TP53 mutation analysis in chronic lymphocytic leukemia: comparison of different detection methods.

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.

Ofatumumab added to dexamethasone in patients with relapsed or refractory chronic lymphocytic leukemia: Results from a phase II study.

The treatment of relapsed/refractory chronic lymphocytic leukemia (CLL) remains a challenging clinical issue. An important treatment option is the use of high-dose corticosteroids. The purpose of this clinical trial was to determine the efficacy and toxicity of an ofatumumab-dexamethasone (O-Dex) combination in relapsed or refractory CLL. The trial was an open-label, multicenter, nonrandomized, Phase II study. The O-Dex regimen consisted of intravenous ofatumumab (Cycle 1: 300 mg on day 1, 2,000 mg on days 8, 15, and 22; Cycles 2-6: 1,000 mg on days 1, 8, 15, and 22) and oral dexamethasone (40 mg on days 1-4 and 15-18; Cycles 1-6). The O-Dex regimen was given until best response, or a maximum of six cycles. Thirty-three patients (pts) were recruited. Twenty-four (73%) pts completed at least three cycles of therapy. The remaining nine pts were prematurely discontinued owing to Grade 3/4 infections (seven pts), disease progression (one pt), or uncontrollable diabetes mellitus (one pt). Overall response rates/complete remissions (ORR/CR) were achieved in 22/5 pts (67/15%). The median progression-free survival (PFS) was 10 months. In pts with p53 defects (n = 8), ORR/CR were achieved in 5/2 pts (63/25%) with a median PFS of 10.5 months. The median overall survival (OS) was 34 months. The Grades 3-5 infectious toxicity in 33% of pts represented the most frequent side effect during the treatment period. In conclusion, the O-Dex regimen shows a relatively high ORR and CR with promising findings for PFS and OS. The study was registered at www.clinicaltrials.gov (NCT01310101).

ATM mutations uniformly lead to ATM dysfunction in chronic lymphocytic leukemia: application of functional test using doxorubicin.

ATM abnormalities are frequent in chronic lymphocytic leukemia and represent an important prognostic factor. Sole 11q deletion does not result in ATM inactivation by contrast to biallelic defects involving mutations. Therefore, the analysis of ATM mutations and their functional impact is crucial. In this study, we analyzed ATM mutations in predominantly high-risk patients using: i) resequencing microarray and direct sequencing; ii) Western blot for total ATM level; iii) functional test based on p21 gene induction after parallel treatment of leukemic cells with fludarabine and doxorubicin. ATM dysfunction leads to impaired p21 induction after doxorubicin exposure. We detected ATM mutation in 16% (22 of 140) of patients, and all mutated samples manifested demonstrable ATM defect (impaired p21 upregulation after doxorubicin and/or null protein level). Loss of ATM function in mutated samples was also evidenced through defective p53 pathway activation after ionizing radiation exposure. ATM mutation frequency was 34% in patients with 11q deletion, 4% in the TP53-defected group, and 8% in wild-type patients. Our functional test, convenient for routine use, showed high sensitivity (80%) and specificity (97%) for ATM mutations prediction. Only cells with ATM mutation, but not those with sole 11q deletion, were resistant to doxorubicin. As far as fludarabine is concerned, this difference was not observed. Interestingly, patients from both these groups experienced nearly identical time to first treatment. In conclusion, ATM mutations either alone or in combination with 11q deletion uniformly led to demonstrable ATM dysfunction in patients with chronic lymphocytic leukemia and mutation presence can be predicted by the functional test using doxorubicin.

Monoallelic and biallelic inactivation of TP53 gene in chronic lymphocytic leukemia: selection, impact on survival, and response to DNA damage.

Deletion of TP53 gene, under routine assessment by fluorescence in situ hybridization analysis, connects with the worst prognosis in chronic lymphocytic leukemia (CLL). The presence of isolated TP53 mutation (without deletion) is associated with reduced survival in CLL patients. It is unclear how these abnormalities are selected and what their mutual proportion is. We used methodologies with similar sensitivity for the detection of deletions (interphase fluorescence in situ hybridization) and mutations (yeast functional analysis) and analyzed a large consecutive series of 400 CLL patients; a subset of p53-wild-type cases (n = 132) was screened repeatedly during disease course. The most common type of TP53 inactivation, ie, mutation accompanied by deletion of the remaining allele, occurred in 42 patients (10.5%). Among additional defects, the frequency of the isolated TP53 mutation (n = 20; 5%) and the combination of 2 or more mutations on separate alleles (n = 5; 1.3%) greatly exceeded the sole deletion (n = 3; 0.8%). Twelve patients manifested defects during repeated investigation; in all circumstances the defects involved mutation and occurred after therapy. Monoallelic defects had a negative impact on survival and impaired in vitro response to fludarabine. Mutation analysis of the TP53 should be performed before each treatment initiation because novel defects may be selected by previous therapies.

