Low-burden TP53 mutations in chronic phase of myeloproliferative neoplasms: association with age, hydroxyurea administration, disease type and JAK2 mutational status.

The multistep process of TP53 mutation expansion during myeloproliferative neoplasm (MPN) transformation into acute myeloid leukemia (AML) has been documented retrospectively. It is currently unknown how common TP53 mutations with low variant allele frequency (VAF) are, whether they are linked to hydroxyurea (HU) cytoreduction, and what disease progression risk they carry. Using ultra-deep next-generation sequencing, we examined 254 MPN patients treated with HU, interferon alpha-2a or anagrelide and 85 untreated patients. We found TP53 mutations in 50 cases (0.2-16.3% VAF), regardless of disease subtype, driver gene status and cytoreduction. Both therapy and TP53 mutations were strongly associated with older age. Over-time analysis showed that the mutations may be undetectable at diagnosis and slowly increase during disease course. Although three patients with TP53 mutations progressed to TP53-mutated or TP53-wild-type AML, we did not observe a significant age-independent impact on overall survival during the follow-up. Further, we showed that complete p53 inactivation alone led to neither blast transformation nor HU resistance. Altogether, we revealed patient's age as the strongest factor affecting low-burden TP53 mutation incidence in MPN and found no significant age-independent association between TP53 mutations and hydroxyurea. Mutations may persist at low levels for years without an immediate risk of progression.

Detailed analysis of therapy-driven clonal evolution of TP53 mutations in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), the worst prognosis is associated with TP53 defects with the affected patients being potentially directed to alternative treatment. Therapy administration was shown to drive the selection of new TP53 mutations in CLL. Using ultra-deep next-generation sequencing (NGS), we performed a detailed analysis of TP53 mutations' clonal evolution. We retrospectively analyzed samples that were assessed as TP53-wild-type (wt) by FASAY from 20 patients with a new TP53 mutation detected in relapse and 40 patients remaining TP53-wt in relapse. Minor TP53-mutated subclones were disclosed in 18/20 patients experiencing later mutation selection, while only one minor-clone mutation was observed in those patients remaining TP53-wt (n=40). We documented that (i) minor TP53 mutations may be present before therapy and may occur in any relapse; (ii) the majority of TP53-mutated minor clones expand to dominant clone under the selective pressure of chemotherapy, while persistence of minor-clone mutations is rare; (iii) multiple minor-clone TP53 mutations are common and may simultaneously expand. In conclusion, patients with minor-clone TP53 mutations carry a high risk of mutation selection by therapy. Deep sequencing can shift TP53 mutation identification to a period before therapy administration, which might be of particular importance for clinical trials.

Detecting minimal residual disease in patients with chronic lymphocytic leukemia using 8-color flow cytometry protocol in routine hematological practice.

INTRODUCTION: Minimal residual disease (MRD) detection has become increasingly important for the assessment of therapy response in chronic lymphocytic leukemia (CLL). However, current MRD analysis methods, both molecular genetic and flow cytometric, are time-consuming and require experienced laboratory staff. METHODS: To reduce the demands of flow cytometric MRD detection in CLL, we have introduced a novel flow cytometric 8-color protocol. The MRD analysis results using this protocol were then compared with the commonly employed 4-color protocol and the molecular genetic (real-time quantitative allele-specific oligonucleotide IGH polymerase chain reaction; RQ-ASO IGH PCR) approach. RESULTS: Forty-two CLL patient samples were repeatedly analyzed after allogeneic stem cell transplantation (n = 20) or after fludarabine-based therapy (n = 22), and 100% concordance was found using both flow cytometric protocols. Furthermore, there was a strong correlation (r = 0.94) between flow cytometric and RQ-ASO IGH PCR results in MRD detection. CONCLUSION: Flow cytometry is less time-consuming, less financially demanding, and moreover, MRD assessment using our novel 8-color protocol is less complicated than the 4-color approach and molecular methods.

Gene expression and cell differentiation in matrix-associated chondrocyte transplantation grafts: a comparative study.

