Most cited article - PubMed ID 29467486
ERIC recommendations for TP53 mutation analysis in chronic lymphocytic leukemia-update on methodological approaches and results interpretation
In chronic lymphocytic leukemia, the reliability of next-generation sequencing (NGS) to detect TP53 variants ≤10% allelic frequency (low-VAF) is debated. We tested the ability to detect 23 such variants in 41 different laboratories using their NGS method of choice. The sensitivity was 85.6%, 94.5%, and 94.8% at 1%, 2%, and 3% VAF cut-off, respectively. While only one false positive (FP) result was reported at >2% VAF, it was more challenging to distinguish true variants <2% VAF from background noise (37 FPs reported by 9 laboratories). The impact of low-VAF variants on time-to-second-treatment (TTST) and overall survival (OS) was investigated in a series of 1092 patients. Among patients not treated with targeted agents, patients with low-VAF TP53 variants had shorter TTST and OS versus wt-TP53 patients, and the relative risk of second-line treatment or death increased continuously with increasing VAF. Targeted therapy in ≥2 line diminished the difference in OS between patients with low-VAF TP53 variants and wt-TP53 patients, while patients with high-VAF TP53 variants had inferior OS compared to wild type-TP53 cases. Altogether, NGS-based approaches are technically capable of detecting low-VAF variants. No strict threshold can be suggested from a technical standpoint, laboratories reporting TP53 mutations should participate in a standardized validation set-up. Finally, whereas low-VAF variants affected outcomes in patients receiving chemoimmunotherapy, their impact on those treated with novel therapies remains undetermined. Our results pave the way for the harmonized and accurate TP53 assessment, which is indispensable for elucidating the role of TP53 mutations in targeted treatment.
- Publication type
- Journal Article MeSH
Patients with chronic lymphocytic leukemia (CLL) exhibit diverse clinical outcomes. An expanding array of genetic tests is now employed to facilitate the identification of patients with high-risk disease and inform treatment decisions. These tests encompass molecular cytogenetic analysis, focusing on recurrent chromosomal alterations, particularly del(17p). Additionally, sequencing is utilized to identify TP53 mutations and to determine the somatic hypermutation status of the immunoglobulin heavy variable gene. Concurrently, a swift advancement of targeted treatment has led to the implementation of novel strategies for patients with CLL, including kinase and BCL2 inhibitors. This review explores both current and emerging diagnostic tests aimed at identifying high-risk patients who should benefit from targeted therapies. We outline existing treatment paradigms, emphasizing the importance of matching the right treatment to the right patient beyond genetic stratification, considering the crucial balance between safety and efficacy. We also take into consideration the practical and logistical issues when choosing a management strategy for each individual patient. Furthermore, we delve into the mechanisms underlying therapy resistance and stress the relevance of monitoring measurable residual disease to guide treatment decisions. Finally, we underscore the necessity of aggregating real-world data, adopting a global perspective, and ensuring patient engagement. Taken together, we argue that precision medicine is not the mere application of precision diagnostics and accessibility of precision therapies in CLL but encompasses various aspects of the patient journey (e.g., lifestyle exposures and comorbidities) and their preferences toward achieving true personalized medicine for patients with CLL.
- Publication type
- Journal Article MeSH
- Review MeSH
In chronic lymphocytic leukemia (CLL), analysis of TP53 aberrations (deletion and/or mutation) is a crucial part of treatment decision-making algorithms. Technological and treatment advances have resulted in the need for an update of the last recommendations for TP53 analysis in CLL, published by ERIC, the European Research Initiative on CLL, in 2018. Based on the current knowledge of the relevance of low-burden TP53-mutated clones, a specific variant allele frequency (VAF) cut-off for reporting TP53 mutations is no longer recommended, but instead, the need for thorough method validation by the reporting laboratory is emphasized. The result of TP53 analyses should always be interpreted within the context of available laboratory and clinical information, treatment indication, and therapeutic options. Methodological aspects of introducing next-generation sequencing (NGS) in routine practice are discussed with a focus on reliable detection of low-burden clones. Furthermore, potential interpretation challenges are presented, and a simplified algorithm for the classification of TP53 variants in CLL is provided, representing a consensus based on previously published guidelines. Finally, the reporting requirements are highlighted, including a template for clinical reports of TP53 aberrations. These recommendations are intended to assist diagnosticians in the correct assessment of TP53 mutation status, but also physicians in the appropriate understanding of the lab reports, thus decreasing the risk of misinterpretation and incorrect management of patients in routine practice whilst also leading to improved stratification of patients with CLL in clinical trials.
