Consensus statement on screening, diagnosis, classification and treatment of endemic (Balkan) nephropathy.

Currently used diagnostic criteria in different endemic (Balkan) nephropathy (EN) centers involve different combinations of parameters, various cut-off values and many of them are not in agreement with proposed international guidelines. Leaders of EN centers began to address these problems at scientific meetings, and this paper is the outgrowth of those discussions. The main aim is to provide recommendations for clinical work on current knowledge and expertise. This document is developed for use by general physicians, nephrologists, urologist, public health experts and epidemiologist, and it is hoped that it will be adopted by responsible institutions in countries harboring EN. National medical providers should cover costs of screening and diagnostic procedures and treatment of EN patients with or without upper urothelial cancers.

Higher apoptotic cell rate in Balkan endemic nephropathy--stereologic analysis.

The aim of this study was to correlate apoptotic cell rate from different nephron segments between control group and groups of patients with Balkan endemic nephropathy (BEN). Kidney specimens of20 patients with clinically and epidemiologically confirmed BEN were compared with biopsy material of 10 patients (group I, non BEN) without glomerular or tubulointerstitial disease. Out of 20 patients with BEN, 10 suffered and died from BEN (group II, BEN) and 10 patients (group III, BEN/CV) suffered from BEN but died from cardiovascular disease. Patient age ranged from 40 to 50 years. The apoptotic cell rate was measured in proximal and distal tubules and in collecting ducts using the 40X objective with a calibrated eyepiece multipurpose M 42 test system according to Weibel. Comparison of all three nephron segments yielded statistically significant differences in volume density of apoptotic cells in proximal tubules and in collecting ducts among all three patient groups (non BEN vs. BEN, non BEN vs. BEN/CV and BEN vs. BEN/CV, P<0.001 all). Statistically significant difference in apoptotic cell rate was also found in distal tubules between non BEN and BEN groups and non BEN and BEN/CV groups, but not between BEN and BEN/CV groups. Our results showed a statistically significant increase of apoptotic cells in all three nephron segments in patients with BEN (BEN and BEN/CV) compared to control group. The highest number of apoptotic cells was found in distal tubules in the groups of patients with BEN and BEN with coexisting cardiovascular disease, suggesting that these cells might be most frequently and most severely injured in patients with BEN.

[Evaluation of morphologically determined apoptotic index]. / Evaluacija morfoloski utvrdenog apoptotickog indeksa.

Stimulated by the huge scientific interest in programmed cell death, the sophisticated but expensive methods of apoptosis detection, occasionally even in various stages of the apoptotic process, have been developed in the last two decades. These methods are quite demanding and time consuming, e.g., TUNEL, flow cytometry and its modifications, immunohistochemistry, hybridization in situ, etc. Irrespective of considerable technical improvements, these methods are associated with a relatively high rate of false-positive and false-negative results. Reading off the results obtained by these methods is mostly left to investigators inexperienced in morphological analysis. It is well known that the basic morphological characteristics of apoptotic cells and apoptotic bodies have been described on the basis of classic light microscopy of histologic hemalaun-eosin (HE) stained slides. As a rule, apoptotic cell has a shrunken, condensed, dark red cytoplasm and dark purple picnotic nucleus, and is surrounded by light, 'hollow' space (halo). Apoptotic bodies are fragments of the disintegrated apoptotic cell and each fragment of dense cytoplasm usually contains a fragment of disintegrated nucleus. It is known that, unlike necrosis, there is no inflammatory reaction in the vicinity of apoptotic cells and apoptotic bodies. During more than 25 years of research into apoptosis, the Zagreb Group for the Study of Apoptosis (Apoptosis Section, Department of Basic Medical Sciences, Academy of Medical Sciences of Croatia) have found it possible to determine the number of apoptotic cells and apoptotic bodies (apoptotic index) in daily routine on classic HE stained histologic slides by counting in 10 large fields under light microscope, following the methodology of mitotic index determination. Of course, the method is not absolutely accurate (the sophisticated and expensive methods mentioned above are not so either), but it is rapid and inexpensive. Thus, we believe that each pathologist should be professionally obliged to determine apoptotic index when assessing mitotic index. Expression of these indices as a mitotic-apoptotic ratio, e.g., 1:1, 1:2, 2:1, etc., may better reflect the growth potential of the examined, usually tumor tissue than determination of the mitotic index alone.