OBJECTIVE: Although scaffold composition and architecture are considered to be important parameters for tissue engineering, their influence on gene expression and cell differentiation is rarely investigated in scaffolds used for matrix-associated autologous chondrocyte transplantation (MACT). In this study we have therefore comparatively analyzed the gene expression of important chondrogenic markers in four clinical applied cell-graft systems with very different scaffold characteristics. METHODS: Residuals (n=165) of four different transplant types (MACI®, Hyalograft®C, CaReS® and Novocart®3D) were collected during surgery and analyzed for Col1, Col2, aggrecan, versican, melanoma inhibitory activity (MIA) and IL-1ß by real-time PCR. Scaffold and cell morphology were evaluated by histology and electron microscopy. RESULTS: Despite the cultivation on 3D scaffolds, the cell differentiation on all transplant types didn't reach the levels of native cartilage. Gene expression highly differed between the transplant types. The highest differentiation of cells (Col2/Col1 ratio) was found in CaReS®, followed by Novocart®3D, Hyalograft®C and MACI®. IL-1ß expression also exhibited high differences between the scaffolds showing low expression levels in Novocart®3D and CaReS® and higher expression levels in MACI® and Hyalograft®C. CONCLUSIONS: Our data indicate that scaffold characteristics as well as culture conditions highly influence gene expression in cartilage transplants and that these parameters may have profound impact on the tissue regeneration after MACT.

[Human genome sequencing--next generation technology or will the routine sequencing of human genome be possible?]. / Sekvenování lidského genomu--technologie nové generace aneb budeme rutinne sekvenovat lidské genomy?

DNA sequencing has become a standard method widely used in molecular genetic analysis of biological materials. Its use in medicine is widespread, especially in diagnostics of inherited disorders and cancer related diseases. Development of DNA diagnostics has been strongly accelerated by publication of the human genome sequence in 2001. During the last few years one can observe rapid development of novel sequencing technologies, which have led to the introduction of so called "New Generation Sequencing". These new technologies based on principles of massive parallel sequencing (e.g. Roche/454, Illumina Genome Analyzer IIx, Life Technologies SOLiD 3 and others) enable a massive increase of sequencing capacity and in parallel also a fundamental decrease of costs. This major technological breakthrough allowed development of the whole-genome sequencing including analyses of individual human genomes. It also started the era of personal genomics. The first sequenced individual human genomes belonged to famous geneticists J. C. Venter (2007) and J. D. Watson (2008), but they were rapidly followed by sequencing analyses of other individuals from various ethnic groups. These studies brought substantial information about interpersonal differences in genome structure (through characterization of nucleotide polymorphisms, DNA deletions and amplifications etc.). Sequencing of cancer cell genomes, e.g. acute myeloid leukemia has already brought first important clinically relevant results. Although currently we are still unable to interpret the relevance of all detected genome variants, it is obvious, that the possibility to sequence individual human genomes represents a fundamental breakthrough not only in DNA diagnostics but also in clinical medicine.

Micro-RNAs miR125b and miR137 are frequently upregulated in response to capecitabine chemoradiotherapy of rectal cancer.

There is increasing evidence that some microRNAs change their levels in reaction to xenobiotic challenge. The aim of this study was to test the possible involvement of micro-RNAs in response to standard anticancer treatment. Tumor biopsies from 35 patients with rectal cancer before therapy and parallel tumor biopsies from 31 patients two weeks after starting preoperative capecitabine chemoradiotherapy were taken. The expression levels of single miRNA species were measured using TaqMan Micro-RNA assays after reverse transcription from isolated total RNAs. Many micro-RNAs (miR10a, miR21, miR145, miR212, miR339, miR361) responded to chemoradiotherapy in individual tumor samples, but there was profound intertumoral variability. However, other two micro-RNAs miR125b, miR137 showed a significant increase in median expression levels after starting therapy in most samples. Moreover, our results for the first time show that higher induced levels of miR125b and miR137 are associated with worse response to the therapy.

Identification of somatic hypermutations in the TP53 gene in B-cell chronic lymphocytic leukemia.

Abnormalities of the TP53 gene are associated with a particularly severe prognosis in patients with B-cell chronic lymphocytic leukemia (B-CLL). This tumor-suppressor is mostly inactivated by the deletion of one and point mutation of the other allele and has not been previously shown to be hypermutated in B-CLL. We identified two patients whose lymphocytes showed repeatedly an extensive proportion of TP53 mutated cells by FASAY analysis (the yeast functional assay) and harbored various TP53 mutations, mostly single-base substitutions, in individual cells. The mutation targeting exhibited characteristic traits of the somatic hypermutation process. In the first patient (harboring the unmutated IgVH locus) a significant bias to point mutations at CG pairs (21/25; P=0.009), their remarkable preference for the RGYW/WRCY motives (28%) and the highest expression of the activation-induced cytidine deaminase (AID) mRNA among the 34 tested B-CLL samples. In the second patient no CG bias was observed but the targeting of point mutations into the RGYW/WRCY motives was even more prominent here (7/16; 44%). Moreover, six out of eight point mutations affecting AT pairs were localized in the WA/TW motives, which are also characteristic for the somatic hypermutations. This patient, who was IgVH-mutated, already did not express any significant amount of the AID transcript. Our findings add a new aspect to the mosaic of the p53 mutability in B-CLL.

