Clinical Impact Of Small TP53 Mutated Subclones In Chronic Lymphocytic Leukemia

Abstract Introduction TP53 mutations are strong predictors of poor survival and refractoriness in chronic lymphocytic leukemia (CLL) and have direct implications for disease management. Clinical information on TP53 mutations are currently limited to lesions that are represented in the majority of CLL cells. Next generation sequencing (NGS) allows sensitive detection of mutations harbored by a small fraction of the tumor cell population. Here we aim at assessing the frequency, evolution during disease course, and prognostic impact of small TP53 mutated subclones in newly diagnosed CLL. Methods The study was based on a consecutive series of 309 newly diagnosed and previously untreated CLL (median age: 71 years; Binet A/B/C: 79/12/9%; unmutated IGHV genes: 35%; clonal TP53/NOTCH1/SF3B1/BIRC3 lesions: 11/11/7/5%; median follow-up: 8.1 years). TP53 mutations (exons 4-8) were screened on peripheral blood (PB) samples (tumor representation 70-98%) by amplicon-based deep-NGS (GSJ, 454 Life Sciences) (average depth: 2660). A bioinformatic algorithm was developed to call TP53 variants out of background noise. By dilution experiments, deep-NGS allowed to detect mutant allele fractions of 0.3%. TP53 variants were considered subclonal if missed by Sanger sequencing, which was performed in parallel. Subclonal TP53 variants were confirmed by duplicate deep-NGS and independently validated by allele specific PCR (AS-PCR). TP53 variant allele frequency (VAF) was corrected for tumor representation. Results Deep-NGS identified 50 subclonal TP53 mutations (VAF 0.3%-11%) in 28/309 (9%) CLL (Fig 1A). All subclonal mutations were non-silent, were missed by Sanger sequencing, and were validated by AS-PCR. The molecular spectrum of subclonal TP53 mutations (i.e. missense/truncating ratio, transition/transversion ratio, distribution across hot spot codons; p>.05; Fig. 1B-D), as well as the residual transactivational activity of mutants toward the p21 promoter (p=.872) were highly consistent with that of fully clonal TP53 mutations reported in CLL (Zenz T, Leukemia 2010). Subclonal TP53 mutations were the sole TP53 genetic event in 15/309 (4.8%) CLL, while in 13/309 (4.2%) cases subclonal TP53 mutations co-existed in the same leukemic population along with a clonal TP53 mutation or with 17p deletion. In cases (n=12) harboring more than one TP53 mutation, the variants mapped on distinct sequencing reads from the same amplicon suggesting that they belonged to different CLL subclones. By combining subclonal TP53 mutations, clonal TP53 mutations and 17p deletion, 50/309 (16%) CLL harbored at least one TP53 defect at diagnosis. Subclonal TP53 mutations were significantly enriched among cases presenting with advanced stage (Binet C: 26%; p=.005) and clonal TP53 abnormalities (37%; p<001). Cases harboring solely subclonal TP53 mutation showed a median overall survival (3.4 years) significantly shorter than TP53 wild type cases (10.8 years; p=.028), and similar to that of cases with clonal TP53 genetic defects (3.1 years; p=.375) (Fig. 1E). By multivariate analysis, cases harboring subclonal TP53 mutations had a significantly increased hazard of death (HR: 2.0; p=.023) after adjusting for age, disease stage, IGHV mutation status, clonal TP53 genetic defects, and lesions of NOTCH1, SF3B1 and BIRC3. Subclonal TP53 mutations showed a similar allele fraction in paired PB and lymph-node CLL cells in 3/5 assessable cases, suggesting a systemic spread of mutated subclones across disease compartments. Among cases harboring solely subclonal TP53 mutations, longitudinal deep-NGS of sequential samples documented the outgrowth of the TP53 variant to a fully clonal level in 57% (4/7) of cases. In all these cases, clonal selection was strongly associated with treatment exposure and development of a chemorefractory phenotype. Conversely, in cases managed by watch-and-wait only, the load of TP53 mutations did not increase during follow-up. Conclusions Small TP53 mutated subclones detected by deep-NGS occur in a significant fraction of newly diagnosed CLL, have the same unfavorable prognostic impact as clonal TP53 defects, and anticipate the development of a chemorefractory phenotype among CLL requiring treatment. Search of minor subclones by deep-NGS should be considered for a comprehensive assessment of TP53 disruption in CLL. D.R. and H.K equally contributed; R.F., R.R. and G.G equally contributed. Disclosures: No relevant conflicts of interest to declare.

