Granular Cell Tumour in a California Kingsnake (Lampropeltis californiae).

A 21-year-old female California kingsnake (Lampropeltis californiae) was presented to the University of Veterinary Medicine, Vienna, Austria, with a space-occupying mass in the caudal abdomen. Following clinical, radiological and sonographical evaluation the mass was removed surgically. Histopathology and transmission electron microscopy confirmed the diagnosis of a granular cell tumour, but immunohistochemical labelling for a range of markers was negative. This lesion is rare in mammals and birds, but has not been reported previously in a reptile.

Rapid screening test of most frequent BRCA1/BRCA2 pathogenic variants in the NGS era.

The average risk of breast cancer in general Slovak population of women is 4-5% and the risk of ovarian cancer is 2%. Probability of breast/ovarian cancer development is higher in individuals carrying a causative germline DNA variant in BRCA1 or BRCA2 gene responsible for hereditary breast/ovarian cancer (HBOC). Although a major proportion of inherited breast/ovarian cancers are due to the mentioned causal mutations, a number of new genes have emerged. Here we describe a rapid, multiplex and comprehensive approach for the detection of pathogenic variants in BRCA1 and BRCA2 genes which most frequently occur in Slovak HBOC population. Analysis comprises the combination of mutation specific methods. Fluorescent PCR amplification followed by fragment analysis for detection of insertions/deletions in exon 11 of BRCA1 gene. Second method is SNaPshot analysis for detection of the most frequent missense and ins/del variants in exons 2, 5, 13, 20 of BRCA1 and exons 11, 23 and 25 of BRCA2 gene. Altogether, we have analyzed 687 samples, 86 (12.5%) in group 1, which fulfilled indication criteria based on the positive family/personal history. Group 2 involved 601 (87.5%) cases, who did not meet the indication criteria and only the screening test was recommended. Using the combined approach, we have identified 47 mutated samples (6.8%), 40 in group 1 (46.5%) and 7 in group 2 (1.1%). However, the presented screening test would not provide complex results of BRCA1/2 gene analysis, it offers testing accessible to a broader spectrum of individuals under the threshold of indication for whole gene analysis. This approach may provide valuable information even in the NGS analysis era.

Evaluation of expression profiles of microRNAs and two target genes, FOXO3a and RUNX2, effectively supports diagnostics and therapy predictions in breast cancer.

Breast cancer (BC) including its progression into bone metastasis is a complex process involving changes in gene expression and function of both, microRNAs (miRNAs) and their target genes. Deregulation of miRNAs has been described as a crucial factor responsible for the initiation and progression of BC, and specific miRNA expression profiles have been found to be associated with particular disease states, histological tumor types, and BRCA1/2 or HER status. BRCA1 tumor suppressor is involved in DNA damage response and repair and epigenetically controls miR-155 expression and its pre-cancerous potential. MiR-155 targets 3´UTR region of multiple components of the pro-oncogenic signaling cascades, including FOXO3a tumor suppressor and RUNX2 transcription factor regulating metastatic potential in BC. We employed qRT-PCR to determine expression level and examine possible regulatory role of selected miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-21, miR-27a and miR-155) and their impact on expression modulation of FOXO3a and RUNX2 in peripheral blood mononuclear cells (PBMCs) in healthy individuals, in women carrying BRCA1 mutations with no disease manifestation, in women carrying BRCA1 mutations after tumor resection and therapy and in women with BC of unknown BRCA1 status in acute stage before tumor resection. Our results showed significant increase of miR-17, miR-19a, miR-21, miR-27, miR-155 and RUNX2 expression in PBMCs in BRCA1 patients and patients in acute stage, while FOXO3a expression was significantly decreased in these patients. MiR-18a and miR-20a expression was not affected. We propose that expressional changes reported in this study could provide significant additive information for early BC diagnosis, disease development prediction and therapy outcome monitoring.

The value of SHOX2 methylation test in peripheral blood samples used for the differential diagnosis of lung cancer and other lung disorders.

Methylation of the cytosine residues within the CpG dinucleotides plays an important role in the fundamental cellular processes, human diseases and even cancer. The DNA methylation represents a very stable sign and therefore may be used as a valuable marker for cancer screening. Epigenetic cancer biomarkers are independent of classical morphology and thus show extensive potential to overcome the limitations of cytology. Several epigenetic cancer markers have been reported to be detectable in body fluids such as bronchial aspirate, sputum, plasma and serum.Short stature homeobox gene 2 (SHOX2) encodes a homeo-domain transcription factor, which has been identified as a close homologue of the SHOX gene and both genes are involved in skeletogenesis and heart development. Methylation of SHOX2 gene has been shown to be present at high prevalence in carcinomas of lung, however may also be used to identify other tumour entities.In the presented study, we have compared suitability of two types of material associated with lung cancer for the detection of SHOX2 methylation. We have confirmed that methylation of SHOX2 gene represents reliable marker of lung malignancies. The parallel tests in the blood plasma revealed that it may represent a good alternative material for testing of the SHOX2 methylation, making the test available to patients who are unable to undergo bronchoscopy.

PTEN sequence analysis in endometrial hyperplasia and endometrial carcinoma in Slovak women.

Phosphatase and tensin homolog (PTEN) is a protein that acts as a tumor suppressor by dephosphorylating the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate. Loss of PTEN function has been implicated in the pathogenesis of a number of different tumors, particularly endometrial carcinoma (ECa). ECa is the most common neoplasia of the female genital tract. Our study evaluates an association between the morphological appearance of endometrial hyperplasia and endometrial carcinoma and the degree of PTEN alterations. A total of 45 endometrial biopsies from Slovak women were included in present study. Formalin-fixed and paraffin-embedded tissue samples with simple hyperplasia (3), complex hyperplasia (5), atypical complex hyperplasia (7), endometrioid carcinomas G1 (20) and G3 (5), and serous carcinoma (5) were evaluated for the presence of mutations in coding regions of PTEN gene, the most frequently mutated tumor suppressor gene in endometrial carcinoma. 75% of the detected mutations were clustered in exons 5 and 8. Out of the 39 mutations detected in 24 cases, 20 were frameshifts and 19 were nonsense, missense, or silent mutations. Some specimens harboured more than one mutation. The results of current study on Slovak women were compared to a previous study performed on Polish population. The two sets of results were similar.

Molecular analysis of EGFR gene in different types of tumor material from NSCLC patients.

Lung carcinoma is the most frequently occurring cancer worldwide and the Non-small Cell Lung Cancer (NSCLC) subtype represents 80% of all diagnosed cases. Epidermal growth factor receptor (EGFR) has an important dual role in NSCLC patients. On one hand, EGFR is frequently mutated in many types of tumors, which leads to deregulation of important downstream pathways including those affecting cell proliferation, differentiation and migration. On the other hand, presence of certain activating mutation leads to increased sensitivity of EGFR to tyrosine kinase inhibitors (TKIs) treatment. Detection of these mutations is essential for identification of NSCLC patients who would profit from such therapy. However, due to the nature of available tumor material and the relatively high number of mutation hot spots, such DNA analysis may be challenging and time consuming. Here we present an approach combining direct sequencing and SNaPshot assay for identification of EGFR mutations in FFPE tissues as well as in rarely analyzed cytological smears. Using this strategy on the set of 450 tested NSCLC samples; we have identified 29 activating mutations and 14 variants, which might be interesting in predicting the efficiency of TKI therapy.

[Treatment strategies of the colorectal anastomosis stricture]. / Strategie lécby striktury kolorektální anastomózy.

INTRODUCTION: The treatment of the stenoses of colorectal anastomoses represents a difficult area of colonic surgery. This is partly connected to the introduction of staplers and an increasing amount of sphincter-preserving surgeries. At our clinic, we solve the stenoses of colorectal anastomoses using a surgical rectoscope with a good effect. MATERIAL AND METHODS: We analysed retrospectively a group of 27 patients with benign stenosis of colorectal anastomosis of the medial and upper rectum who underwent surgery at our clinic in the period between January 2004 and December 2011. From the total amount of 27 patients, in 23 patients the stenosis was caused by the dehiscence of anastomosis, and in 4 patients the stenosis had a different etiology. RESULTS: In the group of 27 patients, in 14 patients the stenosis of colorectal anatomosis was solved radiologically using a high pressure balloon. Total of 13 patients underwent surgery, in 10 of them we used the surgical rectoscope and 3 patients underwent an open abdominal procedure. CONCLUSION: The first method of choice in treatment of the stenoses of the colorectal anastomoses is a balloon dilatation. If this is not successful we can use the surgical rectoscope as a minimally invasive though effective and safe method.

[The expected worldwide burden of oesophagus, stomach and colorectal cancers]. / Ocekávaná zátez nádory jícnu, zaludku a kolorekta ve svete.

According database of Globocan 2008 of total 482 thousand worldwide new esophagel cancers are reported 16.9% cases in more developed and 83.1% in less developed regions, 6.9% in EU, 2.7% in the Eastern Europe; of total 989 thousand new stomach cancers are reported 27.8% in more developed and 72.2% in less developed regions, 8.4% in EU, 7.4% in the Eastern Europe; of total 1.235 milion new colorectal cancers are reported 59% cases in more developed and 41% in less developed regions, 27% in EU, 10.5% in the Eastern Europe. Of total 59,052 all neoplasms (without skin cancers) were reported 10,439 new cases of these three diagnoses in recent Czech Cancer Registry survey, higher by 595 cases than numbers based in Globocan 2008. However, according to these data the Czech males reached the 3rd order and females the 14th order by their cumulative risk of colorectal cancer in the world. The alarming worldwide numbers of new 4.771 milion of these three diagnoses and 3,137 thousands deaths from them, expected in 2030 with a higher risk in population of less developed regions require greater international cooperation and personal responsibility for improving the life-style, which would be failed the expected statistics.

[Risk of colorectal carcinoma in a patient with ulcerative colitis]. / Riziko kolorektálního karcinomu v terénu ulcerózní kolitidy.

Colorectal cancer, in patients with ulcerative colitis, is detected in the resected tissue of approximately 5% of patients, according to the literature. In our set of 82 patients operated on between the years 2000-2009, malignancy was confirmed in 9/82 patients (11%). In two young patients, the peroperative findings showed inoperable generalized carcinoma. The greater incidence of malignity is surely associated with inconsistent dispensarization of these patients.

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.

Complex therapy of advanced colorectal carcinoma.

The present paper reports on a complex therapy of 18 patients with primary unresectable advanced carcinoma of the rectum and rectosigmoid. The results of surgery following complete chemoradiotherapy are evaluated. Radical surgery was successful in 15/18 patients. The authors describe a high incidence of postoperative complications and point out a high erudition of an oncosurgeon necessary for such intervention as well as for the indication of a patient to this extensive operation. (Tab. 2, Ref. 18.)

[Atypical portocaval anastomosis for hemorrhage in portal hypertension]. / Atypické portokavální anastomózy pro krvácení pri portální hypertenzi.

The success of endoscopy in treating esophageal varices and the later introduction of liver transplantation into the algorithm of therapy for liver failure shifted surgery of portal hypertension out of sight of hepatologists and surgeons. This decline from surgical treatment was further confirmed by introduction of TIPS into clinical practice. It is completely out of question that only liver transplantation is the causal solution of decompensated liver disease and a series of reliable and less invasive methods may be selected for acute treatment of bleeding from varices. However, even at the present time the portal-systemic shunt may be used in its own indication in repeatedly bleeding patients with a good liver capacity, where it can play a role by bridging the time to liver transplantation in a way similar to TIPS or even to provide a final solution, which makes it possible to live the life expectancy in adequate comfort without the risk of bleeding complications. However, it is not always possible to place a surgical shunt on some of the main branches of the portal vein. In such cases, devascularization is often successfully applied. Atypical shunts represent an exceptional alternative, because side feeder veins of the portal vein are rarely of sufficient caliber for placing a hemodynamically significant shunt. The authors describe two cases, in which stubborn anemia-causing bleeding events in portal hypertension were treated with left-side epiploic-renal shunt or anastomosis between the mesenteric and left-side iliac vein.

[Mini-invasive treatment of purulent spondylitis associated with a psoas abscess in a child (case report)]. / Miniinvazívne osetrenie purulentnej spondylitídy s psoas abscesom u diet'at'a (kazuistika).

The authors describe a case of a 12 years old boy who suffered from pain in the lumbar region, limitation of dynamics in lumbosacral spine with a gradual deterioration of the condition. The patient was afebrile. The pain was getting worse and the classic radiographs did not show at the beginning any pathological changes. Suspected were mainly the discogenic syndrome, functional vertebrogenous syndrome but also development of a tumour in the region of spine. CT scan revealed structural changes in the region of L3 vertebra and less in the L4 region. At the same time it showed a soft mass pushing the psoas muscle to the left which was described as paravertebral abscess and the structural changes as a suspect L3, L4 spondylitis. The condition of the patient was associated with a high risk of endocarditis as in the childhood he underwent the Senning operation for coarctation of great vessels. Therefore it was necessary to determine etiological agent as quickly as possible. After a mini-invasive surgery with a probational biopsy and subsequent drainage the spondylytis was confirmed also histologically and a coagulation-negative Staphylococcus aureus was cultivated as an etiological agent. At the same time tumour development was eliminated. After 15 weeks the antibiotic therapy was changed as after the removal of drains the microbe was already resistant to the administered antibiotics and therefore the antibiotics were changed with regard to sensitivity and minimum inhibition concentrations. Within 15 weeks after a local drainage by antibiotics and the total antibiotic therapy also inflammation markers gradually decreased and the patient is for a long period without complaints with full range of motion in the lumbosacral spine. In the conclusion the authors emphasize the accurate timing algorithm of the diagnosis in children which may initiate an early and successful treatment.

