Anogenital squamous cell carcinoma in neglected patient.

Skin squamous cell carcinomas (SCCs) are arguably the second most common carcinoma of the skin and are responsible for the majority of non-melanoma skin cancer deaths. Gynecologist treated a Caucasian 56-years old female patient for genital wart with podophyllotoxin cream. She did not achieve complete response and therefore she has interrupted the therapy and the collaboration with the gynecologist. At the time of evaluation the lesion had a size of man's palm in anogenital region and showed characteristic features of neoplasm. The regional lymph nodes have produced infiltrated painful bubo. PCR analysis for HPV proved negative. Histopathology revealed well-differentiated squamous cell keratinizing carcinoma from the tumor as well as from the regional lymph node packet. Staging computed tomography scans proved negative and pelvis scans disclosed regional lymphadenopathy underlying the tumor. Palliative radiation therapy (by linear accelerator) was administered for the oversized tumor to the total TD 50.0Gy. The patient died 6 months after diagnostic assessment from cardio-respiratory failure. Staging computed tomography before her death did not disclose distinct metastases in her inner organs. Well-differentiated squamous cell keratinizing carcinoma could be growing endophytically affecting the underlying adipose tissue and musculature, with spreading into the regional lymph nodes. The rate of metastases into inner organs seems to vary according to the aggressiveness and metastatic behavior of each SCC. The case report calls for attention to the importance of collaboration among various specialists assisting in the diagnosis and management of skin neoplasm (Fig. 5, Ref. 12). Full Text in PDF www.elis.sk.

The detection of circulating tumor cells expressing E6/E7 HR-HPV oncogenes in peripheral blood in cervical cancer patients after radical hysterectomy.

The aim of this study was to establish the sensitive, specific and clinically acceptable method for detection of tumor cells (TCs) circulating in peripheral blood (PB) of cervical cancer patients without the clinically detectable risk of disease progression. The 7.5 ml of PB of healthy donor was spiked with 5 to 100 cells from SiHa or HeLa cell lines. The spiked tumor cells were collected without gradient centrifugation, by standard gradient centrifugation or by modified gradient centrifugation combined with immunomagnetic separation using EpCAM antibody with affinity for epithelial cell adhesion molecule. The number of collected TCs was determined by EpCAM-FITC-staining and their viability was detected by nested RT-PCR amplifying E6/E7 HR-HPV 16 or HR-HPV 18 oncogenes. For the technical validation of this approach the TCs separation and RT-PCRs were repeated several times. The recovery of viable TCs was reproducibly higher using modified gradient centrifugation combined with immunomagnetic separation in comparison with standard approach. The recovery of TCs in low number of spiked TCs (range from 5 - 20 TCs in 7.5 ml of PB) using modified gradient centrifugation was not reproducible. The recovery of TCs in higher number of spiked TCs (25 TCs and more in 7.5 ml of PB) was reproducible with average recovery about 50 %. The sensitivity of nested RT-PCR amplifying E6/E7 oncogenes was decisively influenced by the number of recovered TCs and the amount of cDNA introduced to RT-PCR, as well. Using this approach we were allowed to detect circulating TCs (CTCs) in cervical cancer patients without metastases, thus this procedure might become a tool to early estimation of disease progression. According to our knowledge, this is the first report describing the use of EpCAM antibody for CTCs detection in cervical cancer patients.

Genetic analysis of KRAS mutation status in metastatic colorectal cancer patients.

Colorectal carcinoma (CRC) represents a serious problem worldwide: in the Slovak republic are diagnosed about 2600 new CRC cases annually and its incidence is increasing. Colorectal cancer patients may succumb to the disease because of local recurrence or local formation of metastasis. Therefore, it is necessary to modulate therapeutic algorithm with new methods, leading to early diagnostic of CRC or changing the existing therapeutic procedures. Recent progresses have been made in understanding of EGFR pathway involved in CRC carcinogenesis, especially the role of Ras protein. Mutations in KRAS oncogene are frequently found in human cancers, particularly colorectal, pancreatic, billiary tract and lung tumors. The presence of the KRAS mutations in metastatic colorectal cancer patients correlates with lack of response to the certain epidemal growth factor receptor (EGFR) inhibitor therapies, such as Panitumumab and Cetuximab. Consequently, screening for KRAS mutations status may be used as a prognostic marker, because the CRC patients with KRAS positive tumors have a worse prognosis. The aim of our study was to establish the methods for rapid and sensitive detection of KRAS mutation status in formalin fixed paraffin embedded (FFPE) tissues DNA. We applied Real Time PCR analysis (TheraScreen KRAS Mutation Test Kit) and sequencing analysis (optimised for the analysis of FFPE tissues) to detect somatic mutations in codon 12 and 13 of KRAS gene. Both methods were used concurrently in the panel of DNA isolated from 25 colorectal FFPE tissues tumor. The positive or negative results from all 25 samples were identified by both methods independently. The KRAS mutations were presented in 8 of 25 patients (32%). Our results demonstrate that the Real Time PCR analysis can be used for detection of somatic KRAS mutations in FFPE clinical samples. However, we also recognize that the sequencing analysis of approximately 200bp amplicons may be used for mutations status screening, but with care of method sensitivity.