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Small Subclones Harboring NOTCH1, SF3B1 or BIRC3 Mutations Are Clinically Irrelevant in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v124.21.295.295 ◽

2014 ◽

Vol 124 (21) ◽

pp. 295-295

Author(s):

Davide Rossi ◽

Hossein Khiabanian ◽

Silvia Rasi ◽

Carmela Ciardullo ◽

Lodovico Terzi di Bergamo ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Sanger Sequencing ◽

Lymphocytic Leukemia ◽

Sensitivity Threshold ◽

Consecutive Series ◽

Prognostic Impact ◽

Newly Diagnosed ◽

Tp53 Mutations ◽

Clonal Abundance ◽

Notch1 Mutations

Abstract Introduction. Ultra-deep next generation sequencing (NGS) allows sensitive detection of mutations and estimation of their clonal abundance in tumor cell populations. TP53 mutations identified by ultra-deep NGS significantly impact on survival of chronic lymphocytic leukemia (CLL) patients, independent of their representation in the tumor clone and even if restricted to a small fraction of leukemic cells. Purpose. Here we aim at assessing the frequency, prognostic impact and evolution during disease course of NOTCH1, SF3B1 and BIRC3 mutations identified by ultra-deep NGS in newly diagnosed CLL. Methods. The study was based on a consecutive series of 304 newly diagnosed and previously untreated CLL (median age: 71 years; Binet A/B/C: 79/12/9%; unmutated IGHV genes: 35%; del13q/+12/del11q/del17p: 51/21/8/9%; median follow-up: 8.1 years). By ultra-deep NGS, TP53 mutations (exons 4-8) occurred in 15% of CLL with a median clonal abundance of 18% (range 0.2-95%). NOTCH1 (hg19 g.chr9:139390596-139390829), SF3B1 (exons 14, 15) and BIRC3 (exons 6, 9) mutations were screened on peripheral blood samples by amplicon-based ultra-deep NGS (454 Life Sciences) (average depth: 2437). A bioinformatic algorithm was applied to call non-silent variants out of background NGS noise. Variant calling by the algorithm was validated by Sanger sequencing or, if the variant was below the sensitivity threshold of Sanger sequencing, by both duplicate ultra-deep NGS and allele specific PCR (AS-PCR). Variant allele frequency (VAF) was corrected for tumor representation. Results. Ultra-deep NGS identified 46 NOTCH1 mutations (median VAF 24%; range: 1.4-64%) in 14% (43/304) CLL, 43 SF3B1 mutations (median VAF 16%; range: 0.5-48%) in 11% (35/304) CLL and 37 BIRC3 mutations (median VAF 5.6%; range: 0.2-47%) in 8% (26/304) CLL. In cases harboring more than one mutation (NOTCH1=3; SF3B1=2; BIRC3=5), the variants mapped on distinct sequencing reads from the same amplicon suggesting that they belonged to different CLL subclones. Out of the variants identified by ultra-deep NGS, a significant fraction of NOTCH1 (n=14; median VAF: 3.9%; range: 1.4-9%), SF3B1 (n=25; median VAF: 4.3%; range: 0.5-17%) and BIRC3 (n=30; median VAF: 1.4%; range: 0.2-6%) mutations were missed by Sanger sequencing because their VAF was below the sensitivity threshold of the assay (~10%). The molecular spectrum of these small subclonal mutations was highly consistent with that of mutations that were visible by Sanger sequencing. The overall survival of cases harboring solely small subclonal mutations of NOTCH1, SF3B1 and BIRC3 was similar to that of wild type cases and longer than that of cases with clonal lesions (Fig. 1). Outcome-driven statistical approaches (maxstat and recursive partitioning) were applied to identify the optimal cut-off of the VAF in order to test whether the clonal representation of mutations correlated with CLL survival. Application of these approaches to TP53 mutations failed in identifying a clear cut-off, suggesting that TP53 variants are relevant to the outcome of CLL independent of their VAF. Conversely, in the case of NOTCH1, SF3B1 and BIRC3 mutations, these approaches consistently identified 25%, 35% and 1%, respectively, as the best VAF cut-off values for outcome prediction (Fig. 1), thus suggesting that small subclones harboring NOTCH1, SF3B1 or BIRC3 mutations are clinically irrelevant. By multivariate analysis, NOTCH1 mutations >25% of VAF (HR: 1.8; p=.038), SF3B1 mutations >35% of VAF (HR: 2.9; p=.005) and BIRC3 mutations >1% of VAF (HR: 1.8; p=.044) were significantly associated with an increase in the hazard of death, after adjusting for TP53 lesions (HR: 2.9; p<.001), del11q (HR: 2.2; p=.007), +12 (HR: 1.3, p=.174) and del13q (HR: 0.8; p=.512). Among cases harboring small mutated subclones, longitudinal deep NGS of sequential samples documented the outgrowth of the variants to a clonal level in 1/5 cases for NOTCH1, in 5/12 for SF3B1 and in 3/10 for BIRC3. While selection of NOTCH1 and BIRC3 variants did not associate neither with disease feature nor with treatment, the outgrowth of subclonal SF3B1 mutations was restricted to cases showing unmutated IGHV genes (p=.015). Conclusions. Small mutated subclones of the NOTCH1, SF3B1 and BIRC3 genes appear to be clinically irrelevant. Mutations of NOTCH1 and SF3B1 require a substantial clonal representation to be harmful. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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TP53 Mutation and Survival in Chronic Lymphocytic Leukemia

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.8762 ◽

2010 ◽

Vol 28 (29) ◽

pp. 4473-4479 ◽

Cited By ~ 343

Author(s):

