Prognostic values of ETS-1, MMP-2 and MMP-9 expression and co-expression in breast cancer patients.

The aim of this study was to analyse expression of ETS-1 protein and two gelatinases (MMP-2 and MMP-9) and their possible prognostic value in breast carcinoma patients, as well as correlation of their expression with other known prognostic factors such as tumor size, grade, vascular invasion, steroid receptor values, HER2 values and proliferative index. The expression of MMP-2, MMP-9 and ETS-1 was immunohistochemicaly analysed in 121 consecutive primary breast carcinoma patients who underwent surgery at the Clinical Hospital Centre Zagreb during 2002. Three representative areas from each tumor paraffin blocks were taken and arranged on a recipient paraffin block with predefined coordinates for simultaneous analyses of multiple tissue samples (TMA). ETS-1, MMP-2 and MMP-9 expression and co-expression were correlated with other clinico-pathological parameters and based on the available clinical follow up data survival analysis was performed. The ETS-1 protein is found to be expressed in tumor cell nuclei and cytoplasm as well as in stromal lymphocytes, fibroblasts and endothelial cells. MMP-2 and MMP-9 were found to be expressed in cytoplasm of both, tumor and stromal cells. For our analysis only tumor cell expression was used for statistical analysis. We found 56,2% ETS-1 positive tumors, 77,7% were MMP-2 positive, and MMP-9 was expressed in 90% of primary breast carcinomas. There were no significant correlations between MMP-s expression and other patohistological prognostic factors, but expression of ETS-1 was significantly correlated with higher tumor size and grade, as well as with negative steroid receptors. Co-expression of MMP-2, MMP-9 and ETS-1 was found in 40,5 % of tumors, and more commonly was found in tumors larger than 2 cm, high grade tumors, and steroid receptor negative tumors. In univariate analysis, statistically significant negative impact on overall survival (OS) had tumor size, nuclear and tumor grade, ETS-1 expression in tumor cells, co-expression of ETS-1 either with MMP-2 or MMP-9, as well as co-expression of ETS-1, MMP-2 and MMP-3. Disease free survival (DFS) was significantly shorter in patients with tumors greater than 2 cm, ETS-1 positive tumors, ETS-1 and MMP-2 or MMP-9 co-expressed tumors, and additionally in tumors with ETS-1, MMP-2 and MMP-9 co-expression. These results suggest that expression of ETS-1 as well as MMP-2, MMP-9 and ETS-1 co-expression might be used as a poor prognostic factor in breast cancer patients.

Mutation pattern of KRAS and BRAF oncogenes in colorectal cancer patients.

The aim of this study was to identify KRAS and BRAF gene mutations in colorectal cancer patients and to assess whether they are linked with clinicopathological features. The results of KRAS and BRAF mutation analysis could be used in the selection of patients for anti-EGFR therapy. All specimens were obtained during routine surgery of patients with colorectal carcinoma. The diagnoses were established by standard procedures and confirmed histopathologically. After DNA extraction, KRAS mutations were analyzed using quantitative real-time PCR and BRAF mutations were analyzed using real-time PCR by fluorescence melting curve analysis. Our results show that KRAS gene mutations were detected in 35.6% samples and the most frequent mutation was Gly12Val. BRAF gene mutation Val600Glu was detected in 8.5% samples. Statistical analysis revealed a significant association between the KRAS mutation and Dukes' stage (p=0.034), with the lowest frequency in Dukes'A, and between the KRAS mutation and histological grade (p=0.044), with no KRAS mutation found in poor differentiated tumors. The first data about KRAS and BRAF mutational status in the sample of Croatian population with colorectal cancer shows that the incidence of KRAS and BRAF mutations is within generally valid limits. Prospective studies are to be continued in order to determine whether these mutations contribute to progression of colorectal cancer.

Expression of Toll-like receptor 4 and beta 1 integrin in breast cancer.