[Expression of bcl-2, Ki-67 and p-53 in uveal melanomas]. / Ekspresija bcl-2 onkoproteina, antigena Ki-67 i p-53 proteina u uvejalnim melanomima.

AIM: To investigate expression of proliferative markers bcl-2, Ki-67 and p-53 immunocomplexes in uveal melanoma cells; and to establish whether the intensity of expression correlates with the pathological level of invasion (pT), which would result in prognostic significance. METHODS: Thirty cases of primary uveal melanomas of two different levels of invasion (pT2 and pT3); indirect PAP immunoenzyme method and three step ABC/AP method. The intensity of the reaction was evaluated by a semiquantitative method as negative (-), weakly positive (+), modestly positive (++) and strongly positive (+++). Results were statistically analyzed by Fisher exact test for small examples. RESULTS: The reactivity of Ki-67 and p-53 protein was markedly stronger than bcl-2 in pT2 and pT3 stages. p-53 protein expression showed similar distribution for Ki-67 protein according to pT stages. CONCLUSION: The reactivity of all three markers was stronger in pT3 stage, especially of proliferation markers Ki-67 and p-53. In addition to the fact that they both are reliable indicators of the metastatic potential, Ki-67 is a better marker because there is a well established technique for measuring cell proliferation in all phases of the cell cycle.

Comparison of occurrence of upper urinary tract carcinomas in the region with endemic villages and non-endemic nephropathy region in Croatia.

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial renal disease of a still unknown etiology, associated with an increased frequency of urothelial carcinoma, particularly of the upper urinary tract (UUT). The aim of the study was to compare the occurrence of UUT carcinomas between Brodsko-Posavska Region (BPR) which is the region with endemic villages and the non-endemic region of Zagreb (ZG) in two six-year periods with a 20 year period separating the two, pointing out a possible difference in occurrence regarding war in Croatia (1991-1995). Comparing BPR and ZG regions we found a more then 5 times higher frequency of UUT carcinomas in BPR in the first period and more than 4.5 times higher frequency in the second period. Women in BPR were more frequently affected with UUT carcinomas.

Morphometric analysis of renal arteries in patients with renal cell carcinoma.

The aim of this study was to analyze morphometric parameters of renal arteries (longest diameter and tunica media thickness) in patients with renal cell carcinoma (RCC), to look into their relationship to tumor necrosis and to compare them with morphometric parameters recorded in a control group. We analyzed archival cases of RCC diagnosed in 2003 that also contained routinely sampled specimens of distal segments of renal artery. The control group consisted of specimens from both renal arteries obtained from 16 patients at routine autopsy during 2004-2005. Autopsy, as well as further histological analysis, did not disclose any malignant disease in the control group. Morphometric analysis of diameter and thickness of the renal artery tunica media was performed using Issa 3.1 software (Vamstek 2002, Zagreb, Croatia). The comparison of tunica media thickness showed that renal arteries from RCC cases were significantly thicker compared to distal parts of renal arteries in the control group (p=0.0002). Although renal artery samples from cases with necrotic tumor areas were thicker than those without tumor necrosis, the difference was not statistically significant. It is concluded that significantly thicker tunica media characterizes renal arteries in the group of patients with RCC when compared with the control group.

Periacinar retraction clefting in proliferative prostatic atrophy and prostatic adenocarcinoma.

