Home page of 14th Symposium...
Main topic: 
Dermatopathology
14th Ljudevit Jurak International Symposium on
Comparative Pathology
Zagreb, Croatia
June 6-7, 2003
juraks@kbsm.hr

BIOMARKERS IN MELANOMA. WHAT’S NEW?

Ruiter J D, van Muijen N P G

Department of Pathology, University Medical Center St. Radboud, Nijmegen, Netherlands

Biomarkers in tumors can be divided into differentiation (“lineage”) markers, progression (“stage”) markers and other markers. They consist of DNA, RNA or protein and their detection in tumor material or body fluids may contribute to the assessment of diagnosis and/or prognosis. In melanoma several melanocytic differentiation proteins that are currently used as immunohistochemical markers in diagnostic histopathology and as a target for vaccination purposes have been described. Furthermore, a large number of progression markers that show a preference for one or a few stages of melanoma development have been identified, but most of them lack prognostic information independent of the current dominant prognostic parameters. However, the latter parameters lose power as melanomas are diagnosed in an earlier stage now. Therefore, further research is still needed so as to identify additional progression markers, which may also reveal  new molecules involved in the pathogenesis of tumor invasion and metastasis.
Tumor progression, including metastasis, is only possible through close interaction of the tumor cells with their microenvironment. The tumor microenvironment implies the total functional and structural constellation of neoplastic and non-neoplastic cells and extracellular components, with emphasis on their functional interactions. Molecules derived from the tumor stroma may exert paracrine effects on the adjacent neoplastic cells and they may be shed into the circulation. Based on their tissue distribution molecules derived (partially) from the tumor stroma can also be considered as a progression marker and some of these molecules (e.g. proteinases and related components, cytokines) were shown to have prognostic relevance. Therefore, they should be included in the classification of melanoma progession markers.
Using high through-put (microarray) technology on the DNA (“genomics”), RNA (“transcriptomics”), and/or protein (“proteomics”) level, a  plethora of tumors markers has been found. Pioneering microarray studies in melanoma have identified gene products that may play a role in the pathogenesis of metastasis. The potential value of RNA microarray technology and subsequent sophisticated statistical analysis in the assessment of diagnosis and prognosis has been shown recently in other types of solid tumors, e.g. non-Hodgkin lymphoma, breast cancer and small, round blue-cell tumors in children, but so far not in melanoma. Obstacles for such applications in melanoma may be the requirement for fresh tissue, the amount of tissue needed and the high costs. Therefore, robust techniques suitable for routinely fixed and paraplast embedded tissue should be developed - and  affordable - commercially available testing systems should become available in order to be able to integrate molecular pathology into diagnostic histopathology of melanocytic lesions.
<PROGRAM>

DYSPLASTIC NEVI – MORPHOLOGY AND BIOLOGICAL SIGNIFICANCE

Rudolph P

Department of Pathology, University of Kiel, Germany

The “dysplastic nevus“ was conceived as a hypothetical intermediary between benign nevus and malignant melanoma, based on the assumption that melanocytic lesions progress through sequential stages from benignity to malignancy as some epithelial tumors may do. However, the initial definition of the clinical and histological features of “dysplastic nevi” is blurred, embracing both typical nevi and early stages of melanoma. If rigorous criteria are applied, the “dysplastic nevus” emerges as a characteristic benign melanocytic tumor without signs of incipient malignant transformation. The distinction between “dysplastic” nevi and “common” nevi of junctional or compound type is unwarranted, as it probably reflects a continuum of evolutionary stages in melanocytic nevi. Hence, flat pigmented nevi might be rallied under the term “Clark’s nevus” expounding both clinical and histological attributes. Although the histomorphology of Clark’s nevi is distinctive, irradiation, trauma, special locations, and recurrence after incomplete excision may cause diagnostic problems.
The low frequency of malignant melanomas arising in Clark‘s nevi argues against a premalignant condition or a precursor lesion, the real precursor of malignant melanoma being melanoma in situ. Although Clark‘s nevi may be excised during a period of active growth the proliferative activity is low compared with malignant melanoma, and the proliferative potential appears to be limited by low or absent telomerase activity. Also, no genetic background for a stepwise progression from nevi to melanoma, e.g. in analogy to the adenoma-carcinoma sequence, could be established. The association of high numbers of nevi with an increased risk for malignant melanoma reflects systemic defects in growth regulation or insufficiencies in the immune surveillance, both of which may entail a predisposition to other, non-melanocytic tumors. Because Clark’s nevi account for the overwhelming majority of nevi in adults they cannot be considered as specific markers for melanoma risk. Instead, two recently described entities, the hybrid nevus and the white dysplastic melanocytic nevus, might identify patients at increased risk for malignant melanoma.

* Full article will be published in the next issue of Acta clinica Croatica

http://www.acta-clinica.kbsm.hr
<PROGRAM>


DIFFERENTIAL DIAGNOSIS OF INFLAMMATORY DERMATITIDES

Zelger Bernhard

Department of Dermatovenerology, University of Innsbruck, Austria

<PROGRAM>


GENODERMATOSES (INHERITED DISEASES WITH CUTANEOUS MANIFESTATIONS): MOLECULAR BIOLOGY AND DIAGNOSTICS

Jukić D M

Departments of Dermatology and Pathology, University of Pittsburgh Medical School, UPMC Health System, Shadyside, Pittsburgh, PA, USA

Genodermatoses are a variable group of inherited diseases that initially, or at least very early in life, present with cutaneous findings. They include divergent diseases, which could be divided into, for instance, disorders of pigmentation (example– albinism); disorders of keratinization (example–congenital ichthyosiform erythroderma), etc. The focus of this article is a subset of genodermatoses that are associated with increased risk of various skin neoplasms that develop either very early or later in life. Thus, this subset has often been dubbed as “inherited or heritable cancer syndromes." However, it is important to realize that not all of inherited cancer syndromes have skin manifestations, and that indeed, not all of the genodermatoses have neoplastic associations. The particular diseases this article deals with are Muir-Torre syndrome, Cowden’s disease, and Carney’s complex.

* Full article will be published in the next issue of Acta clinica Croatica

http://www.acta-clinica.kbsm.hr
<PROGRAM>

NEW ASPECTS IN JOINT AND BONE PROCESSES IN PSORIATRIC ARTRITIS (PSA)

Fassbender H G

Zentrum Für Rheuma-Pathologie, Mainz, Germany

* Full article will be published in the next issue of Acta clinica Croatica

http://www.acta-clinica.kbsm.hr

<PROGRAM>

DETECTION OF EBV, HTLV-1, HCV AND BORRELIA BURGDORFERI GENOMES IN SKIN BIOPSIES OF PATIENTS WITH MYCOSIS FUNGOIDES

Miertusova S1, Bonin S1, 2, Stanta G 1, 2

1International Centre for Genetic Engineering and Biotechnology, Area Science Park, Trieste, Italy, 2Department of Clinical, Morphological and Technical Sciences, University of Trieste Italy

Several studies have investigated the possible involvement of viral agents and other microorganisms in ethiopathogenesis of primary cutaneous lymphoma. Our aim was to detect the presence of Epstein- Barr virus (EBV), human T- cell lymphotrophic virus type I (HTLV-1) and hepatitis C virus (HCV) genomes and  Borrelia burgdorferi genome in the samples of mycosis fungoides using polymerase chain reaction/ Southern blot assay. We examined paraffin embedded tissues of skin lesions of 82 patients with mycosis fungoides compared with 33 healthy controls, which were all excisions of homolocalized naevi.
Fourteen of 82 mycosis fungoides  samples (17%) showed positivity for EBV whereas only 1 of 33 controls (3%); p= 0,0431. The incidence of HTLV-1 tax proviral sequence in RNA extracts from 82 mycosis fungoides samples was 42% (35/82), but only 18% in the group of controls (6/33); p= 0,0131. We found HCV- specific RNA in 8 of 82 (10%) cases of mycosis fungoides and in 3 of 33 controls (9%); p= 0,9129. We have detected flagellin gene, the unique conserved region of B. burgdorferi in 17 of 82 (20%) mycosis fungoides samples and in no healthy control; p< 0.0001.
These findings support the idea that EBV, HTLV-1 and Borrelia burgdorferi may be involved in  the ethiopathogenesis of myosis fungoides and indicate that HCV seems not to play a role in it.
<PROGRAM>

CULTURE OF HUMAN KERATINOCYTES AND PRODUCTION OF KERATINOCYTE GRAFTS IN VITRO

Boranić M, Zekušić Marija, Gabrilovac Jelka, Tomičić H, Vrtar Z, Kraus O, Buljat G, Fattorini I, Jakić-Razumović Jasminka

Medical Faculty Osijek, Ruđer Bošković Institute Zagreb, Traumatology Clinics Zagreb, Children’s Clinic Zagreb, Sestre milosrdnice University Hospital Zagreb, Clinical Hospital Center Zagreb

Extensive skin defects after the burn injuries are covered by skin transplants, lyophilized animal skin, artificial tissues made of biodegradable material or the skin epithelial cells (keratinocytes) cultured in vitro. The biotechnological method for the large-scale production of human keratinocytes has been discovered about twenty years ago and today is used in several specialized centers. Keratinocytes obtained from a skin biopsy of 1.5 cm2 may in three weeks yield the progeny sufficient to cover as much as 1.5 m2 which is equivalent to the whole body surface. The period required for the expansion of the autologous keratinocytes of a burned patient is bridged by temporary transplants of allogeneic skin or artificial material. The cultivation of human skin has been started in our Institute and the first cultures have been obtained. Clinical use of the cultured material is planned in collaboration with the plastic surgeons. The biotechnological process for the production of the material suitable for clinical use requires a highly skilled personnel and strict sterility of the working environment.
<PROGRAM>

EXPRESSION OF p53, bcl-2 AND GROWTH HORMONE RECEPTOR IN ACTINIC KERATOSIS AND SQUAMOUS CELL CARCINOMA OF THE SKIN

Stanimirović A, Čupić H, Bošnjak B, Tomas D, Krušlin B,  Belicza M

Ljudevit Jurak University Department of Pathology, Sestre milosrdnice University Hospital, Zagreb, Croatia

INTRODUCTION: Actinic keratosis (AK) and squamous cell carcinoma of the skin (SCC) are becoming extremely common epithelial lesions. Besides skin type and individual susceptibility, the most important causative factor is a long-term UV exposure. Variables such as time of exposure, gender, occupation, place of residence, and especially age significantly influence on prevalence and incidence of AK and SCC. AK was initially considered precancerous or premalignant skin lesion, because it presents no invasion into the dermis, in contrast to SCC. However, AK shares many similarities with SCC: causative factor (UV light), clinical and pathological properties (i.e. localization, atypical keratinocytes, …) as well as molecular abnormalities (i. e. DNA aneuploidy, loss of heterozygosity). Thus, novel investigations consider AK as an early stage in biologic continuum that leads from carcinoma in situ to invasive SCC. Although this opinion is favored by a finding that SCC often develops from AK, there is still no generally accepted agreement about this new postulation.

AIM OF THE STUDY: The relationship between activated protooncogenes and inactivated tumor suppressor genes play a crucial role in the development and progression of AK and SCC. The results of previous studies of genes p53 and bcl-2 in these two entities by imunohistochemical (IHC) techniques are controversial. Recent studies confirmed that growth hormone (GH) is involved in the development of different types of human neoplasms. In order to compare and try to elucidate relationship between AK and SCC we have studied p53, bcl-2 and GH receptor (GHR) expression in AK and SCC.

PATIENTS AND METHODS: Thirty-three specimens of hypertrophic type of actinic keratosis (HAK), 25 of atrophic actinic keratosis (AAK) and 27 specimens of SCC (grade G1), taken from sun-exposed areas in group of patients of both sexes, older than 60 years of age, were investigated. Expression of p53 and bcl-2 was determined by Microwave Streptavidin ImmunoPeroxidase (MSIP) protocol on DAKO TechMateTM Horizon automated immunostainer. Immunohistochemical technique Strept-HRP with the monoclonal antibody MAb263 was used to demonstrate the expression of GHR. The relative proportion of immunoreactive cells was studied.

RESULTS: Detected pattern of p53, bcl-2 and GHR expression in HAK, AAK and SCC are presented in Tables 1-3.