Toll-like receptor (TLR) 4 signaling pathway has been shown to support tumor cell growth in vitro and in vivo. Its stimulation on breast cancer cell lines induces ß1 integrin and promotes tumor invasiveness. However, its role in predicting clinical behavior of tumor is not yet clarified. Therefore, we investigated TLR4 and ß1 integrin expression on 133 primary breast cancer samples by immunohistochemistry and correlated it with overall survival and disease-free survival of patients as well as with clinicopathological characteristics of the tumor. We found higher ß1 integrin expression in invasive lobular cancer in comparison with other tumor types. No significant association of TLR4 and ß1 integrin expression with overall survival or disease-free survival was seen. Therefore, we conclude that expression of these markers is of biological interest but appears to be of little additional use as predictive clinical marker.

The TP53 gene polymorphisms and survival of sporadic breast cancer patients.

The TP53 gene polymorphisms, Arg72Pro and PIN3 (+16 bp), can have prognostic and predictive value in different cancers including breast cancer. The aim of the present study is to investigate a potential association between different genotypes of these polymorphisms and clinicopathological variables with survival of breast cancer patients in Croatian population. Ninety-four women with sporadic breast cancer were retrospectively analyzed. Median follow-up period was 67.9 months. The effects of basic clinical and histopathological characteristics of tumor on survival were tested by Cox's proportional hazards regression analysis. The TNM stage was associated with overall survival by Kaplan-Meier analysis, univariate, and multivariate Cox's proportional hazards regression analysis, while grade was associated with survival by Kaplan-Meier analysis and univariate Cox's proportional hazards regression analysis. Different genotypes of the Arg72Pro and PIN3 (+16 bp) polymorphisms had no significant impact on survival in breast cancer patients. However, in subgroup of patients treated with chemotherapy without anthracycline, the A2A2 genotype of the PIN3 (+16 bp) polymorphism was associated with poorer overall survival than other genotypes by Kaplan-Meier analysis (P = 0.048). The TP53 polymorphisms, Arg72Pro and PIN3 (+16 bp), had no impact on survival in unselected sporadic breast cancer patients in Croatian population. However, the results support the role of the A2A2 genotype of the PIN3 (+16 bp) polymorphism as a marker for identification of patients that may benefit from anthracycline-containing chemotherapy.

TP53 gene polymorphisms and breast cancer in Croatian women: a pilot study.

A case-control retrospective association study was conducted to investigate a possible association of the TP53 polymorphisms, Arg72Pro and PIN3 (+16bp), with sporadic breast cancer in Croatian women. Ninety-five women with breast cancer and 108 age-matched healthy women were analyzed. Arg72Pro polymorphism was detected by TaqMan essay. For designation of PIN3 (+16bp) polymorphism DNA amplification was performed by the polymerase chain reaction (PCR) while the PCR products were detected by capillary electrophoresis. Homozygous genotype of minor allele of the PIN3 (+16bp) polymorphism was associated with sporadic breast cancer (OR = 2.15, 95% confidence interval [CI] 1.80-2.56, p = 0.006). For Arg72 polymorphism, the odds ratio for breast cancer of ArgPro versus reference genotype ArgArg was 0.55 (95% CI 0.30-1.02, p = 0.039) suggesting the protective effect. Although different haplotypes did not influence the susceptibility to the disease, the joint occurrence of genotype combination ProPro/A2A2 frequent in cases, was associated with sporadic breast cancer (OR = 2.20, 95% CI 1.89-2.56, p = 0.021). The study provides evidence of the association of the TP53 gene polymorphisms Arg72Pro and PIN3 (+16bp) with sporadic breast cancer in the Croatian population.

Expression of cyclooxygenase-2 in fine-needle aspirates from breast carcinoma and benign breast diseases.

Numerous studies have demonstrated that the levels of cyclooxygenase (COX), the enzyme that catalyzes the conversion of arachidonic acid to prostaglandin H(2), and of prostaglandins are higher in various tumors and cells during inflammation than in normal tissues. The aim of the present study was to analyze whether COX-2 isoform expression was noticeably higher in fine-needle aspirates (FNA) from breast carcinoma than in FNA from fibroadenoma and fibrocystic breast tissue. COX-2 expression was detected by immunocytochemical (IC) staining and was analyzed by microscopic scoring and computer gray-scale analysis. Evaluation of COX-2 IC positivity in FNA from three groups of patients (nine with breast carcinoma, nine with fibroadenoma, eight with fibrocystic breasts) revealed high COX-2 IC positivity in the majority of patients with breast carcinoma and low or absent COX-2 IC positivity in patients with fibrocystic breast changes. In addition, low or medium COX-2 IC positivity was found in the majority of patients with fibroadenoma, only three of these patients having high COX-2 IC positivity.

