ALBERT

Description: TP53 mutations have been shown to occur in 5–15% of CLL patients and are associated with a poor response to treatment. Because previous studies have examined small cohorts without detailed molecular characterization, the exact correlation of TP53 mutation pattern, allele status and associated molecular genetics has not been possible. We undertook a large scale mutation analysis of TP53 in two centres and pooled the data to characterize the mutation pattern of TP53 in CLL. Two methods of mutational analysis (DHPLC exons 2–11 / FASAY assay) were used. We found 110 mutations in 106 patients with CLL. As expected, most mutations were located in the DNA binding domain of p53. Transitions were found in 54 of 110 mutations (9/110 at CpG sites), while transversions, small deletions and especially insertions were less common (33/110; 22/110; 1/110 resp.). Cytogenetics by FISH were available for 100/106 patients. Most mutations were accompanied by deletion of the other allele (17p-)(n=67). Nonetheless, TP53 mutations were also found in the good prognostic subgroups with normal karyotype (n=6) and 13q- as the sole abnormality (n=15). 17 patients with TP53 mutations had a mutated VH status vs. 79 with an unmutated VH status. We quite frequently observed a deletion 11q- (16/100), accompanying evenly the abnormalities of one as well as of both TP53 alleles (deletion/mutation). It indicates that this deletion may not be a simple alternative to p53 inactivation, as it has been proposed previously. Chemotherapy before mutation analysis was noted in 43/92 patients and was more frequent in those with aberration of one TP53 allele without reaching statistical significance (64% vs. 51% of previously treated patients bearing both inactivated alleles). The transitions, transversions and small deletions were evenly distributed among both untreated and treated patients. When comparing the predicted functional activity of the mutated TP53 in different cytogenetic subgroups (http://p53.free.fr/Database/p53_recomendations.html) we observed significantly lower residual activity of the missense mutations in the 17p- subgroup compared to the patients with 13q- (single) and a TP53 mutation (median 3,1 vs. 10,4 p=0,037). The codons most frequently mutated were at positions 209, 220, 234, suggesting that the classical hot spots (codons 175, 248, 273) may not be commonly mutated in CLL. The very low number of mutations which were identified at CpG dinucleotides implies a relatively negligible contribution of endogenous mutability at methylated cytosine. This is manifested also by a finding of only two mutations at the very prominent, heavily methylated hot-spot at position 248Arg. We assume that some other mechanisms of mutagenesis may play a relevant role in B-CLL. The current study characterizes the mutational profile of TP53 mutations in CLL. While the classical hot spot mutants were not observed at high frequencies we could identify 3 codons that made up for 12,7% of the mutations. The majority of mutations are accompanied by the inactivation of the other allele (deletion 17p). For the first time, the analysis of a large cohort of TP53 mutations will allow to investigate potential differences in the clinical course of monoallelic vs. biallelic aberrations of TP53.