DISCUSSION AND CONCLUSION: Nuclear staining for P53 protein was detected in 91% of HAK and 100% AAK specimens, as well in 89% of SCC specimens. This finding is among the highest percentages of P53-positive AK and SCC determined in up-to-date investigations. Highly increased expression of gene p53 in AK and SCC from sun-exposed areas indicates its major role in photocarcinogenesis.
Expression of BCL-2 protein was detected in all investigated (100%) AK specimens of both types. On the other hand, only 66% of investigated SCC specimens showed bcl-2 expression. Relatively decreased expression of bcl-2 in SCC -  in comparison to AK - suggests that there are certain molecular differences between these two lesions. This finding points out to possible role of other pro- and anti-apoptotic genes of BCL-2 family (i.e. bax, bad, bcl-x) in cell protection from apoptosis. There is a need for further investigations to confirm this hypothesis.
We had detected GHR positivity in 36% of HAK specimens, while 80% of AAK specimens were positive. This is the first report of GHR positivity in AK. Detected finding of significantly increased GHR expression in atrophic AK compared to hypertrophic AK, indicates that atrophic AK might have higher proliferative potential. This opinion is favored by finding of an increased GHR expression in SCC (93%).
In our investigation we have determined the great similarity in expression of investigated immunohistochemical markers (tumor suppressor gene p53, oncogene bcl-2 and GHR) between AK and SCC.  Our results support novel opinions that AK is not precancerosis but the initial stage of SCC. We suggest that further investigations of other immunohistochemical markers in different types of AK and different grades of SCC are necessary to confirm these new attitudes completely.


TABLES
 

Table 1. Expression of p53, bcl-2 and GHR in hypertrophic actinic keratosis (n=33).

IHC expression p53 bcl-2 GHR
- 3 9% 0 0% 21 64%
+ 8 24% 30 91% 4 12%
++ 8 24% 0 0% 4 12%
+++ 14 43% 3 9% 4 12%
Total positive 30 91% 33 100% 12 36%

(-) no positive cells
(+) less than 10% positive cells
(++) 10 to 25% positive cells
(+++) more than 25% positive cells

Table 2. Expression of p53, bcl-2 and GHR in atrophic actinic keratosis (n=25).

IHC expression p53 bcl-2 GHR
- 0 0% 0 0% 5 20,0%
+ 18 72,0% 22 88,0% 12 48,0%
++ 6 24,0% 3 12,0% 8 32,0%
+++ 1 4,0% 0 0% 0 0%
Total positive 25 100% 25 100% 20 80,0%

(-) no positive cells
(+) less than 10% positive cells
(++) 10 to 25% positive cells
(+++) more than 25% positive cells

Table 3. Expression of p53, bcl-2 and GHR in squamous cell carcinoma of the skin (n=27).

IHC expression p53 bcl-2 GHR
- 3 11% 9 33% 2 7%
+ 8 30% 17 63% 2 7%
++ 11 41% 1 4% 5 19%
+++ 5 18% 0 0% 18 67%
Total positive 24 89% 18 67% 25 93%

(-) no positive cells
(+) less than 10% positive cells
(++) 10 to 25% positive cells
(+++) more than 25% positive cells


REFERENCES:
Ginarte M, García-Cabarello T, Fernández-Redondo V, Beiras A, Toribio J. Expression of growth hormone receptor in benign and malignant cutaneous proliferative entities. J Cutan Pathol 2000; 27:276-82.
Lincoln DT, Sinowatz F, Temmim-Baker L, Baker HI, Kölle S, Waters MJ. Growth hormone receptor expression in the nucleus and cytoplasm of normal and neoplastic cells. Histochem Cell Biol 1998; 109:141-59.
Lober BA, Lober CW. Actinic keratosis is squamous cell carcinoma. South Med J 2000; 93:650-5.
Nagano T, Ueda M, Ichihashi M. Expression of p53 protein is an early event in ultraviolet light-induced cutaneous squamous cell carcinogenesis. Arch Dermatol 1993; 129: 1157-61.
Nakagawa K, Yamamura K, Maeda S, Ichihashi M. bcl-2 expression in epidermal keratinocytic diseases. Cancer 1994; 74: 1720-4.
Onodera H, Nakamura S, Sugai T. Cell proliferation and p53 protein expression in cutaneous epithelial neoplasms. Am J Dermatopathol 1996;18: 580-8.
Salasche SJ. Epidemiology of actinic keratoses and squamous cell carcinoma. J Am Acad Dermatol 2000;42:S4-S7
Shimizu T, Oga A, Murakami T, Muto M. Overexpression of P53 protein associated with proliferative activity and histological degree of malignancy in solar keratosis. Dermatology 1999;199: 113-8.
Tucci MG, Offidani A, Lucarini G, Simonelli L, Amati S, Cellini A, et al. Advances in the understanding of malignant transformation of keratinocytes: an immunohistochemical study. J Eur Acad Dermatol Venereol 1998;10: 118-24.
Wikonkal NM, Berg RJ, van Haselen CW, Horkay I, Remenyik E, Begany A, Hunyadi J, van Vloten WA, de Gruijl FR. bcl-2 vs. p53 expression and apoptotic rate in human nonmelanoma skin cancers. Arch Dermatol 1997;133:599-602.
<PROGRAM>


SMALL VESSEL VASCULITIS OF THE SKIN WITH IgA-POSITIVE IMMUNE DEPOSITS

Pižem J, Perković Tatjana, Jurčić Vesna, Vizjak Alenka

Institute of Pathology, Faculty of Medicine University of Ljubljana, Slovenia

AIMS: To evaluate the significance of skin vascular IgA-positive immune deposits in differentiating Henoch-Schönlein purpura (HSP) from other types of immune-complex small-vessel vasculitides.
METHODS: Among 695 skin biopsies, analysed by light and immunoflorescence microscopy techniques in the period from 2001-2002, 70 were found to have IgA-positive vascular immune deposits and were included in the study. The clinical diagnoses were vasculitis, HSP, or purpura.
RESULTS: The median age of patients was 59 years (range 8 - 80 years). Only 3 patients were younger than 16 years. Histological findings were leucocytoclastic vasculitis (43/70), perivascular mononuclear or mixed cell inflammatory infiltrate (15/70) and no changes (12/70). Granular immune deposits, demonstrated in superficial vessels in 70 cases and in deep dermal vessels in 9, stained positive for IgA and C3 in all cases, for IgM in 29, for IgG in 5, for C1q in 6, and for fibrin/fibrinogen in 68. They were IgA-dominant in 56 and IgM-dominant in 14 cases. Complement component C1q was detected in 5 IgM-dominant and in one IgA-dominant case. The deep vessels were significantly more often involved by inflammation (28/70) then by immune deposits (9/70). Leucocytoclastic vasculitis was more frequently found in the IgM-dominant group. There was a strong clinical suggestion for drug induced vasculitis in one IgM-dominant C1q-positive case and a diagnosis of mixed cryoglobulinemic vasculitis, based on serologic finding of mixed IgA, IgG and IgM cryoglobulins was established in one IgA-dominant C1q positive case.
DISCUSSION AND CONCLUSION: The majority of cases with IgA-positive skin vasculitis show predominance of IgA in vascular immune deposits and most probably, even in the older age group, represent HSP. Dominant IgM, accompanying IgA in vascular deposits, may be rarely found in late lesions of HSP and presumably represent secondary nonspecific deposition. Frequently, despite inflammation, no immune deposits can be found in the deep vessels, probably as a consequence of their clearance by inflammatory cells. The positivity for C1q, indicating the classical pathway of complement activation, can be demonstrated also in IgA-dominant vascular immune deposits and suggests other types of immune-complex vasculitis, such as cryoglobulinemic and drug induced. The clinical and serologic data as well as biopsy histologic and immunofluorescence findings should be considered in the final diagnosis of skin small vessel vasculitis.

REFERENCES:
Jennette JC, Falk RJ, Andrassy K, et al: Nomenclature of systemic vasculitides.
Proposal of an International Consensus Conference. Arthritis Rheum 1994, 37:187-192
Jennette JC, Falk R. Small-vessel vasculitis. New Engl J Med 1997; 20: 1512-1523
<PROGRAM>


GENE EXPRESION PROFILES OF HEPATOCELLULAR CARCINOMAS

Denk H

Department of Pathology, University of Graz, Austria

* Full article will be published in the next issue of Acta clinica Croatica

http://www.acta-clinica.kbsm.hr
<PROGRAM>

ZOONOTIC DERMATITIDES

Del Piero F

University of Pennsylvania, School of Veterinary Medicine, Department of Pathobiology and Department of Clinical studies New Bolton Center, Philadelphia

Zoonoses are those diseases and infections which are naturally transmitted between vertebrate animals and man. Viruses, bacteria, fungi protozoa, helminthes and arthropods can be transmitted directly or indirectly from animals to humans. A restricted number of agents are able to cause skin disease. The etiologic agent can be passively transmitted to the human host or can be actively inoculated in the skin with bites and scratches or arthropod bite. Although all humans are at risk for these skin diseases, some job categories are associated with greater exposure. Immunodeficient human patients are at particular risk of infection by zoonotic agents and, for them, the outcome can be easily fatal. Here we described the lesions caused by zoonotic agents able to induce dermatitis in humans.
 

* Full article will be published in the next issue of Acta clinica Croatica

http://www.acta-clinica.kbsm.hr
<PROGRAM>

NEW OUTBREAK OF ENDEMIC UROPATHY IN “CRNAC POLJE” FOCUS, CROATIA: A PRELIMINARY REPORT

Božić Z1, Matijašević Maja2, Duančić V3

1Urologist, Private Practice, Zagreb, Croatia, 2General Practice Unit, Davor, Croatia, 3Department of Epidemiology, Medical Centre for Prevention and Rehabilitation of Heart Diseases, Zagreb, Croatia

The authors previously defined Panonian-Balkan endemic uropathy (PBEU) as a single rural household environmental disease of the entire urinary tract due to shared living conditions and nourishment, with imprecisely determined but usually long latency. Clinical manifestations, including chronic renal failure (CRF) due to the tubulointerstitial nephropathy, renal cell carcinoma and urotheliomas observed with a significantly higher frequency in the affected population, represent a complex, however one unique nosological entity which can be found in a single patient.
The follow-up performing in an endangered part of Croatia recently detected an unexpected outbreak of PBEU in the villages Davor and Orubica (3255 inhabitants), belonging to almost forgotten “Crnac Polje” endemic focus on the Sava river-bank. In 2002 the prevalence rate of endemic terminal stage CRF cases and CRF cases with associated endemic urotheliomas has risen to 12/3255 and 8/3255 respectively, or alternatively to crude prevalence rates of 369/100 000 and 246/100 000. The prevalence of exclusively terminal stage CRF endemic nephropaths in Davor and Orubica population (12/3255) was significantly higher (?2=17.3 ; p=0.0000) in comparison with the total prevalence of terminal stage CRF patients in the rural area of Sesvete, north-west Croatia (12/11 481). The prevalence of exclusively endemic urothelioma cases in Davor and Orubica population (8/3255) was also significantly higher (?2=19.9 ; p=0.0000) in comparison with the total prevalence of patients with urotheliomas in the rural area of Sesvete (4/11 481). Screening based on the WHO diagnostic criteria for PBEU has never been conducted in the observed villages making possible the recognition of more, still undetected cases. The latest literature estimations suggesting that “PBEU is decreasing in incidence and prevalence in Croatia” appear to be premature like all other so far.
<PROGRAM>

NEONATICIDE AND INFANTICIDE IN THE EAST CROATIA OSIJEK COUNTY

Marcikić M1, Dumenčić B1, Matuzalem Elizabeta1, Marjanović Ksenija2, Ugljarević M2

1Department of Pathology and Forensic Medicine, Medical Faculty, Osijek, Croatia and 2Department of Pathology General Hospital Vukovar, Croatia

