unmutated IGHV; 12 mutated), and we assessed post-treatment samples for the presence of CLL clonotype-associated oligoclonality. Two of 63 pts exhibited clonal evolution in post-treatment samples. One patient with unmutated CLL was MRD negative for over 7 years following allogeneic hematopoietic cell transplant (HCT), and subsequently became MRD positive with the evolved clonotype (differing by 1 nucleotide from the index clonotype) leading to clinical relapse 9 months after MRD positivity, while the original index clone remained undetectable. The patient was treated with ibrutinib upon clinical relapse and continues to have detectable MRD with the same evolved CLL clonotype (Fig 1A). In a second patient with mutated IGHV, we observed several evolved clonotypes in the dx sample. Multiple evolved clonotypes, including 5 that exhibited a significant increase in their frequency relative to the index clonotype, were present in the follow-up sample after treatment with fludarabine and rituximab (Fig 1B). These evolved clonotypes differed from the index clonotype by 1-4 nucleotides, but otherwise shared CDR3 identity, excluding independently arisen B cell clonotypes. Conclusions: We observed evidence of clonal evolution at Ig loci in a small subset (3.2%) of pts with CLL undergoing treatment. The presence of evolution in pts with CLL indicates that either the SHM mechanism, including the AID enzyme, remains active after neoplastic transformation, or the evolved clonotypes arose through a mechanism distinct from SHM. These evolved CLL clonotypes may have a selective advantage, and may be useful as surrogate markers for other oncogenic mutations providing resistance to therapy. Additional cases are under investigation and updated results will be presented. Figure 1. CLL clonal evolution during therapy. MRD levels of two related Ig clonotypes, expressed as leukemia molecules per million leukocytes in peripheral blood, are shown at multiple time points following allogeneic HCT (A). In another patient undergoing conventional treatment, the level of each individual evolved clonotype as a fraction of the total CLL molecules is plotted at dx and post treatment time points. The index clone, evolved clones with increasing levels post-treatment, and evolved clones with decreasing levels post-treatment are shown in red, blue, and white, respectively (B). Figure 1A. Figure 1A. Figure 1B. Figure 1B. Disclosures Moorhead: Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Development of Molecular Mutations during the Evolution of Therapy-Related MDS and Therapy-Related AML

Blood ◽

10.1182/blood.v124.21.4624.4624 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4624-4624

Author(s):

Wolfgang Kern ◽

Manja Meggendorfer ◽

Tamara Alpermann ◽

Andreia de Albuquerque ◽

Claudia Haferlach ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Gene Panel ◽

