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

Anaplastic large-cell lymphoma (ALCL) is a T-cell malignancy predominantly driven by the oncogenic anaplastic lymphoma kinase (ALK), accounting for approximately 15% of all paediatric non-Hodgkin lymphoma. Patients with central nervous system (CNS) relapse are particularly difficult to treat with a 3-year overall survival of 49% and a median survival of 23.5 months. The second-generation ALK inhibitor brigatinib shows superior penetration of the blood-brain barrier unlike the first-generation drug crizotinib and has shown promising results in ALK+ non-small-cell lung cancer. However, the benefits of brigatinib in treating aggressive paediatric ALK+ ALCL are largely unknown. We established a patient-derived xenograft (PDX) resource from ALK+ ALCL patients at or before CNS relapse serving as models to facilitate the development of future therapies. We show in vivo that brigatinib is effective in inducing the remission of PDX models of crizotinib-resistant (ALK C1156Y, TP53 loss) ALCL and furthermore that it is superior to crizotinib as a second-line approach to the treatment of a standard chemotherapy relapsed/refractory ALCL PDX pointing to brigatinib as a future therapeutic option.

• Keywords
• ALCL, PDX, brigatinib, crizotinib, tyrosine kinase inhibitors,
• MeSH
• Anaplastic Lymphoma Kinase MeSH
• Lymphoma, Large-Cell, Anaplastic * drug therapy   pathology   MeSH
• Child MeSH
• Heterografts MeSH
• Protein Kinase Inhibitors therapeutic use   MeSH
• Crizotinib pharmacology   therapeutic use   MeSH
• Humans MeSH
• Neoplasm Recurrence, Local drug therapy   MeSH
• Lung Neoplasms * drug therapy   MeSH
• Carcinoma, Non-Small-Cell Lung * MeSH
• Organophosphorus Compounds pharmacology   therapeutic use   MeSH
• Receptor Protein-Tyrosine Kinases therapeutic use   MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anaplastic Lymphoma Kinase MeSH
• brigatinib MeSH Browser
• Protein Kinase Inhibitors MeSH
• Crizotinib MeSH
• Organophosphorus Compounds MeSH
• Receptor Protein-Tyrosine Kinases 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

Chronic lymphocytic leukemia is associated with a highly heterogeneous disease course in terms of clinical outcomes and responses to chemoimmunotherapy. This heterogeneity is partly due to genetic aberrations identified in chronic lymphocytic leukemia cells such as mutations of TP53 and/or deletions in chromosome 17p [del(17p)], resulting in loss of one TP53 allele. These aberrations are associated with markedly decreased survival and predict impaired response to chemoimmunotherapy thus being among the strongest predictive markers guiding treatment decisions in chronic lymphocytic leukemia. Clinical trials demonstrate the importance of accurately testing for TP53 aberrations [both del(17p) and TP53 mutations] before each line of treatment to allow for appropriate treatment decisions that can optimize patients' outcomes. The current report reviews the diagnostic methods to detect TP53 disruption better, the role of TP53 aberrations in treatment decisions and current therapies available for patients with chronic lymphocytic leukemia carrying these abnormalities. The standardization in sequencing technologies for accurate identification of TP53 mutations and the importance of continued evaluation of TP53 aberrations throughout initial and subsequent lines of therapy remain unmet clinical needs as new therapeutic alternatives become available.

• MeSH
• Chromosome Deletion * MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell diagnosis   genetics   therapy   MeSH
• Humans MeSH
• Chromosomes, Human, Pair 17 genetics   MeSH
• Mutation * MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Disease-Free Survival MeSH
• Prognosis MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Tumor Suppressor Protein p53 MeSH

In chronic lymphocytic leukemia (CLL), TP53 gene defects, due to deletion of the 17p13 locus and/or mutation(s) within the TP53 gene, are associated with resistance to chemoimmunotherapy and a particularly dismal clinical outcome. On these grounds, analysis of TP53 aberrations has been incorporated into routine clinical diagnostics to improve patient stratification and optimize therapeutic decisions. The predictive implications of TP53 aberrations have increasing significance in the era of novel targeted therapies, i.e., inhibitors of B-cell receptor (BcR) signaling and anti-apoptotic BCL2 family members, owing to their efficacy in patients with TP53 defects. In this report, the TP53 Network of the European Research Initiative on Chronic Lymphocytic Leukemia (ERIC) presents updated recommendations on the methodological approaches for TP53 mutation analysis. Moreover, it provides guidance to ensure that the analysis is performed in a timely manner for all patients requiring treatment and that the data is interpreted and reported in a consistent, standardized, and accurate way. Since next-generation sequencing technologies are gaining prominence within diagnostic laboratories, this report also offers advice and recommendations for the interpretation of TP53 mutation data generated by this methodology.

• MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell genetics   MeSH
• Genes, p53 genetics   MeSH
• Humans MeSH
• DNA Mutational Analysis methods   MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Practice Guideline MeSH
• Geographicals
• Europe MeSH

Chronic lymphocytic leukemia is a disease with up-regulated expression of the transmembrane tyrosine-protein kinase ROR1, a member of the Wnt/planar cell polarity pathway. In this study, we identified COBLL1 as a novel interaction partner of ROR1. COBLL1 shows clear bimodal expression with high levels in chronic lymphocytic leukemia patients with mutated IGHV and approximately 30% of chronic lymphocytic leukemia patients with unmutated IGHV. In the remaining 70% of chronic lymphocytic leukemia patients with unmutated IGHV, COBLL1 expression is low. Importantly, chronic lymphocytic leukemia patients with unmutated IGHV and high COBLL1 have an unfavorable disease course with short overall survival and time to second treatment. COBLL1 serves as an independent molecular marker for overall survival in chronic lymphocytic leukemia patients with unmutated IGHV. In addition, chronic lymphocytic leukemia patients with unmutated IGHV and high COBLL1 show impaired motility and chemotaxis towards CCL19 and CXCL12 as well as enhanced B-cell receptor signaling pathway activation demonstrated by increased PLCγ2 and SYK phosphorylation after IgM stimulation. COBLL1 expression also changes during B-cell maturation in non-malignant secondary lymphoid tissue with a higher expression in germinal center B cells than naïve and memory B cells. Our data thus suggest COBLL1 involvement not only in chronic lymphocytic leukemia but also in B-cell development. In summary, we show that expression of COBLL1, encoding novel ROR1-binding partner, defines chronic lymphocytic leukemia subgroups with a distinct response to microenvironmental stimuli, and independently predicts survival of chronic lymphocytic leukemia with unmutated IGHV.

• MeSH
• Survival Analysis MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell classification   diagnosis   genetics   mortality   MeSH
• Humans MeSH
• Mutation MeSH
• Cell Movement MeSH
• Cell Polarity MeSH
• Prognosis MeSH
• Wnt Signaling Pathway MeSH
• Receptor Tyrosine Kinase-like Orphan Receptors metabolism   MeSH
• Immunoglobulin Heavy Chains genetics   MeSH
• Transcription Factors metabolism   MeSH
• Immunoglobulin Variable Region genetics   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• COBLL1 protein, human MeSH Browser
• ROR1 protein, human MeSH Browser
• Receptor Tyrosine Kinase-like Orphan Receptors MeSH
• Immunoglobulin Heavy Chains MeSH
• Transcription Factors MeSH
• Immunoglobulin Variable Region MeSH

Treatment options for TP53-mutated lymphoid tumors are very limited. In experimental models, TP53-mutated lymphomas were sensitive to direct inhibition of checkpoint kinase 1 (Chk1), a pivotal regulator of replication. We initially tested the potential of the highly specific Chk1 inhibitor SCH900776 to synergize with nucleoside analogs (NAs) fludarabine, cytarabine and gemcitabine in cell lines derived from B-cell malignancies. In p53-proficient NALM-6 cells, SCH900776 added to NAs enhanced signaling towards Chk1 (pSer317/pSer345), effectively blocked Chk1 activation (Ser296 autophosphorylation), increased replication stress (p53 and γ-H2AX accumulation) and temporarily potentiated apoptosis. In p53-defective MEC-1 cell line representing adverse chronic lymphocytic leukemia (CLL), Chk1 inhibition together with NAs led to enhanced and sustained replication stress and significantly potentiated apoptosis. Altogether, among 17 tested cell lines SCH900776 sensitized four of them to all three NAs. Focusing further on MEC-1 and co-treatment of SCH900776 with fludarabine, we disclosed chromosome pulverization in cells undergoing aberrant mitoses. SCH900776 also increased the effect of fludarabine in a proportion of primary CLL samples treated with pro-proliferative stimuli, including those with TP53 disruption. Finally, we observed a fludarabine potentiation by SCH900776 in a T-cell leukemia 1 (TCL1)-driven mouse model of CLL. Collectively, we have substantiated the significant potential of Chk1 inhibition in B-lymphoid cells.