Identification and functional characterization of new missense SNPs in the coding region of the TP53 gene.

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.

An unusual loss of EGFR gene copy in glioblastoma multiforme in a child: a case report and analysis of a successfully derived HGG-02 cell line.

PURPOSE: The aim of this study was to perform a detailed cytogenetic and molecular genetic analysis of a tumor taken from a 14.5-year-old boy with glioblastoma multiforme who showed an atypical clinical course. METHODS: Formalin-fixed, paraffin embedded tumor tissue and the corresponding HGG-02 cell line derived from this tumor were analyzed using fluorescence in situ hybridization (FISH), G-banding, multiplex ligation-dependent probe amplification (MLPA), functional analysis of separated alleles in yeast (FASAY), immunohistochemistry (IHC), and immunocytochemistry (ICC). RESULTS: Mutation of the p53 gene and hypermethylation of the MLH1 gene were detected by FASAY and MLPA, respectively. Cytogenetic analysis showed a polyploid karyotype with extensive heterogeneity in chromosome number. Using FISH, we identified a very unusual genetic change - a loss of EGFR gene copy in both the tumor tissue and the HGG-02 cell line. In accordance with the cytogenetic findings, IHC and ICC did not demonstrate overexpression of EGFR in the tumor tissue or HGG-02 cells. CONCLUSIONS: Despite his very poor prognosis, the patient experienced 34 months of event-free survival after surgery and adjuvant radiotherapy and chemotherapy. The detected loss of the EGFR gene copy may contribute to the unusual biological features of this tumor, but the forthcoming detailed expression analysis of cancer regulatory pathways is necessary to better understand this tumor phenotype.

Presence of heterozygous ATM deletion may not be critical in the primary response of chronic lymphocytic leukemia cells to fludarabine.

OBJECTIVES: Abnormalities of the TP53 or ATM, cooperating tumor-suppressor genes, significantly worsen the treatment options for chronic lymphocytic leukemia (CLL) patients. Although the aberrations seem to be mutually exclusive in this leukemia, inactivation of the former gene leads to worse prognosis. We tested the in vitro sensitivity of the CLL samples with heterozygous ATM deletion to fludarabine and combination of fludarabine and rituximab; the responses were compared with the TP53-abnormal and wild-type (wt) cells to delimitate relative significance of ATM deletion. METHODS: In vitro analysis was performed on fifty-nine characterized CLL samples using viability assay WST-1. Western blot and real-time RT-PCR were used to monitor the activation of the ATM/p53 pathway. RESULTS AND CONCLUSIONS: At the clinically relevant concentration of fludarabine, TP53-abnormal samples exhibited markedly higher resistance to fludarabine than the remaining CLL samples (P = 0.012); cohort with ATM deletion was not more resistant than wt cells. A similar induction of the p53 protein and its downstream target genes PUMA and BAX in ATM-deleted and wt cells confirmed that the former subgroup has preserved a critical pro-apoptotic response. Proportions of the samples, which had been sensitized to fludarabine by rituximab pretreatment, were insignificantly lower (P = 0.22) in the TP53-abnormal and ATM-deleted subgroups compared to the wt cases (30%; 29%; 50%, respectively). The presence of ATM (11q22-23) deletion in the CLL cells should not be considered an indication of resistance to fludarabine or its combination with rituximab.

Detection and Functional Analysis of TP53 Mutations in CLL.

Chronic lymphocytic leukemia (CLL) represents a prototype disease in which TP53 gene defects lead to inferior prognosis. Here, we present two distinct methodologies which can be used to identify TP53 mutations in CLL patients; both protocols are primarily intended for research purposes. The functional analysis of separated alleles in yeast (FASAY) can be flexibly adapted to a variable number of samples and provides an immediate functional readout of identified mutations. Amplicon-based next-generation sequencing then allows for a high throughput and accurately detects subclonal TP53 variants (sensitivity <1% of mutated cells).

ATM and TP53 mutations show mutual exclusivity but distinct clinical impact in mantle cell lymphoma patients.

Mantle cell lymphoma (MCL) is characterized by the hallmark t(11;14)(q13;q32) translocation, leading to cyclin D1 over-expression. Additionally, disrupting the DNA damage response pathway through ATM or TP53 defects plays an important role in MCL pathogenesis. Using deep next-generation sequencing we analyzed the mutual composition of ATM and TP53 mutations in 72 MCL patients, and assessed their impact on progression-free survival (PFS) and overall survival (OS). Mutated ATM and TP53 alleles were found in 51% (37/72) and 22% (16/72) of the cases examined, respectively, with only three patients harboring mutations in both genes. Only a mutated TP53 gene was associated with the significantly reduced PFS and OS and the same output was observed when ATM and TP53 defective groups included also sole deletions 11q and 17p, respectively. Determining the exact ATM/p53 pathway dysfunction may influence the selection of MCL patients for innovative therapies based on the targeted inhibition of selected proteins.

An unusual p53 mutation detected in Burkitt's lymphoma: 30 bp duplication.

Burkitt's lymphomas (BL) are aggressive rapidly growing tumors typified by a high c-myc expression resulting from t(8;14)(q24;q32), t(2;8)(p12;q24) or t(8;22)(q24;q11) translocations. Alterations of the p53 tumor suppressor are also relatively frequent in BL. Several approaches have been adopted for detection of the p53 aberrations such as immunohistochemical analyses, immunoblotting, DNA sequencing, fluorescence in situ hybridization (FISH), and functional assays. We used these methods to characterize the p53 mutation in tumor cells of a 53-year-old male suffering from Burkitt's lymphoma. By immunohistochemical analyses, we detected high levels of the p53 protein in the tumor tissue. Immunoblotting showed a higher molecular weight of the p53 protein overexpressed in the tumor tissues than that of the standard p53 protein. Similarly, the molecular weight of the PCR product prepared by amplification of the tumor p53 cDNA was higher than that of the standard p53 cDNA. Functional analyses of separated alleles in yeast evidently revealed that the tumor p53 protein was transcriptionally non-functional. The yeast colonies expressing this p53 variant possessed a unique phenotype in that they were red with many white spots on their surface. Sequencing of the tumor cDNA revealed a duplication of the 30 bp region of the p53 gene (g.12155_12184dup30) leading to a repeat of 10 amino acids (Pro-77 to Ala-86) in the p53 protein. Further analyses showed that the mutation was unstable in yeast cells. The FISH analyses did not confer loss of the p53-specific locus 17p13.

Retinoic acid enhances differentiation of v-myb-transformed monoblasts induced by okadaic acid.

Differentiation of various leukemic cells can be induced by liganded retinoic acid receptors and protein phosphatase inhibitors. In this study, we explored the effects of okadaic acid (OA), the phosphatase inhibitor, and retinoic acid (RA) in v-myb-transformed monoblasts BM2. OA induced differentiation of BM2 monoblasts into macrophage-like cells, as documented by analyses of cell morphology, cell cycle, phagocytic activity, non-specific esterase activity, production of reactive oxygen species and expression of vimentin and Mo-1. In contrast to many other leukemic cell lines, BM2 cells do not respond to retinoic acid. However, once exposed to OA and RA simultaneously, BM2 cells differentiate along monocyte/macrophage pathway more efficiently. We conclude that RA enhances differentiation of v-myb-transformed monoblasts induced by protein phosphorylation.

Complex analysis of the TP53 tumor suppressor in mantle cell and diffuse large B-cell lymphomas.