Description: Introduction Mutations in NOTCH1 and especially TP53 genes represent potent drivers of chronic lymphocytic leukemia (CLL) progression and chemo-refractoriness. Although a coexistence of these mutations was reported in CLL, a molecular basis of this phenomenon has not been described yet. To clarify this issue, we performed a detailed analysis of CLL patients with parallel NOTCH1 and TP53 mutations including single cancer cell examination. Methods TP53 mutations were determined based on FASAY analysis coupled with direct sequencing. In a collected cohort of 111 TP53 -mutated patients a presence of hot spot c.7544_7545delCT NOTCH1 mutation was assessed using direct gDNA sequencing. In NOTCH1 -TP53 -mutated patients with available material, the mutations' coexistence was tested in single flow-sorted CD19+ cells (cancer cell proportion 〉 80 %) using multiplex PCR followed by direct sequencing. Results The NOTCH1 mutation was detected in 19/111 (17 %) of the TP53 -mutated patients. Eleven of the NOTCH1-TP53 -mutated patients carried single TP53 mutation; multiple TP53 mutations were detected in 8 of them. Based on gDNA sequencing, the NOTCH1 and TP53 mutation coexistence in the same cancer cells was evident in 4/19 of the NOTCH1-TP53-mutated patients, as at least one of the gene mutations occurred in 100 % of the DNA. In the remaining 15 NOTCH1-TP53 -mutated patients the clonal composition was not possible to assess using sequencing data only and therefore a single cell analysis was performed in 8 of them with available material. Remarkably, irrespective of the mutation proportion, in all of these patients the NOTCH1 mutation was always present together with at least one of the detected TP53 mutations. Considering both the DNA sequencing and single cell analysis data, the 12 patientswith proven NOTCH1-TP53 mutation coexistence might be stratified into three groups with different clonal composition: i) patients with NOTCH1 and single TP53 mutations showing a comparable mutation proportion (n = 3), in which both gene mutations were always detected in the same cells and never occurred separately; ii) patients with either NOTCH1 or TP53 mutation predominance (n = 6), in which the predominant mutation was present separately as well as in combination with the coexisting mutation(s) in individual cells; iii) patients with NOTCH1 and multiple TP53 mutations showing different mutation proportion (n = 3), in which NOTCH1 mutation was present together with one of the detected TP53 mutations in the same cells, while the other TP53 mutations occurred separately. In two of the NOTCH1-TP53 -mutated patients who received intensive chemo-immunotherapy, the consecutive samples were available for single cell analysis. In one of these patients only single TP53 mutation was detected at first time point. In relapse after rituximab-dexamethasone treatment the clone carrying the original TP53 mutation expanded in parallel with another NOTCH1-TP53-mutated clone. Different situation was noticed in the second patient, in which the NOTCH1-TP53-mutated clone detected at first time point diminished after alemtuzumab treatment, while another TP53-mutated-NOTCH1-wild-type clone expanded in relapse. Conclusion We have shown that in NOTCH1-TP53 -mutated patients the mutations often coexist in the same CLL cells. These patients exhibit a considerable clonal heterogeneity that may be further influenced by chemo-immunotherapy. This study was supported by IGA NT/13493 and NT/13519, MUNI/A/1180/2014, CZ.1.05/1.1.00/02.0068. Disclosures Mayer: Janssen: Research Funding.

Description: The treatment of relapsed/refractory chronic lymphocytic leukemia (CLL) remains a challenging clinical issue despite remarkable improvements in prognostication and therapy. One emerging treatment option for relapsed/refractory CLL is the use of high-dose corticosteroids. High-dose methylprednisolone or dexamethasone combined with rituximab are active in relapsed/refractory CLL, but serious infections are frequent and progression-free survival (PFS) is short. The purpose of this clinical trial was to determine the efficacy and toxicity of ofatumumab-dexamethason (O-dex) combination in relapsed or refractory CLL population. The trial was an open-label, multi-center, non-randomized, phase II study. The O-dex regimen consisted of intravenous ofatumumab (Cycle 1: 300mg on day 1, 2000mg on days 8, 15, 22; Cycles 2-6: 1000mg on days 1, 8, 15, 22) and oral dexamethasone (40mg on days 1-4 and 15-18; Cycles 1-6). Premedication consisted of glucocorticoid, paracetamol, and antihistamine before each ofatumumab infusion. All patients received allopurinol, omeprazol, co-trimoxazole, and fluconazole for prophylaxis of tumor lysis syndrome and infections. The O-dex regimen was given for a minimum of 3 cycles, until best response, or a maximum of 6 cycles. Between July 2010 and December 2012, 32 