The spectrum and incidence of BRCA1 pathogenic mutations in Slovak breast/ovarian cancer families.

Pathogenic germline mutations in BRCA1 and BRCA2 account for the majority of hereditary breast/ovarian cancer cases. The analysis of BRCA1 gene was carried out in 156 breast/ovarian cancer families: 82 families with strong family history and 59 families with medium family history. Generally, 31 families and 71 cases with BRCA1 pathologic mutations (14 different types) were identified in this study by combination of SSCP and direct sequencing techniques. Using approved systematic nomenclature numbering, c.5266dupC (8 families, 21 cases), c.181T>G (5 families, 11 cases), c.68_69delAG (3 families, 5 samples) and c.843_846del4 (3 families, 4 samples) were the most frequently found mutations in BRCA1 gene. Altogether these 4 mutations accounted for 61.3% of all detected pathogenic mutations in BRCA1. One novel mutation c.1166delG was detected in one family (4 cases). Frame-shift mutations were found in 21 families (46 cases), nonsense mutations in 4 families (8 cases) and missense mutations in 6 families (17 cases). Even though the 4 most frequent mutations account for 61.3% of all detected pathogenic mutations, screening of the whole BRCA1 coding region is necessary, due to the large scale of low frequency disease causing mutations in breast/ovarian cancer families in Slovakia.

New approach to human high-risk papillomavirus (HR-HPV) genotyping.

Human high-risk papillomaviruses (HR-HPVs) are involved in the induction of invasive cervical cancer. The aim of this study was to introduce a simple, semi-automated and reproducible approach suitable for HR-HPV detection in clinical practice. The procedure is based on DNA isolation, nested polymerase chain reaction, single strand conformational polymorphism and evaluation of HR-HPV genotypes with Gel-Pro software. The clinical performance of the new approach was assessed in two different patient materials: 1) cervical smears with cytological classification Pap2-3 or Pap3 lacking nuclear atypia (anisonucleosis and polychromasia) or koilocytotic atypia and without any previous therapy 2) formalin-fixed, paraffin-embedded cervical carcinoma and lymph node sections. Using the new approach we detected HR-HPV DNA in 64% patient samples cytologically classified as Pap2-3 or Pap3 respectively and in 80% formalin-fixed, paraffin-embedded lymph node sections histologically classified as lymph nodes without carcinoma cell infiltration. The combination of methods described in this study results in increased sensitivity of HR-HPV identification allowing detection of HPV DNA in a very small amount of target DNA so that it can be widely used in distinguishing the pre- malignant lesions and in determination of invading carcinoma cells to lymph nodes in patients with advanced cervical cancer. The new approach is useful in unambiguous HR-HPV genotyping even in double-HPV infection.

c-Ha-ras BamHI RFLP in human urothelial tumors and point mutations in hot codons.

High-molecular-weight DNAs from 30 bladder and renal cell carcinomas (RCC) were isolated and the c-Ha-ras gene BamHI RFLP was examined. Amplification of c-Ha-ras with normal localization with regard to the size of alleles was found only in one case. One of the normally localized c-Ha-ras allele termed RCC c-Ha-ras of a length of about 6.6 kbp was cloned and an oncogene-activating point mutation was identified using two restriction enzymes. After comparison of CfrI and Cfr10I cleavage maps of RCC c-Ha-ras to complete nucleotide sequences of EJ/T24 c-Ha-ras oncogene and its normal counterpart, a point mutation was identified within codon 11 or 12. The use of CfrI and Cfr10I is of value for clinical practice in identification of point mutations in c-Ha-ras PCR product in neoplasia accompanied by somatic mutation of c-Ha-ras. The correlation among c-Ha-ras allele, amplification/loss, presence of point mutation and progression of neoplasia is discussed.

Molecular and biological properties of hamster tumor cell lines transformed with B77 virus: expression of v-src does not correlate with metastatic potential.

Three hamster tumor cell lines (B77Hep, B77H1De and ML cl 3.1) were investigated with the aim to determine some biological and molecular properties of cells which are connected with invasive and metastatic ability. Except B77H1De all cell lines exhibited high metastatic capacity in syngeneic adult animals. Analysis of cell lines revealed a relationship between metastatic ability and growth properties (growth rate, saturation density and colony formation in soft agar). Southern blot analysis of genomic DNA samples from high metastatic cell line (B77Hep) and low metastatic (B77H1De) showed that the metastatic potential of these cell lines did not depend on the number of integrated proviral copies. Northern blot analysis was used to determine the level of mRNA encoded by v-src gene in B77Hep and B77H1De cell lines. We found a good correlation between the number of integrated proviral copies and the level of v-src gene expression in investigated cell lines, but not with their metastatic potential. No proviral sequences were found in genomic DNA isolated from ML cl 3.1 cell line. In cell lines used in this study we found differences in expression of endogenous proto-oncogenes c-myc and c-fos.

Identification and expression of human c-Ha-ras and c-sis sequences in NIH3T3 transformants.

High-molecular-weight DNAs from 5 bladder carcinomas were used in transfection of mouse NIH3T3 cells. The manifestation of heterologous oncogene(s) expression in NIH3T3 cells was morphological transformation very often accompanied by changes in growth characteristics of recipient cells. In DNA samples from secondary NIH3T3 transformants human c-Ha-ras and c-sis sequences were identified. In some secondary transformants these sequences were expressed. On the basis of change of the growth characteristics of some secondary transformants we could expect the integration and expression of another human gene(s) for growth factor or growth factor receptor or even activation of mouse genes. We did not manage to identify any Alu sequences in some secondary transformants carrying human c-Ha-ras sequences. On the other hand, it has not been revealed yet that BamHI DNA fragments carrying c-Ha-ras gene contained any Alu sequence. So, the identification of Alu sequences does not have to be the first step in investigation of DNA samples from NIH3T3 transformants.

[Detection of cellular proto-oncogenes and oncogenes]. / Detekcia bunkových proto-onkogénov a onkogénov.

Detection of proto-oncogenes in normal cells and oncogenes in cells of solid tumors and in leukemic cells confirmed the assumption on the genetic basis of neoplastic cell transformation. Proto-oncogenes constitute a specific group of genes involved in physiological processes of the cell. Inappropriately expressed forms of proto-oncogenes are referred to as oncogenes. Proto-oncogenes and oncogenes were detected either by means of animal oncogenic viruses, in in vitro transfection experiments, or by means of some cytological methods revealing chromosomal abnormalities. Proto-oncogenes were detected in cells of phylogenetically very distant species, suggesting their importance in the regulation of the cell cycle.

Restricted replication of the E5"14" clone of Langat TP21 virus (from the tick-borne encephalitis complex) in CNS of subadult mice.

Subadult ICR mice were infected with the low virulent Langat virus TP21 E5 strain clone "14" belonging to the tick-borne encephalitis (TBE) complex by subcutaneous (s.c.) and intracerebral (i.c.) routes. From 5 to 6 days post infection (p.i.), no virus was detected in cultured brain fragments of mice, which received 10(6) ic LD50 into interscapular area. Acute lethal encephalitis with lesions confined to the vicinity of the inoculation area (parietal cortex, basal ganglia, thalamus) has developed in all mice, which received greater than or equal to 3 PFU of the virus by i.c. route. However, no virus was recovered from the cultured fragments of brain stem and cerebellum of these animals, although direct isolation attempts were regularly positive from brain cortex and basal ganglia. Survivors, which did not succumb to i.c. administration of approximately equal to 1 ic LD50 (0.3 PFU) of the attenuated Langat strain were autopsied between 53-74 days p.i. Attempts to isolate the virus from cultured fragments of brain cortex and basal ganglia remained negative despite of the presence of focal residual histological lesions in g. hippocampi in 15% of of animals examined.