For the lay person no crime is more difficult to comprehend than the killing of child by their own parents. This is a retrospective study of neonaticide and infanticide in the East Croatia, Osijek County from 1980 to 2002. A judicial records of infanticide cases stored in County Court were analyzed for the circumstances surrounding the offence, personal data, witness testimony, mothers` motivation, autopsy findings, and the legal procedures. Twenty four babies were discovered in various places during investigation. The victims were almost equally divided between boys (12) and girls (11). For one baby gender was unknown. The mean weight of babies were 2733 grams. Neonaticide rate were approximately 8 per 100 000 live born.  The perpetrators preferred rubbish-heap (33.4%), burying in soil (16.7%), various premises in or around house (16.7%) and garbage can (12.5%) as a place for hiding the dead baby. The most dominant cause of death in sixteen cases of live birth was mechanical asphyxia (37.5%) with equal distribution of smothering, stuffing the mouth with rags and strangulation. The other frequent causes of death were placing the child in plastic bag and abandonment (25%), brain injury (25%), and wounding by a sharp weapon (12.5%). The cause of death for six babies remained unknown due to advanced decomposition. Two babies were stillborn. The age of accused mothers varied from 16 to 33 years. Most of them were unmarried (60%) and had limited formal education. They usually kept  pregnancy a secret (73%) and gave birth (93%) out of public health service. The mother lived in terror of shame and guilt that accompany conception without marriage. A fear (60%) seemed to be a pronounced motivating factor for committing the crime.
Data on court procedure were available in fifteen cases. Mothers were officially indicted in all cases for infanticide under Croation Penal Code. The perpetrator remained unidentified in nine suspicious crimes.
Finally, the court convicted ten mothers for infanticide. Juries were often unwilling to punish the woman. This trend was a consequence of the belief that the woman who has killed her child has constructed enough guilt by the act and it is already the punishment. Nine mothers were put on probation from one to three years. Only one mother  was sentenced to  ten months of imprisonment.
A continuous  effort is needed for providing a better „sexual education”, better communication within families and more outreach by pregnancy service providers to prevent future neonaticide. A desparate young woman should be advised that there is an alternative to neonaticide in her community. Hence, more research and further attention to this problem is warranted.
<PROGRAM>

SIDE ARM HOMICIDE IN THE ITALIAN PROVINCE OF TRIESTE BETWEEN 1953 AND 2002

Nardi U, Tomasella F, Gongolo F, Peretti A, Costantinides F

Department of Science of Public Health – Forensic Pathology Unit, University of Trieste School of Medicine

The authors present a complete overview of the phenomenon of side arm homicide,  basing the study on the data collected at the Forensic Pathology Unit of the University of Trieste School of Medicine. Side arms are the most frequently used homicidal method in the town and province of Trieste in a considered study period of 50 years from 1953 to 2002.
The analysis of the collected data shows that the town and province of Trieste are communities still very well disjoined from a reality in which crimes against life are yet very uncommon occurrences. This conclusion is well supported by the fact that it has not been possible to define a category of potential victims, which is on the contrary quite easy among societies where organised crime is consistent and widespread.
The popularity of side arm as homicide method in our province supports the hypothesis that these crimes are often not aforethought murders, perpetrated by subjects often afflicted by recorded mental health problems, acting during an insanity raptus. Knives are not only actually the easiest weapons to obtain in a household, but are very easily used as well, even by the subjects not accustomed to handle weapons.

* Full article will be published in the next issue of Acta clinica Croatica

http://www.acta-clinica.kbsm.hr
<PROGRAM>

CELL PROLIFERATION AND p53 PROTEIN EXPRESSION IN PROLIFERATIVE SKIN DISEASES

Batinac Tanja1, Zamolo Gordana2, Gruber F1, Lipozenčić Jasna3, Jonjić Nives2

1Department of Dermatovenerology, Clinical Hospital Center, Rijeka, Croatia
2Department of Pathology, Medical Faculty, University of Rijeka, Rijeka Croatia
3Department of Dermatovenerology, Clinical Hospital Center, Zagreb, Croatia

AIM OF THE STUDY: The aim of this study is to elucidate the role of the p53 tumor-suppressor protein in the pathogenesis of different hyperproliferative, non-malignant and malignant skin diseases, and association between p53 overexpression and cell proliferation. We also investigated the influence of aging on p53 and Ki-67 protein expression.

MATERIAL AND METHODS: Hundred and fifty skin specimens, consisting of 30 cases of each: normal skin (NS), psoriatic skin (PS), keratoacanthomas (KA), basal cell carcinomas (BCC), squamous cell carcinomas (SCC) were examined immunohistochemicaly to assess p53 and Ki-67 protein expression.

RESULTS: p53 immunostaining of NS, PS, KA, BCC and SCC was positive in 39.0%, 46.7%, 66.7%, 80% and 86.7% cases, respectively. P53 and Ki-67 positive cells were present in basal (NS) and suprabasal layers (PS), and not only in cancer nests of KA, BCC and SCC, but also in dysplastic and even morphologically normal epidermis adjoining cancers. The positivity of p53 and Ki-67 protein differed significantly among the groups, with no differences in p53 expression between NS and PS, and in Ki-67 expression between PS and KA. Within all groups, there was a significant correlation between p53 and Ki-67 protein expression.

CONCLUSION: Our findings suggest that p53 overexpression occurs widely in neoplastic and non-neoplastic skin lesions. It is associated with the cell proliferation in the normal, as well as in the changed epithelium. P53 accumulation is an age related process and significantly related to sun exposure, especially in NS and PS, as well as in KA and SCC. We also suggest that p53 overexpression begins in the early stage of carcinogenesis, before the appearance of malignancies in skin, and may be a useful predictor for the detection of nonmelanocytic skin cancer.

REFERENCES:
1. Heenen M., Ann Dermatol Venereol 1997;124:452.
2. Liang SB, et al., Virchows Arch 1999;434:193.
3. Hannuksela-Svahn A, et al., Acta Derm Venerol 1999;79:195.
<PROGRAM>


EXPRESSION OF CATHEPSIN D AND G IN CUTANEOUS MALIGNANT MELANOMA

Lipozenčić Jasna, Jakić-Razumović Jasminka1, Batinac Tanja2, Pašić A, Šitum Mirna3, Herman S

Department of Dermatology and Venerology and 1Department of Pathology Zagreb University Hospital Center, Zagreb, Croatia; 2Department of Dermatovenerology, Clinical Hospital Center, Rijeka, Croatia; 3Department of Dermatology and Venerology , Sestre milosrdnice University Hospital , Zagreb, Croatia

AIM OF THE STUDY: To elucidate the prognostic significance of cathepsin D and G expression in malignant melanoma. Tumor invasion and metastases are associated with proteolytic action of various proteases. Increased levels of cathepsin D and G have been observed in primary and metastatic cancer types. The correlation between cathepsin D tissue concentration and aggressiveness of tumors has been detected in melanoma patients.

MATERIAL AND METHODS: The study was performed on a group of 18 patients with histopathological proven primary cutaneous malignant melanoma. There were 18 patients aged between 22 and 66 years. All tumors were thinner than 5 mm (Breslow Index from I to V) and Clark level from I to IV. No previous treatment and no evidence of infection disease 2 month before. Cathepsin D and G analysis in all 18 patients was performed by standard immunohistochemically staining using monoclonal antibodies anti-cathepsin D and anti-cathepsin G.

RESULTS: Cathepsin D was proved in all 18 tumors and macrophages and in 17 patients in surrounding epidermis. Cathepsin G was proven in 11 tumors; in macrophages in all 18 samples, but Cathepsin G in surrounding epidermis was found only in 2 patients.

CONCLUSION: Our results indicate that cathepsin D and G are expressed at high levels by melanoma cells. The extremely high expression of cathepsin D was in the three of our patients in tumors with progression of the disease over a 36-month follow-up period. It seems that there is a significant correlation between cathepsin D and cathepsin G expression with disease free interval.
<PROGRAM>


DIRECT IMMUNOFLUORESCENCE AS A DIAGNOSTIC TOOL IN DERMATOLOGY

Marinović B, Lakoš-Jukić I

Department of Dermatology and Venerology, Zagreb Unviersity Hospital Center, Zagreb, Croatia

Immunofluorescence has been used for about half a century, both to investigate the pathophysiology of skin disorders and to help physicians in the diagnosis of various cutaneous disorders, especially bullous diseases and connective tissue diseases. Direct immunofluorescence (DIF) is a histologic stain that helps detect immunoglobulins and complement components within biopsy specimens. Interpretation of DIF specimen takes into account several parameters, including site of immune deposition, class of immunoglobulin or other type of deposit, intensity of immune deposits and morphology of deposition, i.e. linear, granular, shaggy, etc.
The most common patterns of DIF findings are deposition of IgG, IgA and/or C3 found in the intercellular spaces of the epithelium (pemphigus diseases) or in the basement membrane zone (subepidermal blistering diseases, lupus erythematosus). These immunofluorescent findings are valuable in confirming a diagnosis that is suspected by clinical or histologic examination, especially in the group of subepidermal blistering diseases where clinical and histologic findings may quite frequently overlap.
<PROGRAM>

EXPRESSION OF AMINOPEPTIDASE N (APN; EC 3.4.11.2; CD13) AND NEUTRAL ENDOPEPTIDASE (NEP; EC 3.4.24.11; CD10) ON CULTURED HUMAN KERATINOCYTES

Gabrilovac Jelka1, Buza-Vidas Barbara1, Zekušić Marija1, Breljak Davorka1, Kraus O2, Boranić M1

1Ruđer Bošković Institute, Division of Molecular Medicine, Zagreb, Croatia; 2Sestre milosrdnice University Hospital Zagreb, Clinical Hospital Center Zagreb

By secreting cytokines and growth factors keratinocyte actively participate in immune response and inflammation. Recently membrane-bound aminopeptidases have been proposed as additional mechanism of cell-to-cell interaction, as well as a negative loop in controlling concentration of peptide signalling molecules. In this study we examined expression of two membrane-bound peptidases: aminopeptidase N (APN; EC 3.4.11.2; CD13) and neutral endopeptidase (NEP; EC 3.4.24.11; CD10)  on nonstimualted cultured human keratinocytes obtained from healthy skin.  Membrane expression of CD13 and CD10 at the population level was analysed by FACS and at the single cell level by means of fluorescent microscope. Functional properties of CD13 and CD10 were examined by testing their enzymatic activity in the presence of selective substrates and inhibitors. The data were compared to those obtained on cultured nonstimulated human skin fibroblasts expressing both CD13/APN and CD10/NEP. The data obtained have shown that cultured, nonstimulated keratinocytes from normal healthy skin express CD13. At the population level 31.7 % ? 2.8% of keratinocytes (n = 3) are CD13+, as compared to fibroblasts which are 100% CD13+ (n = 3). On the per cell basis, the density of CD13 is several times lower on keratinocytes than on fibroblasts. Distribution of CD13 on keratinocytes is homogenous throughout the membrane, appearing in a typical ring-like fluorescence, whereas distribution of CD13 on fibroblasts is predominantly patchy or polarized. Membrane CD13 expression on keratinocytes was associated with significant enzyme  activity, which on the basis of substrate (Ala-pNA and Ala-?NA) and inhibitor (bestatin, actinonin) selectivity could be ascribed to aminopeptidase N. APN activity on keratinocytes was several times lower than on fibroblasts, and could be abrogated to comparable level in both cell types by the APN inhibitor actinonin. In contrast to membrane CD13 associated with APN enzyme activity, neither membrane CD10, nor its enzyme (NEP) activity could be found on the same keratinocyte samples. These data suggest that detection of  CD13/APN is not likely to be due to its activation during the cell cultivation, but rather represents its constitutive expression. Presence of CD13 with catalytic activity on keratinocytes may be relevant for their communication with surrounding cells in the course of immune response and inflammation.
<PROGRAM>

IMMUNOFLUORESCENT DIAGNOSTIC OF AUTOIMMUNE  BULLOUS DISEASES

Kotrulja Lena

Sestre milosrdnice University Hospital, Department of Dermatovenereology, Zagreb

Immunofluorescence (IF) was introduced in dermatology in 1960s and has since then contributed greatly to the development  of contemporary concepts and knowledge of bullous and connective tissue disease, at the same time becoming an important diagnostic tool in routine practice. Histological finding in differential diagnosis of autoimmune bullous disease has only orientational role and IF finding is indispensable for definitive diagnosis.
Direct immunofluorescence (DIF) is  histological stain used to detect antigens such as immunoglobulins, complement and fibrin deposited in the skin utilizing fluoresceinated antibodies specific to the antigen. Indirect immunofluorescence (IIF) is a serologic technique based on the capacity of circulating antibodies to bind to the corresponding target antigen.
Apart from  the diagnostic importance, this reaction has a substantial prognostic value, particularly for pemphigus cases, since it is well known that the titers of circulating pemphigus antibodies correlate with the disease activity. Thus, IIF is performed at regular intervals as an important part of the follow-up of these patients.
In this paper we emphasize the clinical and specific DIF and IIF findings of  the most important  autoimmune bullous diseases such as pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus,  drug induced pemphigus, IgA pemphigus (intraepidermal IgA neutrophilic dermatosis and subcorneal pustular dermatosis), bullous pemphigoid, cicatricial pemphigoid, pemphigoid (herpes) gestationis, linear IgA bullous dermatosis, and epidermolysis bullosa acquisita.
We present two case reports of the patients who, with their clinical, histological and immunofluorescent (DIF and IIF) findings, present a  typical example of pemphigus foliaceus and bullous pemphigoid.
<PROGRAM>

