[Two Approaches to Cancer Development]. / Dva pohledy na vývoj nádoru.

BACKGROUND: The somatic mutation theory explaining the process of carcinogenesis is generally accepted. The theory postulates that carcinogenesis begins in a first renegade cell that undergoes gradual transformation from a healthy to a fully malignant state through the accumulation of genetic and epigenetic "hits". This theory focuses specifically on mutations and genetic aberrations, and their impact on cells. It considers tumors as populations of sick cells that lose control of their own proliferation. The theory was put forward by Robert Weinberg and Douglas Hanahan, and is the predominant view in current cancer biology. By contrast, the tissue organization field theory proposed by Carlos Sonnenschein and Ana Soto considers loss of physiological structure and function by a tissue as key events in tumor development. According to this theory, tumors arise at a tissue rather than at a cellular level. It is based on a presumption that proliferation status, rather than quiescence, is the default position of cells in multicellular organisms. AIM: The article aims to provide answers to following questions: Are the views of proponents of the somatic mutation theory (the reductionists) and proponents of the tissue organization field theory (the organicists) incompatible and incommensurable, even when the mainstream of tumor biology has shifted its attention from tumor cells toward the tumor microenvironment? Where to find a third interconnecting systemic approach? Is it useful to be aware of the controversy between reductionists and organicists? What this awareness contributes to? How do these alternative views influence practical oncology and tumor biology in general? CONCLUSION: Whether the true position is held by reductionists or organicists is unimportant. What is important is to be aware of the existence of these two concepts because this knowledge makes the way we think about tumor origin and development, and how we set up and interpret our experiments, more precise. KEY WORDS: carcinogenesis - mutation - cell - tissues - cell proliferation - cell quiescenceThis study was supported by grant of Internal Grant Agency of the Czech ministry of Health No. NT/13784-4/2012.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 29. 7. 2015Accepted: 27. 4. 2016.

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.

[Brazilian story of the R337H p53 mutation]. / Brazilský príbeh mutace p53 R337H.

The p53 tumor suppressor is an evergreen of molecular oncology. Since its discovery in 1979, it has been subjected to intensive investigation. The p53 protein is composed of "only" 393 amino acid residues, and function of almost each of them has been addressed in detail. Somatic mutations are extremely frequent, they can be found almost in each of the p53 codons and in all types of tumors. Inherited p53 mutations are rare but very penetrant, and they are typically associated with development of a broad spectrum of tumors. However, in 2001, the p53 research provided an unexpected discovery: the R337H allele was found in southern Brazil. This allele was atypically associated with only one type of tumor -  childhood adrenocortical carcinoma and it exhibited low penetrance. Therefore, new data on functioning and impact of the R337H mutation were highly desired. The results obtained during a few following years helped to elucidate not only this specific p53 variant but also provided insight into general principles of mutant p53 variants function. It also turned out that all R337H alleles that are very frequent in southern Brazil originate from one common ancestor.

[Doublehit lymphomas -  review of the literature and case report]. / Lymfomy se dvema zásahy -  prehled literatury a kazuistika.

UNLABELLED: Blymphocytes are cells of the immune system responsible for the antibody  mediated immune response. As estimated, a human body can produce as much as 1011 specific antibodies. There are no specific genes coding for every individual antibody in the human genome. Discrepancy between the huge diversity of antibodies and limited coding capacity of the genome is solved by combination of unique arrangement of genetic information for immunoglobulin and unique genetic and somatic processes providing this wide spectrum of antibodies. On one side, these mechanisms represent a life protecting source of a wide spectrum of antibodies but at the same time, they can be life threatening by raising the risk of a serious tumor disease, the B cell lymphoma. Double hit lymphomas represent a specific group of B cell lymphomas often featuring concurrent rearrangements of BCL2 and MYC genes. Activation of the MYC oncogene, typical for Burkitt lymphoma (BL), causes strong stimulation of cell proliferation. High activity of BCL 2, typical for follicular lymphoma, induces resistance to apoptosis. Concurrent damage of regulation of apoptosis and proliferation is probably responsible for the typical clinical manifestation of double hit lymphomas - aggressive course, resistance to conventional chemotherapy, high-risk of early relapse, short overall survival, frequent extranodal and central nervous system involvement. Recently, these lymphomas have attracted a strong attention of researchers as they provide sharp insights into processes of lymphocytes maturing and lymphomas development and highlight the double edged nature of mechanisms allowing the antibody broad diversity. CASE REPORT: Fifty  three year  old man was diagnosed with B cell lymphoma unclassifiable with features intermediate between diffuse large B cell lymphoma (DLBCL) and BL, based on morphology and immunophenotype. Fluorescent in situ hybridization analysis revealed double hit lymphoma diagnosis as the tumor cells bear t(14;18) translocation concurrently with the MYC gene rearrangement. The patient died five months after dia-gnosis.