AIMS: To evaluate the presence and extent of periacinar retraction clefting in proliferative prostatic atrophy and carcinoma in radical prostatectomy specimens. METHODS: Atrophic foci and neoplastic glands were analysed in specimens from 50 patients who underwent radical prostatectomy. Analysed atrophic glands were classified in two main groups, proliferative atrophy (PA) and proliferative inflammatory atrophy (PIA); each group was subclassified into simple atrophy (SA) and postatrophic hyperplasia (PAH). According to the presence and extent of periacinar retraction clefting, atrophic and neoplastic glands were classified as: group 1, glands without clefts or with clefts affecting 50% of the circumference in <50% of examined glands; and group 3, glands with clefts that affected >50% of the circumference in >or=50% of examined glands. RESULTS: Forty-four (88.0%) atrophic foci were without periacinar clefts or clefts were present in less than half of the gland circumference (group 1). In 6 (12.0%), atrophic foci clefts affected >50% of gland circumference (groups 2 and 3). Forty-five (90.0%) carcinomas were with clefts which affected more than 50% of gland circumference (groups 2 and 3); and in five carcinomas only, clefts were not found or affected <50% of gland circumference (group 1). CONCLUSION: Results indicate that periacinar retraction clefting represents a reliable criterion in differential diagnosis between proliferative atrophy and carcinoma.

Different types of atrophy in the prostate with and without adenocarcinoma.

OBJECTIVES: The purpose of this study was to evaluate, according to a classification proposed by a working group, the extent and type of atrophy lesions in radical prostatectomy specimens obtained from patients with prostatic carcinoma and benign prostatic hyperplasia (BPH), and to compare the prevalence and types of atrophy between two investigated groups. METHODS: Histologic analysis of 1096 slides from 50 patients with carcinoma and 277 slides from 31 patients with BPH was performed to evaluate, according to the new prostatic atrophy classification, the number of foci and type of atrophic lesions. RESULTS: Age, Gleason grade, and TNM showed no significant correlation with the number of proliferative atrophy (PA) and proliferative inflammatory atrophy (PIA) foci (p>0.05). PIA was significantly more frequent in prostates with carcinoma (1.63 vs 1.27 atrophic lesions per slide) (p<0.001), whereas PA displayed an increased frequency in BPH (2.28 vs 0.76 atrophic lesions per slide) (p<0.001). CONCLUSIONS: We confirmed that PA and PIA are common findings in prostates with and without carcinoma, but the question of whether inflammation produces tissue damage and PA or whether some other insult induces the tissue damage and atrophy directly, with inflammation occurring secondarily, is still unresolved.

Periacinar Clefting and p63 Immunostaining in Prostatic Intraepithelial Neoplasia and Prostatic Carcinoma.

The aim of the present study was to correlate the presence and extent of retraction clefting and the expression of p63 in neoplastic glands and glands with prostatic intraepithelial neoplasia (PIN) in needle core biopsies. We analyzed needle core biopsies from 28 patients with PIN and 41 patients with adenocarcinoma. Neoplastic glands and those with PIN were analyzed on high power field (400x) and classified in three groups according to the extent of clefting. Immunohistochemical staining was performed following Microwave Streptavidin ImmunoPeroxidase (MSIP) protocol on DAKO TechMate Horizon automated immunostainer. Periacinar retraction clefting was significantly more prominent in prostatic carcinoma compared to PIN (p<0.0001) and nonneoplastic glands (p<0.0001). There was no difference between normal glands and PIN regarding clefting (p=0.8064). p63 was positive around the whole circumference in 12 out of 28 cases with PIN, and discontinuously positive in remaining 16 PIN cases suggesting initial disruption of the basal cell layer. p63 immunostaining was also positive in all nonneoplastic glands, and negative in all carcinomas. We conclude that retraction clefting was associated with cancer and lack of basal cells, but not with PIN. The relationship between clefting and p63 immunostaining in prostatic cancer should be further analyzed.

[Determination of grade of urothelial carcinoma according to WHO 2004 classification]. / Odredivanje gradusa urotelnog karcinoma prema klasifikaciji Svjetske zdravstvene organizacije iz 2004.