- MeSH
- Leukemia, Lymphocytic, Chronic, B-Cell * genetics diagnosis MeSH
- Humans MeSH
- Mutation * MeSH
- DNA Mutational Analysis methods standards MeSH
- Tumor Suppressor Protein p53 * genetics MeSH
- High-Throughput Nucleotide Sequencing * methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Names of Substances
- Tumor Suppressor Protein p53 * MeSH
- TP53 protein, human MeSH Browser
Recent evidence suggests that the prognostic impact of gene mutations in patients with chronic lymphocytic leukemia (CLL) may differ depending on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status. In this study, we assessed the impact of nine recurrently mutated genes (BIRC3, EGR2, MYD88, NFKBIE, NOTCH1, POT1, SF3B1, TP53, and XPO1) in pre-treatment samples from 4580 patients with CLL, using time-to-first-treatment (TTFT) as the primary end-point in relation to IGHV gene SHM status. Mutations were detected in 1588 (34.7%) patients at frequencies ranging from 2.3-9.8% with mutations in NOTCH1 being the most frequent. In both univariate and multivariate analyses, mutations in all genes except MYD88 were associated with a significantly shorter TTFT. In multivariate analysis of Binet stage A patients, performed separately for IGHV-mutated (M-CLL) and unmutated CLL (U-CLL), a different spectrum of gene alterations independently predicted short TTFT within the two subgroups. While SF3B1 and XPO1 mutations were independent prognostic variables in both U-CLL and M-CLL, TP53, BIRC3 and EGR2 aberrations were significant predictors only in U-CLL, and NOTCH1 and NFKBIE only in M-CLL. Our findings underscore the need for a compartmentalized approach to identify high-risk patients, particularly among M-CLL patients, with potential implications for stratified management.
- MeSH
- Leukemia, Lymphocytic, Chronic, B-Cell * genetics MeSH
- Phenotype MeSH
- Humans MeSH
- Mutation MeSH
- Myeloid Differentiation Factor 88 genetics MeSH
- Prognosis MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Myeloid Differentiation Factor 88 MeSH
BACKGROUND: Telomeres are protective structures at chromosome ends which shorten gradually with increasing age. In chronic lymphocytic leukemia (CLL), short telomeres have been associated with unfavorable disease outcome, but the link between clonal evolution and telomere shortening remains unresolved. METHODS: We investigated relative telomere length (RTL) in a well-characterized cohort of 198 CLL patients by qPCR and focused in detail on a subgroup 26 patients who underwent clonal evolution of TP53 mutations (evolTP53). In the evolTP53 subgroup we explored factors influencing clonal evolution and corresponding changes in telomere length through measurements of telomerase expression, lymphocyte doubling time, and BCR signaling activity. RESULTS: At baseline, RTL of the evolTP53 patients was scattered across the entire RTL spectrum observed in our CLL cohort. RTL changed in the follow-up samples of 16/26 (62%) evolTP53 cases, inclining to reach intermediate RTL values, i.e., longer telomeres shortened compared to baseline while shorter ones prolonged. For the first time we show that TP53 clonal shifts are linked to RTL change, including unexpected RTL prolongation. We further investigated parameters associated with RTL changes. Unstable telomeres were significantly more frequent among younger patients (P = 0.032). Shorter telomeres were associated with decreased activity of the B-cell receptor signaling components p-ERK1/2, p-ZAP-70/SYK, and p-NFκB (P = 0.04, P = 0.01, and P = 0.02, respectively). CONCLUSIONS: Our study revealed that changes of telomere length reflect evolution in leukemic subclone proportion, and are associated with specific clinico-biological features of the explored cohort.