Roscovitine-induced apoptosis of H1299 cells depends on functional status of p53.

Roscovitine, an inhibitor of cyclin-dependent kinases, is promising anticancer agent. Its antiproliferative and cytotoxic effects can be mediated by the p53 signaling pathway. To define the role of p53 in roscovitine-induced cell response, we prepared H1299/p53 cell lines inducibly expressing specific variants of p53 (p53wt and hotspot R175H, temperature-dependent P98A, A159V, S215G, Y220C, Y234C mutants). In the presence of roscovitine, each cell line variant behaved in specific way reflecting activity of the p53 protein. Roscovitine decreased production of the cell cycle inhibitor p21 and induced apoptosis. This effect was the most efficient in cells expressing p53wt protein with full activity. The cell expressing partially and conditionally active p53 mutants responded to roscovitine less efficiently. The cells expressing p53 mutants A159V and Y234C were very sensitive to roscovitine but their response was clearly temperature-dependent. The cells expressing P98A, S215G and Y220C p53 mutants exhibited only weak sensitivity to roscovitine and underwent apoptosis in low frequency. In principle, each td p53 mutant responded to roscovitine in distinct way. We showed clearly that the impact of roscovitine on H1299 cells depends on functional status of p53 they produce. This suggests that patients with tumors exhibiting specific p53 variants can benefit from the roscovitine therapy.

TP53 mutation profile in chronic lymphocytic leukemia: evidence for a disease specific profile from a comprehensive analysis of 268 mutations.

The TP53 mutation profile in chronic lymphocytic leukemia (CLL) and the correlation of TP53 mutations with allele status or associated molecular genetics are currently unknown. We performed a large mutation analysis of TP53 at four centers and characterized the pattern of TP53 mutations in CLL. We report on 268 mutations in 254 patients with CLL. Missense mutations appeared in 74% of cases compared with deletions and insertions (20%), nonsense (4%) and splice site (2%) mutations. The majority (243 of 268) of mutations were located in the DNA-binding domain. Transitions were found in 131 of 268 mutations, with only 41 occurring at methylated CpG sites (15%), suggesting that transitions at CpGs are uncommon. The codons most frequently mutated were at positions 175, 179, 248 and 273; in addition, we detected a common 2-nt deletion in the codon 209. Most mutations (199 of 259) were accompanied by deletion of the other allele (17p-). Interestingly, trisomy 12 (without 17p-) was only found in one of 60 cases with TP53 mutation (without 17p-) compared with 60 of 16 in the cohort without mutation (P=0.006). The mutational profile was not different in the cohorts with and without previous therapy, suggesting that the mechanism underlying the development of mutations may be similar, independent of treatment.

[Lymphoma of the small intestine]. / Lymfomy tenkého streva.

A series of eight small intestine lymphomas comprised two cases of follicular lymphoma (FL), one anaplastic large cell lymphoma (ALCL) ALK negative, and five cases of diffuse large B-cell lymphoma. The lymphomas were diagnosed by routine hematoxylin-eosin staining, immunohistochemistry and the FISH method for translocation t(14;18). Immunohistochemistry revealed that the diffuse large B-cell lymphomas were of the non-germinal center type (non GC-DLBCL). In most cases, the tumors formed solid well-circumscribed nodules or resulted in diffuse infiltration of the intestinal wall. In one case of follicular lymphoma, microscopic foci of tumor were found in the intestinal mucosa which spread far from the primary nodule and probably beyond the resection border. It is difficult to ascertain whether this phenomenon represents colonization of pre-existing non-neoplastic follicles by lymphoma or spreading of the tumor within the same tissue. In this case, surgical removal of the lymphoma is problematic.