• Keywords
• SCH900776, TP53, checkpoint kinase 1/Chk1, chronic lymphocytic leukemia, nucleoside analogs,
• MeSH
• Apoptosis MeSH
• B-Lymphocytes cytology   MeSH
• Cell Cycle MeSH
• Checkpoint Kinase 1 antagonists & inhibitors   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell drug therapy   genetics   metabolism   MeSH
• Cytarabine administration & dosage   MeSH
• Deoxycytidine administration & dosage   analogs & derivatives   MeSH
• Gemcitabine MeSH
• Humans MeSH
• Mitosis MeSH
• Mutation MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Nucleosides genetics   MeSH
• Cell Proliferation MeSH
• Pyrazoles pharmacology   MeSH
• Pyrimidines pharmacology   MeSH
• Drug Screening Assays, Antitumor MeSH
• Signal Transduction MeSH
• Cell Survival MeSH
• Vidarabine administration & dosage   analogs & derivatives   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Checkpoint Kinase 1 MeSH
• CHEK1 protein, human MeSH Browser
• Cytarabine MeSH
• Deoxycytidine MeSH
• fludarabine MeSH Browser
• Gemcitabine MeSH
• MK-8776 MeSH Browser
• Tumor Suppressor Protein p53 MeSH
• Nucleosides MeSH
• Pyrazoles MeSH
• Pyrimidines MeSH
• TP53 protein, human MeSH Browser
• Vidarabine MeSH

• MeSH
• Adenine analogs & derivatives   MeSH
• Alleles MeSH
• Bridged Bicyclo Compounds, Heterocyclic therapeutic use   MeSH
• Quinazolinones therapeutic use   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell diagnosis   drug therapy   genetics   pathology   MeSH
• Molecular Targeted Therapy MeSH
• Gene Expression MeSH
• Class I Phosphatidylinositol 3-Kinases antagonists & inhibitors   genetics   metabolism   MeSH
• Gene Frequency MeSH
• Humans MeSH
• Biomarkers, Tumor genetics   metabolism   MeSH
• Tumor Suppressor Protein p53 deficiency   genetics   MeSH
• Piperidines MeSH
• Prognosis MeSH
• Agammaglobulinaemia Tyrosine Kinase MeSH
• Antineoplastic Agents therapeutic use   MeSH
• Purines therapeutic use   MeSH
• Pyrazoles therapeutic use   MeSH
• Pyrimidines therapeutic use   MeSH
• Sulfonamides therapeutic use   MeSH
• Protein-Tyrosine Kinases antagonists & inhibitors   genetics   metabolism   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH
• Editorial MeSH
• Names of Substances
• Adenine MeSH
• Bridged Bicyclo Compounds, Heterocyclic MeSH
• Quinazolinones MeSH
• Class I Phosphatidylinositol 3-Kinases MeSH
• ibrutinib MeSH Browser
• idelalisib MeSH Browser
• Biomarkers, Tumor MeSH
• Tumor Suppressor Protein p53 MeSH
• PIK3CD protein, human MeSH Browser
• Piperidines MeSH
• Agammaglobulinaemia Tyrosine Kinase MeSH
• Antineoplastic Agents MeSH
• Purines MeSH
• Pyrazoles MeSH
• Pyrimidines MeSH
• Sulfonamides MeSH
• TP53 protein, human MeSH Browser
• Protein-Tyrosine Kinases MeSH
• venetoclax MeSH Browser

TP53 gene defects represent a strong adverse prognostic factor for patient survival and treatment resistance in chronic lymphocytic leukemia (CLL). Although various methods for TP53 mutation analysis have been reported, none of them allow the identification of all occurring sequence variants, and the most suitable methodology is still being discussed. The aim of this study was to determine the limitations of commonly used methods for TP53 mutation examination in CLL and propose an optimal approach for their detection. We examined 182 CLL patients enriched for high-risk cases using denaturing high-performance liquid chromatography (DHPLC), functional analysis of separated alleles in yeast (FASAY), and the AmpliChip p53 Research Test in parallel. The presence of T53 gene mutations was also evaluated using ultra-deep next generation sequencing (NGS) in 69 patients. In total, 79 TP53 mutations in 57 (31 %) patients were found; among them, missense substitutions predominated (68 % of detected mutations). Comparing the efficacy of the methods used, DHPLC and FASAY both combined with direct Sanger sequencing achieved the best results, identifying 95 % and 93 % of TP53-mutated patients. Nevertheless, we showed that in CLL patients carrying low-proportion TP53 mutation, the more sensitive approach, e.g., ultra-deep NGS, might be more appropriate. TP53 gene analysis using DHPLC or FASAY is a suitable approach for mutation detection. Ultra-deep NGS has the potential to overcome shortcomings of methods currently used, allows the detection of minor proportion mutations, and represents thus a promising methodology for near future.

• MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell genetics   MeSH
• Adult MeSH
• Genes, p53 * MeSH
• In Situ Hybridization, Fluorescence MeSH
• Polymorphism, Single Nucleotide MeSH
• Middle Aged MeSH
• Humans MeSH
• Mutation * MeSH
• Oligonucleotide Array Sequence Analysis MeSH
• Aged MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Chromatography, High Pressure Liquid MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH