D. Baličević's research while affiliated with University Clinical Hospital Center "Sestre Milosrdnice" and other places

Background/aim: Colorectal cancer is a major public health problem. The adenoma-carcinoma sequence offers potential for screening and surveillance. We tested the clinical behavior and diagnostic utility of connexin 43 (CX43) in connection with pathohistological risk. Patients and methods: Immunohistochemical expression of CX43 in colonic adenomas and surrounding mucosa from 87 patients was determined. Results: CX43 expression was higher in mucosa surrounding adenomas with high-grade dysplasia (p=0.047), larger adenomas (p=0.015) and villous adenomas (p=0.02). No difference of CX43 expression in adenomas according to grade of dysplasia was found (p=0.87). CX43 expression in adenomas was dependent on the patient's hemoglobin level (p=0.002), family history of colorectal cancer (p=0.009) and statin therapy (p=0.049). Conclusion: CX43 expression in mucosa surrounding adenoma could be an additional factor indicative of malignant potential. CX43 expression in colonic adenoma seems to be closely related to family history of colorectal cancer, statin therapy and hemoglobin level.

OBJECTIVE OF THE STUDY: Family history of hereditary colorectal cancer and common variants in the genome are recognized as a risk factor in more than 20% of colorectal cancer cases at the population level. Mutation of the APC gene is early event in colorectal carcinogenesis, and can be found in 60 to 82% of adenomatous polyps. Connexin 43 (Cx43) gene is a target for APC mutant genes. Wnt signaling pathway and beta-catenin transcription enhanced Cx43 transcription, which has the possibility of feedback regulation. Cx43 or Cx43 parts act directly on the protein transcription in the cell, and in process of colorectal carcinogenesis acts as a tumor suppressor (Picture 1). To our knowledge, Cx43 expression related to hereditary predisposition to colorectal cancer so far was not defined. METHODS AND PROCEDURES: Eighty seven patients with endoscopically resected colorectal adenomatous polyps were included in the study. In all patients two biopsies from surrounding mucosa were taken. Cx43 expression was determined by immunohistochemical staining, and was assessed according to a 2-point scale: negative, <10% Cx43 positive cells; positive, ≥10% Cx43 positive cells. Cx43 expression was correlated with family history of colon cancer, and polyps characteristic. RESULTS: Cx43 expression was positive in all adenomas of the patients who have a positive family history of colon cancer among first-degree relatives (15/15, 100%), regardless of endoscopic and histopathological features of adenomas. In patients without family history for colon cancer, Cx43 was positive in 65.3% of adenomatous polyps (47/72), which was statistically significant (p=0.007)(Picture 2). Difference of Cx43 expression in colonic mucosa in corellation to family history of colon cancer was not found. CONCLUSIONS BASED ON THE RESULTS: Our data suggests connection of the Cx43 expression in colorectal adenomatous polyps and patients positive family history of colon cancer among first-degree relatives. These completely new data make the significance of the Cx43 as a tumor suppressor in the process of colorectal carcinogenesis even more interesting. This role may be independent from the APC gene function. Given the relatively small number of patients and the fact that we were not measuring APC expression in adenomatous polyps, this results require further research. 1. Kanczuga-Koda L, Sulkowski S, Koda M, Sobaniec-Lotowska M, Sulkowska M. Expression of connexins 26, 32 and 43 in the human colon--an immunohistochemical study. Folia Histochem Cytobiol. 2004;42(4):203-7. 2. Kanczuga-Koda L, Koda M, Sulkovski S, Wincewicz A, Zalewski B, Sulkowska M. Gradual Loss of Functional Gap Junction within Progression of Colorectal Cancer – A Shift from Membranous Cx32 and Cx43 Expression to Cytoplasmic Pattern During Colorectal Carcinogenesis. In vivo 2010;24:101-108. 3. Biscanin A, Ljubicic N, Pavic I, Balicevic D, Pavic T, Budimir I, Troskot B. Connexin 43 Expression in patients with colorectal ademoatous polyps (Abstract). UEG Journal 2013;1:Suplement 1:231-2. 4. Han Y, Zhang PJ, Chen T, Yum WS, Pasha T, Furth EE. Connexin43 Expression Increases in the Epithelium and Stroma along the Colonic Neoplastic Progression Pathway: Implications for Its Oncogenic Role. Gastroenterol Res Pract 2011;10:1155. 5 Sirnes S, Bruun J, Kolberg M, i sur. Connexin43 acts as a colorectal cancer tumor suppressor and predicts disease outcome. Int J Cancer 2012;131:570-81.

To assess the frequency and forms of pulmonary tuberculosis at autopsy in a high-traffic hospital in the capital city of a country with a low tuberculosis incidence. We performed a retrospective search of autopsy data from the period 2000 to 2009 at Sestre Milosrdnice University Hospital Center, Zagreb, Croatia. We also examined patients' records and histological slides. Of 3479 autopsies, we identified 61 tuberculosis cases, corresponding to a frequency of 1.8%. Active tuberculosis was found in 33 cases (54%), 23 of which (70%) were male. Of the 33 active cases, 25 (76%) were clinically unrecognized and 19 (76%) of these were male. Clinically undiagnosed tuberculosis accounted for a substantial proportion of active tuberculosis cases diagnosed at autopsy. Autopsy data may be an important complement to epidemiological data on tuberculosis frequency.

Aim To assess the frequency and forms of pulmonary tu-berculosis at autopsy in a high-traffic hospital in the capital city of a country with a low tuberculosis incidence. Methods We performed a retrospective search of autopsy data from the period 2000 to 2009 at Sestre Milosrdnice University Hospital Center, Zagreb, Croatia. We also exam-ined patients' records and histological slides. Results Of 3479 autopsies, we identified 61 tuberculosis cases, corresponding to a frequency of 1.8%. Active tu-berculosis was found in 33 cases (54%), 23 of which (70%) were male. Of the 33 active cases, 25 (76%) were clinically unrecognized and 19 (76%) of these were male. Conclusion Clinically undiagnosed tuberculosis account-ed for a substantial proportion of active tuberculosis cases diagnosed at autopsy. Autopsy data may be an important complement to epidemiological data on tuberculosis fre-quency.

Liver disease associated with diabetes mellitus (DM) is common and usually takes the form of simple steatosis or nonalcoholic steatohepatitis (NASH). Harrison et al in 2006 described changes in liver biopsies in insulin-dependent diabetes mellitus, characterized by dense perisinusoidal fibrosis not necessarily located in a zonal pattern, thickening of hepatic arteriole in concentric layers of basement membrane and lacking other features commonly seen in NASH such as steatosis and hepatocellular ballooning, called it diabetic hepatosclerosis (DHS). Patients with long-standing diabetes mellitus who underwent liver biopsy can show rare form of nonalcoholoc fatty liver disease. We present typical changes of DHS in 40-year old male patient who underwent liver biopsy to evaluate fatty changes in liver. The laboratory findings showed slightly elevated liver transaminases, alkaline phosphatase was elevated and oral glucose tolerance test showed slightly increased levels. Mild increase of kreatinin was found and body mass index was not increased. Family and patient history of DM was negative, and patient denied alcohol abuse. He underwent liver biopsy. Hematoxylin-eosin and trichrome staining (Mallory, Orcein) slides of liver biopsy were evaluated for the degree of fatty liver changes. We did not find any fatty liver change and hepatocellular ballooning, neither. Liver biopsy slides showed non zonal dense perisinusoidal fibrosis, slight perivenular fibrosis and marked thickening of branches of hepatic artery in concentric layer fashion. DHS diagnosis was taken into observation but without any confirmative diagnosis from clinicians. DHS may represent a hepatic form of microvascular disease in DM; the prevalence of 12% in the autopsy series suggests it is not uncommon, but in the majority of the cases, it was clinically silent and can represent difficulties when analyzing liver biopsies.