[Long-term follow-up of patients with extensive ulcerous colitis with emphasis on the occurrence of dysplastic changes and colorectal carcinoma]. / Dlouhodobé sledování nemocných s extenzivním typem ulcerózní kolitidy se zamerením na výskyt dysplastických zmen a kolorektálního karcinomu.

The chronic course of idiopathic intestinal inflammations is characterized also by a relatively high incidence of complications, local ones affecting the entire large intestine, as well as extraintestinal ones in remote organs. In the submitted paper the authors present the results of a six-year follow-up focused on the incidence of dysplastic changes of the intestinal mucosa and colorectal carcinoma in 46 patients from a group of 220 patients followed up on a long-term basis at the Second Medical Clinic of the Faculty Hospital and Medical Faculty Olomouc because of the extensive type of ulcerative colitis. The incidence of these complications is not high in our group and is only slightly higher than in the general population. As it is however a very serious disease and so far an optimal algorithm for its follow up has not been elaborated, we feel it is useful to investigate this high risk group of patients after 6-24-month intervals according to the confirmed grade of dysplasia of the intestinal mucosa.

Radiotherapy of early stages Hodgkin's disease. 10 years experience of the Masaryk Memorial Cancer Institute.

Radiotherapy and chemotherapy, alone or in combination, are curative treatment methods in early stages of Hodgkin's disease (HD). The choice of treatment depends on the stage of the disease, histological type and localization of the tumor, as well as on other prognostic factors. A retrospective study was conducted including 145 patients with clinical Stages I and II of HD according to Ann Arbor classification, all treated in the Masaryk Memorial Cancer Institute in Brno during the years 1985 through 1994. 80 patients were males (55%) and 65 patients females (45%). The age of the patients ranged from 11 to 77 years, with an average of 34.8 years. 41 patients were diagnosed with Stage IA tumor, 1 patient with Stage IB, 75 patients with Stage IIA and 28 with Stage IIB disease. The histological types of the disease were lymphocyte predominant in 23 patients, nodular sclerosis in 49 patients, mixed cellularity in 65 cases and lymphocyte depletion in 8 cases. 91 patients were treated with radiotherapy alone. In this group 14 patients relapsed within the radiation field (15%) and 25 outside the radiation field (28%). 39 patients were treated with combination of radiotherapy and chemotherapy. In this group relapse occurred within the radiation field in 3 patients (8%) and outside the radiation field in 7 patients (18%). 15 patients were given chemotherapy alone, 7 patients from this group experienced a relapse. The five-year survival was 81% in patients with Stages IA and IIA disease, 65% in Stages IB and IIB disease. The five-year survival in the patients who relapsed was 56%. Radiotherapy remains the curative method of choice in highly selected group of patients with early stages of Hodgkin's disease. The results of radiotherapy alone are unsatisfactory in unselected clinical Stage I--II patients because of the presence of patients with adverse prognostic factors, particularly B symptomatology, mixed cellularity/lymphocyte depletion histology, higher age. These patients are candidates for combined treatment. Modern equipment and meticulous treatment are conditions crucial for the outcome of curative radiotherapy in patients with Hodgkin's disease. Combination chemotherapy is very effective in the treatment of relapse following the primary radiotherapy.

[Surgical treatment of a complication of Crohn's disease of the duodenum]. / Chirurgická lécba komplikací Crohnovy choroby duodena.

In 1995-1997 at the First Surgical Clinic, Medical Faculty, Palacký University and Faculty Hospital Olomouc three patients were treated on account of obstruction of the pyloroduodenal region caused by Crohn's disease. The condition was treated by anastomosis, possibly with vagotomy, depending on the site of stenosis. Clinical, laboratory and endoscopic control examinations after a 6-month to 3-year interval revealed good results of this approach.

[Initial experience with treatment of choledocholithiasis using shock waves]. / První zkusenosti s lécbou choledocholitiázy rázovou vlnou.

From March 1996 till August 1997 at the First Surgical Clinic in Olomouc 18 patients with concrements in the biliary pathways were treated by shock waves. All patients were older than 68 years; residual choledocholithiasis was involved which could not be eliminated during ERCP because of the size of the size of the stone. In 125 instances treatment was successfully terminated, i.e. by crushing the stones and their elimination. Only in three patients it was necessary to resolve the situation by surgery. The authors confirmed that it is possible to treat by this method patients with choledocholithiasis and avoid the risk of surgery in particular in older patients.