Extranodal sinus histiocytosis (Rosai-Dorfman disease) of the brain parenchyma.

Rosai-Dorfman Disease (RDD) is an idiopathic histiocytic proliferation affecting lymph nodes. Although extranodal involvement has been reported in diverse sites, manifestation in the central nervous system (CNS) is extremely rare, particularly in the brain parenchyma. A 39-year-old male presented with an isolated well-circumscribed brain mass in the right temporal lobe, preoperatively thought to be a meningioma. Histology and immunohistochemistry confirmed that the lesion was RDD. The intraparenchymal brain location of RDD appears to have a benign course. Although the adjuvant therapy is a treatment of choice, surgical resection seems to be the appropriate treatment modality. From the clinical point of view RDD might be an important intracerebral entity because it may mimic other lesions, particularly other histiocytic disorders.

Benign mesenchymal liver hamartoma in an adult male: a case report and review of the literature.

Mesenchymal hamartoma of the liver is an uncommon benign lesion seen almost exclusively in children younger than two years of age. The first case was reported in 1903, and until now fewer than 200 cases have been reported. A 38-year-old male complaining of dull subcostal pain lasting for almost 10 months was found on abdominal ultrasound to have a focal lesion of the liver. He was referred to our Unit where imaging procedures (ultrasound and computed tomography) revealed a solid lesion, 8 centimeters in diameter, located in the IVb and V segments of the liver. There were no clear margins between the lesion and the gallbladder wall. Alpha-fetoprotein and carcinoembryonic antigen were within normal limits and carbohydrate antigen 19-9 was minimally elevated. There was no evidence of metastatic disease. Ultrasound-guided biopsy of the lesion was done and pathology report of a biopsy specimen suggested that the tumor was probably a benign mesenchymal hamartoma. The patient underwent a formal bi-segmentectomy (IVb and V segments) with cholecystectomy. Definitive pathology report confirmed the diagnosis of a benign mesenchymal hamartoma.

Long-term results of conventional-dose salvage chemotherapy in patients with refractory and relapsed Hodgkin's disease (Croatian experience).

BACKGROUND: The aim of this study was to analyze outcome of patients with Hodgkin's disease (HD) in whom first-line chemotherapy with mustine/vincristine/procarbazine/prednisone (MOPP) had failed. PATIENTS AND METHODS: From January 1982 to December 1989 among 210 patients treated with MOPP and radiotherapy to initial bulky sites, 65 patients were primary refractory to or relapsed after initial treatment. RESULTS: Twenty-nine of 65 patients (44%) were primary refractory to initial chemotherapy, 20 relapsed within 12 months after complete remission (CR) and 16 relapsed after CR that lasted more than 12 months. Patients with primary refractory HD and early relapse (<12 months after CR) were treated with doxorubicin/bleomycin/vinblastine/darcarbazine. In patients with late relapse (>12 months after CR) MOPP was repeated. The median follow-up for all patients was 115 months. The overall response rate was 63%. Thirty-three patients (51%) achieved a second CR and eight patients (12%) partial response. Remission rate was greatest in patients with late relapse (CR >12 months) (75 versus 55% for early relapse versus 35% for primary refractory HD) (P <0.01). At 10 years, overall and failure-free survival rates were 21 and 16%, respectively. Patients who were in first remission longer than 12 months had a superior overall survival (37 versus 18% for early relapse) and failure-free survival (24 versus 10% for early relapse). No patient with primary refractory HD was alive beyond 52 months after initial treatment failure (P <0.01). Main prognostic factors were duration of the first remission and tumor bulk at relapse. CONCLUSIONS: Our results confirm previous observations that a significant proportion of patients with HD who experience induction treatment failure cannot be cured with conventional treatment and probably need more aggressive therapy.

Atopic dermatitis: immunophenotyping of inflammatory cells in skin lesions.