Complex analysis of cyclin D1 expression in mantle cell lymphoma: two cyclin D1-negative cases detected.

BACKGROUND AND AIM: The cytogenetic and diagnostic hallmark of mantle cell lymphoma (MCL) is translocation t(11;14)(q13;q32), resulting in overexpression of cyclin D1. Cyclin D1 expression was analysed in 32 cases of MCL. METHODS: The t(11;14) translocation was detected by fluorescence in situ hybridisation, level of cyclin D1 mRNA by competitive RT-PCR, and level of cyclin D1 and D2 proteins by immunohistochemistry and/or immunoblotting. RESULTS: In 30 cases, the presence of translocation t(11;14), a high level of cyclin D1 mRNA, and a high level of the cyclin D1 protein were confirmed. Two cyclin D1-negative cases overexpressing cyclin D2 were detected by immunoblotting. CONCLUSIONS: There are rare cyclin D1-negative cases of MCL overexpressing cyclin D2. Anti-cyclin D1 antibodies with low specificity can bind both cyclin D1 and cyclin D2, thus providing false cyclin D1-positive signals in immunohistochemical analysis.

[Our experience in using fluorescence in situ hybridization FISH-Uro Vysion in diagnostics of urothelial carcinoma]. / Nase zkusenosti s metodou fluorescencní in situ hybridizace pri detekci uroteliálního karcinomu.

UNLABELLED: Urothelial carcinoma is a disease at high risk of recurrence after the initial therapy (70-80%) and with the tendency to progression accomplishing the recurrence (30%). Long lasting monitoring of patients with urothelial carcinoma is necessary. Cystoscopy and cytology are currently the primary modalities used to detect and monitor urothelial carcinoma. However, cytology has relatively poor sensitivity especially in well differentiated tumors. Cystoscopy is an invasive and relatively expensive method. Therefore, methods improving detection of urothelial carcinoma from urine specimens are employed. Uro Vysion (Vysis) fluorescence in situ hybridization (FISH) for improved detection of urothelial carcinoma was evaluated. MATERIALS AND METHODS: Bladder tumor progression is accompanied by increased chromosomal instability and aneuploidy of chromosomes 3, 7, 17 and loss of locus 9p21. A total of 124 patients were analyzed at Dpts. of Urology and Pathology, Faculty Hospital in Brno. Cytologically analyzed urine specimens were tested by FISH and simultaneously cystoscopy was employed including biopsy for histological examination. RESULTS: FISH analysis was positive in 35 cases, including 5 cases with negative biopsy and cytology. Negative FISH result was detected in 24 cases where the malignant status was determined. The sensitivity of FISH in our series was 58.9% and the specificity 88.1%. CONCLUSIONS: FISH is a relatively simple, speedy and non invasive diagnostic method. It detects the symptoms of malignity on the molecular level, which leads to earlier diagnosis and therapy and, hence, to potential extended survival. FISH makes it possible to take decision in cases of atypical or unclear cytological finding. The FISH method using the Uro Vysion kit appears as a prospective non invasive method capable of early UK detection, with a higher sensitivity than the standard cytology of urine.

[Immunohistochemical detection of the gene p53 and p21 expression in cells of the malignant melanoma of the uvea]. / Imunohistochemická detekce exprese genu p53 a p21 v bunkách maligního melanomu uvey.