Employment Equity ◽

Time Points ◽

Number Of Patients ◽

Patients At Risk ◽

Previously Treated ◽

Equity Ownership ◽

Time Point

Abstract Introduction: Therapy-related myelodysplastic syndrome (t-MDS) and acute myeloid leukemia (t-AML) develop after the application of chemotherapy for malignancies in a significant number of patients (pts). Mutations in TP53 have been described recently to be present even before chemotherapy for the prior malignancy and thus also before any sign of t-MDS or t-AML. Data suggested that chemotherapy selected the TP53mutated clone which evolved to t-MDS/t-AML. More comprehensive genetic analyses, however, have been lacking so far. Aim: To identify molecular mutations by a comprehensive gene panel in pts at t-MDS/t-AML diagnosis and to backtrack them to prior time points. Patients and Methods: We searched our database for pts diagnosed with t-MDS or t-AML for whom in addition ≥1 prior peripheral blood or bone marrow sample from assessment of a previously treated malignancy was stored. Diagnosis of t-MDS and t-AML was performed by cytomorphology, cytochemistry and cytogenetics according to WHO classification 2008 in all cases. A total of 11 pts were identified (3/8 females/males; median age at t-MDS/t-AML diagnosis 72 years, range 50-81 years). 8 pts had t-MDS and 3 had t-AML. All pts had received chemotherapy for CLL before. All pts underwent mutation analysis at t-MDS/t-AML diagnosis by a 26 gene panel targeting ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3-TKD, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PHF6, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1, and WT1. The library was generated with the ThunderStorm (RainDance Technologies, Billerica, MA) and sequenced on MiSeq instruments (Illumina, San Diego, CA). Specific mutations identified at t-MDS/t-AML diagnosis were selectively analyzed in prior samples of the respective patients. Mutations were considered for this analysis only if they were present at t-MDS/t-AML diagnosis at mutation loads clearly higher than residual CLL infiltration. Accordingly, mutations were excluded from this analysis if their load was in the range of residual CLL infiltration or lower. One not yet described genetic variant was also excluded. Results: 13 mutations were identified at t-MDS/t-AML diagnosis in 8/11 pts. While in 3 pts no mutations were found, 5 pts had 1 mutation, 2 had 2, and 1 had 4 mutations. Mean number of mutations per pt was 1.6. TP53 was mutated most frequently (n=5), RUNX1 was mutated in 2 pts, and FLT3-TKD, IDH2, KRAS, NPM1, NRAS, and U2AF1 in 1 pt each. Mean mutation load was 27% (range 4-48%) while mean CLL infiltration at the same time point was 2% (range 0-4%). Thus, the attribution of the described mutations to t-MDS/t-AML is highly likely. We then analyzed a total of 13 samples (8 bone marrow, 5 peripheral blood) drawn prior to t-MDS/t-AML diagnosis from the 8 pts for the respective mutations identified at t-MDS/t-AML diagnosis. In 5/8 patients the respective specific mutations identified at t-MDS/t-AML diagnosis were found in at least one prior sample. Genes found mutated in the prior samples were TP53 in 2 cases and IDH2, KRAS, NPM1, RUNX1, and U2AF1 in 1 case each. Mutation loads in general were lower in prior samples as compared to samples at t-MDS/t-AML diagnosis (median 54-fold lower, range 1.5 to 205-fold), except for one sample with a similar load at both time points which both times was clearly higher than the residual CLL infiltration (50% and 42% vs. 9% and 4%). Specifically, in 3/4 patients with samples available from the time point of CLL diagnosis all of these mutations (n=4) were not detectable at a sensitivity level of 1% while in 1 patient 2 mutations were not detectable and a U2AF1mutation was identified with a 1.9% load. This further supports the concept of these mutations being related to a pre-malignant clone which in the majority of cases might have been present at undetectable levels at the time point of CLL diagnosis or which even developed only during chemotherapy and later evolved into t-MDS/t-AML. The mean interval from first detection of the respective mutations to t-MDS/t-AML diagnosis was 10 months (range 4-25 months). Conclusions: Mutational screening applying a 26 gene panel identified molecular mutations in the majority of pts. These mutations were present up to 2 years before t-MDS/t-AML diagnosis. Further studies focusing on patients at risk of t-MDS/t-AML should clarify the role of early molecular screening helping to potentially improve diagnosis and management of t-MDS/t-AML. Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. de Albuquerque:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Increased Eligibility for Attempting Treatment-Free Remission (TFR) with Frontline Nilotinib (NIL) vs Imatinib (IM) in Patients with Philadelphia Chromosome-Positive (Ph+) Chronic Myeloid Leukemia (CML): Results of a Stochastic Simulation Model

Blood ◽

10.1182/blood.v126.23.5154.5154 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5154-5154

Author(s):

Francois-Xavier Mahon ◽

An Tran-Duy ◽

Raechelle G. Ocampo ◽

David Ray ◽

Estella Mendelson ◽

...

Keyword(s):