- Keywords
- BCR signaling, Chronic Lymphocytic Leukemia, Clonal evolution, TP53, Telomere,
- MeSH
- Leukemia, Lymphocytic, Chronic, B-Cell genetics MeSH
- Clonal Evolution genetics MeSH
- Middle Aged MeSH
- Humans MeSH
- Mutation MeSH
- Tumor Suppressor Protein p53 genetics MeSH
- Proto-Oncogene Proteins c-bcr metabolism MeSH
- Signal Transduction MeSH
- Telomerase genetics MeSH
- Telomere ultrastructure MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Tumor Suppressor Protein p53 MeSH
- Proto-Oncogene Proteins c-bcr MeSH
- Telomerase MeSH
- TP53 protein, human MeSH Browser
Patients with chronic lymphocytic leukemia (CLL) bearing TP53 mutations experience chemorefractory disease and are therefore candidates for targeted therapy. However, the significance of low-burden TP53 mutations with <10% variant allele frequency (VAF) remains a matter for debate. Herein, we describe clonal evolution scenarios of low-burden TP53 mutations, the clinical impact of which we analyzed in a "real-world" CLL cohort. TP53 status was assessed by targeted next-generation sequencing (NGS) in 511 patients entering first-line treatment with chemo- and/or immunotherapy and 159 patients in relapse before treatment with targeted agents. Within the pretherapy cohort, 16% of patients carried low-burden TP53 mutations (0.1% to 10% VAF). Although their presence did not significantly shorten event-free survival after first-line therapy, it affected overall survival (OS). In a subgroup with TP53 mutations of 1% to 10% VAF, the impact on OS was observed only in patients with unmutated IGHV who had not received targeted therapy, as patients benefited from switching to targeted agents, regardless of initial TP53 mutational status. Analysis of the clonal evolution of low-burden TP53 mutations showed that the highest expansion rates were associated with fludarabine, cyclophosphamide, and rituximab regimen in both first- and second-line treatments (median VAF increase, 14.8× and 11.8×, respectively) in contrast to treatment with less intense treatment regimens (1.6×) and no treatment (0.8×). In the relapse cohort, 33% of patients carried low-burden TP53 mutations, which did not expand significantly upon targeted treatment (median VAF change, 1×). Sporadic cases of TP53 mutations' clonal shifts were connected with the development of resistance-associated mutations. Altogether, our data support the incorporation of low-burden TP53 variants in clinical decision making.
- MeSH
- Leukemia, Lymphocytic, Chronic, B-Cell genetics therapy MeSH
- Adult MeSH
- Immunotherapy MeSH
- Kaplan-Meier Estimate MeSH
- Clonal Evolution * drug effects MeSH
- Middle Aged MeSH
- Humans MeSH
- Mutation drug effects MeSH
- Tumor Cells, Cultured MeSH
- Tumor Suppressor Protein p53 genetics MeSH
- Antineoplastic Combined Chemotherapy Protocols therapeutic use MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Tumor Suppressor Protein p53 MeSH
- TP53 protein, human MeSH Browser
The paradigm of first-line treatment of chronic lymphocytic leukaemia (CLL) is currently undergoing a radical change. On the basis of several randomised phase III trials showing prolongation of progression-free survival, chemoimmunotherapy is being replaced by treatment based on novel, orally available targeted inhibitors such as Bruton tyrosine kinase inhibitors ibrutinib and acalabrutinib or bcl-2 inhibitor venetoclax. However, the use of these agents may be associated with other disadvantages. First, with the exception of one trial in younger/fit patients, no studies have so far demonstrated benefit regarding the ultimate endpoint of overall survival. Second, oral inhibitors are extremely expensive and thus currently unavailable due to the absence of reimbursement in some countries. Third, treatment with ibrutinib and acalabrutinib necessitates long-term administration until progression; this may be associated with accumulation of late side effects, problems with patient compliance, and selection of resistant clones. Therefore, the identification of a subset of patients who could benefit from chemoimmunotherapy would be ideal. Current data suggest that patients with the mutated variable region of the immunoglobulin heavy chain (IGHV) achieve fairly durable remissions, especially when treated with fludarabine, cyclophosphamide, and rituximab (FCR) regimen. This review discusses current options for treatment-naïve patients with CLL.