Thorsten Zenz ◽

Barbara Eichhorst ◽

Raymonde Busch ◽

Tina Denzel ◽

Sonja Häbe ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

High Performance ◽

Tp53 Mutation ◽

Prospective Trial ◽

Progression Free Survival ◽

Complete Response ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Tp53 Mutations ◽

The Impact

Purpose The precise prognostic impact of TP53 mutation and its incorporation into treatment algorithms in chronic lymphocytic leukemia (CLL) is unclear. We set out to define the impact of TP53 mutations in CLL. Patients and Methods We assessed TP53 mutations by denaturing high-performance liquid chromatography (exons 2 to 11) in a randomized prospective trial (n = 375) with a follow-up of 52.8 months (German CLL Study Group CLL4 trial; fludarabine [F] v F + cyclophosphamide [FC]). Results We found TP53 mutations in 8.5% of patients (28 of 328 patients). None of the patients with TP53 mutation showed a complete response. In patients with TP53 mutation, compared with patients without TP53 mutation, median progression-free survival (PFS; 23.3 v 62.2 months, respectively) and overall survival (OS; 29.2 v 84.6 months, respectively) were significantly decreased (both P < .001). TP53 mutations in the absence of 17p deletions were found in 4.5% of patients. PFS and OS for patients with 17p deletion and patients with TP53 mutation in the absence of 17p deletion were similar. Multivariate analysis identified TP53 mutation as the strongest prognostic marker regarding PFS (hazard ratio [HR] = 3.8; P < .001) and OS (HR = 7.2; P < .001). Other independent predictors of OS were IGHV mutation status (HR = 1.9), 11q deletion (HR = 1.9), 17p deletion (HR = 2.3), and FC treatment arm (HR = 0.6). Conclusion CLL with TP53 mutation carries a poor prognosis regardless of the presence of 17p deletion when treated with F-based chemotherapy. Thus, TP53 mutation analysis should be incorporated into the evaluation of patients with CLL before treatment initiation. Patients with TP53 mutation should be considered for alternative treatment approaches.

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Monoallelic TP53 inactivation is associated with poor prognosis in chronic lymphocytic leukemia: results from a detailed genetic characterization with long-term follow-up

Blood ◽

10.1182/blood-2008-04-154070 ◽

2008 ◽

Vol 112 (8) ◽

pp. 3322-3329 ◽

Cited By ~ 272

Author(s):

Thorsten Zenz ◽

Alexander Kröber ◽

Katrin Scherer ◽

Sonja Häbe ◽

Andreas Bühler ◽

...

Keyword(s):

Tp53 Mutation ◽

Direct Sequencing ◽

Clonal Evolution ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Clone Size ◽

Tp53 Mutations ◽

Long Term Follow Up ◽

Serial Samples

AbstractThe exact prognostic role of TP53 mutations (without 17p deletion) and any impact of the deletion without TP53 mutation in CLL are unclear. We studied 126 well-characterized CLL patients by direct sequencing and DHPLC to detect TP53 mutations (exons 2-11). Most patients with 17p deletions also had TP53 mutations (81%). Mutations in the absence of 17p deletions were found in 4.5%. We found a shorter survival for patients with TP53 mutation (n = 18; P = .002), which was more pronounced when analyzed from the time point of mutation detection (6.8 vs 69 months, P < .001). The survival was equally poor for patients with deletion 17p plus TP53 mutation (7.6 months, n = 13), TP53 mutation only (5.5 months, n = 5), and 17p deletion only (5.4 months, n = 3). The prognostic impact of TP53 mutation (HR 3.71) was shown to be independent of stage, VH status, and 11q and 17p deletion in multivariate analysis. Serial samples showed evidence of clonal evolution and increasing clone size during chemotherapy, suggesting that there may be patients where this treatment is potentially harmful. TP53 mutations are associated with poor sur-vival once they occur in CLL. The de-monstration of clonal evolution under selective pressure supports the biologic significance of TP53 mutations in CLL.

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Prognostic Impact of NOTCH1 Hotspot Mutation in TP53-Mutated Patients with Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v124.21.3283.3283 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3283-3283

Author(s):

Barbara Kantorova ◽

Jitka Malcikova ◽

Veronika Navrkalova ◽

Jana Smardova ◽

Kamila Brazdilova ◽

...

Keyword(s):