patients (pts.) were recruited at 3 centers. Basic patient characteristics at the start of O-dex therapy were as follows: median age 66 years (range, 50-77); 24 males, 8 females; median CIRS score 7 (0-15); median previous treatment lines 3 (1-10); 30 (94%) pts. were pretreated with fludarabine and 12 (38%) with alemtuzumab; Rai III/IV stage was present in 20 (63%) pts.; 6 (19%) pts. had bulky lymphadenopathy; IgVH genes were unmutated in 30 (94%) pts.; del 11q was present in 6 (19%) and p53 defects (del 17p and/or TP53 mutation) in 8 (25%) pts. The median number of O-dex cycles administered was 6 (1-6). Twenty two (69%) pts. completed at least 3 cycles of therapy. The remaining 9 patients were prematurely discontinued due to CTCAE grade 3/4 infections (7 pts.), disease progression (1 pt.), or uncontrollable diabetes mellitus (1 pt.). Overall responses/complete remissions (ORR/CR) were achieved in 22/5 pts. (69/16%). One patient achieved minimal residual disease negativity (measured by 4-color flow cytometry) at the end of therapy. Median PFS was 10 months. In patients with p53 defects, ORR/CR were achieved in 5/2 pts. (63/25%). The Median PFS was 10.5 months for this subgroup. Median overall survival (OS) has not yet been reached. During therapy, CTCAE grade 3/4 toxicity consisted of bacterial infections (25%), ofatumumab infusion-related side-effects (9%), neutropenia (9%), hyperglycemia (6%), and anemia (3%). No reactivation of herpetic viral infection was observed during the course of therapy. Nine patients died during the follow-up as a result of disease progression (6 pts.), infections (2 pts.), or complications after allogeneic stem cell transplantation (1 relapsed pt.). The median CD20 antigen density in CLL cells was 4766 (881-18515) MESF (molecules of equivalent soluble fluorochrome) units at baseline. At the end of therapy, CD20 density had significantly decreased (median 821 MESF; 443-2637); nevertheless, it was high once more at relapse (median 6619 MESF; 628-21359). In vitro testing of malignant pts. cells sensitivity to dexamethasone and ofatumumab did not show synergistic or additive effect of these compounds in majority of patients. Also, in vitro testing did not clearly predict the outcome of O-dex therapy. Conclusions The O-dex regimen shows relatively high ORR and CR, with promising findings for PFS and OS (including pts. harboring p53 defects), as compared to published data on rituximab plus dexamethasone regimen or ofatumumab in monotherapy. The infectious toxicity in 1/4 of pts. represents the most frequent side effect for this regimen. The study was registered at www.clinicaltrials.gov (NCT01310101). Disclosures: Doubek: GlaxoSmithKline: Research Funding. Mayer:Roche: Consultancy, Research Funding; Glaxo: Consultancy, Research Funding.

Description: 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.

Description: Abstract 3911 Background The adverse prognostic significance of p53 aberrations (gene deletion at locus 17p13.1 and/or TP53 mutations) has been already proven in chronic lymphocytic leukemia (CLL). In contrast to the standardized examination of the gene deletion by interphase FISH, various methodologies with different detection efficiency are applied for mutation analysis. To reduce inter-laboratory variability, the European Research Initiative on CLL (ERIC) has recently released recommendations for p53 mutational testing (Pospisilova et al., 2012). However, the optimal detection methodology has not been established yet. Aim To compare molecular-biological methods for exact determination of TP53 mutational status in CLL patients. Methodology The analyzed cohort included 100 high-risk CLL patients with unfavorable disease prognosis represented by unmutated IgVH gene status, 17p and 11q deletions and/or chemotherapy resistence. Mutational screening of TP53 gene was performed in all patients by the combination of the following methods: (1) direct Sanger sequencing (DNA and/or cDNA), (2) denaturing high-performance liquid chromatography (DHPLC; Varian), (3) functional analysis (FASAY), (4) CLL custom resequencing microarray (Affymetrix), (5) Roche AmpliChip p53 Test (Roche Molecular Systems). In the selected samples, the presence of mutations was confirmed by ultra-deep next generation sequencing (NGS; GS Junior System, Roche). Results The parallel p53 analysis using all five above mentioned detection techniques revealed totally 66 mutations in 47/100 patients. The predominant proportion of the identified alterations was represented by prognostically adverse missense substitutions (67%), mainly localized in p53 DNA-binding domain (5–8 exons). Other clinically relevant sequencing variants included frameshift mutations (15%), splice-site mutations (8%), nonsense mutations (6%) and in-frame deletions (4%). Although the used detection methods reached comparable sensitivity (with the exception of