PIGMENTED MAMMARY PAGET DISEASE IN A 65 YEAR OLD MAN - CASE REPORT

Monsef Ali Reza, Eghbalian Fatemeh

Dermatopathology Dept., Sina hospital, Hamedan University of Medical Sciences Hamedan, Iran

Mammary Paget disease results from intraductal mammary Paget carcinoma, which  extends to the epidermis of the nipple and areola through a lactiferous duct or from invasive breast carcinoma that reaches the epidermis via direct extension from the dermis. Pigmented mammary Paget disease is a rare clinicopathologic variant of mammary Paget disease. It may mime malignant melanoma both clinically and histopathologically. The involvement of the dermo-epidermal junction by neoplastic cells of the mammary Paget carcinoma seems to be a prerequisite for development of the clinical pigmentation. We report a case of pigmented mammary Paget disease. He was a 65-year-old male complaining of an erythematous, pigmented scaly plaque around his right nipple measuring 8x6 cm  with mild fissuring from two years ago. The histopathology revealed pagetoid spread of large cells in the epidermis with pigmentation of basal cells and melanocytes. The IHC confirmed the mammary Paget disease and exclude malignant melanoma and squamous cell carcinoma.
<PROGRAM>

AN INDOLENT SUBCUTANEOUS NODULE OF THE FACE? - CASE REPORT

Puizina-Ivić N, Stipić T

Department of Dermatovenerology, Clinical Hospital Split, Split, Croatia

A 67-year-old woman was admitted at the Department of Dermatology in the Clinical Hospital Split, because of one nontender and indolent nodule in the right temporal region. The nodule was first noticed two months before, it was not visible, but palpable 1,5 cm in diameter, firm, slightly movable and painless. She had no other symptoms. She was treated with corticosteroid ointments but without any improvement. Under the suspicion of atheroma, cyst or benign tumor, an excisional biopsy was done. Histologic examination revealed a transverse and longitudinal section of immature female worm of Dirofilaria with numerous eosinophils in surrounding tissue. Now it is sure that dirofilariasis exists in Croatia as well as infected dogs and its vectors.
<PROGRAM>

DERMATITIS ARTEFACTA – CASE REPORT

Gregurek-Novak T1, Novak-Bilić G 1, Vučić Majda2

University Department of Dermatology1 and “Ljudevit Jurak” University Department of Pathology2 Sestre milosrdnice University Hospital, Zagreb, Croatia

INTRODUCTION: Self-induced factitial dermatitis, or dermatitis artefacta, is a rare and difficult condition to diagnose and treat. The patient is friendly but bewildered and the relatives are angry and frustrated. Because of a lack of diagnostic stringency, quoted female-to-male ratios range from 3:1 to 20:1, with the highest incidence of onset in late adolescence to early adult life. Most patients have a personality disorder; borderline features are common. The patient's denial of psychic distress and negative feelings aroused in healthcare personnel make the management difficult. There is a variety of means that patients are using to cause the skin changes.

CASE REPORT: We report a case of 72 years old woman with characteristic symmetric changes under breast and right inguinal region. The clinical expression was a very symmetric, partly composed of hemorrhagic round lesions and partly of scars, which were «cigarette paper»-like. Patient had myocardial infarct 6 months earlier and diagnosed diabetes mellitus one year before. Routine laboratory data were within normal ranges. Special consultations with psychiatrist, internist and diabetologist excluded the possibility of side effects due to the use of medication. We made skin biopsy (inguinal lesion) and hystopathology revealed edema in the dermis and strong extravagation of red blood cell suggesting mechanical irritation on the skin. The skin lesions were covered for 24 hours in occlusion techniques. In few days under medical control, the skin lesions improved by 50 %. The patient was discharged from the hospital under psychiatric and family care.

DISCUSSION: Typical symmetric clinical findings, histology findings, consultations with psychiatric and family were proof for the diagnosis of dermatitis artefacta. The patient has many problems in family, lives alone, far away from the family and she wants to have every day care and someone to pay attention to her. Our patient was in older age group, in which dermatitis artefacta is not common. In such cases team work and cooperation of dermatologist, hystopathologist, psychiatrist and patient's family is necessary. Research studies are necessary to document more accurately the expectable cause, treatment outcome and prognosis for this group of patients.

REFERENCES:
Farrier JN, Mansel RE. Dermatitis artefacta of the breast: a series of case reports.
Eur J Surg Oncol 2002;28:189-92.
Joe EK, Li VW, Magro CM, Arndt KA. Diagnostic clues to dermatitis artefacta. Cutis 1999;63:209-14.
Koblenzer CS. Dermatitis artefacta. Clinical features and approaches to treatment. Am J Dermatol 2000;1:47-55.
Antony SJ, Mannion SM. Dermatitis artefacta revisited. Cutis 1995;55:362-4.
<PROGRAM>


GRANULOMA ANNULARE AND NECROBIOSIS LIPOIDICA; CLINICAL AND HISTOPATOLOGICAL RESEMBLANCES AND DIFFERENCES. CASE REPORT.

Meštrović-Štefekov Jelena1, Šitum Mirna1, Zarubica Jelena2, Kotrulja Lena1, Vučić Majda2

1Department of Dermatology and Venerology and 2Ljudevit Jurak University Department of Pathology, Sestre milosrdnice University Hospital, Zagreb

Two granulomatous diseases of unknown etiology - granuloma annulare and necrobiosis lipoidica are very easy to diagnose in their typical clinical forms, but in some rarer variants (or/and localization) may be difficult to distinguish.
Both diseases have been related to diabetes mellitus (about 20% of patients with disseminated form of granuloma annulare and 40% patients with necrobiosis lipoidica).
Typically, granuloma annulare was characterized with confluent papules arranged in a ring, mostly in the back of hand and foot, but also fingers, toes and elbows (over a joint). In less typical forms, for instance plague form, granuloma annulare may clinically resemble to necrobiosis lipoidica, but also other granulomatous diseases of unknown etiology.
Necrobiosis lipoidica typically consist of yellow-brown, indurate plaques with an atrophic and slightly depressed center and a well-defined raised erythematous edge. The legs, particularly the shins, are the most common sites of involvement with symmetric and bilateral distribution. Typical form of necrobiosis lipoidica is a one-look diagnosis. About 15% of those lesions are estimated to appear elsewhere on the body and usually causes problem in differential diagnosis, especially if the patient has no diabetes mellitus. In those cases, other granulomatous diseases, primary granuloma annulare and sarcoidosis (scalp lesions) must be excluded.
These atypical, rare clinical variants of granuloma annulare and necrobiosis lipoidica may be  distinguished by histopathology, but also histological overlaps may occur.
Histologically, differences between these two diagnoses are mostly in a number of multinucleate giant cells, presence or absence of palisading histyocites and changes on blood vessels in the middle and lower dermis.
We report a case of acral and truncal multilocular annular and serpiginous lesion clinically indicative for disseminated form of granuloma annulare, in a diabetic, 70-year old male patient, the histopathological study of which showed focal collagen degeneration surrounding with large number of multinucleate giant cells of foreign body types that is histopathological characteristic of necrobiosis lipoidica.
<PROGRAM>

SCARRING ALOPECIA AS A MANIFESTATION OF DIFFERENT SKIN CHANGES

Oremović L, Lugović Liborija, Nola Ivana, Sjerobabski-Masnec Ines, Ožanić-Bulić S

Department for Dermatology and Venereology, Sestre milosrdnice University Hospital, Zagreb

BACKGROUND: All scarring processes can damage the hair follicle, leading to hair loss and by definition irreversible alopecia. Histologically, it is characterized by dermal scarring, often relatively deep, along with absent or reduced hair follicles and reduced numbers of erector pili muscles. The most common causes of scarring alopecia are discoid lupus erythematosus (DLE), lichen planus, pseudopelade, chronic folliculitis, sarcoidosis, morphea, mucinosis follicularis, etc.

OBJECTIVE: The purpose of this study was to examine the causes of scarring alopecia by anamnestic data and different tests, particularly by pathohistologic examination of patients in our Clinic during 12-month period. We also analysed the obtained data, different examinations and previous treatment in each patient.

METHODS: A total of 10 patients with clinically scarring alopecia were involved in this study. The examination included anamestic data, duration, previous treatment, light pull test, trichogram, antinuclear antibodies, allergic tests, etc.

RESULTS: Skin biopsies and pathohistologic examination of the patients showed different skin disorders - pseudopelade, lichen planus, discoid lupus erythematosus (DLE), chronic folliculitis etc. There were no important medical or dermatological associations with other disorders or related medical conditions.

CONCLUSION: The pathohistologic examination of most scarring alopecia patients showed different skin disorders (pseudopelade, lichen planus, DLE, chronic folliculitis) although some patients showed nonspecific histology. This experience is going to take us to more complex investigation to extend our knowledge and we hope that this type of alopecia may attract more attention and investigation in the future.
<PROGRAM>


SUBCUTANEOUS PANNICULITIS-LIKE T-CELL LYMPHOMA - CASE REPORT

Troskot N1, Lugović Liborija1, Vučić Majda2, Ožanić-Bulić S1

1Department for Dermatology and Venereology and 2Ljudevit Jurak University Department of Pathology, Sestre milosrdnice University Hospital, Zagreb

A 45-year-old male, with a 6-month history of tender erythematous subcutaneous skin lesions and with no signs of systemic symptoms is presented. The patient denied exposure to trauma or thick bite. The patient noticed erythematous tender lesion on his left pectoral region, which was gradually enlarging and spreading centrifugally. It appeared livid in colour, and significantly hard on palpation. The cytopunction of the lesion indicate a mesenchymal tumour. Borrelia burgdorferi serologic test was negative. The lesion showed significant deterioration in respect of clinical signs as well as the worsening in pain followed by restriction in motion of the left shoulder. The result of the  biopsy showed Morphea (Sclerodermia circumscripta). On patient`s arrival at our Clinic, the area affected was livid-brown in colour, 5 cm in size, hard and tender on palpation with central postoperative scar 1 cm in diameter.  In the   infraumbilical region, there were two subcutaneous nodular lesions of 1,5 cm, covered with erythematous-livid skin. In the  left pectoral region, similar skin changes – 1 cm in size - were also present.  No axillary lymph nodes were detected. During hospitalisation two biopsies were taken from the subcutaneous lesions on the abdominal skin,  and the pathohistological analysis indicated primary to panniculitis in complex of Morphea. Results of the tumorous tissue obtained from the patient's back, as well as the immunohistochemistry  showed Subcutaneous panniculitis-like T-cell lymphoma. Specific haematological analysis and medical treatment was continued.
<PROGRAM>

PATHOHISTOLOGIC VERIFICATION OF CLINICALLY SUSPECTED PSORIASIS VULGARIS SKIN CHANGES

Lugović Liborija1, Šitum Mirna1, Soldo-Belić A1, Vučić Majda2

1Department for Dermatology and Venereology and 2Ljudevit  Jurak University Department of  Pathology, Sestre milosrdnice University Hospital, Zagreb

Psoriasis vulgaris is a skin disease connected with keratinocyte hiperproliferation and abnormal epidermal differentiation. The classic histological features that are called psoriasiform, delineate the fully developed scaly silvery plaque and include uniform elongation of the rete ridges, a thinned suprapapilary plate, mounds of parakeratosis and several accumulations of neutrophils. There is exocytosis of erythrocytes and lymphocytes from the dilated and tortuous papillary vessels, producing the so-called squirting papillae. This study was performed to compare the equality of clinical and pathohistologic picture of psoriasis vulgaris, what may be of importance for the treatment of patients skin changes. Fifty patients were examined in our Clinic and were included in the study. The patients with skin picture suspected on psoriasis vulgaris were included in this study. The biopsies of skin changes on predilection sites typical for psoriasis were performed.
The obtained results showed that most of the patients with clinical pictures suspected on psoriasis vulgaris had histological verified pictures typical for psoriasis vulgaris. Among the  examined patients, there were many patients with skin changes suspected on psoriasis vulgaris in whom histologic analysis showed another clinical picture (e.c. dermatitis psoriasiformis, dermatitis subacuta, dermatitis eczematoides). Although most of the patients with clinical suspected psoriasis vulgaris had pathohistologically verified psoriasis vulgaris, there were also many patients in whom pathohistologic picture showed other dermatoses, mostly dermatitis psoriasiform. This experience is especially important to keep in mind when choosing a kind of therapy.
<PROGRAM>

HISTOLOGICAL TYPES OF  POLYPOID CUTANEOUS MELANOMA II

Knežević F, Petrovečki M, Duančić V, Horvat-Knežević Anica, Kostović K

University Hospital for Tumors - Department of Pathology, Dubrava University Hospital, Institute for Cardiovascular Prevention and Rehabilitation, Faculty of Science, Department of Animal Physiology; Sestre milosrdnice University Hospital

In the study of 645 cases of primary cutaneous melanoma, we found 147 (22.8%) polypoid melanomas. By analysing the growth phases, the type of the neoplastic melanocytes and  the epidermis appearance in this series we defined 2 (1.4%) acral lentigo maligna melanomas (ALMM), 4 (2.8%) lentigo maligna melanomas (LMM), 42 (28.6%) superficially spreading melanomas (SSM) and 99 (67.2%) nodular melanomas (NM). Based on these results, the authors classified the polypoid cutaneous melanoma as a morphological variant with different histological types (ALMM, LMM, SSM, NM) all of which display an exophytic vertical phase of growth. The majority of the polypoid cutaneous melanoma are of nodular histological type.