BACKGROUND: The ever increasing incidence of atopic dermatitis (AD) has stimulated many researchers to use various diagnostic procedures to obtain new data to help elucidate the pathogenesis of the disease. AIMS: To perform cell immunophenotyping and to analyze the presence of inflammatory cell-surface markers in the biopsies of skin lesions from 15 AD patients and five healthy subjects. METHODS: Immunohistochemical analysis was performed in a group of AD patients and compared with that in a control group of healthy subjects. Avidin-biotin immunoperoxidase staining of paraffin-embedded, 4 microm skin sections, with semiquantitative counting of cells labeled with anti-CD3, anti-CD8, anti-CD20, anti-HLA-DR (HLA, human leukocyte antigen), and anti-immunoglobulin E (anti-IgE) primary antibodies, was used. RESULTS: The results of AD skin analysis showed a greater infiltration of CD3+ lymphocytes, especially of CD4+ subtype, compared with CD8+ lymphocytes. AD skin biopsy specimens also showed a higher intraepidermal HLA-DR+ Langerhans' cell count, the presence of HLA-DR on lymphocytes in the dermis, and higher intraepidermal expression of IgE+ cells compared with healthy controls. CONCLUSIONS: A statistically significant difference (P < 0.05) was found between the two groups for intradermal and intraepidermal CD3, CD4, and HLA-DR, intradermal CD8, and intraepidermal IgE+ cells. Immunophenotyping was found to be a useful diagnostic method in AD patients.

Trastuzumab in the treatment of advanced breast cancer: single-center experience.

A significant number of women with advanced breast cancer fail to respond to standard-dose chemotherapy. From the beginning of 1999, 17 women with HER2 positive advanced breast cancer received Herceptin as monotherapy or in combination with paclitaxel or other non-anthracyclines. Eight (47%) women previously received high-dose chemotherapy followed by haematopoiesis stem cell rescue. Three women received Herceptin alone, eleven Herceptin plus paclitaxel and three Herceptin and some of the other non-anthracyclines (CCNU, cisplatin and gemcitabine). In the group of patients who received Herceptin monotherapy, one has partial response (PR), one stable disease (SD) and in the third patient the disease progressed. Out of three patients who received Herceptin in combination with other non-anthracyclines, two have SD and one progressed. In the group of 11 women who received Herceptin + Taxol, 7 (64%) patients achieved PR, 2 (18%) SD, and 2 (18%) had disease progression. Grade 3-4 neutropenia has been observed in four (23%) women. Febrile neutropenia was observed in two cases and resolved completely when antibiotics were introduced. Other grade 3 toxicity that has been noted is peripheral neuropathy in three (18%) patients, diarrhoea in four (23%) and onycholysis in one (6%). Serial heart ultrasound showed no significant decline in left ventricular ejection fraction. According to our preliminary experience, Herceptin therapy showed promising results in women with metastatic breast cancer.

Assessment of the prognostic impact of the Epstein-Barr virus-encoded latent membrane protein-1 expression in Hodgkin's disease.

We have examined expression of the Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1) in the malignant Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin's disease (HD) and its impact on response to treatment and on survival. Paraffin tissue from 100 adult immunocompetent patients with HD were analysed using immunohistochemistry to identify LMP1 expression. According to the Rye classification, 8% of patients had lymphocyte predominance (LP) subtype, 48% had nodular sclerosis (NS) disease, 37% were of the mixed cellularity (MC) subtype and 7% were of the lymphocyte depletion (LD) subtype. During the five year follow-up period 27 patients died and 74 patients achieved a complete remission. Patients with LD subtype were older (P = 0.03), less frequently achieved complete remission (P = 0.01), had shorter disease-free survival (P = 0.01) and overall survival (P = 0.002) compared with the other subtypes of HD. LMP1 expression was found in the tumour cells of 26% of cases of HD. LMP1 expression was less common in NS disease than in the other subtypes (P = 0.05), whereas an association between MC subtype and LMP1 expression was not found (P = 0.22). Using the log-rank test there were no differences in overall survival or disease-free survival based on EBV status either when all patients were analysed or when LD and LP subtypes were excluded. However, the presence of EBV was associated with significantly longer disease-free survival in the subgroup of patients 35 years old, but this difference was not statistically significant (P = 0.11). A similar trend was observed in patients > 50 years old. Analysis of the impact of LMP1 expression in patients who had different stage and B symptoms status showed that expression of EBV was associated with longer disease-free survival (P = 0.019) in early stage (1 + 2) patients without B symptoms. Significant differences in the other subgroups based on EBV status was not found. Factors adversely affecting the likelihood to achieve a complete remission were: absence of LMP1 expression (OR 6.4, 95% Cl 1.07-38.5, P = 0.04), age (OR 1.68, 95%Cl 1.15-2.5, P = 0.007) and subtype of HD (OR 3.32, 95%Cl 1.11-9.94, P = 0.03). Age and subtype of HD had an independent impact on overall survival (P = 0.01). We conclude that expression of LMP1 in HRS cells has a favourable impact on prognosis for HD patients, but that this effect may be restricted to young adult patients and those with early stage disease.