Several classifications of transitional/urothelial cell tumors have been proposed during last few years in order to standardize nomenclature, and criteria for grading and invasion. They also aimed to avoid the term cancer for neoplasms that very rarely invade, recur, and/or cause death of the patient. As a result of these efforts a new WHO classification emerged in the year 2004. Instead of the term transitional, the use of urothelial neoplasms was recommended. In the group of noninvasive urothelial neoplasms, a new category of tumor of low malignant potential was added. Three-tier grading of papillary noninvasive tumor was substituted by low and high-grade category. Criteria for the grades are cited in the classification but are somewhat imprecise and difficult to apply. On the basis of the data from the literature and our own experience, in the transitional period we recommend the use of WHO 1973 simultaneously with the new one. Problems of the identification of lamina propria invasion are still not solved, and therefore the use of additional histochemical and immunohistochemical methods should be recommended in difficult cases.

The correlation between the tumor necrosis and renal artery changes in renal cell carcinoma.

Necrosis, cysts, hemorrhage, and calcification represent common findings in renal cell carcinoma. Different lesions, including arteriosclerosis or fibromuscular dysplasia, or both, may involve the main renal artery. This study analyzed the relationship between the presence and extent of necrosis in renal cell carcinoma with renal artery changes in a consecutive series of 112 patients (71 men, 41 women) with mean renal cell carcinoma of 7.7 cm (range, 2 to 20 cm). Necrosis was seen macroscopically and confirmed microscopically in 88 cases (78.6%), with 64 tumors having less than 50% and 24 more than 50% necrosis. Fibromuscular dysplasia was found in 41 patients (36.6%; 17 men, 24 women) and atherosclerotic changes in 21 patients (18.8%; 18 men, 3 women). The results suggest that necrosis of renal cell carcinoma was significantly more common in women with associated fibromuscular dysplasia (especially type I) and men with atherosclerotic changes of renal artery.

[Role of yeasts in diabetic foot ulcer infection]. / Uloga kvasaca u infekciji rane dijabetickog stopala.

AIMS: The aim was to assess the incidence of isolation of individual fungal species and interpret the meaning of fungal isolates from foot ulcers of 509 diabetic outpatients using mycologic and histopathologic methods. Another aim was to explore risk factors for the development of fungal infections in foot ulcer. METHODS: Fungus isolation was made on selective media and their identification by standard mycologic methods. Histopathologic diagnosis of fungal ulcer infections was made on PAS-stained histopathologic preparations and imprint preparations (PAS and Papanicolaou staining) of foot wound biopsy specimens. RESULTS: Fungal and mixed foot ulcer infections were found in 14.9% of diabetic patients. In 33.8% of patients, these infections were confirmed by a finding of fungal elements in histopathologic preparations of ulcer biopsy specimens, as follows: in 16.9% of patients, by finding fungal elements in imprint preparations of ulcer biopsy specimens and by isolation fungus from the swab of the same ulcer; in 2.3% by fungus isolation from ulcer biopsy specimens; in 36.9% by fungus isolation from ulcer swabs in pure culture and/or in a large number of colonies and/or from several ulcers on the foot of the same patient. More than 89% of patients had a single foot ulcer with fungal or mixed infection, big toe and the plantar-metatarsal region in one foot or both feet being the most common sites of ulcer. Fifteen species from the genera Candida, Cryptococcus, Trichosporon and Rhodotorula were the causative agents of fungal and mixed foot ulcer infections. C. parapsilosis (in 61.5% of patients), and C. albicans and C. tropicalis (in 10.8% of patients each) were the most common causes of these infections. The presence of yeasts and/or dermatophytes in the toe web of the same or other foot, or of both feet, did not influence the incidence of fungal and mixed foot ulcer infections. Patient sex and age, type and length of diabetes, or clinical picture of diabetic foot did not affect it either. In IDDM patients, the risk factor for the development of fungal and mixed foot ulcer infections was ulcer infection lasting for more than 13 weeks, whereas in NIDDM patients the length of ulcer infection did not contribute to the incidence of fungal and mixed foot ulcer infection. DISCUSSION: Our results and other reports suggest that Candida species are the most common fungal isolates (between 93.2% and 100% of all fungal isolates) from diabetic foot ulcer, with C. parapsilosis being the most common causative agent of fungal and mixed infection. From diabetic foot ulcer, bacterial isolation was 5 times as common as that of yeasts (327 vs. 65 patients). Nevertheless, this investigation showed fungal isolates, originating not only from a primarily sterile ulcer sample (biopsy specimen) but also from foot ulcer swabs to be the causative agents (not ulcer colonizers or contaminants) of the foot ulcer infection. The pathogen c effect of yeasts in foot ulcer is indicated by the severity of clinical finding, chronic course of infection, and infection progression despite antibiotic therapy. Equally indicative are microbiologic diagnostic parameters (isolation in pure culture, and/or isolation in a large number of colonies, and/or isolation from several ulcers in the foot of the same patient). CONCLUSIONS: In diabetic patients at highest risk of developing fungal and mixed foot ulcer infections (IDDM patients with ulcer infection persisting for more than 13 weeks, and NIDDM patients with the clinical picture of deep ulcer and abscess in the plantar region, irrespective of the duration of ulcer infection), routine bacteriologic diagnosis should be supplemented with targeted mycologic and histopathologic methods.