REFERENCES:
McGovern VJ et al. Prognostic significance of a polypoid configuration in malignant melanoma. Histopathology 1983;7:663-72.
Manci E et al. Polypoid melanoma, a virulent variant of the nodular growth pattern. Am J Clin Pathol 1981;75(6):810-15.
Reed KM et al. Prognosis for polypoidal melanoma is determined by primary tumor thickness. Cancer 1986;57(6):1201-3.
Knežević F. Polipoidni melanom kože (magistarski rad) Zagreb, Republika Hrvatska: Sveučilište u Zagrebu; 1990.
Knežević F et al. Histological types of polypoid cutaneous melanoma. Croat Med J 1992;33(4):220-4.
Knežević F. Multifaktorijalna analiza polipoidnog melanoma kože (doktorska disertacija) Zagreb, Republika Hrvatska: Sveučilište u Zagrebu; 2002
<PROGRAM>


A MULTIFACTORIAL ANALYSIS OF POLYPOID CUTANEUS MELANOMA I (Influence of Examined Parameters on Disease Free Survival)

Knežević F, Duančić V, Horvat-Knežević Anica, Petrovečki M

University Hospital for Tumors, Department of Pathology; Institute for Cardiovascular Prevention and Rehabilitation, Faculty of Science, Department of Animal Physiology, Dubrava University Hospital

The basic goal of our study is to determine the dominant, mutually independent prognostic parameters in polypoid cutaneous melanoma and their influence on the disease free survival.
645 primary melanoma cases have been investigated and 147 (22.8%) polypoid cutaneous melanoma cases have been found.
23 morphological-clinical data among them have been statistically evaluated with MedCalc and SPSS programs, t-test and ?2 test, Kaplan-Meier method and Cox regression test. Statistical inference have been carried out with 95% reliability, i.e. p < 0.05.
A single-factor analysis of the parameters examined has shown: that the disease free survival is relatively influenced by sex (p = 0.024), clinical stages of the disease, clinical stage I, clinical stage II (p < 0.001); and the thickness of the tumor in mm (p = 0.016).
A multifactorial analysis of the parameters examined has shown that the disease free survival is independently influenced by the clinical stages of the disease and clinical stage I (p < 0.001).
In clinical stage I 38% of polypoid melanoma patients have 24 months of disease free survival and ca. 22% have 72 months of disease free survival.

REFERENCES:
Balch CM et al. An analysis of prognostic factors in 4000 patients with cutaneous melanoma. ln: Balch CM et al. Cutaneous melanoma. Clinical management and treatment results worldwide 1st ed.. Philadelphia: J.B.Lippincott Company;1985, pp. 321–52.
Cox DR. Regression model and life tables. J Royal Stat Soc 1972;B34:187–220.
Balch CM et al. A multifactorial analysis of melanoma III. Prognostic factors in melanoma patients with lymph node metastases (stage II). Ann Surg 1981;193:377–88.
Balch CM et al. A multifactorial analysis of melanoma. IV. Prognostic factors in 200 melanoma patients with distant metastases (stage III). J Clin Oncol 1983;1:126–34.
Day CL et al. A multivariate analysis of prognostic factors for melanoma patients with lesion ? 3.65 mm in thickness. The importance of revealing alternative Cox models. Ann Surg 1982;195:44–9.
Drzewiecki KT et al. Malignant melanoma. Changing trends in factors influencing metastasis-free survival from 1964 to 1982. Cancer 1990;65:362–6.
Knežević F. Multifaktorijalna analiza polipoidnog melanoma kože (doktorska disertacija) Zagreb, Republika Hrvatska: Sveučilište u Zagrebu; 2002.
<PROGRAM>


A MULTIFACTORIAL ANALYSIS OF POLYPOID CUTANEOUS MELANOMA II  (Influence of  Examined Parameters on Cumulative Survival Rates)

Knežević F, Duančić V, Petrovečki M, Horvat-Knežević Anica

University Hospital for Tumors, Department of Pathology; Institute for Cardiovascular Prevention and Rehabilitation, Dubrava University Hospital, Faculty of Science, Department of Animal Physiology

The basic goal of our study is to determine the dominant, mutually independent prognostic parameters in polypoid cutaneous melanoma and their influence on the cumulative survival rates.
645 primary melanoma cases have been investigated and 147 (22.8%) polypoid cutaneous melanoma cases have been found.
23 morphological-clinical data among them have been statistically evaluated with MedCalc and SPSS programs, t-test and ?2 test, Kaplan-Meier method and Cox regression test. Statistical inference have been carried out with 95% reliability, i.e. p < 0.05.
A single-factor analysis of the parameters examined has shown that the cummulative survival rates is relatively influenced by clinical stages, clinical stage I, clinical stage II, degree of malignancy to Clark (p<0.00l) and M/V in mm2  of the tumor tissue ( p=0.016).
A multifactorial analysis od the parameters examined has shown that the overall survival is independently influenced by the clinical stages of the disease and clinical stage I ( p<0.001). In the clinical stage I  64% of the patients live 5 years and 44% ten years. In the clinical stage II 18% of the patients live one year while patients in the clinical stage III die within one year.
As for the prognosis of polypoid cutaneous melanoma patients the most important independent prognostic parameter is the  clinical stage of the disease in the moment  when the diagnosis of melanoma is being made.

REFERENCES:
Balch CM et al. An analysis of prognostic factors in 4000 patients with cutaneous melanoma. ln: Balch CM et al. Cutaneous melanoma. Clinical management and treatment results worldwide 1st ed. Philadelphia: J.B.Lippincott Company;1985, pp. 321–52.
Cox DR. Regression model and life tables. J Royal Stat Soc 1972;B34:187–220.
Balch CM et al. A multifactorial analysis of melanoma III. Prognostic factors in melanoma patients with lymph node metastases (stage II). Ann Surg 1981;193(3):377–88.
Balch CM et al. A multifactorial analysis of melanoma. IV. Prognostic factors in 200 melanoma patients with distant metastases (stage III). J Clin Oncol 1983;1(2):126–34.
Day CL et al. A multivariate analysis of prognostic factors for melanoma patients with lesion ? 3.65 mm in thickness. The importance of revealing alternative Cox models. Ann Surg 1982;195(1):44–9.
Lipponen PK et al. Volume corrected mitotic index (M/V index) in human bladder cancer; relation to histological grade (WHO), clinical stage (UICC) and prognosis. Scand J Urol Nephrol 1990;24:39–45.
Haapasalo H et al. Prognosis of ovarian carcinomas: prediction by histoquantitative methods. Histopathology 1989; 15:167–78.
Haapasalo H, Pesonen E. Volume corrected mitotic index (M/V - INDEX). The standard of mitotic activity in neoplasms. Pathol Res Pract 1989;185:551–54.
Knežević F. Multifaktorijalna analiza polipoidnog melanoma kože (doktorska disertacija) Zagreb, Republika Hrvatska: Sveučilište u Zagrebu; 2002.
<PROGRAM>


SKIN TUMORS OF THE PERIORBITAL REGION AND EYELIDS IN THE 1998-2002 PERIOD

Vučić Majda, Talan-Hranilović Jasna, Vucelić Vesna, Rožanković Sanda, Iveković Renata1

Ljudevit Jurak University Department of Pathology and 1Department of Ophthalmology, Sestre milosrdnice University Hospital, Zagreb, Croatia

The aim of this study was to determine the prevalence, year distribution and localisation of the skin tumours and precancerous skin lesions in the periorbital region and eyelids during the 1998-2002  period among biopsy specimens in the Ljudevit Jurak University Department of Pathology . We also analysed their relationship with sex and age of the patients. During the observed period there were total number of 286 tumors and precancerous lesions. The most common was basal cell carcinoma with slightly female prevalence. Other analysed lesions; nevus lesions, precancerous lesions and squamous cell carcinoma were found with significantly higher female prevalence. The most frequent localisation for all lesions was on the eyelids probably as the consequence of UV irradiation.  Average age for all lesions in time of diagnoses was 65 years for males and 64 years for females. All patients with skin lesions should be advised of the risk of recurrent or new tumors. Prevention remains the most important in minimising the morbidity and mortality due to lesions in this region. Exposure to ultra-violet (UV), especially UV-B, radiation is the most common cause for genetic abnormalities in cells and provoked factor in oncogenesis of skin tumors.

* Full article will be published in the next issue of Acta clinica Croatica

http://www.acta-clinica.kbsm.hr
<PROGRAM>

PRIMARY SYNCHRONOUS UVEAL AND SKIN MALIGNANT MELANOMA - CASE REPORT

Talan-Hranilović Jasna 1, Vučić Majda 1, Iveković R 2, †Kušić V 2

1Ljudevit Jurak University Department of Pathology, 2University Department of Ophthalmology, Sestre milosrdnice University Hospital, Zagreb, Croatia

Simultaneous occurrence of primary uveal melanoma and skin melanoma is rare. We presented a 66 year old female patient with primary synchronous choroidal and skin malignant melanoma on right upper-arm. The patient has a negative family history for cutaneous or uveal melanomas and did not display the dysplastic nevus syndrome phenotype. Six years after enucleation of left bulbous and skin excision of malignant melanoma, the patient presented with lymphatic metastasis in right axillary lymph nodes and two years after those metastases in mesenteric lymph nodes and solitary nodules in spleen. After nine years, the patient developed metastatic inquinal lymph node. Metastatic spread was due to lymphatic dissemination of more biologically aggressive primary skin malignant melanoma. Ten years have passed after the initial presentation of the tumors.

* Full article will be published in the next issue of Acta clinica Croatica

http://www.acta-clinica.kbsm.hr
<PROGRAM>

CLINICAL CASE OF ACRAL HEMORRHAGIC DARIER'S DISEASE IS NOT CAUSED BY MUTATIONS IN EXON 15 OF THE ATP2A2 GENE

Pećina-Šlaus Nives1, Milavec-Puretić Višnja2, Nikuševa-Martić Tamara1, Kubat M3, Fischer-Žigmund Martina 1, Lipozenčić Jasna2

1Department of Biology, Zagreb University School of Medicine, Zagreb, Croatia, 2Department of Dermatovenereology, Clinical Hospital, Zagreb University School of Medicine, 3Department of Forensic Medicine, Zagreb University School of Medicine.

Dyskeratosis follicularis (Darier's Disease, DD, MIM#124200) is a genetic skin disorder characterized by the loss of adhesion between epidermal cells (acantholysis) and pathogenetic changes of keratinization with variant forms of cutaneous phenotype. Recently it has been shown that DD cause mutations in the ATP2A2 gene, at 12q24.1. This gene (GenBank accession nos M23115 and M23114) encodes the sarco/endoplasmic reticulum calcium-pumping ATPase (SERCA2), which is highly expressed in keratinocytes.  We report on a severe case of the acral hemorrhagic type of DD with an unusual manifestation involving Staphylococcal sepsis. The patient was treated systemically with infusions, oral antibiotics, and retinoids. Antiseptics, keratolytic ointments and creams were given topically to promote epithelization. His condition improved dramatically after 14 days of treatment. All erosions of the trunk, extremities, neck, and head had epithelized. Mutations in exon 15 of the ATP2A2 gene are reported to be the most consistent mutations associated with the acral hemorrhagic type of DD (Ruiz-Perez, et al. Hum Molec Genet 1999; 8: 1621-1630; Sakuntabhai, et al. Nat Genet 1999; 21: 271-277). In order to establish genetic background of our patient`s clinical phenotype we investigated exon 15 of the ATP2A2 gene. By direct sequencing of the PCR amplified
exon 15 of the ATP2A2 gene we did not detect any mutation in our patient`s DNA (skin biopsy and blood), nor did we detect mutations in 4 members of his family. Our results show that mutations in exon 15 of the ATP2A2 gene are not a necessary prerequisite for acral hemorrhagic type of DD. Our finding support the variability of clinical manifestations of DD and a lack of genotype/phenotype consistency.
<PROGRAM>

NEVUS DYSPLASTICUS - PRECURSOR OF MELANOMA MALIGNUM?