Overall Survival ◽

Chronic Lymphocytic Leukemia ◽

Tp53 Mutation ◽

Direct Sequencing ◽

Lymphocytic Leukemia ◽

The Other ◽

Wild Type ◽

Tp53 Mutations ◽

Time To First Treatment ◽

Hotspot Mutation

Abstract Introduction A presence of activating mutations in NOTCH1 gene has been recently associated with reduced survival and chemo-immunotherapy resistance in chronic lymphocytic leukemia (CLL). However, a prognostic significance of the NOTCH1 mutations with respect to TP53mutation status has not been fully explained yet. Methods An examined cohort included 409 patients with CLL enriched for high risk cases; in 121 patients consecutive samples were investigated. To determine the TP53 mutation status, a functional analysis of separated alleles in yeast (FASAY, exons 4-10) combined with direct sequencing was performed; the ambiguous cases were retested using an ultra-deep next generation sequencing (MiSeq platform; Illumina). The presence of NOTCH1 hotspot mutation (c.7544_7545delCT) was analyzed using direct sequencing complemented by allele-specific PCR in the selected samples. In several patients harboring concurrent NOTCH1 and TP53 mutations, single separated cancer cells were examined using multiplex PCR followed by direct sequencing. A correlation between mutation presence and patient overall survival, time to first treatment and other molecular and cytogenetic prognostic markers was assessed using Log-rank (Mantel-cox) test and Fisher's exact test, respectively. Results The NOTCH1 and TP53 mutations were detected in 16% (65/409) and 27% (110/409) of the examined patients, respectively; a coexistence of these mutations in the same blood samples was observed in 11% (19/175) of the mutated patients. The detected increased mutation frequency attributes to more unfavorable profile of the analyzed cohort; in the TP53-mutated patients missense substitutions predominated (75% of TP53 mutations). As expected, a significantly reduced overall survival in comparison to the wild-type cases (147 months) was observed in the NOTCH1-mutated (115 months; P = 0.0018), TP53-mutated (79 months; P < 0.0001) and NOTCH1-TP53-mutated patients (101 months; P = 0.0282). Since both NOTCH1 and TP53 mutations were strongly associated with an unmutated IGHV gene status (P < 0.0001 and P = 0.0007), we reanalyzed the IGHV-unmutated patients only and interestingly, the impact of simultaneous NOTCH1 and TP53 mutation presence on patient survival was missed in this case (P = 0.1478). On the other hand, in the NOTCH1 and/or TP53-mutated patients significantly reduced time to first treatment was identified as compared to the wild-type cases (41 months vs. 25 months in NOTCH1-mutated, P = 0.0075; 17 months in TP53-mutated, P < 0.0001; and 18 months in NOTCH1-TP53-mutated patients, P = 0.0003). The similar results were observed also in the subgroup of the IGHV-unmutated patients, with the exception of patients carrying sole NOTCH1 mutation (P = 0.2969). Moreover, in the NOTCH1-TP53-mutated patients an increased frequency of del(17p)(13.1) was found in comparison to the TP53-mutated patients only (72% vs. 56%); this cytogenetic defect was not detected in the patients with sole NOTCH1 mutation. Our results might indicate, that NOTCH1 mutation could preferentially co-selected with particular, less prognostic negative type of TP53 defects. Notably, in our cohort the NOTCH1 mutation predominated in the patients harboring truncating TP53 mutations localized in a C-terminal part of the TP53 gene behind the DNA-binding domain (P = 0.0128). Moreover, in one of the NOTCH1-TP53-mutated patients the analysis of separated cancer cells revealed a simultaneous presence of NOTCH1 mutation and TP53 in-frame deletion in the same CLL cell. In contrast, in the other examined NOTCH1-TP53-mutated patient the concurrent NOTCH1 mutation and TP53 missense substitution (with presumed negative impact on patient prognosis) were found in different CLL cells. Conclusions The parallel presence of NOTCH1 hotspot mutation might be detected in a significant proportion of TP53-mutated patients and it seems to be associated with less prognostic unfavorable TP53 mutations. Nevertheless, these preliminary data should be further confirmed in a large cohort of patients. This study was supported by projects VaVPI MSMT CR CZ.1.05/1.1.00/02.0068 of CEITEC, IGA MZ CR NT13493-4/2012, NT13519-4/2012 and CZ.1.07/2.3.00/30.0009. Disclosures Brychtova: Roche: Travel grants Other. Doubek:Roche: Travel grants Other.

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A comprehensive study of TP53 mutations in chronic lymphocytic leukemia: Analysis of 1287 diagnostic and 1148 follow-up CLL samples

Leukemia Research ◽

10.1016/j.leukres.2010.12.016 ◽

2011 ◽

Vol 35 (7) ◽

pp. 889-898 ◽

Cited By ~ 10

Author(s):

Sona Pekova ◽

Oldrich Mazal ◽

Radek Cmejla ◽

David W. Hardekopf ◽

Radek Plachy ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Tp53 Mutations ◽

Comprehensive Study

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Clinical Impact of Clonal and Subclonal TP53, SF3B1, BIRC3, and ATM Mutations in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v126.23.4138.4138 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4138-4138

Author(s):

Ferran Nadeu ◽

Julio Delgado ◽

Cristina Royo ◽

Tycho Bauman ◽

Tatjana Stankovic ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Allele Frequency ◽