Feasibility study of NeoMend, a percutaneous arterial closure device that uses a nonthrombogenic bioadhesive.

OBJECTIVE: The aim of this prospective single-center phase I feasibility study was to investigate the safety and efficacy of a novel vascular sealing device, the NeoMend Arterial Closure Device, that uses a bioadhesive after percutaneous endovascular procedures. SUBJECTS AND METHODS: In 26 consecutive patients, the sealing device was deployed at the femoral artery access site immediately after a catheterization procedure using a 6-French (1.91-mm) sheath. Patients were followed up at 24 hr with Doppler sonography of the treated femoral artery puncture site, and at 1 week and 1 month by a telephone interview. RESULTS: Successful hemostasis was achieved with the NeoMend Arterial Closure Device in 21 (88%) of 24 patients. One major complication required surgery: formation of puncture site hematoma and pseudoaneurysm 3 days after the intervention after successful primary hemostasis. Two device failures required crossover to manual compression, which was done without further complications. The mean time to hemostasis was 7.0 +/- 4.5 min. Mean time to ambulation was 6.0 hr. At follow-up, the patients did not report any puncture-site-related complaints. Doppler sonography of the puncture sites revealed three insignificant hematomas of less than 20 mL and patent common femoral vessels without stenoses. CONCLUSION: The NeoMend Arterial Closure Device appears to achieve rapid hemostasis with the potential of early ambulation after arterial punctures with a 6-French sheath. The device is an alternative in situations in which suture- or collagen-mediated devices show high complication rates.

Interleukin-10-deficient mice and inflammatory bowel disease associated cancer development.

Interleukin-10-deficient mice develop colitis and colorectal cancer similar to the inflammatory bowel disease associated cancer in humans. The aim of this study was to identify possible mutations of oncogenes and tumour suppressor genes involved in tumorigenesis in Interleukin-10 (IL-10)-deficient mice. Twenty colon carcinomas from IL-10-deficient mice were screened for mutations in the K-ras and p53 genes by 'cold' single-strand-conformation polymorphism. Immunohistochemical staining was performed to detect mutations in the proteins P53, APC and MSH2, and the transforming growth factor beta type II receptor. Microsatellite instability was analysed at eight chromosomal loci and plasma levels of transforming growth factor beta1 (TGF-beta1) were also measured. At 9 weeks, 14% of the animals developed colorectal cancer, and at 10-31 weeks the incidence of carcinoma was 65%. No mutations were detected in the analysed oncogene and tumour suppressor genes. Plasma TGF-beta1 levels in IL-10-deficient mice 10-31 weeks old were higher than in wild-type littermates e.g. 45.7 +/- 4.6 ng/ml versus 19.8 +/- 4.5 ng/ml (P<0.01). No alterations in K-ras, p53, APC: and Msh2 genes suggests that other genes are involved in the development of these tumours. Elevated TGF-beta1 plasma levels correspond to the high incidence of dysplasia and cancer. Normal expression of the TGF-beta II receptors hints at genetic alterations in other members of the TGF-beta receptor signal transduction pathway.

Incidence of nephrotic syndrome in patients with glomerulonephritis.

The occurrence of nephrotic syndrome was studied in different types of glomerulonephritis. In 284 consecutive adult cases the diagnosis was confirmed by the histological evaluation of percutaneous renal biopsy. Nephrotic syndrome was more frequent in minimal change glomerulonephritis, in focal sclerosis and in membranous nephropathy. The incidence of glomerulonephritis decreases, while the relative incidence of nephrotic syndrome increases with age. Glomerulonephritis is more frequent in men whereas the relative incidence of nephrotic syndrome is higher in women.

Inhibiting effect of foetal renal antigen on leukocyte migration in human glomerulonephritis.

In 141 cases of glomerulonephritis confirmed by renal biopsy it was demonstrated that foetal glomerular basement membrane antigen caused a migration inhibition most frequently in minimal change glomerulonephritis. Cellular hypersensitivity was less common in membranous nephropathy, membranoproliferative (I-III) glomerulonephritis, IgA nephropathy and lupus nephritis. The correlation between LMT positivity and the occurrence of renal tissue IF activity was a linear one, but in one type, minimal change glomerulonephritis, there was no such correlation. The occurrence of LMT positivity does not show any considerable difference in glomerulonephritis with and without nephrosis.