Inflammatory pseudotumor of the trachea.

Primary tracheal tumors are extremely rare, and the majority of them are malignant. Inflammatory pseudotumor is a benign, tumorlike lesion, most likely of a reactive nature. Its basic morphologic characteristic is spindle cell (myoblasts and fibroblasts) proliferation with a variable number and type of inflammatory cells. A case of intratracheal inflammatory pseudotumor in a 14-year-old boy is presented together with a review of all similar lesions in the available literature. The discussion includes the presentation of tracheal tumors, their basic morphologic and immunohistochemical characteristics, and treatment modalities that are available. The surgeon must exercise caution not to perform radical surgery based on the initial pathologic diagnosis from the intraoperative frozen section, because the prognosis of these benign lesions generally is excellent. This is the second reported case of intratracheal inflammatory pseudotumor successfully endoscopically vaporized using a CO(2) laser, which is an excellent alternative in cases in which surgical treatment is feasible. J Pediatr Surg 36:631-634.

Comparison of histopathologists' workloads in two pathology departments in Croatia.

AIM: To calculate workloads of Departments of Pathology and staff histopathologists at two Croatian University Hospitals by means of the Suvarna and Kay's Kim Unit (KU) activity index. METHOD: The total number of specimens and KU activity/year were calculated to compare staff pathologists' workload in two Pathology Departments, one at Split and the other at Zagreb University Hospital. The individual specimen types were assigned a difficulty score on a scale of 1-5 units (KUs), depending on the time needed for specimen dissection and macroscopic description, number of sections and stains required, and time spent on microscopy of an "average case". KU activity was calculated for all pathologists individually in terms of histology (1-5 KUs), autopsy (10 KUs), supervision of residents, and outside consultations (2 KUs). RESULTS: According to calculated Kim Unit activity index, pathologists' workload in two investigated pathology departments was not equally spread. In the Split Department of Pathology the distribution of workload was more uniform than in the Zagreb Department of Pathology. The average workload of both institutions was 4,562 KUs. CONCLUSION: KU activity index is a very useful method for assessing average pathologists' workload. It may be also used in hospital administration for predicting changes in service and number of working pathologists.

Event-related potentials in medical workers with long-term exposure to xylene.

The effects of chronic exposure to xylene on cognitive ability were studied in a group of 35 medical workers occupationally exposed to low-level concentrations of xylene for at least five years by using event-related potentials (ERPs), and compared with a control group of 21 subjects. The exposure to xylene was confirmed through determination of m-methylhippuric acid, a reliable biological indicator of xylene exposure, in pre- and post-shift urine. A dose-effect relationship between log m-methylhippuric acid and ERP log latency (p = 0.032), and the ERP amplitude (p = 0.047) was statistically significant. The group of medical workers showed significantly longer ERP log latency (p < 0.001) than did the control group with respect to factors of exposure to smoking, education and age as covariates. For the ERP amplitude the difference was found not to be significant (p = 0.263), probably due to high between subject variability. The cognitive impairment may occur in workers chronically exposed to xylene.

Histopathological features of gastritis before and after treatment for Helicobacter pylori.