Mutations and deletions of the tumor suppressor TP53 gene are the most frequent genetic alterations detected in human tumors, though they are rather less frequent in lymphomas. However, acquisition of the TP53 mutation was demonstrated to be one of the characteristic markers in mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) and prognostic value of the TP53 status has been recognized for these diseases. We present the complex analysis of the TP53 aberrations in 57 cases of MCL and 131 cases of DLBCL. The TP53 status was determined by functional analyses in yeast (FASAY) followed by cDNA and gDNA sequencing. The level of the p53 protein was assessed by immunoblotting and loss of the TP53-specific locus 17p13.3 was detected by FISH. Altogether, we detected 13 TP53 mutations among MCL cases (22.8%) and 29 TP53 mutations in 26 from 131 DLBCL cases (19.8%). The ratio of missense TP53 mutations was 76.9% in MCL and 82.8% in DLBCL. The frequency of TP53 locus deletion was rather low in both diseases, reaching 9.3% in MCL and 15.3% in DLBCL. The presence of TP53 mutation was associated with shorter overall survival (OS) and progression-free survival (PFS) in MCL. Among DLBCL cases, the TP53 mutations shortened both OS and PFS of patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) and decreased both OS and PFS of patients with secondary DLBCL disease.

Expression of D-type cyclins in mantle cell and diffuse large B-cell lymphomas.

D-type cyclins are involved in cell cycle regulation and play an important role in the pathogenesis of lymphomas. Aberrant expression of cyclin D1 is associated with mantle cell lymphoma (MCL) and serves as a diagnostic marker of MCL. Analysis of cyclin D expression in tumor tissues of patients with diffuse large B-cell lymphoma (DLBCL) which comprises a heterogeneous group of tumors may contribute to their stratification. We analyzed expression of cyclin D1, D2, and D3 mRNAs in 30 MCL and 104 DLBCL patients using qRT-PCR and addressed their significance for disease outcome. We confirmed a high level of cyclin D1 mRNA in 29 MCL cases (97%). One case (3%) was identified as positive for cyclin D2. Expression of cyclin D1 was limited to MCL and did not occur in DLBCL. Overexpression of cyclin D2, which is rare in MCL, occurred more frequently in DLBCL (11 cases, 10.6%). We showed that high expression of cyclin D2 in DLBCL cases de novo decreased the overall survival rate (P=0.016) and progression-free survival (P=0.009). The expression pattern of cyclin D3 was similar in both types of studied lymphomas and it did not affect the disease outcome.

Complex analysis of the p53 tumor suppressor in lung carcinoma.

Lung cancer is the leading cause of cancer-related deaths worldwide. The p53 tumor suppressor is a transcription factor controlling expression of its target genes in response to various stress stimuli. Mutations of the TP53 gene occur very frequently in lung carcinomas and they play an important role in both oncogenic transformation of lung epithelial cells and lung carcinoma progression. We determined the TP53 status in 42 samples of squamous cell lung carcinoma (SQCC) and 56 samples of lung adenocarcinoma (AC) by the functional analysis FASAY and its variant called split assay. Altogether, we detected 64 TP53 mutations in 63 patients and analyzed them by cDNA and gDNA sequencing. The TP53 mutations were found in 76.2% (32/42) of SQCC cases, and 55.4% (31/56) of ACs. Immunoblotting revealed the p53 protein accumulation in 18 samples (42.9%) among SQCC cases and 19 samples (33.9%) among AC cases. Using fluorescence in situ hybridization we detected loss of the TP53-specific 17p13.3 locus in 23 from 41 analyzed SQCC samples (56.1%) and in 20 from 54 analyzed AC samples (37.0%). We did not find any statistically significant differences in overall and disease-free survival in relation to TP53 status.

Analysis of p53 status in human cell lines using a functional assay in yeast: detection of new non-sense p53 mutation in codon 124.

p53 tumor suppressor is a sequence-specific DNA-binding protein that controls the expression of many genes in response to diverse stress stimuli. p53 gene is often mutated in human cancer and in cancer cell lines. Several methods are available for identification of p53 mutations, including functional analysis of separated alleles in yeast (FASAY). FASAY distinguishes yeast colonies expressing functional p53 protein from colonies producing a dysfunctional p53 protein simply on the basis of color. We analyzed the p53 status of 26 human cell lines of different tissue origin using the functional assay in yeast. Wild-type p53 was found in six cell lines and various p53 aberrations in the remaining twenty. FASAY detected temperature-sensitive p53 mutations in breast cancer cell line, BT474, and leiomyosarcoma cell line, SK-LMS-1, and two independent p53 point mutations in SK-UT-1 and SK-LMS-1 leiomyosarcoma cell lines. In addition, the assay revealed that a recombination occurred between the two mutated p53 alleles producing a stable ratio of p53 wild-type alleles (11.7 or 2.7% respectively). In the case of acute myeloid leukemia cell line, ML-1, we detected both wild-type and heterozygous p53 status, depending on the source of the cell line. In Hs913T, HL60 and Saos-2 cell lines, FASAY failed to assess p53 status due to a large deletion/rearrangement of the p53 gene. In acute lymphoid leukemia HPB cell line, we disclosed unknown non-sense mutation in codon 124 of the p53 gene.