Lymphocytic Leukemia ◽

Molecular Characteristics ◽

Prognostic Impact ◽

Driver Genes ◽

Germline Variants ◽

Tp53 Mutations ◽

Mutational Status ◽

The Impact

Abstract Genomic studies have provided a complete profile of somatic mutations in chronic lymphocytic leukemia (CLL). These comprehensive approaches have revealed a relatively large number of mutated genes, the adverse prognostic value of some of which has been demonstrated in a number of reports. Recent studies have shown the clinical relevance of TP53 mutations at very low allele frequency. The presence and prognostic impact of minor mutated clones of other CLL driver genes and their clonal dynamics in the evolution of the disease is not well known. The goal of this study was to explore the presence of clonal and subclonal mutations of TP53, SF3B1, BIRC3, and ATM using an ultra-deep next-generation sequencing (NGS) strategy, to define the evolution of these subclones in different time-points of the disease, and to determine their influence in the outcome of the patients. Samples from 363 untreated CLL cases were included in this study. Copy number alterations were investigated by high density SNP-arrays or by quantitative PCR in 341 and 16 cases, respectively. Targeted ultra-deep NGS of TP53 (exons 4-10), ATM (exons 2-63), BIRC3 (exons 2-9), and SF3B1 (exons 14-16 and 18), including splicing sites, was performed using the Access-Array system (Fluidigm) and sequenced in a MiSeq equipment (Illumina). This methodology combined with a robust bioinformatic analysis based on well-known available tools allowed the identification of mutations down to 0.3% of variant allele frequency (VAF). Results obtained were fully verified by orthogonal techniques. Twelve per cent of VAF was used as threshold for the classification of clonal or subclonal mutations since 12% was the cut-off for detection of mutations by Sanger sequencing. Deletions of 11q comprising ATM or BIRC3 were found in 7% of the cases and were associated with mutations of the other ATM allele in 19/26 (73%) cases and BIRC3 in 3/23 (13%). Deletions of 17p were found in 19 (5%) cases and co-existed with TP53 mutations in 15 (79%) of them. Regarding the mutational status of the studied genes, TP53 mutations were present in 11.6% of patients (7.2% clonal, 4.4% subclonal), ATM mutations in 10% (7% clonal, 1% subclonal, 2% germline mutations considered pathogenic), SF3B1 mutations in 12% (7% clonal, 5% subclonal), and BIRC3 mutations in 4% (2% clonal, 2% subclonal). These subclonal mutations had similar molecular characteristics to their respective high-allele frequency mutations supporting a comparable pathogenic effect. In this regard, clonal and subclonal SF3B1 mutations were associated with shorter time to first treatment (TTT) independently of IGHV mutations. Clonal and subclonal TP53 mutations predicted for shorter overall survival (OS) together with the IGHV mutational status, although the impact of isolated TP53 mutations (i.e. without 17p deletion) on OS was not so evident, as has been the case in other studies. In addition, the outcome of patients with clonal and subclonal BIRC3 mutations showed a similar significant shorter OS. Regarding ATM, the effect of isolated subclonal ATM mutations could not be evaluated because of their low number, but ATM mutations as a whole had a significant impact on TTT even in the absence of 11q deletions. This study also reinforces the need to study the germline of the patients to fully characterize the ATM mutations observed in the tumors. Of note, germline variants previously described as pathogenic were associated with 11q deletions, confirming the hypothesis already suggested that these germline variants may influence disease progression through loss of the otherallele. Clonal dynamics was examined in longitudinal samples of 45 CLL patients. We confirmed the expansion of most TP53 mutated clones after therapy. However, both TP53 and SF3B1 mutations expanded also before any therapy in some patients, indicating that progressive dynamics of these clones is not only dependent on therapy selection. On the contrary, small ATM mutated clones seemed to be more stable. Although the number of cases is limited, we observed that clonal evolution in longitudinal samples had an unfavorable impact on OS. In conclusion, this study shows the presence of a high number of subclonal mutations of different driver genes in CLL and provides insights on the impact of these mutations on the outcome of the patients. These findings suggest that the characterization of the subclonal architecture may be relevant for a better management of CLL patients. Disclosures No relevant conflicts of interest to declare.

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Lymphocyte Doubling Time As A Key Prognostic Factor To Predict Time To First Treatment In Early-Stage Chronic Lymphocytic Leukemia

Frontiers in Oncology ◽

10.3389/fonc.2021.684621 ◽

2021 ◽

Vol 11 ◽

Author(s):

Fortunato Morabito ◽

Giovanni Tripepi ◽

Riccardo Moia ◽

Anna Grazia Recchia ◽

Paola Boggione ◽

...

Keyword(s):

Prognostic Factors ◽

Chronic Lymphocytic Leukemia ◽

Doubling Time ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Newly Diagnosed ◽