direct sequencing), some inconsistent results were observed. In comparison with DNA-based methodologies, the FASAY failed in recognition of nonsense mutations leading to RNA degradation (nonsense-mediated decay phenomenon). On the other hand, the technical aspects of chip arrays have not facilitated the proper determination of deletions and insertions. From this perspective, DHPLC in connection with direct sequencing enabled the most specific recognition of the present gene alterations. Using this methodic combination, 57/66 mutations covering all mutation types were clearly identified. Nevertheless, for the correct evaluation of the biological importance and the clinical consequences of the detected mutations, the DNA screening should be supplemented with functional analysis. Conclusion The heterogeneous biological properties of TP53 mutations require sensitive and specific detection methodology. Although many different methods are currently used for mutation analysis, each of them has some advantages and shortcommings. The combination of DNA testing with functional analysis offers the most efficient tool for improved prediction of the disease course and the response of patients to therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abnormalities of TP53 and ATM genes are well established adverse prognostic markers in CLL. Mutations in splicing factor SF3B1 have recently been described as recurrent and predominantly subclonal aberration. Previous studies inconsistently suggested mutual exclusivity or partial overlap with TP53 mutations. Concerning ATM defects, the association between SF3B1 mutations and del(11q) was reported, but relation to ATM mutations remains unclear. The aims were: (a) to assess association between SF3B1 mutations and the most adverse classic genetic lesions represented by TP53 mutations and del(11q); (b) to investigate association between SF3B1 mutations and ATM mutations in a subset of ATM-characterized patients, and (c) to delineate SF3B1 mutation profile and proportion. We used Sanger sequencing of SF3B1 hot-spot exons 14-16, FASAY analysis of TP53 exons 4-10 and resequencing microarray for ATM mutation detection (all 62 coding exons). We analyzed unfavorable cohort of 338 patients characterized by prevalence of unmutated IGHV (72%). At the time of analysis, 82.5% of the patients were previously untreated. We observed SF3B1 mutation in 17.5% (59/338), TP53 mutation in 20% (68/338), and del(11q) in 27.5% (93/338) of cases. All these genetic defects were significantly more frequent in treated patients (SF3B1: P=0.008, TP53: P

Description: 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.

Description: Abstract 4256 Background Imatinib resistance in chronic myeloid leukemia (CML) patients is correlated with mutations in BCR-ABL tyrosine kinase domain in about half of the cases. Additional mechanisms related to imatinib resistance are under investigation. As TP53 gene is an important tumor suppressor that triggers apoptosis in response to DNA damage, its inactivation is responsible for chemotherapy resistance in cancer. Its role in cellular response to targeted biological treatment is currently unresolved. Inactivation of p53 by deletion and/or mutation in the TP53 gene is observed in CML patients during progression to accelerated phase (AP) and blast crisis (BC). It was suggested that BCR-ABL inhibition by imatinib induce the p53 response and therefore p53 inactivation may play role in resistance to targeted treatment (Wendel et al., 2006; Yamamoto et al., 2008). On the other hand, imatinib-induced p53 independent pro-apoptotic mechanism was described recently (Liu et al., 2009). Aim We investigated the relationship between imatinib resistance and abnormalities in the TP53 gene and additional genomic changes. Methods RNA and genomic DNA were isolated from peripheral blood mononuclear cells of CML patients that either fail to achieve or lost the major cytogenetic response (MCyR). TP53 mutational status was examined using functional analysis of separated alleles in yeast (FASAY). In defined cases direct sequencing of genomic DNA was used. Genomic changes were detected by conventional metaphase cytogenetics and CGH microarrays 4×44K (Agilent). Copy number analyses were performed using MEV software. Results FASAY analysis to detect functional state of TP53 gene was performed in 16 imatinib-resistant CML patients and in one Ph+ B-ALL patient. Six of these patients were negative for BCR-ABL mutations. Four patients were examined at the time of blast crisis (two patients with myeloid BC and two patients with lymphoid BC). All examined patients carried functional TP53 gene. As FASAY is based on RNA analysis, it is not able to detect some mutations leading to nonsense-mediated RNA decay; therefore, in six patients without BCR-ABL mutation, direct sequencing of TP53 gene from genomic DNA was performed. Also