Treatment Discontinuation ◽

Molecular Response ◽

Employment Equity ◽

Transcript Levels ◽

Time Points ◽

Pharmaceutical Corporation ◽

Rutgers University ◽

Equity Ownership ◽

Simulated Cohort ◽

Time Point

Abstract Background : Tyrosine kinase inhibitors (TKIs) have dramatically improved outcomes in patients with Ph+ CML, in part by allowing achievement of sustained, low levels of BCR-ABL1 transcripts (quantified on the International Scale [IS]). TFR studies (eg, STIM, ENESTfreedom) evaluate whether some of these patients can stop TKI therapy and maintain a therapeutic molecular response off treatment. Here, we evaluated BCR-ABL1IS transcript levels of patients treated with frontline NIL 300 mg twice daily or IM 400 mg once daily for ≥ 1 year to predict time to TFR eligibility according to the ENESTfreedom trial criteria. Methods : A statistical model was developed to predict the probability of future premature treatment discontinuation (due to adverse events, progression, or suboptimal response) and BCR-ABL1IS transcript levels at any time point after 1 year of treatment. The 5-year data from the ENESTnd clinical trial, in which BCR-ABL1IS transcript levels were assessed every 3 months, were the basis for this model. Probabilities of premature treatment discontinuation were modeled using parametric survival methods; early molecular response (EMR; BCR-ABL1IS ≤ 10% at 3 months) status was used as a predictor. For patients remaining on treatment, a second-order Markov chain model was used to predict probabilities of BCR-ABL1IS transcript levels being in each of 5 clinically relevant categories (≤ 0.0032% [MR4.5 ], > 0.0032% to ≤ 0.01% [MR4 ], > 0.01% to ≤ 0.1%, > 0.1% to ≤ 10%, and > 10%) at any time point after 1 year of therapy. Probabilities were a function of EMR status, the proportion of previous BCR-ABL1IS observations at or below MR4, and BCR-ABL1IS categories from the previous 2 assessments. A simulated cohort of 1000 patients was created to match the distribution of EMR status and BCR-ABL1IS categories in the first year of therapy in each of the trial populations (NIL or IM). Premature treatment discontinuation and BCR-ABL1IS categories were randomly drawn at each 3-month interval based on their corresponding predicted probabilities. Time to eligibility criteria for TFR was defined as: last BCR-ABL1IS assessment of MR4.5, none of the prior 3 assessments worse than MR4, and no more than 2 of the prior 3 assessments between MR4 and MR4.5. Results : For years 2 to 5 of the ENESTnd trial, the observed distribution of BCR-ABL1IS categories over time had reasonable agreement with the computer-simulated cohort. Simulation results (Figure) demonstrated that more patients on NIL than on IM were eligible for TFR by year 5 (52% vs 38%, respectively) and by year 10 (72% vs 64%, respectively; P < .0001 for both time points). Conclusion: Patients in our simulated cohort received a minimum of 3 years of frontline treatment with NIL or IM prior to TFR eligibility evaluation, similar to the current consensus in clinical disease management. Treatment with NIL resulted in significantly more patients becoming eligible for TFR by all time points vs treatment with IM. These data suggest that TFR as a therapeutic goal may be more attainable with NIL than IM. Studies evaluating the duration of TFR are presently being conducted. Disclosures Mahon: Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; ARIAD: Consultancy; Pfizer: Consultancy. Tran-Duy:Pharmerit International: Consultancy. Ray:Novartis Pharmaceutical Corporation/Rutgers University: Other: I am currently a fellow with Rutgers University, conducting my "field" experience at Novartis.. Mendelson:Novartis Pharmaceutical Corporation: Employment, Equity Ownership. Buchbinder:Novartis Pharmaceutical Corporation: Employment, Equity Ownership. Edrich:Novartis Pharma AG: Employment. Snedecor:Pharmerit International: Employment, Other: Institution received payment to conduct this study. Saglio:Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis Pharmaceutical Corporation: Consultancy, Honoraria.

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Comprehensive Analyses of Mantle Cell Lymphoma with a 17 Gene Panel Reveal an Accumulation of TP53 Mutations in SOX11 Negative Patients

Blood ◽

10.1182/blood.v124.21.3012.3012 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3012-3012

Author(s):