Prognostic Role ◽

Mutational Status ◽

Binet Stage ◽

Time To First Treatment

The prognostic role of lymphocyte doubling time (LDT) in chronic lymphocytic leukemia (CLL) was recognized more than three decades ago when the neoplastic clone’s biology was almost unknown. LDT was defined as the time needed for the peripheral blood lymphocyte count to double the of the initial observed value. Herein, the LDT prognostic value for time to first treatment (TTFT) was explored in our prospective O-CLL cohort and validated in in two additional CLL cohorts. Specifically, newly diagnosed Binet stage A CLL patients from 40 Italian Institutions, representative of the whole country, were prospectively enrolled into the O-CLL1-GISL protocol (clinicaltrial.gov identifier: NCT00917540). Two independent cohorts of newly diagnosed CLL patients recruited respectively at the Division of Hematology in Novara, Italy, and at the Hospital Clinic in Barcelona, Spain, were utilized as validation cohorts. In the training cohort, TTFT of patients with LDT >12 months was significantly longer related to those with a shorter LDT. At Cox multivariate regression model, LDT ≤ 12 months maintained a significant independent relationship with shorter TTFT along with IGHV unmutated (IGHVunmut) status, 11q and 17p deletions, elevated β2M, Rai stage I-II, and NOTCH1 mutations. Based on these statistics, two regression models were constructed including the same prognostic factors with or without the LDT. The model with the LTD provided a significantly better data fitting (χ2 = 8.25, P=0.0041). The risk prediction developed including LDT had better prognostic accuracy than those without LDT. Moreover, the Harrell’C index for the scores including LDT were higher than those without LDT, although the accepted 0.70 threshold exceeded in both cases. These findings were also confirmed when the same analysis was carried out according to TTFT’s explained variation. When data were further analyzed based on the combination between LDT and IGHV mutational status in the training and validation cohorts, IGHVunmut and LDT>12months group showed a predominant prognostic role over IGHVmut LTD ≤ 12 months (P=0.006) in the O-CLL validation cohort. However, this predominance was of borden-line significance (P=0.06) in the Barcelona group, while the significant prognostic impact was definitely lost in the Novara group. Overall, in this study, we demonstrated that LDT could be re-utilized together with the more sophisticated prognostic factors to manage the follow-up plans for Binet stage A CLL patients.

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Other Cancers in Long-Term Survivors of Chronic Lymphocytic Leukemia: Incidence and Prognostic Relevance

Blood ◽

10.1182/blood.v124.21.3323.3323 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3323-3323 ◽

Cited By ~ 2

Author(s):

Lorenzo Falchi ◽

Michael Keating ◽

Susan Lerner ◽

Xuemei Wang ◽

Kplola Y Elhor Gbito ◽

...

Keyword(s):

Bladder Cancer ◽

Chronic Lymphocytic Leukemia ◽

Conflicts Of Interest ◽

Observation Time ◽

Lymphocytic Leukemia ◽

P Value ◽

Prognostic Impact ◽

Long Term Survivors

Abstract Introduction. The clinical course of chronic lymphocytic leukemia (CLL) is mostly indolent. About one third of the patients are managed with lifelong watch-and-wait (WW) and those who receive therapy often achieve a durable remission. As a result, the majority of patients with CLL will live with their disease for long periods of time, and be exposed to several complications, including the occurrence of other cancers (OC). Patients with CLL may have an increased incidence in OC. Published reports indicate an incidence of 3-27%, mostly in treated patients, however, very little is known on OC in patients with CLL not requiring therapy. Furthermore, observation time in published studies is limited to <5 years, and the incidence of OC in patients followed for longer than 10 years is unknown. We, therefore, studied the incidence and prognostic impact of OC in treatment-naïve patients with CLL followed for ≥10 years. Methods. We reviewed our database and identified all patients with CLL untreated at the time of referral. We selected long-term survivors (LTS), defined as patients with a follow-up ≥10 years, and analyzed the incidence and prognostic impact of OC in this population. Non-melanoma skin cancers were excluded since these were diagnosed and treated promptly in virtually all cases and felt not to have prognostic impact. Standardized incidence ratios (SIR) were calculated for OC occurring after the diagnosis of CLL that were reportable to the Surveillance, Epidemiology and End Results program.The estimated overall survival (OS) according to the presence of OC was plotted considering OC as a time-dependent covariate. Results. We identified 797 LTS of CLL seen at our institution between 1957 and 2003. Median age was 56 years (24-88). 57% of patients were males. Median follow-up for the entire population is 154 months (120-485). We recorded 383 OC in 286 (36%) patients. 76/286 (26%) patients had >1 OC (62 had 2 OC, 10 had 3, 2 had 4, 1 had 5 and 1 had 6).The firstOC preceded or was diagnosed concomitantly with CLL in 100 patients (35%), while in the remaining 186 (65%) it occurred later during the course of the disease. 570 patients (71%) required treatment for CLL. Median time to treatment was 18 months (0-454). In treated patients, the cumulative frequency of OC was 205/570 (36%) and in WW patients 81/227 (36%). The SIR for all OC was 1.2 (p = .034). Males and patients younger than 60 years had a significantly higher incidence of OC (SIR 1.31 and 1.27, respectively). Among OC types, secondary leukemia, melanoma and head and neck cancers had the highest observed-to-expected ratio. Surprisingly, lung, digestive tract, and bladder cancer had a lower-than-expected incidence (table). 474 patients (59%) are alive. 222/570 (39%) treated patients and 101/227 (44%) WW patients have died. The median OS was longer in patients without OC (279 months) vs. those with OC (189 months). Independent predictors of shorter survival in multivariate analysis included higher creatinine, the presence of OC, and older age. Discussion. This is the first study to address the incidence of OC in LTS of CLL, including WW patients. In our population, the frequency of OC is similar in treated and WW patients. Although the incidence of OC in LTS of CLL is higher compared to matched general population, the incidence of lung, digestive and bladder cancer is lower than expected. Reasons of this finding remain to be identified.The occurrence of OC is an independent predictor of shorter survival, thus constituting a relevant competing risk of mortality in LTS of CLL. Variable Observed Expected Person-years SIR (O/E) 95% CI for O/E P -value Overall 148 123.34 10956 1.20 1.01 – 1.40 0.034 Male 96 73.4 5885 1.31 1.06 – 1.58 0.013 Female 52 49.93 5071 1.04 0.78 – 1.36 0.67 Age ≥60 years 60 54.33 3416 1.10 0.84 – 1.42 0.44 Age <60 years 88 69.02 7540 1.27 1.02 – 1.57 0.027 OC type Prostate 28 25.92 11809 1.08 0.72 – 1.56 0.64 Lung 20 29.08 11942 0.69 0.42 – 1.06 0.04 Breast 19 18.60 11855 1.02 0.62 – 1.59 0.96 Melanoma 16 4.23 11926 3.78 2.16 – 6.14 0.00 Leukemia 15 4.27 12009 3.51 1.96 – 5.79 0.00 Non-Hodgkin lymphoma 6 6.38 11996 0.94 0.34 – 2.05 1.00 Digestive 16 40.4 11937 0.40 0.23 – 0.64 0.00 Colon 8 19.42 11972 0.41 0.18 – 0.81 0.006 Pancreas 2 4.83 12024 0.41 0.05 – 1.49 0.18 Rectal 3 8.69 12011 0.34 0.07 – 1.00 0.05 Bladder 3 11.18 11993 0.27 0.05 – 0.78 0.009 Multiple Myeloma 2 1.98 12012 1.01 0.12 – 3.64 1.00 Lip 3 0.02 12015 150 31.00 – 438.5 0.00 Salivary gland 2 0.03 12026 66.66 8.00 – 240.06 0.00 Disclosures No relevant conflicts of interest to declare.