Synchronous anorectal melanoma.

Anorectal melanoma is a very rare tumor with poor prognosis. Rectal bleeding is the most frequent symptom and surgical treatment ranges from local excision to radical abdominoperineal resection. We report a case of a 75-years-old male patient who presented with a history of recurrent rectal bleeding, and whose histopathological diagnosis was melanoma. Macroscopically, we found two distinct tumors in anorectal region, 0.5 cm and 1.5 cm from dentate line. The first one was pedunculated, on a thin stalk, measuring 1 cm in greatest diameter, and the second one was sessile and nodular measuring up to 2.8 cm in largest diameter. Microscopic examination and immunohistochemical analysis of both tumors confirmed the diagnosis of melanoma. This case represents multiple synchronous primary melanoma of the anorectal region, with a possibility that one of the lesions is primary melanoma and the second one is a satellite lesion.

Of mice and men: teratomas and teratocarcinomas.

Teratomas and teratocarcinomas are tumors containing tissue derivatives of all three germ-layers. They can be induced by transplantation of animal embryos to ectopic microenvironment. Development of malignant teratocarcinomas depends on embryonic stage, species-specificity and immunological competence of the host. In the man, teratomas and teratocarcinomas usually represent a subtype of germ-cell tumors but sacrococcygeal teratomas arise from the remnants of the pluripotent primitive streak. Undifferentiated embryonal carcinoma (EC) cells are responsible for the malignancy of experimental mouse teratocarcinomas. Mouse EC cells injected to the adult give rise to tumors and upon injection to early embryos to differentiated tissues--thus resembling normal mouse embryonic stem cells (mESC). Epigenetic changes rather than mutations are associated with transformation of mESC to EC cells. Human EC and ES cell-lines (hESC) contain chromosomal abnormalities and can form teratocarcinoma after transplantation. ES cells are among those proposed for cell replacement therapy in the man. Suicide gene introduction should be recommended prior to their use in vivo to ablate them in case of malignant transformation.

TP53, Bcl-2 and growth hormone receptor expression in cutaneous squamous cell carcinoma.

The incidence and prevalence of premalignant and malignant skin lesions including squamous cell carcinoma (SCC) of the skin are increasing worldwide. The aim of the study was to determine TP53, Bcl-2 and growth hormone receptor (GHR) expression in SCC and to investigate relative importance of these proto-oncogenes in its biological behavior. Expression of TP53, Bcl-2 and GHR was determined by immunohistochemistry in 27 SCC specimens and adjacent perilesional skin. The relative proportion of immunoreactive cells was counted with semiquantitative method. TP53 positivity was detected in 24 (89%), Bcl-2 in 18 (67%) and GHR in 25 (93%) of 27 SCC specimens investigated. In comparison with perilesional skin, TP53 and GHR positivity was significantly increased, and Bcl-2 positivity significantly decreased in SCC. Increased TP53 expression in SCC lesions implies that Tp53 mutation is an early and crucial event in its development. Increased GHR expression suggests a role of growth hormone in the development of SCC.