AIM: To examine by means of immunohistochemistry the expression of the tumor suppressing gene p53 and gene p21 in cells of malignant melanoma of the uvea from formalin-paraffin material from patients, who were during the period 2000 - 2006 surgically treated due to malignant melanoma of the uvea at the Department of Ophthalmology in the University Hospital in Brno (Brunn), Czech Republic, E.U., and to correlate the results of the immunohistochemical detection with clinical signs of the tumor of each patient. METHODS: Twenty-nine malignant melanomas of the uvea were examined by means of monoclonal antibody DO-1 (Novocastra company) and all 29 samples of malignant melanoma of the uvea were immunohistochemically examined for the p21 gene expression by means of the monoclonal antibody SX 118 (DAKO company). We evaluated the percentage of positive nuclei and the intensity of the staining in immunohistochemically detected p53 and p21 genes expression. RESULTS: Results suitable for evaluation we obtained in 28 samples of malignant melanomas, one sample was not suitable for evaluation due to extremely high presence of melanin pigment. In 3 patients, weak nuclear p53 gene expression was detected in 5-15% of cells, in 1 patient, the very weak intensity of staining in 5-15% of cells was found. In three patients, in 5-15% of cells, weak expression of p21 gene, and in one patient, very weak expression of p21 gene in 5-15% of cells (in all 4 cases, the p53 expression was established) were found. In one of those 4 patients with p53 gene expression it was the malignant melanoma of the iris, in one of them it was malignant melanoma of the ciliary body, and in 2 of them it was malignant melanoma of the choroid. CONCLUSION: The expression of the p53 gene and the expression of the gene p21 were established in 4 out of 28 patients (14.3%). From the above-mentioned results we can assume that stabilizing mutations of p53 gene are rare in the melanoma of the uvea. The proved expression p53 in 4 patients is probably result of the expression of the standard (wild-type) p53 gene, especially according to the ability to induce the expression of p21 gene. In our group, there were not proved marked nuclear accumulation of p53, which would suggest the presence of p53 gene mutation.

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.

Detailed mapping of chromosome 17p deletions reveals HIC1 as a novel tumor suppressor gene candidate telomeric to TP53 in diffuse large B-cell lymphoma.

Deletions in the short arm of chromosome 17 (17p) involving the tumor suppressor TP53 occur in up to 20% of diffuse large B-cell lymphomas (DLBCLs). Although inactivation of both alleles of a tumor suppressor gene is usually required for tumor development, the overlap between TP53 deletions and mutations is poorly understood in DLBCLs, suggesting the possible existence of additional tumor suppressor genes in 17p. Using a bacterial artificial chromosome (BAC) and Phage 1 artificial chromosome (PAC) contig, we here define a minimally deleted region in DLBCLs encompassing approximately 0.8 MB telomeric to the TP53 locus. This genomic region harbors the tumor suppressor Hypermethylated in Cancer 1 (HIC1). Methylation-specific PCR demonstrated hypermethylation of HIC1 exon 1a in a substantial subset of DLBCLs, which is accompanied by simultaneous HIC1 deletion of the second allele in 90% of cases. In contrast, HIC1 inactivation by hypermethylation was rarely encountered in DLBCLs without concomitant loss of the second allele. DLBCL patients with complete inactivation of both HIC1 and TP53 may be characterized by an even inferior clinical course than patients with inactivation of TP53 alone, suggesting a functional cooperation between these two proteins. These findings strongly imply HIC1 as a novel tumor suppressor in a subset of DLBCLs.

Analysis of transactivation capability and conformation of p53 temperature-dependent mutants and their reactivation by amifostine in yeast.

The p53 gene is often mutated during cancer development. Frequency and functional consequences of these mutations vary in different tumor types. We analysed conformation and temperature dependency of 23 partially inactivating temperature-dependent (td) p53 mutants derived from various human tumors in yeast. We found considerable differences in transactivation capabilities and discriminative character of various p53 mutants. No correlations in transactivation rates and conformations of the td p53 proteins were detected. Amifostine-induced p53 reactivation occurred only in 13 of 23 td mutants, and this effect was temperature dependent and responsive element specific. The most of the p53 mutations (10/13) reactivated by amifostine were located in the part of the p53 gene coding for hydrophobic beta-sandwich structure of the DNA-binding domain.

[Waldenström macroglobulinemia--clinical manifestations and differential diagnosis and prognosis of the disease]. / Waldenströmova makroglobulinemie: klinické projevy a diferentiální diagnostika a prognóza nemoci.

Waldenström macroglobulinemia is defined by the presence of IgM type monoclonal immunoglobulin and histological prove of lymphoplasmocytary lymphoma in the bone marrow. Clinical symptoms of the disease depend on the pressure on the bone marrow by the malignant clone with subsequent cytopenia, extramedullary infiltration and toxic manifestations of monoclonal immunoglobulin. 6q deletion and absence of translocation in the sphere of the heavy immunoglobin chain gene, which is otherwise typical for other lymphoproliferative disorders, is the typical cytogenetic abnormality. The text describes the symptoms of the disease and the issues of its differential diagnosis.