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The TP53 Codon72 Polymorphism Is Associated with TP53 Mutations in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v118.21.1783.1783 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1783-1783 ◽

Cited By ~ 1

Author(s):

Vera Grossmann ◽

Valentina Artusi ◽

Susanne Schnittger ◽

Frank Dicker ◽

Sabine Jeromin ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Mutation Rate ◽

Tp53 Mutation ◽

Genome Project ◽

Lymphocytic Leukemia ◽

Wild Type ◽

Employment Equity ◽

Tp53 Mutations ◽

Equity Ownership ◽

Exon 4

Abstract Abstract 1783 TP53 is one of the most important cell-cycle regulator genes and its tumor suppressor activity is fundamental in cellular responses. Mutations in TP53 are known to influence clinical outcome in diverse diseases. In particular, a relationship between TP53 mutations and a poor prognosis has been established in chronic lymphocytic leukemia (CLL), which is one of the most commonly diagnosed lymphoid malignancies in Western countries. Thus far, it has been demonstrated that TP53 mutations are associated with codon72 polymorphism in different diseases e.g. breast cancer, lung cancer, head and neck squamous cell carcinoma, and that this variant could determine cancer susceptibility. In this study, we investigated the overall TP53 mutation rate in 511 CLL and focused on the codon72 polymorphism (rs1042522) in exon 4 (transcript-ID: ENST00000269305). We initially examined the published available 1000 Genome Project results of the European cohort: from a total of 283 genomes analyzed, 137 showed an ARG/ARG genotype (48%), 124 an ARG/PRO genotype (43%) and 22 a PRO/PRO genotype (7.7%). Secondly, in order to determine a potential association between this polymorphic variant and mutations in the TP53 gene, we investigated 511 thoroughly characterized patients with CLL, all diagnosed by immunophenotyping in our laboratory. For molecular analyses, all cases were analyzed for TP53 mutations (exon 4 to exon 11) either by DHPLC and subsequent Sanger sequencing (n=210/511), or using a sensitive next-generation amplicon deep-sequencing assay (n=301/511) (454 Life Sciences, Branford, CT). We observed the occurrence of the three distinct genotypes (ARG/ARG, ARG/PRO, PRO/PRO) of codon72 in the CLL cohort and detected ARG/ARG as the most common genotype (63%), followed by ARG/PRO (31.7%), and PRO/PRO (5.3%); very similar to the distribution of the codon72 polymorphism in the 1000 Genome Project data. Moreover, mutations in TP53 were detected in 63/511 patients resulting in an overall mutation rate of 12%, which reflects the expected mutation rate in this disease. Importantly, as already demonstrated in other malignancies, we here present that also in CLL patients harboring a PRO/PRO genotype a significantly higher frequency of TP53 mutations (9/27, 33%) was observed compared to ARG/ARG (41/321, 13%, P=.037) and ARG/PRO (13/163, 8%, P=.012). With respect to the clinical outcome we confirmed a generally poor survival for the TP53 mutated cases as compared to TP53 wild-type patients (n=23 vs. 189 with clinical data available, alive at 7 years: 29.6% vs. 88.1%; P<.001). Moreover, the impact of the three distinct genotypes on outcome was analyzed. However, no correlation was detectable, neither in the cohort of TP53 mutated cases (P=.225) nor in the TP53 wild-type patients (P=.190). In summary, we demonstrated a significant association between the codon72 allelic variant and TP53 mutation rate in our CLL cohort. Patients with a PRO/PRO genotype showed a significantly higher frequency of TP53 mutations than all other genotypes. However, no prognostic impact of codon72 allelic variant was observed, neither in the TP53 wild-type nor in the TP53 mutated cohort. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Artusi:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Boeck:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment.