[Second primary malignant tumors in patients with primary renal cell carcinoma]. / Ucestalost drugih primarnih tumora u bolesnika s primarnim karcinomom bubreznih stanica.

INTRODUCTION: Among multiple primary cancers in the same patient, renal cell carcinoma occurring either synchronously or metachronously is one of the most common. AIM: The aim of the study was to determine the frequency of second primary malignant tumor in patients with primary renal cell carcinoma. MATERIAL AND METHODS: Between 1992 and 2000, 447 patients underwent nephrectomy for primary renal tumor at the Department of Urology, Sestre milosrdnice University Hospital. Out of 447 patients 310 were registered at Cancer Registry of the Republic of Croatia. There were data on 297 patients with renal cell carcinoma, 197 male and 104 female patients, age group 24-91 (mean 59.9) years. RESULTS: Data on 24 (8.1%) patients with second primary malignant tumors were found in Hospital Registry and Croatian Cancer Registry during the study period. There were 13 male patients, age range 47-80 (median 65.1) years, and 11 female patients, age range 51-70 years (median 61.1) years with 26 second primary tumors. One male patient presented with four different primary tumors (kidney, prostate, urinary bladder and colon). The patients most commonly presented with prostate and colon carcinoma. The second malignant primary tumors occurred most commonly as antecedent to renal cell carcinoma, i.e. in 11 (42.3%) cases. CONCLUSION: Our results indicate that during the clinical follow-up of patients treated for primary renal cell cancer, a possibility of second primary cancers should always be considered. In order to upgrade detection of multiple cancer, the quality and completeness of cancer notification and registration from hospitals as well as collaboration between the National Cancer Registry and hospital cancer registries should be improved.

Multiple metastases to the small bowel from large cell bronchial carcinomas.

AIM: Metastases from lung cancer to gastrointestinal tract are not rare at postmortem studies but the development of clinically significant symptoms from the gastrointestinal metastases is very unusual. METHODS: Formalin-fixed, paraffin-embedded tissues were cut into 5 microm thick sections and routinely stained with hematoxylin and eosin. Some slides were also stained with Alcian-PAS. Antibodies used were primary antibodies to pancytokeratin, cytokeratin 7, cytokeratin 20, epithelial membrane antigen, vimentin, smooth muscle actin and CD-117. RESULTS: We observed three patients who presented with multiple metastases from large cell bronchial carcinoma to small intestine. Two of them had abdominal symptoms (sudden onset of abdominal pain, constipation and vomiting) and in one case the tumor was incidentally found during autopsy. Microscopically, all tumors showed a same histological pattern and consisted almost exclusively of strands and sheets of poorly cohesive, polymorphic giant cells with scanty, delicate stromas. Few smaller polygonal anaplastic cells dispersed between polymorphic giant cells, were also observed. Immunohistochemistry showed positive staining of the tumor cells with cytokeratin and vimentin. Microscopically and immunohistochemically all metastases had a similar pattern to primary anaplastic carcinoma of the small intestine. CONCLUSION: In patients with small intestine tumors showing anaplastic features, especially with multiple tumors, metastases from large cell bronchial carcinoma should be first excluded, because it seems that they are more common than expected.

Correlation of periacinar retraction clefting in needle core biopsies and corresponding prostatectomy specimens of patients with prostatic adenocarcinoma.

One of the underemphasized supportive criteria for the diagnosis of prostatic cancer is the presence of retraction clefting around neoplastic glands. We analyzed a series of 152 prostatic cancer cases to determine the frequency, extent, and correlation of periacinar retraction clefting between needle core biopsies (NCB) and corresponding matched radical prostatectomy (RP) specimens. Clefting was significantly more frequent in neoplastic compared to nonneoplastic acini in NBC and RP (p<0.05). There was no significant difference in the frequency of retraction clefting in neoplastic acini between NCB and corresponding RP (p>0.05). We have also found a concordance in matched RP and NCB (Kappa=0.582). We conclude that periacinar retraction clefting appears more frequently in neoplastic acini and could serve as a reliable criterion in the diagnosis of prostatic adenocarcinoma.