by this approach no TP53 mutation was detected. Alterations of the p53 pathway were found in only 2 patients, both in lymphoid BC: one patient without BCR-ABL mutation had +8 and the deletion of TP53 locus accompanied by i(17p). Second patient with BCR-ABL mutation T315I carried heterozygous deletion of 9p with biallelic loss of 9p21. In this locus two important tumor suppressor genes CDKN2A and CDKN2B are localized. CDKN2A alternative transcript contains an alternate open reading frame (ARF) that functions as a stabilizer of the tumor suppressor protein p53. In other two patients in chronic phase that did not reach MCyR and had no BCR-ABL mutations additional genomic changes potentially connected to imatinib resistance were found: 1) +der(16) coupled with amplification of 16(p11-q12), where ABCC11 and ABCC12 genes are localized. The proteins encoded by these genes are members of the superfamily of ATP-binding cassette (ABC) transporters responsible for multidrug resistance. 2) del(3)(p13p21), involving locus 3p14 where multiple tumor suppressors are localized (e.g. FHIT, ADAMTS9, LRIG1). Chromosomal region 3p14 was shown to be often deleted in different types of human cancers. Conclusions Imatinib resistance in CML patients is probably not associated with TP53 inactivation. Alterations of the p53 pathway occur within transformation to more advanced stages, what is in concordance with previous findings. Genomic aberrations potentially influencing response to imatinib treatment may be found in some patients. Larger patients' cohorts are required to identify relevant recurrent genomic aberrations involved in imatinib resistance. This work was supported with grants NR9858-4/2008 and NR9305-3/2007 provided by IGA MH CR of Czech Republic, and MSM0021622430 provided by MEYS of Czech Republic Disclosures: No relevant conflicts of interest to declare.

Description: Background: Defects in the p53 gene predispose B-CLL patients for an inferior outcome, particularly a resistance to treatment by conventional chemotherapeutics. Very little data exist, however, about the efficacy of monoclonal antibody rituximab on B-CLL cells bearing p53 abnormalities. One reason for that might be a methodological - rituximab alone does not have virtually any effect on the viability of B-CLL cells when cultivated in vitro, unless an active human plasma is added. After that, however, the cells are quickly lysed by complement, what is a process independent on p53. Aims: We used an in vitro system (not containing the active human plasma) to monitor a rituximab activity on B-CLL cells with p53 inactivation in relation to subsequently used nucleoside analog fludarabine, which demonstrably acts through the p53 in B-CLL cells. Methods: The p53 abnormalities in blood samples of B-CLL patients were detected by FISH and by functional yeast analysis coupled to sequencing, as described previously (Trbusek et al., Leukemia2006, 20: 1159–1161). Vitally frozen samples were used in all cases, after 24h pre-cultivation. Mononuclear cells were cultivated for 72h with or without 20μg/ml of rituximab and subsequently for another 48h with four different concentrations of fludarabine (40μg/ml–0,625μg/ml). The cell viability was determined by a WST-1 assay. A sensitization effect of rituximab pretreatment was determined by an ANOVA analysis, with the value p=0,05 being a threshold for a statistical significance. To monitor an apoptosis (a suppossed mechanism of fludarabine action), the Western blotting was used for the caspase-3 cleavage, which was proved previously to occur in drug-treated B-CLL cells. Results: In the subgroup of eleven p53-wt samples the three cases manifested sensitization by rituximab for fludarabine activity, one case showed an oposite (antagonistic) effect, while there was no significant difference for another seven samples. Among ten p53-mutated samples there was just one case exhibiting no influence of rituximab pretreatment (with the p53 alleles being deletion / 281 Asp→Glu), one sample manifested with antagonistic effect (del / 220 Tyr→Cys), while the remaining eight cases showed a statistically significant sensitization by rituximab (del / truncated protein aa 314, del / no protein, del/ wt protein, del / 248 Arg→Gln, del / 249 Arg→Gly, del / del aa 252, del / del aa 252–254, and a composed mutant 281Asp→Asn / 254 Ile→Thr). We noticed a statistically significant potentiation also in three out of four ATM-deleted samples (ATM is the p53-regulatory kinase). An apoptosis occured after fludarabine addition both in pretreated and control cells, as evidenced by the caspase-3 cleavage in some (but not all) samples. Conclusions: We show, to our konwledge for the first time, that rituximab can significantly sensitize the B-CLL cells bearing different types of p53 mutations to fludarabine. This result warrants further investigation of the mechanism behind.