Malignant fibrous histiocytoma of the parotid gland.

We described a rare malignant fibrous histiocytoma of the parotid gland (MFH) in a 63-year-old woman. During six months the tumour size became 10 cm in diameter with skin ulceration. The tumour was examined morphologically, by immunohistochemistry and molecular biology methods - FASAY and CGH. The histology revealed a storiform-pleomorphic type of MFH with high mitotic rate. The FASAY method identified a non-mutated p53 gene. The chromosomal changes were identified by the CGH method and 6 cytogenetic changes were found in the tumour cells (deletions at 8p12-p22, 13q32-qter, 14q24-qter, and gains of chromosomal material at 5p, 8q12-q23, and Xq25-qter). The patient died shortly after the beginning of chemotherapy. Autopsy revealed brain and cerebellar haemorrhage. No other tumour foci were proved. In view of short course of disease we lack the data about the influence of the non-mutated p53 gene on the prognosis and therapy.

Discriminating functional and non-functional p53 in human tumours by p53 and MDM2 immunohistochemistry.

Mutation and/or loss of the TP53 tumour suppressor gene is the single most common genetic abnormality in human cancer. The majority of TP53 mutations lead to stabilization of the protein, so that immunohistochemical staining for p53 can suggest mutation status in many cases. However, various false-positive and false-negative situations mean that simple immunostaining for p53 is not informative in a substantial number of tumours. In the present study, a series of 119 human cancers were immunostained using a highly sensitive technique that detects the low levels of wild-type protein expressed in normal cells, such that homozygous gene deletion or non-sense TP53 mutation can be identified by an absence of staining. TP53 gene status was also assessed using FASAY as a genetic/functional screen and in selected cases by direct sequencing. A quantitative scoring system was employed to assess p53 levels, and p53 post-translational modification was evaluated using antibodies that detect specific phosphorylation sites. Phosphorylated p53 correlated with total p53 levels and did not improve the prediction of TP53 mutation status. The transcriptional activity of TP53 was determined by staining for two downstream target genes, p21(WAF1) and MDM2, and statistical correlations between MDM2/p21(WAF1) and p53 were found in tumours with wild-type, but not mutant TP53. Measurement of staining for p53 and MDM2 accurately identifies the TP53 status of tumours. This simple and cost-effective method, applicable to automated staining and quantitation methods, improves the identification of TP53 status over standard methods for p53 immunostaining and provides information about tumour p53 phenotype that is complementary to genotyping data.

c-Fos but not v-Fos protein induces programmed cell death of v-myb-transformed monoblasts.

c-Fos and v-Fos belong to a group of proteins forming the transcription factor AP-1 that is important for regulation of proliferation, differentiation and programmed cell death in multiple cell types. In this study, we examined the role of c-Fos and v-Fos proteins in v-myb-transformed BM2 monoblasts. We show that while the v-Fos protein prolongs the G0G1 phase of the BM2 cell cycle, c-Fos leaves the cell cycle unaffected and, rather, induces programmed cell death. The apoptosis-promoting activity of the c-Fos protein is markedly enhanced in cells cultivated under serum-free conditions. c-Fos-induced apoptosis of BM2 cells occurred in the presence of Bcl-2 and was not dependent on the transcription activation function of the c-Fos protein. No differentiation-promoting activity of the Fos proteins was observed. The effects of Fos proteins on BM2 cells differ from those induced by Jun proteins, suggesting differential roles of individual components of the AP-1 transcription factor in regulation of essential cellular processes.

Gamma irradiation results in phosphorylation of p53 at serine-392 in human T-lymphocyte leukaemia cell line MOLT-4.

Exposure of human leukaemia MOLT-4 cells to ionizing irradiation led to apoptosis, which was detected by flow cytometric analysis and degradation of the nuclear lamina. The multiple signalling pathways triggered by either membrane or DNA damage play a critical role in radiation-induced apoptosis. The response to DNA damage is typically associated with the p53 protein accumulation. In this study, we proved that the transcriptionally active p53 variant occurs in the MOLT-4 cells and its abundance alteration is triggered in the gamma-irradiated cell population concomitantly with phosphorylation at both the serine-392 and serine-15 residues. The p21 upregulation followed the p53 phosphorylation process in irradiated MOLT-4 cells.