Šitum Mirna, Papić Marijana, Bučan Željana, Nola Ivana

Department of Dermatology, Sestre milosrdnice University Hospital

BACKGROUND: Nevus dysplasticus is an acquired melanocytic nevus, typically flat, and with allegedly specific histological features. There are some disagreements among the authors. Some authors prefer to use the term atypical nevus for lesions that attract clinical attention and reserve dysplastic nevus for those lesions with proven histological changes. Histological features of dysplastic nevi according to Clark are (1) persistent lentiginous melanocytic hyperplasia; (2) atypical melanocytic hyperplasia (melanocytic nuclear atypia); (3) lamellar fibroplasia; (4) concentric eosinophilic fibroplasia; and (5) sparse patchy lymphocytic infiltrates, whereas according to Ackerman criteria for dysplastic nevi are silhouette of a benign neoplasm (e.g., symmetric, sharply circumscribed, relatively flat base), with only a slight, if any, elevation, in its center, and nests of melanocytes with small, oval, monomorpheus nuclei confined to the dermoepidermal junction and the upper part of the dermis.
We classified dysplastic nevus as small, dark, flat, sharply bordered lesion measuring a few millimeters in diametar. They are located most commonly on the trunk and tend to increase in number during adolescence, pregnancy, following intense exposure to sun and immunosuppression. Larger dysplastic nevi, may be up to 10-15 mm in diametar, multicolored, contain central elevated or dark nodule, white areas of regression or have an erythematous border. Thus, they can be very difficult to distinguish from melanoma malignum.
It is still unclear whether these multiple nevi are precursors of melanoma malignum or markers for melanoma malignum susceptibility.
AIM: Aim of the study was to find out the total number of melanoma malignum in patients from Department of Dermatology of Sestre milosrdnice University Hospital and whether melanoma malignum develop from pigmented lesions or de novo.

MATERIAL AND METHODS: In this retrospective trial we used files and pathohistological documentation of 661 patients in Department Dermatology with following diagnoses: solitary, sporadic nevi, multiple nevi, dysplastic nevus syndrome and melanoma malignum.

RESULTS: Total number of patients with sporadic dysplastic nevi is 208; among them 103 has histologicaly verified diagnosis, and 105 has typical clinical features of dyspalstic nevi and are in observation.
The overall number of patients with multiple dysplastic nevi is 149; among them 90 have histological verification and 59 have typical clinical features and are in observation.
Dysplastic nevi syndrome was established in 129 patients: among them 108 have histological verified diagnosis and 21 have clinical features of syndrome and we are observing them.
The total number of patients with histological diagnosis of melanoma malignum is 175.
Our study showed that melanoma malignum developed from solitary and multiple dysplastic nevi in 24 %, from dysplastic nevus syndrome in 17 %, from pigmented nevi in 31%, from lentigo maligna in 3 % and de novo in 25 % of cases.

CONCLUSION: Based on our study, we conclude that melanoma malignum develops more frequently from dysplastic nevi (41%) rather then other lesions such as pigmented nevi (31%), lentigo maligna (3%) or de novo (25%).
<PROGRAM>


CLINICAL AND HISTOPATHOLOGICAL PARAMETERS AS PREDICTORS OF CLINICAL OUTCOME FOR PATIENTS WITH VULVAR CARCINOMA

Blažanović A1, Dotlić S2, Morović A3, Milošević M3, Munivrana H3

1Department of Pathology, General Hospital Vinkovci, Vinkovci, Croatia, 2Department of Pathology, Clinical Hospital Center Zagreb, 3Medical University, Zagreb, Croatia

AIM OF THE STUDY: The aim of this study was to determine the most important clinical and pathohistological parameters that influence the prognosis and outcome of  patients with invasive squamous carcinoma of the vulva in order to offer them the most suitable form of therapy. Moreover, it was designed to assess the clinical significance of DNA content analysis by flow cytometry.

PATIENTS AND METHODS: Forty-three cases of squamous cell carcinoma of the vulva, diagnosed between 1978 and 1996 were selected. In order to assess the influence of the various prognostic factors on survival, the study included clinical and
pathohistological parameters as well as the results of flow cytometric DNA analysis of paraffin-embedded tumor samples from all cases. Clinicopathological parameters focused on the age of patients, clinical stage of the disease, menstrual status, diameter and localization of the tumor, histological grade, nuclear grade, combined histological and nuclear grade, depth of tumor invasion, tumor growth pattern, the presence of giant cells in the tumor and the form of therapy, while flow cytometric DNA analysis comprised  DNA ploidy, proliferative activity and DNA index.

RESULTS: Five-year survival of our selected population of patients was 62,3 + 7,8%.
The results of univariate statistical analysis confirmed that statistically significant prognostic parameters included the age of patients (p=0,033), clinical stage of the disease (p=0,014), histological grade (p=0,047), nuclear grade (p<0,001) and the depth of invasion (p<0,001). On the other hand, the results of multivariate statistical analysis showed that only combined histological and nuclear grade (p=0,015) and the depth of tumor invasion (p<0,001) can be considered independent, statistically significant prognostic parameters.

CONCLUSION: The age of patients at the time of diagnosis, clinical stage of the disease, histological grade and nuclear grade are considered to be the parameters of crucial relevance for the prognosis of patients with squamous cell carcinoma of the vulva. However, only combined histological and nuclear grade, as well as the depth of tumor invasion were proven to be independent statistically significant parameters relevant for the outcome of patients with this disease.
<PROGRAM>


NON-MELANOMA SKIN CANCERS AND PRECANCEROUS SKIN LESIONS IN THE 1996 – 2002 PERIOD

Vučić Majda, Rožanković Sanda, Vucelić Vesna, Belicza Mladen, Šitum Mirna1

Ljudevit Jurak University Department of Pathology and 1Department of Dermathology, Sestre milosrdnice University Hospital, Zagreb, Croatia

Non-melanoma skin cancers and precancerous skin lesions have a significant morbidity although with relatively low mortality rates in geriatric population. These lesions developed especially on every day sun exposed skin regions. The aim of this study was to determine the prevalence, age and sex distribution of non-melanoma skin cancers and precancerous skin lesions among biopsy specimens collected during seven years (1996-2002) in the Ljudevit Jurak University Department of Pathology.  Their relationship with sun exposure on different body regions has also been analyzed. During the observed period there were 2486 basal cell carcinoma, 419 squamous cell carcinoma and 468 precancerous skin lesions. Basal cell carcinoma was more common in males then in females with ratio 1:0,9 as well as squamous cell carcinoma with male to female ratio 1:0,8. Precancerous skin lesions were more frequent in the female population with male to female ratio 1:1,3. Maximal incidence for booth types of non-melanoma tumours was between 70 and 79 years in both sexes while precancerous skin lesions appeared one-decade erlier. It has also been found that all analyzed skin lesions appear in 60-70% on skin of the head, which is the body region almost permanently exposed to the sun.

* Full article will be published in the next issue of Acta clinica Croatica

http://www.acta-clinica.kbsm.hr
<PROGRAM>

CUTANEOUS PSEUDOLYMPHOMAS

Pešut A, Gašparov S, Džebro S, Šitić S, Milković M, Dominis M

Department of Clinical Pathology and Cytology, Merkur University Hospital, Zagreb, Croatia

Cutaneous pseudolymphomas (CPL) are heterogeneous group of benign reactive T- or B- cell lymphoproliferative processes of diverse causes that simulate cutaneous lymphomas clinically and/or histopathologically. Depending on the predominant cell type in the infiltrate, according to histologic, immunophenotypic and molecular analyses, they are divided into T- and B- cell pseudolymphomas. The differentiation between CPL and primary cutaneous lymphomas (PCL) is often very difficult, but it is important because it has therapeutic consequences.

Aims of this study are to show experiences in CPL in our practice in the past 9 years (1995-2003). 33 cases of CPL were diagnosed and classified according to EORTC and WHO classifications of primary cutaneous lymphomas.

 Samples were sent to our department as suspected cases of PCL. Specimens were surgical biopsies, paraffin- embedded tissue and sections of skin. We used routinely fixed paraffin- embedded tissue sections stained with HE, PAS, Giemsa, Gomori and panel of antibodies for immunohistochemical analyses. According to the above mentioned classifications 21 of the cases were classified as B- cell and 10 cases as T-cell pseudolymphomas, respectively. In two patients skin lesions were skin manifestations in the course of rheumatoid arthritis.
 

REFERENCES:

Jaffe E.S. et al. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Haemathopoietic and Lymphoid Tissues. IARC Press: Lyon 2001
Cerroni L. et al. An Illustrated Guide to Skin Lymphoma. Press: Blackwell Science Ltd. 1998
<PROGRAM>


ORAL MANIFESTATIONS OF AUTOIMMUNE DISEASES

Nola Ivana, Kostović K, Kotrulja Lena, Sjerobabski-Masnec Ines, Lugović Liborija, Meštrović-Štefekov Jelena

Sestre milosrdnice University Hospital, Department of Dermatovenereology Zagreb, Croatia

The oral mucous membrane has properties in common with the skin because both originate from the ectoderm. Reactibility related both to skin and mucosa is the reason for transition of pathological process from skin to mucosa and vice versa. Collagen tissue diseases, bullous dermatoses and diseases with possible immunopathogenesis are only some of the skin diseases, which occur relatively frequently in the region of oral cavity. Pemphigus is a group of bullous disorders characterized by the development of autoantibodies against desmocollins and desmogleins in the epidermis, giving rise to superficial erosions and blister on both epidermal and mucosal surfaces. The most common variety of pemphigus is pemphigus vulgaris, that usually begins with shallow erosions and easily ruptured blisters on mucosal surfaces (in over 50% of cases). Oral lesions in patients with bullous pemphigoid are less frequent than in pemphigus but should be sought. Mucous membranes are affected in about 20% of patients with epidermolysis bullosa dystrophica. Also, in patient with systemic scleroderma the soft tissue of the mouth and palate are affected, as well as, the mouth gradually shrinks. The oral mucosa is involved in 15-25% of the cases of discoid lupus erythematosus, and in 30-45% of the patients with systemic lupus erythematosus. The diagnosis should be confirmed by histopathology, direct and indirect immunofluorescence and by confirmation of circulating autoantibodies.
<PROGRAM>

DISTRIBUTION AND AMOUNT OF CATHEPSIN B IN ACUTE KIDNEY INJURY INDUCED BY GENTAMICIN

Svara T, Juntes P, Pogačnik M

University of Ljubljana, Veterinary Faculty, Ljubljana,  Slovenia

Cathepsin B is a lysosomal enzyme important for degradation of proteins. The largest amount of cathepsin B was found in kidney, in the epithelial cells of proximal convoluted tubules. Only few references about the role of cathepsin B in kidney diseases were found. The aim of our study was to establish in what way the amount and distribution of cathepsin B changes in tubules during acute kidney injury and if there is any connection between the degree of kidney injury and the amount of cathepsin B in damaged tubules. The hypothesis was explored on the model of acute kidney injury induced by application of nephrotoxic antibiotic gentamicin. Gentamicin was injected at a dose of 40 mg /kg body weight (first treated group) and 80 mg /kg body weight (second treated group) for 14 days. Control groups received injection of physiological saline only. Pathohistological examination was done on tissue sections of kidneys stained with haematoxylin and eosin. Monoclonal antibodies against humane cathepsin B (product of Krka, catalogue number CATHBM1) were applied on formalin - fixed, paraffin - embedded tissue sections of kidneys. Results of immunohistochemical examination were statistically processed.
Application of gentamicin provoked vacuolisation of proximal convoluted tubules in the first treated group. In the second treated group, necrosis of proximal convoluted tubules was observed, as well as the signs of regeneration. In both treated groups weaker positive reaction for cathepsin B was noticed in comparison to the same tubules of control groups. Positive reaction for cathepsin B was also statistically weaker in the proximal convoluted tubules of the second treated group in comparison to the first treated group (P < 0.05). Decrease in positive immunohistochemical reaction was larger in outer renal cortex, where pathohistological lesions were more expressed. At the same time, stronger positive immunohistochemical reaction for cathepsin B was found in proximal straight tubules, but the differences were not statistically significant (P > 0.05). A comparison between the treated groups showed a significantly stronger reaction for cathepsin B in the second treated group in comparison to the first treated group (P < 0.05).
The results suggest that during acute kidney injury the amount of cathepsin B decreases in damaged tubules, but at the same time, according to the degree of injury, its amount in proximal straight tubules increases and probably proximal straight tubules take over the function of damaged segments of the proximal tubules.