AIM: To assess five main histological features of gastritis in gastric mucosa colonized with Helicobacter pylori before and after the treatment. METHODS: Histologic assessment of H. pylori-associated gastritis was performed according to the Sydney classification before and after the treatment in 97 patients. Two additional parameters - the presence of lymphocytic aggregates and coccoid forms of bacteria - were also analyzed. Helical and coccoid forms of H. pylori were detected by immunohistochemistry in biopsies after the treatment. RESULTS: Whereas acute epithelial damage was quickly repaired, some of the local responses to bacteria, e.g., lymphoid aggregates and intestinal metaplasia, persisted after treatment. Higher H. pylori and cocci density was found before and after treatment in patients with intestinal metaplasia (p=0.020). Correlation between H. pylori and mucosal atrophy was found only after treatment (p=0.009). Immunohistochemical staining was more sensitive in detecting of H. pylori than Giemsa staining (p=0.007) in cases where, using only Giemsa staining, it was not possible to distinguish coccoid forms of H. pylori from other cocci. CONCLUSION: After treatment, H. pylori-associated gastritis showed reduction of acute and chronic inflammation, but lymphoid aggregates and intestinal metaplasia persisted. Immunohistochemistry of different forms of H. pylori may be a valuable technique in monitoring the success of the treatment.

Mutual predictive value of c-erbB-2 overexpression and various prognostic factors in ductal invasive breast carcinoma.

AIMS AND BACKGROUND: Breast carcinoma is a heterogeneous disease, the prognosis of which correlates with various prognostic factors. The aim of this study was to assess the prognostic significance of c-erbB-2 overexpression in breast carcinoma patients in association with other known prognostic factors. METHODS & STUDY DESIGN: The relationship between immunohistochemical expression of the c-erbB-2 oncoprotein and various established prognostic factors such as tumor size, axillary node status, estrogen and progesterone receptor status, DNA ploidy, proliferation index, cathepsin D expression and histological grade in invasive ductal breast carcinoma is presented in this study. RESULTS: Of the 93 ductal invasive carcinomas 22 (23.7%) were grade I, 51 (54.8%) grade II, and 20 (21.5%) grade III, and the majority (78: 83.9%) were 2-5 cm in diameter. Tumor metastases were identified in one or more lymph nodes in 55 (59.1%) patients, the remaining 38 (40.9%) patients being lymph node negative. According to the DNA histograms 40 (43.0%) tumors were aneuploid and 53 (57.0%) were diploid, and the majority of tumors had more than 4% of cells in the S phase of the cell cycle (83.9%). Expression of c-erbB-2 as shown by immunohistochemical intense membrane staining was present in 49 (52.7%) tumors. Cathepsin D-positive cytoplasmic granular staining and cathepsin D-positive stromal macrophages were found in 60 (64.5%) and 72 (77.4%) tumors, respectively. Univariate analysis showed that overall survival correlated significantly with axillary lymph node involvement and with estrogen and progesterone receptor status for each of the receptors separately and for their coexpression, and only marginally with c-erbB-2 overexpression. In mulitivariate analysis only axillary lymph node metastases and coexpression of estrogen and progesterone receptors were found to be independent and significant prognostic factors. CONCLUSIONS: When patients were stratified according to c-erbB-2 expression it was shown that those with c-erbB-2 overexpression and grade II tumors, tumor size greater than 2 cm, high content of aneuploid cells and cathepsin D-positive stromal macrophages had a shorter long-term survival than c-erbB-2 negative patients.

[Skin cell culture: utilization in plastic surgery and laboratory studies]. / Kultura koznih stanica: primjena u plasticnoj kirurgiji i laboratorijskom istrazivanju.