- Keywords
- acalabrutinib, chemoimmunotherapy, chronic lymphocytic leukaemia, direct inhibitors, ibrutinib, obinutuzumab, prognosis, rituximab, venetoclax,
- Publication type
- Journal Article MeSH
- Review MeSH
Infrequent and rare genetic variants in the human population vastly outnumber common ones. Although they may contribute significantly to the genetic basis of a disease, these seldom-encountered variants may also be miss-identified as pathogenic if no correct references are available. Somatic and germline TP53 variants are associated with multiple neoplastic diseases, and thus have come to serve as a paradigm for genetic analyses in this setting. We searched 14 independent, globally distributed datasets and recovered TP53 SNPs from 202,767 cancer-free individuals. In our analyses, 19 new missense TP53 SNPs, including five novel variants specific to the Asian population, were recurrently identified in multiple datasets. Using a combination of in silico, functional, structural, and genetic approaches, we showed that none of these variants displayed loss of function compared to the normal TP53 gene. In addition, classification using ACMG criteria suggested that they are all benign. Considered together, our data reveal that the TP53 coding region shows far more polymorphism than previously thought and present high ethnic diversity. They furthermore underline the importance of correctly assessing novel variants in all variant-calling pipelines associated with genetic diagnoses for cancer.
- MeSH
- Genes, p53 genetics MeSH
- Polymorphism, Single Nucleotide genetics MeSH
- Humans MeSH
- Mutation, Missense genetics MeSH
- Tumor Suppressor Protein p53 genetics MeSH
- Neoplasms genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Tumor Suppressor Protein p53 MeSH
Next-generation sequencing (NGS) has transitioned from research to clinical routine, yet the comparability of different technologies for mutation profiling remains an open question. We performed a European multicenter (n=6) evaluation of three amplicon-based NGS assays targeting 11 genes recurrently mutated in chronic lymphocytic leukemia. Each assay was assessed by two centers using 48 pre-characterized chronic lymphocytic leukemia samples; libraries were sequenced on the Illumina MiSeq instrument and bioinformatics analyses were centralized. Across all centers the median percentage of target reads ≥100x ranged from 94.2- 99.8%. In order to rule out assay-specific technical variability, we first assessed variant calling at the individual assay level i.e., pairwise analysis of variants detected amongst partner centers. After filtering for variants present in the paired normal sample and removal of PCR/sequencing artefacts, the panels achieved 96.2% (Multiplicom), 97.7% (TruSeq) and 90% (HaloPlex) concordance at a variant allele frequency (VAF) >0.5%. Reproducibility was assessed by looking at the inter-laboratory variation in detecting mutations and 107 of 115 (93% concordance) mutations were detected by all six centers, while the remaining eight variants (7%) were undetected by a single center. Notably, 6 of 8 of these variants concerned minor subclonal mutations (VAF <5%). We sought to investigate low-frequency mutations further by using a high-sensitivity assay containing unique molecular identifiers, which confirmed the presence of several minor subclonal mutations. Thus, while amplicon-based approaches can be adopted for somatic mutation detection with VAF >5%, after rigorous validation, the use of unique molecular identifiers may be necessary to reach a higher sensitivity and ensure consistent and accurate detection of low-frequency variants.