Alport syndrome (AS) was first described in 1927 as hereditary disorded characterized by a combination of nephritis and deafness (1). AS is basement membrane disease affecting approximately 1 in every 5,000 people maifested with juvenile to adult onset progressive glomerulonephritis (2). Two forms have been recognized; dominant X-linked and recesive autosomal form that comprimised about 15% of all cases of AS. Renal transplantation has proven to be the best treatment for patients with end-stage renal disease. It is generally successful in patient with AS and therapy with cyclosporin-A (CsA) has been shown to be effective at halting the progression of renal failure. Gingival enlargement, occuring in a range of 25-81% of the patients, is a severe pathology that interferes with normal oral functions such as mastication and speech, and causes profound psychological problems to the patients (3). Periodical examination of the oral tissue are consequent for these patients with a low quality of life. A 35-year old male with AS was addmited at Department of Periodontology complaining of tumor-like bump on gums above the upper left molar developing in last 6 months. The simptoms of AS were identified at the age of 11 years. Genetic testing confirmed AS (Syndrome Epstein subtype with macrothrombocytopenia; a de novo mutation, recesive form). 10 years ago he had a kidney transplantation (allotransplant, mother) due to renal insufficiency. Since then he is continuously taking corticosteroids and CsA. Clinical examination revealed generalised hyperplasia of the papillae and soft tissue overgrowth of the attached gingiva in pedicle like shape at the upper left second molar measuring 10x10 mm (Figure 1). Gingival overgrowth created pseudopockets. The mechanical therapy was performed and detailed instructions in oral hygiene were given. In addition, chlorhexidine gluconate 0.2 % solution and azithromycin were prescribed. Six week later control exam revealed significantly reduced hyperplasia of the papillae on molars and premolars. Tumor-like bump atached to gingiva retained the size was surgicaly removed and sent for histopathological analysis (Figure 2). Histopathological analysis revealed epitehlial hyperplasia and fibrosis observed under light microscopy (Figure 3). Pathologist conected these changes to the effect of CsA on gingival tissue and recomended continous follow up. Conclusion Gingival overgrowth in Alport syndrome caused by the immunosupressive drugs can be successfully treated using debridement and surgical removal, supported by meticulous plaque control. Maintance of good oral hygiene with regular visits to the dentist can limit gingival hyperplasia. Gingival biopsy easily done under local anesthesia is less risky than renal and skin biopsies and tissue specimens that obtained from the intraoral region do not cause esthetic problems.

Lichen planus (LP) is an inflammatory condition of the skin but can also affect the mouth and genital region. Oral lichen planus (OLP) may occur on its own or in combination with LP of the skin or genitals. It is thought to affect 1 to 2% of the population. Typically it affects middle-aged and elderly women although it can affect men, and rarely children. The cause of OLP is not known in most instances, but it is assumed that the immune system is responsible for the majority of idiopathic OLP. Occasionally, it can be caused by certain medicines and materials in dental fillings. In most patients oral lichen planus is not serious, but up till now OLP is not curable. It can be treated to reduce symptoms. There is about a 1% risk to cancerous change over a 10-year period. The problem exists in identifying lesions that will actually transform into cancer. Tumor markers are used to identify cancer, but in some instances it can suggest potentially malignant lesion. We tried to evaluate OLP lesions by immunohistochemistry markers (epidermal growth factor receptor (EGFR), bcl-2, Ki67) comparing it to subepithelial band inflammatory infiltrate. OLP patients were divided into smoking and nonsmoking groups. Histologically there was no difference in these two groups. We found that the mean age at which OLP was diagnosed is lower in OLP patient with smoking habits (48.7±10.6) versus mean age of nonsmoking OLP patients (55.8±11.5). Spearman’s correlation test for immunohistochemical EGFR expression in two groups did not show any difference (p=0.4), but comparing EGFR expression in relation to subepithelial inflammatory infiltrate we found strong difference (p<0.01). Testing two populations (smoking and nonsmoking group), we found that immunohistochemical expression of EGFR, bcl-2 and Ki67 in squamous epithelium in relation to subepithelial inflammatory infiltrate shows very strong difference in OLP smoking group patients (p=0.001). The importance of maintaining a high standard of oral hygiene has already been emphasized. Some tooth pastes may aggravate oral lichen planus, in which case dentist may suggest an alternative. Avoiding spicy, acidic or salty foods is advisable. In view of the small risk of cancerous change in OLP, it is important that patient are ensure that their mouth is checked on a regular basis by a dentist or oral specialist, so that any early changes can be spotted. Smoking and excessive amounts of alcohol are previously established main risk factors for mouth cancer. In conclusion, we can not fail to notice; that the younger age at which diagnosis of OLP in a smoking group was established is one additional risk factor for carcinoma development in next ten years. These findings implicate the possibility of cancer development in younger age than it is statistically reported for oral carcinoma.