Vladimíra Vranová

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.

Missense Mutations Located in Structural p53 DNA-Binding Motifs Are Associated With Extremely Poor Survival in Chronic Lymphocytic Leukemia

PURPOSE There is a distinct connection between TP53 defects and poor prognosis in chronic lymphocytic leukemia (CLL). It remains unclear whether patients harboring TP53 mutations represent a homogenous prognostic group. PATIENTS AND METHODS We evaluated the survival of patients with CLL and p53 defects identified at our institution by p53 yeast functional assay and complementary interphase fluorescence in situ hybridization analysis detecting del(17p) from 2003 to 2010. RESULTS A defect of the TP53 gene was identified in 100 of 550 patients. p53 mutations were strongly associated with the deletion of 17p and the unmutated IgVH locus (both P < .001). Survival assessed from the time of abnormality detection was significantly reduced in patients with both missense (P < .001) and nonmissense p53 mutations (P = .004). In addition, patients harboring missense mutation located in p53 DNA-binding motifs (DBMs), structurally well-defined parts of the DNA-binding domain, manifested a clearly shorter median survival (12 months) compared with patients having missense mutations outside DBMs (41 months; P = .002) or nonmissense alterations (36 months; P = .005). The difference in survival was similar in the analysis limited to patients harboring mutation accompanied by del(17p) and was also confirmed in a subgroup harboring TP53 defect at diagnosis. The patients with p53 DBMs mutation (at diagnosis) also manifested a short median time to first therapy (TTFT; 1 month). CONCLUSION The substantially worse survival and the short TTFT suggest a strong mutated p53 gain-of-function phenotype in patients with CLL with DBMs mutations. The impact of p53 DBMs mutations on prognosis and response to therapy should be analyzed in investigative clinical trials.

Identification of rare complete BRCA1 gene deletion using a combination of SNP haplotype analysis, MLPA and array-CGH techniques

The Large genomic rearrangements (LGR) in BRCA1/2 represent a substantial proportion of disease-causing changes. In our pilot study we demonstrate the specific case of the Slovak breast/ovarian cancer family, where BRCA1 analysis revealed the discrepancy of SNPs haplotypes and primarily indicated the presence of LGR. Initially, the analysis of all exons of BRCA1 was based on the combination of SSCP and sequencing techniques. After the abnormal SNPs haplotypes identification, MLPA analysis was performed. The results were finally proved with array-comparative genomic hybridization (array-CGH). The hemizygous status of 9 SNPs identificated in BRCA1 indicated a possible occurrence of LGR. The MLPA results showed reduction of peaks levels for each BRCA1 exon. Array-CGH method displays a single deletion signal for BRCA1 gene among set of 287 gene probes. Totally, 8 members of the family where analysed, in 3 of them the deletion was confirmed. Two of the LGR carriers suffered with unilateral and bilateral breast cancer respectively; the third carrier of the LGR was affected with an ovarian cancer. We have discovered rare BRCA1 germline LGR affecting complete gene. According to our knowledge, this is the first Slovak family with LGR in BRCA1 gene and second time, when the complete deletion of BRCA1 gene was described worldwide. Concerning the family history it is evident, that clinical effect of LGR is comparable with small deletions/insertions and substitutions and leads to the loss-off gene function. In conclusion, it is also important to note that DNA analysis of BRCA1/2 genes should be performed in all affected members of breast/ovarian cancer families concurrently, since the discrepancy in the SNPs haplotypes may indicate the presence of LGR.

Unique Combination of 22q11 and 14qter Microdeletion Syndromes Detected Using Oligonucleotide Array-CGH

We report an infant with a unique combination of 22q11 deletion syndrome and 14q terminal deletion syndrome. The proband had clinical symptoms compatible with diagnosis of 22q11 deletion syndrome: microcephaly, micrognathia, high-arched palate, hypertelorism, short palpebral fissures, square nasal root, prominent tubular nose, hypoplastic nasal alae, bulbous nasal tip, dysplastic low-set ears, short philtrum, and heart defect, but no cell-mediated immunodeficiency typical for the syndrome. G-banding and fluorescence in situ hybridization analyses revealed a karyotype 45,XY,der(14)t(14;22)(q32.3;q11.2),-22.ish del(14)(q32.33)(D14S1420-),del(22)(q11.2q11.2)(N25-). Subsequent analyses disclosed a translocation between chromosomes 14 and 22 in the proband’s mother with a deleted 14q telomere. Using comparative genome hybridization on oligonucleotide-based microarray (array-CGH), the deletion at 22q11.21 in the size of ∼4.25 Mb was revealed in the proband as well as the deletion of the telomeric area at 14q32.33qter (∼3.24 Mb) in the proband and his mother. However, both the proband and his mother showed mild symptoms (microcephaly, thin lips, carp-shaped mouth) typical for patients with the described terminal 14q deletion syndrome.