Description: Tumor suppressor p53 is a powerful transcription factor responsible for cell cycle regulation, which can bind about 300 different promoter elements in the human genome. Disruption of p53 function by mutation or deletion belongs to the most frequent alterations observed in human cancers, in B-CLL ranging from 10 to 15%. In our cohort of 230 B-CLL patients, we detected 10.5% patients with Tp53 mutation, 67% of these exhibited a point mutation (single nucleotide switch) largely combined with deletion of the second allele. The detected mutations are mostly localized in the DNA binding domain of p53 gene and, in accordance with the IARC database, are clustered into two hot-spot regions between amino acids 234–249 (32% of the mutations) and 273–282 (25% of the mutations). B-CLL patients with mutant Tp53 have mostly non-mutated IgVH (95%) and a significantly worse prognosis (17% of patients with Tp53 mutation and only 3% of patients having wt-p53 died within 2 years after diagnosis). In addition, 92% of patients with mutated p53 required therapy; in the non-mutated group this number represented only 54% of the respective patients. Due to these findings detection of p53 status became an essential part of B-CLL diagnostics in our laboratory. However, recent results suggest existence of significant differences in the properties of specific mutants, especially regarding their DNA-binding ability to activate particular promoters (e.g. Pospisilova et al., Mol. Cancer Res. 2: 296–304, 2004). Presence of mutation need not represent a total loss of protein activity but, to the contrary, some amino acid substitutions can evoke a gain of function of the respective p53 mutant. Therefore, it seems to be very important to determine the specific type of amino acid substitution in a particular B-CLL patient and detect ability of the respective p53 mutant to induce cell cycle arrest and/or apoptosis. We used functional protein arrays bearing p53 mutants to determine their DNA-binding activity towards promoter sequences of different p53-response genes such as mdm2, p21/waf1, pig3 or bax. Several analyzed p53 mutants displayed DNA-binding ability similar or even higher than wt-p53 towards all studied promoters (e.g. mutant Arg337Cys) suggesting gain of function. Interestingly, not only the amino acid localization but also type of substitution in the same position can influence protein activity. The most frequent mutational hotspots in B-CLL and majority of other cancers, Arg248 and Arg273, displayed significantly higher DNA-binding activity of mutant variants Arg248Gln and Arg273His in comparison to Arg248Trp and Arg273Cys. These experimental results were validated by protein modeling clearly demonstrating respective structural constraints, such as prevented binding of Trp248 to the promoter region DNA groove for sterical and hydrogen-bonding reasons. In conclusion, our results of B-CLL patient analysis demonstrate importance of the presence of p53 mutation for B-CLL treatment and prognosis. We also document the significance of localization and type of amino acid substitution for p53 mutant activity. These factors represent a potentially valuable marker for grading of p53 mutant functionality in B-CLL patients and tumor prognosis.