REFERENCES:
Eisenberger U et al. Renal Physiol Biochem 1995;18:89
Olbricht CJ et al. Kidney Int 1991;39:639
Olbricht CJ, Euro J Chem Clin Biochem 1992;30:675
<PROGRAM>


ANALYSIS OF DEVELOPMENTAL POTENTIAL OF POSTIMPLANTATION RAT EMBRYO IN METABOLICALLY POOR CONDITIONS BY USING COMBINED IN VITRO AND IN VIVO TECHNIQUES

Belovari Tatjana1, Stević Nataša1, Gajović S2, Kostović-Knežević Ljiljana2

1Dpt. of Histology and Embriology, School of Medicine, University of Osijek, Osijek, Croatia, 2Croatian Institute for Brain Research, Zagreb University School of Medicine, Zagreb, Croatia

In vitro cultures are used in experimental embryology for investigation of developmental processes, regulatory factors and embryotoxic substances. However, differentiation is restricted in in vitro conditions, even in the optimal cultivation medium with serum (1). Subsequent grafting under the kidney capsule shows that differentiation can be improved in richer environment (2). Since serum-free cultures are used for precise investigations, the question is how metabolically poor conditions affect developmental potential. In rat embryo cultivated 14 days in serum-free medium only epidermis and cartilage differentiates well, while other differentiated tissues are rare or absent. Epidermis retains its developmental potential during cultivation in serum-free medium (3).
To analyze stability of in vitro restriction of developmental potential, rat embryos were cultivated in serum-free medium and then transplanted in vivo.
Fisher rat embryos (E 9.5) were cultivated for 14 days in Eagle's minimal essential medium (MEM) alone and with 50% rat serum, as a control. The resulting teratomas were transplanted under the kidney capsule. After 14 days grafts were processed for histology, serially sectioned and analysed with light microscope.
Regardless of the presence of the serum epidermis with derivatives, respiratory and gut epithelium developed well in grafts. Cartilage and enchondral ossification were found. However, neural and adipose tissue, glandular epithelia, skeletal and smooth muscles differentiation was statistically lower in grafts precultivated in serum-free medium.
It can be concluded that although the differentiation of that embryo cells in the serum-free conditions was restricted, cells retained to significant extent their developmental potential, which could be revealed after in vivo transplantation.

REFERENCES:
Cockroft DL. Int J Dev Biol 1997; 41: 127.
Škreb N. et al. J Embryol Exp Morph 1977; 42: 127.
Bulić-Jakuš F. et al. Cells Tissues Organs 2001; 169: 134.
<PROGRAM>


THE EFFECT OF LEVAMISOLE ON GUT MUCOSA OF WEANED PIGS VACCINATED AGAINST COLIBACILLOSIS

Božić F1, Smolec O1, Lacković G2, Valpotić I1, Sabočanec R1

1Faculty of Veterinary Medicine & 2Faculty of Science, University of Zagreb, Zagreb, Croatia

Apart from its anthelmintic activity in domestic food animals, levamisole may be used as an adjuvant for preventive vaccines but information on its potential adjuvant activity in hosts vaccinated against gastrointestinal pathogens is generally lacking. In the present study we aimed to test whether levamisole acts in weaned pigs vaccinated against colibacillosis by priming the lymphocytes in the gut mucosa. Ten weaned piglets were used and allocated into 2 equal groups. The experimental group was intramuscularly primed with levamisole in immunostimulatory dose of 2.5 mg/kg given daily - in 3 consecutive days - and the control group received saline according to the same schedule. Both groups were orally vaccinated with the vaccinal Escherichia coli strain on day 0. Seven days later all pigs were infected with the virulent E. coli strain and sacrificed on post-challenge day 6. Histological examination of the gut mucosa did not reveal significant morphological changes in the controls. However, in the vaccinated weaned pigs primed by levamisole jejunal villi were shortened and their lamina propria was infiltrated with mononuclear cells. In the latter pigs, jejunal submucosa was oedematous but hyperplasia of the crypts was not generally observed. The immunohistochemical in situ staining results showed that priming by levamisole of the vaccinated weaned pigs selectively recruits and activates T-cells in the villous epithelium as well as in the ileal Peyer’s patch, a primary lymphoid organ generating B-lymphocytes. Our overall data  suggest that levamisole appeared to stimulate the effector sites of the gut immune system in the vaccinated weaned pigs, serving as a critical component in the establishment of protective immunity against enterotoxigenic E. coli-induced clinical disease.
<PROGRAM>

DAILY MORTALITY AND DAILY AMBIENT POLLUTANT CONCENTRATIONS IN ZAGREB AND LJUBLJANA 1995-1997.

Pavlović M1, Gale I2

1Institute for medical research and occupational health, Zagreb, 2Institute of public health of the Republic of Slovenia, Ljubljana

The data on daily mortality and specific daily mortality - respiratory and cardiovascular diseases (ICD 10) - were collected in Zagreb and Ljubljana for the period of 1096 days – from 1995-1997. In the same period   (average daily) temperature, relative humidity and pressure were analyzed. Additionally, we collected data on daily cases of flu in Zagreb. In the same period daily average concentrations of nitrogen oxides (NO2), sulfur dioxide (SO2) and black smoke (BS) were measured for both cities, due to analysis according to APHEA II EC protocol.
The aim of this study was to analyze the relationship between daily data on air-pollution and general and specific mortality in both cities.
In Zagreb, the correlation between daily NO2 and SO2 concentrations and daily general mortality and cardiovascular mortality was statistically significant. In the period from 1995-1997 standardized general mortality (Ljubljana), cardiovascular mortality (Zagreb) and respiratory mortality (Ljubljana) were statistically different, as well as daily concentrations of NO2, SO2 and BS.
Results because of a vital data frequency for Zagreb and Ljubljana indicate a longer follow-up.
<PROGRAM>

INFLUENCES ON HELICOBACTER SP. INFECTION OF DOGS IN SLOVENIA

Gombač M, Černe M, Pogačnik M

University of Ljubljana, Veterinary Faculty, Ljubljana, Slovenia

Helicobacters have been identified and isolated from dogs, but their prevalence and influence of different parameters on infection in different populations of dogs are still unknown.
The aim of our study was to establish the prevalence of Helicobacter infection in dogs in Slovenia and to evaluate the influence of epidemiological parameters (gender, age, breed, nutrition, region and housing condition) on infection.
In our research we included 213 randomly chosen dogs of 44 different breeds from all parts of Slovenia, which died or had been euthanised. Both genders were included in the age from 9 days to 15 years. They lived indoors or outdoors and were still suckling or were fed either with commercially prepared food or home made food.
Using Toluidin blue staining method on necropsy taken stomach tissue samples we detected Helicobacter in 92,4% of dogs in all examined parts of stomach i.e. fundus, corpus and antrum. Helicobacters were located in mucus layer, covering the stomach epithelium, gastric pits and in gastric gland’s lumen.
The determination of effects of gender, age, breed, nutrition, region and housing condition on infection with Helicobacters was made calculating the prevalence of infection and statistically evaluating the results.
We established that age and feeding regimen affect the infection, whereas gender, breed, location and housing condition do not. The rate of infection was 33,3% in dogs up to 2 months of age and 94,4% in dogs older than 2 months. The infection rate of dogs fed with home prepared food was 95,9% and in dogs fed with commercially prepared food 91,5%. All suckling puppies were uninfected.

REFERENCES:
Happonen I et al., J Vet Med A 1996;43:305
Hermanns W et al., J Comp Pathol 1995;112:307
Peyrol S et al., J Submicrosc Cytol Pathol 1998;30:425
<PROGRAM>


INCIDENCE OF SQUAMOUS METAPLASIA IN TRANSITIONAL CELL CARCINOMA OF URINARY BLADDER

Baličević D1 , Novosel Irena2 ,  Pirkić A1

1Ljudevit Jurak University Department of Pathology, Sestre milosrdnice University Hospital, Zagreb, Croatia
2Department of Pathology, «Dr Ivo Pedišić» County Hospital, Sisak, Croatia

Squamous metaplasia in transitional cell carcinoma of urinary bladder is considered  as an adverse prognostic factor.  These patients have weaker response on therapy and  lower survival rate.
In our study we used pathohistological  data of 1781  patients operated for urinary  bladder cancer in the Department of Urology of Sestre milosrdnice University Hospital  during the period from 1989 to 2000.  The aim of our study was to determine the incidence of squamous metaplasia in the  bioptic material  according to histologic grade and growth pattern.
Squamous metaplasia was found in 5.7% of patients. According to growth pattern, this phenomenon is more frequent in patients with solid growth pattern. In papillary cancers, the higher incidence of squamous metaplasia was determined in G1 and G2 histologic grade, while in the solid growth pattern, the most of the cases with squamous metaplasia or over 80%  were of G3 histologic grade.
Muscle layer invasion  was present in high percentage of both, papillary and solid urothelial cancers with squamous metaplasia, which imply the high malignant potential of cancers with foci of squamous metaplasia.

* Full article will be published in the next issue of Acta clinica Croatica

http://www.acta-clinica.kbsm.hr
<PROGRAM>

MYOFIBROBLASTS IN INVASIVE AND IN SITU UROTHELIAL CARCINOMA OF THE BLADDER

Zarubica Jelena, Gabelić Tereza, Bulimbašić Stela, Turčić Marijana, Trnski D, Krušlin B

General Hospital Pula, Medical Faculty, University of Zagreb, Sveti duh General Hospital, Ljudevit Jurak University Department of Pathology, Sestre milosrdnice University Hospital, Zagreb, Croatia

INTRODUCTION
Invasive carcinomas are usually accompanied with stromal response in which  myofibroblasts may have an important role. Such a reaction may not be present in in situ carcinomas that are in majority of cases associated with lymphocytic reaction.
AIM
The aim of this study was to analyze the presence and quantity of myofibroblasts in in situ and invasive urothelial carcinoma of the bladder.
PATIENTS AND METHODS
Urological pathology registry at the Ljudevit Jurak University Department of Pathology was canvassed for patients with both,  in situ and invasive urothelial  carcinoma. Relevant patient data including age, sex, tumor grade and stage of  the disease were analyzed. All cases were examined by routine histology. In this study we analyzed the expression of alpha smooth muscle actin and vimentin as imunohistochemical markers for myofibroblasts. The intensity of reaction was graded semiquantitatively and expresssed as (-) for negative, (+) for weak staining, (++) for moderate and (+++) for strong staining.
RESULTS
In the period from 1998-2002 there were 1243 biopsies with the diagnosis of urothelial carcinoma. Thirty-three biopsies with urothelial carcinoma in situ were found. We identified 10 patients with both in situ and invasive carcinoma. All of them were males ranging in age from 61-77 years (mean age 70.2 years).  Imunohistochemical analysis revealed that in cases of invasive urothelial carcinoma reactive stromal cells are alpha smooth muscle actins positive indicating the myofibroblast phenotype. In areas with in situ carcinoma the intensity of SMA reaction was moderate in 3 cases, weak in 5 cases and negative in 2 cases and intensity of vimentin reaction was strong in 2 cases, moderate in 7 cases and weak in 1 case. In areas with invasive carcinoma the intensity of SMA reaction was strong in 5 cases and mild in 5 cases and intensity of vimentin reaction was strong in all of 10 cases.
CONCLUSION
We found that the expression of myofibroblastic markers was strong in stroma of invasive carcinoma but absent or weak in in situ urothelial carcinoma.
This finding may indicate a role of myofibroblasts in the stromal reaction to invasion but may also be useful in diagnosis of in situ versus invasive urothelial carcinoma. Further studies on larger groups of tumors are certainly needed.
<PROGRAM>

MYOID CELL HYPERPLASIA IN AN INFERTILE PATIENT WITH AZOOSPERMIA: A CASE REPORT

Ježek D1, Mužić V1, Krhen I2, Knežević N2, Podobnik P1, Mareković Z2

1Institute of Histology & Embryology, 2Urology Clinic, Medical School, University of Zagreb, Zagreb, Croatia

AIM OF THE STUDY: In the testis, myoid (peritubular) cells are situated within the lamina propria. These cells are thought to contract at intervals of 12-15 min. in order to “push” sperms towards the epididymis. The aim of the present study is to present a case of a male infertility with hyperplasia of myoid cells.