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EGR2 Mutations in Chronic Lymphocytic Leukemia: A New Bad Player

Blood ◽

10.1182/blood.v126.23.4126.4126 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4126-4126

Author(s):

Daniel Noerenberg* ◽

Emma Young* ◽

Viktor Ljungström ◽

Larry Mansouri ◽

Karla Plevova ◽

...

Keyword(s):

Overall Survival ◽

General Practice ◽

Chronic Lymphocytic Leukemia ◽

Deep Sequencing ◽

Research Funding ◽

Sanger Sequencing ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Bcr Signaling ◽

The Uk

Abstract *Contributed equally as first authors. **Contributed equally as senior authors. Recurrent mutations within EGR2, a versatile transcription factor involved in differentiation of hematopoietic cells, were recently reported in 8% of advanced-stage chronic lymphocytic leukemia (CLL) patients, where they appear to be associated with a worse outcome. EGR2 is activated through ERK phosphorylation upon B-cell receptor (BcR) stimulation, and we have previously shown that EGR2 -mutated CLL patients display altered expression of EGR2 down-stream target genes compared to wildtype (wt) patients, thereby pointing to a pathogenic role for EGR2 mutations in dysregulating BcR signaling. To gain further insight into the incidence and prognostic impact of EGR2 mutations in CLL, we screened samples from a well-characterized series of 1430 patients, either by Sanger sequencing (n=1019) or targeted deep-sequencing (n=370), both covering the recently reported EGR2 hotspot in exon 2. In addition, whole-exome data was available for an additional 43 patients. Different cohorts were included in our analysis ranging from 'general practice' CLL (33% IGHV-unmutated (U-CLL), 6% TP53 -aberrant (TP53abn), n=693), to adverse-prognostic CLL (89% U-CLL, 26% TP53abn, n=325), patients belonging to clinically aggressive stereotyped subsets #1-3 & #5-8 (n=342), patients relapsing after FCR therapy (n=41) and Richter transformed cases (n=31), thus reflecting the heterogeneous nature of CLL. Nineteen EGR2 mutations were detected by Sanger sequencing, while 22 additional mutations were identified with deep-sequencing using a 5% variant allele frequency (VAF) cutoff (median 39%, range 5.6-63.9%, median coverage 43,000X). With the exception of one in-frame deletion, all mutations were missense alterations located within the three zinc-finger domains. Significant enrichment of EGR2 mutations was observed in adverse-prognostic (18/325, 5.5%) and FCR-relapsing (4/41, 9.8%) CLL compared to the 'general practice' cohort (18/693, 2.6%, Figure 1A). A surprisingly low frequency was observed among clinically aggressive stereotyped subsets (5/342, 1.5%), although the cause for this observation is currently unknown. Finally, 2/31 (6.5%) cases with Richter transformation carried an EGR2 mutation. Of the 4 FCR-relapsing, EGR2 -mutated cases with available overtime samples, all demonstrated a significant expansion of the EGR2 -mutated clone at relapse (VAF-increase between 15-41%). In addition, subclonal levels of EGR2 hotspot mutations (VAF 0.5-5%) were detected in an additional 13/370 (3.5%) cases by deep-sequencing. The majority of EGR2 -mutated CLL patients (32/39, 82%) concerned U-CLL and the following aberrations co-occurred: 11q-deletions (n=10), TP53abn (n=6), NOTCH1 (n=3)or SF3B1 (n=3) mutations. EGR2 -mutated patients displayed a significantly worse overall survival compared to wt patients (median survival 59 vs. 141 months, p=0.003, using a conservative 10% VAF cutoff), and a poor outcome similar to cases with TP53abn (Figure 1B). In multivariate analysis (n=583), EGR2 status remained an independent factor (p=0.038), along with stage (p=0.048) and IGHV status (p<0.0001), while TP53abn and del(11q) showed borderline significant values (p=0.069 and p=0.059, respectively). To investigate the impact of EGR2 mutations in a homogeneously treated patient cohort, EGR2 mutation analysis of the UK CLL4 trial is underway. To date, 8/247 patients have been identified as EGR2 -mutated by deep-sequencing and they show a decrease of their median overall survival (42 vs. 77 months) compared to wt patients; however, this did not reach statistical significance, probably due to the low number of EGR2 -mutated cases. Final results of the UK CLL4 trial will be presented at the ASH meeting. In summary, EGR2 -mutant cases appear to constitute a novel poor-prognostic subgroup of CLL, with mutations occurring either as disease-initiating aberrations, i.e. in cases where mutations were found in the entire clone, or as subclonal driver events linked to progressive disease. The latter is reflected by the enrichment of EGR2 mutations in aggressive CLL and the association of EGR2 mutations with an overall dismal prognosis. Considering the potential role of mutated EGR2 in altering BcR signaling, it will be particularly relevant to study the efficacy of BcR inhibitors in this patient group. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Langerak: Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam); InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics.; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL. Schuh:Acerta Pharma BV: Research Funding. Strefford:Roche: Research Funding.

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