Skin is the largest organ of the human body (8-10 kg, 1.5-2.0 m2, 10(11) cells of epidermal, mesenchymal and neural origin). Although endowed with remarkable regeneration ability, the recovery after major injuries viz. burns requires appropriate surgical treatment, temporary coverage of defects and supportive measures. Large defects are covered with viable transplants of autologous or allogeneic skin, frozen or lyophilized human and animal skin, bioartificial tissues made of synthetic or biodegradable materials, sheets of keratinocytes cultured in vitro. The use of autotransplants is limited by the size of preserved skin areas as well as by general condition of the patient. Allotransplants collected from cadavers or volunteers are rejected after 1 or 2 weeks and thus afford only temporary coverage. Grafts of human or animal skin devitalized by lyophilization or freezing in glycerol accommodate connective tissue and blood vessels ingrowing from the graft bed but eventually dissolve. Artificial skin consists of collagen, chondroitin or similar fiber network (substituting the dermis) covered by semipermeable silicon foil (substituting the epidermis). After healing in, the silicon foil is peeled off and replaced by skin autotransplants or autologous keratinocytes grown and expanded in vitro. The technique for massive production of human keratinocytes, invented some twenty years ago, has been applied for clinical purposes by several specialized centers. During the culture period of approximately three weeks the keratinocyte population may enlarge five to ten thousandfold. Keratinocytes obtained from a 1.5 cm2 piece of skin (half of a postal stamp) may thus yield progeny sufficient for the coverage of 1.5 m2, which is almost the total body surface. The period required for culturing autologous keratinocytes is bridged by temporary transplants and vigorous supportive treatment of the patient. Cultured keratinocytes display all essential features of keratinocytes in situ. They divide and differentiate, express membrane structures required for intercellular communication and reception of signals regulating cell division and differentiation, secrete cytokines. In addition to clinical application, the culture of human keratinocytes is a convenient and useful model for studies of cellular biology. This review is illustrated by first examples of keratinocyte cultures grown in our laboratory.

Autopsy findings and clinical diagnoses: retrospective study of 3,117 autopsies.

AIM: To determine the extent of agreement between clinical and autopsy findings. METHODS: A 14-year retrospective study (1982-1995) of autopsies from the Departments of Internal Medicine and Surgery was performed at the Zagreb University Hospital Center, Croatia. The autopsy findings were compared with the clinical diagnoses. RESULTS: The autopsy rate among hospital deaths ranged between 23% and 33%. The overall rate of major discrepancies was 11.6% for all autopsy reports. The most common cause of death were cardiovascular diseases (40.9%), followed by malignancies (25.2%) and infections (12.9%). Among all cardiovascular diseases, myocardial infarction was the most frequently diagnosed (17.9%) and was misdiagnosed by clinicians in 16.5% of the cases. Incorrectly diagnosed malignancies were found in only 5.7% of the cases; hematological and lymphoid malignancies (48.8%) were the most common neoplasms and were usually confirmed before death. Infections were found in 46.9% of all autopsies. Bacterial pneumonias and peritonitis were overlooked in 67.5% and 23. 5% of the cases, respectively, in which they existed together with another serious condition. CONCLUSIONS: Modern technology has not improved the overall accuracy of clinical diagnoses. When an autopsy should be performed is still a matter of discussion.

Organotypic skin cultures: a human model for basic studies.

AIM: To produce organotypic skin cultures using human skin samples as a source of keratinocytes and fibroblasts. METHODS: Keratinocytes and fibroblasts from human skin samples were separated by warm trypsine and collagenase, respectively. Keratinocytes were plated in tissue culture dishes in keratinocyte serum free medium supplemented with epidermal growth factor and bovine pituitary extract, and were grown until confluence. Fibroblasts were cultured in Dulbecco's medium (DMEM) supplemented with fetal bovine serum and hydrocortisone. A mixture of fibroblasts, rat dermal collagen type I, E tissue culture medium, and reconstitution buffer were used as a dermal equivalent. Keratinocytes were plated on the top of the dermal equivalent and cultured for 10 days in organotypic culture dishes on stainless steel grids in the supplemented DMEM medium. The cultures were fixed in formaline, embedded in paraffin, stained with hematoxylin and eosin, and immunohistochemically stained with a nti-cytokeratin and anti-HLA-DR antibody. RESULTS: Cultured keratinocytes in organotypic skin cultures expressed the majority of the cytokeratins seen in the normal stratified epithelium. Consistent with previous studies, organotypic skin cultures did not show antigen-presenting Langerhans cells. CONCLUSION: Human skin from patients who underwent thoracic surgery can be used to produce organotypic skin cultures. This artificial skin can serve as a basis for future basic science studies and as a skin transplantation model.