High frequency of temperature-sensitive mutations of p53 tumor suppressor in acute myeloid leukemia revealed by functional assay in yeast.

The tumor suppressor p53 is a transcription factor that participates in control of many cellular functions. All these activities are mediated by direct binding of the p53 tetramer to specific target sequences in promoters of directed genes. Lack of p53 function is often connected with development of cancer, but the frequency of p53 mutations is low in almost all types of leukemia. The aim of this study was to assess the frequency of mutations in the p53 gene in leukocytes of patients with acute myeloid leukemia (AML) using the FASAY functional analysis and to assess the relationship between the presence of p53 mutation and disease outcome. The following observations were made: i) the presence of p53 mutations was detected in 13 of 62 tested AML cases (21%) and in 1 of 4 tested myelodysplastic syndrome (MDS) cases by FASAY; ii) the presence of p53 mutation was shown to be a poor prognostic/predictive factor in AML (p=0.03/0.002); iii) although there is a statistically significant relationship between the presence of p53 mutation and p53 protein accumulation (p=0.05), not all samples having p53 mutation exhibited p53 protein accumulation; iv) five of 13 p53 mutations detected in the leukocytes of AML patients (38.5%) and the mutation detected in the leukocytes of the MDS patient (altogether 6/14-42.9%) were partially inactivating ts mutations. The high frequency of the ts p53 mutations in our study; and a novel modification in performing FASAY, are discussed; v) different ts mutations differ in the level of their temperature sensitivity and in their responsiveness to the cytoprotective drug amifostine.

Analysis of tumor suppressor p53 status in head and neck squamous cell carcinoma.

Head and neck cancer belongs to the most common types of cancer in both males and females with a mortality rate of approximately 50%. More than 90% of head and neck cancers are squamous cell carcinoma (HNSCC). Carcinogenesis of this disease involves activation of proto-oncogenes and inactivation of tumor suppressor genes. Among them, aberrations of p53 tumor suppressor gene are common events. The aim of this study was to assess the frequency of the tumor suppressor p53 aberrations in Czech population by using a functional test in yeast (FASAY) and by two immunochemical methods. We compared results of the methods and assessed the relationship between the presence of p53 aberration and some clinico-pathological parameters. The following observations were made: i) the accumulated p53 protein was detected in 33 of 50 tested samples (66%) by immunohistochemical analysis and in 27 of 49 tested samples (55.1%) by immunoblotting; ii) the presence of p53 mutation was detected in 36 of 50 tested samples (72%); iii) 6 of 36 p53 mutations detected by FASAY were temperature sensitive (16.7%); iv) 2 independent p53 mutations were found in at least 2 of the 36 positive cases; v) no statistically significant relationship was found between p53 aberration and overall survival.

Determination of optimal conditions for analysis of p53 status in leukemic cells using functional analysis of separated alleles in yeast.

Tumor suppressor p53 is transcription factor that participates in control of many cellular functions. Somatic mutations of the p53 gene are frequently detected in human cancers. Several methods can be used for identification of p53 mutations, including FASAY - functional analysis of separated alleles in yeast. FASAY distinguishes yeast colonies expressing functional p53 protein from colonies producing a dysfunctional p53 protein simply on the basis of color. The validity of the method depends on a low background level. There are several sources of background as PCR-induced point mutations, low quality of RNA and alternative splicing of intron 9 affecting the p53 carboxy-terminus. In the present work we show that FASAY can be successfully used for analysis of mRNA isolated from blood samples that were collected and stored for 24 hours at 0 degrees C without undesired increase of background. We also measured fidelity of several commonly used DNA polymerases and determined the most suitable kinds of Pfu DNA polymerases for FASAY. Reaction conditions described in this report allow routine analysis of p53 status in leukemic cells using FASAY.