Manja Meggendorfer ◽

Wolfgang Kern ◽

Claudia Haferlach ◽

Torsten Haferlach ◽

Susanne Schnittger

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

Gene Mutations ◽

Gene Panel ◽

Prognostic Impact ◽

Employment Equity ◽

Mutational Status ◽

Mantle Cell ◽

Sox11 Expression ◽

Equity Ownership

Abstract Introduction: Mantle cell lymphoma (MCL) belongs to the mature B-cell neoplasms and is characterized by t(11;14)(q13;q32)/IGH-CCND1 rearrangement resulting in overexpression of CyclinD1 that is encoded by CCND1. CCND1 is a weak oncogene, requiring additional cooperating oncogenic events. MCL show mainly an aggressive course of disease, although a subset of patients have been identified with an indolent clinical course. This has been associated with a mutated IGHV status and the lack of SOX11 expression. However, some additional gene mutations have been identified in recent years, but the underlying biological heterogeneity is still under debate. Also the prognostic impact of SOX11 remains controversially discussed. Aim: To analyze 1) recently identified molecular mutations in CCND1, UBR5, WHSC1 for their specificity for MCL, 2) in more detail SOX11 negative MCL to get more insights in this group of MCL by sequencing a 17 gene panel. Patients and Methods: In total 184 patients with mature B-cell neoplasms were investigated. All cases were diagnosed according to WHO classification by cytomorphology, immunophenotyping and cytogenetics. 81 cases were diagnosed as MCL, 78 as chronic lymphocytic leukemia/prolymphocytic (CLL/PL), and 25 as CLL. The cohort comprised 67 females and 117 males. CyclinD1 and SOX11 expression levels were quantified by real time PCR. The 17 gene panel included ATM, BIRC3, BRAF, CCND1, FBWX7, IGHV, KLHL6, KRAS, MYD88, NOTCH1, NRAS, POT1, SF3B1, TP53, UBR5, WHSC1, and XPO1. Next generation sequencing was performed on MiSeq instruments (Illumina, San Diego, CA), except for CCND1, UBR5, WHSC1, and IGHV mutational status, which were analyzed by Sanger sequencing. The latter 4 genes and gene expression levels were analyzed in the total cohort, while the 17 gene panel was applied to 26 MCL patients only (13 SOX11 negative and 13 SOX11 positive cases matched for CCND1 mutations and IGHV mutation status). Results: 23/184 (13%) patients had CCND1 mutations, while only 3/184 patients (2%) carried a UBR5 mutation, and 4/184 patients (2%) a WHSC1 mutation. Of note, CCND1 and UBR5 mutations occurred exclusively in MCL patients, WHSC1 mutations were found in MCL and CLL/PL. Therefore CCND1 mutation was, as expected, specific for MCL in comparison to the other 2 mature B-cell neoplasms (p<0.001). Of 81 MCL patients 68 (84%) showed SOX11 overexpression, 23 (28%) had CCND1 mutations and 3 (4%) each had UBR5 and WHSC1 mutations, respectively. The IGHV mutational status was evaluable in 73/81 cases, revealing 32/73 (44%) patients with a mutated IGHV status. A negative correlation of CCND1 mutations and SOX11 overexpression was found: 8/13 (62%) SOX11 negative patients were CCND1 mutated as compared to 15/68 (22%) SOX11 positive patients (p=0.007). Furthermore, CCND1 mutations were more frequent in patients with a mutated IGHV status than in those with unmutated status (15/32 (47%) vs. 8/41 (20%), p=0.021). Accordingly, SOX11 overexpression occurred more often in patients with an unmutated than with a mutated IGHV status (88% vs. 75%; n.s.). Regarding clinical data, more males were SOX11 positive (51/54, 94% vs. 17/27, 63%; p=0.001), and correspondingly more females were CCND1 mutated (12/27, 63% vs. 11/54, 20%; p=0.036). To get more insight in the SOX11 negative patients (n=13) we addressed all these cases and a SOX11 positive control group, matched for IGHV mutational status and CCND1 mutations (n=13), by comprehensive mutational analyses. Overall, beside CCND1 the most frequently mutated gene was TP53 (8/26, 31%), followed by ATM (6/26, 23%), BIRC3 (2/21, 10%), and KRAS (2/26, 8%). No mutations were detected in any of the other genes analyzed. Addressing differences in gene mutations between SOX11 negative and SOX11 positive cases revealed that TP53 mutations were found more frequently in SOX11 negative cases (6/13, 46% vs. 2/13, 15%; n.s.), while ATM mutations were more frequent in SOX11 positive cases (5/13, 39% vs. 1/13, 8%; n.s.). Conclusions: 1) CCND1 mutations are specifically found in MCL, correlate with SOX11 negativity and IGHV mutated status, and are more frequent in females. 2) TP53 is frequently mutated in SOX11 negative patients and its prognostic impact has to be further evaluated. 3) Thus, the differences in clinical course between SOX11 positive and negative MCL patients might correlate with a different spectrum of additional molecular alterations. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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