MATERIALS AND METHODS: A 36-year old patient has suffered form azoospermia due to the unilateral testicular tumour. One testicle (in which the tumour was located)  has been removed. An irradiation therapy was meant to follow after the unilateral orchiectomy.  A biopsy of the remaining testicle was indicated in order to preserve sperms before the irradiation. Small pieces of the testicular tissue obtained by the open testicular biopsy were immediately fixed in 5.5% buffered glutaraldehyde followed by a postfixation with 1% OsO4. After dehydration, tissue was embedded in Durcopan. Semithin sections were performed by a Reichert ultramicrotome and stained with toluidine blue.

RESULTS: Testicular biopsy revealed hyperplasia of myoid cells. Instead of 5-7 layers of these cells, the lamina propria consisted of 7-14 layers of peritubular cells. Seminiferous tubules displayed a “mixed atrophy”. However, in some seminiferous tubules elongated spermatids and sperms were found.

CONCLUSION: Hyperplasia of myoid cells could be the cause of the male infertility and azoospermia.
<PROGRAM>


ADENOMATOID TUMOR OF FALLOPIAN TUBE

Labinac-Peteh Loredana1, Matković-Bilin M1, Pirkić A1, Končar M2

1Ljudevit Jurak University Department of Pathology, 2University Department of Gynecology and Obstetrics, Sestre milosrdnice University Hospital, Zagreb, Croatia

Although it is rare, adenomatoid tumor is the most frequent benign primary tumor of Fallopian tube. In female genital tract, similar lesions appear in the uterus and rarely in the ovary. Usually it has asymptomatic course and represent incidental finding during a routine pathomorphological adnexal examination. Previously it was confused with adenoma, leiomyoma or mesonephroid tumor and cases of ovarian lymphangioma that were reported earlier probably were adenomatoid tumor in fact. Today it is considered to be of mesothelial origin based on microscopic, ultrastructural and immunohistochemical analysis.
On gross examination tumor has nodular pattern, its size is usually one to 2 cm  and it is  yellow or whitish gray on the cut surface. Greater tumors may displace the tubal lumen eccentrically and it may grow in an infiltrating manner. On microscopic examination tumor is composed of branching tubular or cystic spaces lined by epithelial-like cells that are of mesothelial profile and surrounded by fibrovascular stroma.
51-years old nulipara was admitted to Department of Gynecology and Obstetrics, Sestre milosrdnice University Hospital, because of the single cyst of the left ovary, measuring 80 mm in diameter. Hysterectomy with bilateral salpingoophorectomy was performed and cyst having smooth surface and thin fibrous wall in the left ovary was found. Histologically it was determinated as a simple cyst. There were no particular changes in the right adnexa. One endometrial polyp of hyperplastic type, one mural leiomyoma and adenomyosis were found in the uterus.
The left tube was 60 mm long and 5 mm wide containing well circumscribed round yellowish nodule of medium firm consistency measuring 6:5 mm, which was situated under serosa and partially in the muscular layer. Tubal lumen was strongly displaced by the tumor.
Histologically tumor was composed of many irregular clefts or tubular partially cystic dilated spaces covered mainly by flat to cubical epithelial-like cells. There were no blood cells in described spaces. An anastomosing fibrovascular partially hyalinized stromal network among tumor clefts and tubules was shown.
Findings of performed immunohistochemical analysis revealed the mesothelial immunophenotype of the tumor and were as follows: expressions of EMA (epithelial membrane antigen) as well as of cytokeratin 8 (LMW) were negative and faint mosaically positive reaction of cytokeratin MNF 116 (PAN) was observed. Expressions of CD34 and of FVIII were negative in tumor cells, but positive in the vascular stroma. Expression of vimentin was strongly positive and intense equally in tumor cells as well as in the stroma.
Presented adenomatoid tumor of Fallopian tube represents incidental finding and its practical importance is in differential diagnosis vs. tubal adenocarcinoma. These tumors may also be the cause of extrauterine pregnancy.
<PROGRAM>

IS THERE A CORRELATION BETWEEN HER-2/NEU STATUS AND GRADE OF THE PRIMARY BREAST CANCER?

Vučić Majda, Leniček Tanja, Čupić H, Tomas D, Krušlin B, Belicza M

Ljudevit Jurak University Department of Pathology, Sestre milosrdnice University Hospital, Zagreb, Croatia

HER-2/neu amplification/overexpression is a marker of poor prognosis in breast cancer. Patients whose tumors show overexpression of HER-2/neu have shortened disease-free and overall survival. The aim of this study was to determinate immunohistochemically HER-2/neu overexpression in 121 cases of primary breast cancer. In addition HER-2/neu status is correlated with hormone receptor status, grade and pTNM stage. The median age of both groups of HER-2/neu negative and HER-2/neu positive patients was about 60 years.  In all the HER-2/neu groups (negative, 1+, 2+ and 3+) estrogen and progesterone receptor were strongly positive in a high percentage of cases. Also in both groups of HER-2/neu negative and HER-2/neu positive invasive ductal carcinoma the most common grades were 2, but in HER-2/neu 3+ group, there were also 30% of cases having grade 3. Our results support a correlation between HER-2/neu overexpression and higher grade of breast cancer. The two most common pTNM stages were T1N0MX and T2N1MX. HER-2/neu receptor is an important therapeutic target and testing for HER-2/neu status is now recommended as a part of the routine breast cancer diagnosis. The role of new biomarkers, such as HER2/neu and clinical value of its determination must be confirmed by prospective clinical studies.

* Full article will be published in the next issue of Acta clinica Croatica

http://www.acta-clinica.kbsm.hr
<PROGRAM>

COLORECTAL CANCER TRENDS BY AGE AND SEX DISTRIBUTION, ANATOMIC SUBSITE AND SURVIVAL (1989 - 2002)

Tomas D, Belicza M, Baličević D, Brezovečki-Biđin Dora, Ciglar D, Leniček Tanja, Dokozić D, Dujmović A, Radotić Vladna, Gladić Vedrana, Krušlin B

Ljudevit Jurak University Department of Pathology, Sestre milosrdnice University Hospital, Zagreb, Croatia

Recent epidemiological studies have suggested that the anatomic distribution of colorectal carcinoma, especially in developed countries may have undergone a distal to proximal shift over several decades, which has been attributed variously to environmental and genetics factors as well as preventive measures. The aim of the study was to compare some colorectal cancer features (age and sex distribution, anatomic localization, and survival) during fourteen years, in order to assess the possible changing trends of these disease during the observed period and to compare observed data with our previous study published in 1985 as well as with similar colorectal cancer features reported worldwide. The mean age of patients with right-sided carcinomas was slightly higher than in patients with left-sided colorectal carcinomas (65.9 vs. 65.2). Sex distribution showed male predominance (57.3% vs. 42.7%). Males and females had similar anatomic distribution. Recto-sigmoid was the most common site (77.9%) followed by transverse colon cancers (6.8%), ascending colon cancers (6.5%), cancers in cecum (6.2%) and descending colon cancers (2.6%). In the last four years of the observed period  (1999 to 2002) the incidence of right-sided cancers was increased compared to the previous period. Our study showed a continuing trend of the increased incidence of right-sided carcinomas that is similar with reports from western European countries and North America.

* Full article will be published in the next issue of Acta clinica Croatica

http://www.acta-clinica.kbsm.hr
<PROGRAM>

TREND OF INCREASING AGE OF PATIENTS WITH INTRACRANIAL TUMORS BY HISTOLOGICAL SUBTYPES DURING 14-YEAR PERIOD (1989-2002)

Rumboldt Tihana, Vučić Majda, Talan-Hranilović Jasna, Belicza M

Ljudevit Jurak University Department of Pathology, Sestre milosrdnice University Hospital, Zagreb, Croatia

The aim of this study was to analyse distributions and proportions of intracranial tumors from the data obtained in the Ljudevit Jurak University Department of Pathology from 1989 to 2002. The data from our computer database were analyzed according to the histological diagnosis, patient age (four groups), and sex.
There were 2403 intracranial tumors, out of which 667 (27,8%) were malignant gliomas, 593 (24.7%) meningiomas, 328 (13.6%) pituitary adenomas, and 159 (6.6%) schwannomas. Metastatic intracranial tumors were diagnosed in 326 (13.6%) patients. We found a statistically significant trends of increasing incidence over time in the oldest age group (over 65 years of age) for meningiomas in women, and for malignant gliomas for both sexes.
The observed distributions and proportions of intracranial tumors are predominantly in accordance with the recent European, North American, and Japanese reported studies.

* Full article will be published in the next issue of Acta clinica Croatica

http://www.acta-clinica.kbsm.hr
<PROGRAM>

MELANOCYTIC NAEVI (COMPOUND AND SPITZ) – CASE REPORT

Bogoeva B, Kostadinova S, Ilievski B, Kocmanovska S

Institute of Pathology, Faculty of Medicine, Skopje, Macedonia

Benign melanocytic lesions comprise a wide spectrum of tumors including melanocytic naevi. Some of them have the features important for the differential diagnosis with malignant melanoma.
The aim of this study is to present specific variants of melanocytic naevi: compound and Spitz naevus (benign juvenile melanoma).
MATERIAL AND METHODS: We analyzed 30 cases of benign melanocytic lesions. In this series we found 3 compound and 1 Spitz naevus. Standard histopathological stainings of paraffin sections were done. Immunohistochemical stainings for S-100 protein and HMB-45 have been used.
CASE REPORT: Compound naevi were elevated, usually pigmented papules and polyps with average diameter of 8 mm. Histologically, almost all dermal elements were symmetrical and had homogenous growth patterns. There were typically round or epithelioid melanocytes in the papillary or superficial zones and fusiform melanocytes in the deeper portions. Cytoplasmic pigmentation varied from lesion to lesion. Immunohistochemically, melanocytes showed cytoplasmic and nuclear positivity for S-100 protein.
The Spitz naevus was discrete, well-demarcated, typically skin-colored macule with a diameter of 8 mm from an 8-year-old girl. Histologically it was a compound lesion. The epidermal portion had regular spaces composed of spindle and epithelioid melanocytes. Solitary melanocytes were presented in the higher portion of the epidermis (pagetoid spread). The dermal portion was composed of nests of spindle and large epithelioid cells. There were small groups and single melanocytes in the deeper spaces. The epithelioid melanocytes had abundant eosinophilic cytoplasm without melanin, being similar to myoblastoid cells. The nuclei were circular with small apparent nucleoli. The spindle cells were usually more uniform and followed the surface line of the adnexal epithelium. Mitotic figures were absent. Immunohistochemically melanocytes were positive for S-100 protein and weakly positive for HMB-45.
CONCLUSION: Some melanocytic naevi may have features associated with malignancy, which can make the histological distinction between them very difficult. The use of valid differential diagnostic criteria greatly increases the possibility of correct diagnosis.
REFERENCES: Barnhill RL. et al. Atypical Spitz Nevi/Tumors: Lack of consensus for diagnosis, discrimination from melanoma and prediction of outcome. Human Pathology 1999; 30:513-20.
Expert opinion. What criteria reliably distinguish melanoma from benign melanocytic lesions? Histopathology 2000; 37:464-72.
Okun MR. Histological demarcation of lateral borders: an unsupportable criterion for distinguishing malignant melanoma from Spitz naevus and compound naevus. Histopathology 1998; 33:158-62.
Fletcher CDM. Diagnostic histopathology of tumors. Churchill Livingstone, London. 1995. (Tumors of the Skin).
<PROGRAM>

SKIN METASTASIS OF HEAD AND NECK SQUAMOUS CELL CARCINOMA

Jakovina K, Đanić D, Jakovina Tratinčica, Fuštar-Preradović LJ

Department of Otorhinolaryngology and Cervicofacial Surgery, Department of pathology and forensic medicine, General Hospital Slavonski Brod

Metastasis to skin sites from squamous cell carcinoma of the mucosa of head and neck are very rare. Skin metastases may represent the first clinical evidence of the malignant disease, loco-regional recurrence or distant metastasis. Patients with skin metastases have very poor prognosis. Herein we report three new cases and the first patient with multiple skin metastases of the squamous cell carcinoma from the cervical part of the oesophagus above and below the level of the diaphragm.
<PROGRAM>